U.S. patent application number 16/664199 was filed with the patent office on 2020-04-16 for compositions and methods for detecting human pegivirus 2 (hpgv-2).
The applicant listed for this patent is Abbott Laboratories The Regents of the University of California. Invention is credited to Michael Berg, Kevin Cheng, Charles Chiu, Kelly Coller, George Dawson, Kenn Forberg, Matthew Frankel, John R. Hackett, JR., Deanna Lee.
Application Number | 20200115767 16/664199 |
Document ID | / |
Family ID | 54929879 |
Filed Date | 2020-04-16 |
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United States Patent
Application |
20200115767 |
Kind Code |
A1 |
Chiu; Charles ; et
al. |
April 16, 2020 |
COMPOSITIONS AND METHODS FOR DETECTING HUMAN PEGIVIRUS 2
(HPgV-2)
Abstract
Provided herein are compositions, methods, and kits for
detecting human Pegivirus 2 (HPgV-2). In certain embodiments,
provided herein are HPgV-2 specific nucleic acid probes and
primers, and methods for detecting HPgV-2 nucleic acid. In other
embodiments, provided herein are HPgV-2 immunogenic composition
compositions, methods of treating a subject with immunogenic HPgV-2
peptides, and methods of detecting HPgV-2 subject antibodies in a
sample.
Inventors: |
Chiu; Charles; (San
Francisco, CA) ; Lee; Deanna; (San Francisco, CA)
; Berg; Michael; (Abbott Park, IL) ; Dawson;
George; (Abbott Park, IL) ; Coller; Kelly;
(Abbott Park, IL) ; Cheng; Kevin; (Abbott Park,
IL) ; Hackett, JR.; John R.; (Abbott Park, IL)
; Frankel; Matthew; (Abbott Park, IL) ; Forberg;
Kenn; (Abbott Park, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Abbott Laboratories
The Regents of the University of California |
Abbott Park
Oakland |
CA
CA |
US
US |
|
|
Family ID: |
54929879 |
Appl. No.: |
16/664199 |
Filed: |
October 25, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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15893120 |
Feb 9, 2018 |
10501816 |
|
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16664199 |
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15642992 |
Jul 6, 2017 |
9938589 |
|
|
15893120 |
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|
14752262 |
Jun 26, 2015 |
9777340 |
|
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15642992 |
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62018282 |
Jun 27, 2014 |
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62107782 |
Jan 26, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2333/183 20130101;
A61K 39/12 20130101; G01N 33/56983 20130101; C12N 2770/24021
20130101; C12N 2770/24034 20130101; G01N 2469/20 20130101; C07K
14/005 20130101; C12Q 1/701 20130101; A61P 31/14 20180101; C12N
2770/24022 20130101; A61K 49/085 20130101 |
International
Class: |
C12Q 1/70 20060101
C12Q001/70; A61K 49/08 20060101 A61K049/08; A61K 39/12 20060101
A61K039/12; C07K 14/005 20060101 C07K014/005; G01N 33/569 20060101
G01N033/569; A61P 31/14 20060101 A61P031/14 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under Grant
No. R01-HL105704 awarded by the National Institutes of Health. The
Government has certain rights in the invention.
Claims
1. A method for generating an immune response directed against a
human Pegivirus 2 (HPgV-2) infection in a subject comprising:
administering to a subject a composition comprising attenuated or
inactivated HPgV-2 particles, and/or an antigenic portion of said
HPgV-2, thereby generating an immune response in the subject
directed against said HPgV-2.
2. The method of claim 1, wherein said antigenic portion of said
HPgV-2 comprises a peptide, wherein said peptide comprises at least
a portion of said HPgV-2 selected from the group consisting of: the
S protein, the E1 protein, and the E2 protein.
3. The method of claim 1, wherein said antigenic portion of said
HPgV-2 comprises a peptide, wherein said peptide comprises at least
a portion of said HPgV-2 selected from the group consisting of: the
X protein, the NS2 protein, and the NS3 protein.
4. The method of claim 1, wherein said antigenic portion of said
HPgV-2 comprises a peptide, wherein said peptide comprises at least
a portion of said HPgV-2 selected from the group consisting of: the
NS4A protein, and the NS4B protein.
5. The method of claim 1, wherein said antigenic portion of said
HPgV-2 comprises a peptide, wherein said peptide comprises at least
a portion of said HPgV-2 selected from the group consisting of: the
NS5A protein, and the NS5B protein.
6. An immunogenic composition suitable for administration to a
subject comprising: a composition comprising attenuated or
inactivated HPgV-2 particles, and/or an antigenic portion of the
HPgV-2.
7. The composition of claim 6, wherein said antigenic portion of
said HPgV-2 comprises a peptide, wherein said peptide comprises at
least a portion of said HPgV-2 selected from the group consisting
of: the S protein, the E1 protein, and the E2 protein.
8. The composition of claim 6, wherein said antigenic portion of
said HPgV-2 comprises a peptide, wherein said peptide comprises at
least a portion of said HPgV-2 selected from the group consisting
of: the X protein, the NS2 protein, and the NS3 protein.
9. The composition of claim 6, wherein said antigenic portion of
said HPgV-2 comprises a peptide, wherein said peptide comprises at
least a portion of said HPgV-2 selected from the group consisting
of: the NS4A protein, and the NS4B protein.
10. The composition of claim 6, wherein said antigenic portion of
said HPgV-2 comprises a peptide, wherein said peptide comprises at
least a portion of said HPgV-2 selected from the group consisting
of: the NS5A protein, and the NS5B protein.
Description
[0001] The present application a continuation of U.S. application
Ser. No. 15/893,120, filed Feb. 9, 2018, which is a divisional of
U.S. application Ser. No. 15/642,992, filed Jul. 6, 2017, now U.S.
Pat. No. 9,938,589, issued Apr. 10, 2018, which is a continuation
of U.S. application Ser. No. 14/752,262, filed Jun. 26, 2015, now
U.S. Pat. No. 9,777,340, issued Oct. 3, 2017, which claims priority
to U.S. Provisional application Ser. No. 62/018,282, filed Jun. 27,
2014, and U.S. Provisional application Ser. No. 62/107,782, filed
Jan. 26, 2015, all of which are herein incorporated by reference in
their entireties.
FIELD OF THE INVENTION
[0003] Provided herein are compositions, methods, and kits for
detecting human Pegivirus 2 (HPgV-2). In certain embodiments,
provided herein are HPgV-2 specific nucleic acid probes and
primers, and methods for detecting HPgV-2 nucleic acid. In other
embodiments, provided herein are HPgV-2 immunogenic compositions,
methods of treating a subject with immunogenic portions of HPgV-2,
and methods of detecting HPgV-2 specific subject antibodies in a
sample.
BACKGROUND
[0004] Within the family Flaviviridae, viruses belonging to the
genus Hepacivirus have been shown to cause hepatitis (Hepatitis C
virus (HCV) and GB virus (GBV-B)). The newly defined genus
Pegivirus contains viruses similar to Hepaciviruses in genome
organization but distinct in tropism and associated pathogenicity
(Stapleton et al., J Gen Virol 2011: 92: 233-246). All members of
the family Flaviviridae contain a positive sense, single stranded
RNA genome of about 10 kb, that encodes for a single long open
reading frame (ORF) polyprotein of about 3,000 amino acids
(Lindenbach et al., Flaviviridae: The Viruses and Their
Replication. Chapter 33. In Fields Virology Fifth Edition, (Knipe
et al., Eds.) Wolters Kluwer/Lippincott Williams and Williams,
Philadelphia Pa. Pages 1101-1152). The polyprotein is cleaved into
smaller functional structural and nonstructural (NS) components by
a combination of host and viral proteases. The viral structural
proteins are encoded at the amino terminal portion of the genome
and include envelope glycoproteins and a nucleocapsid. While HCV
and GBV-B encode a nucleocapsid protein, HPgV-1 does not appear
encode a nucleocapsid protein in the polyprotein. Phylogenetic
analysis show distinct evolutionary lineages between the genera but
conserved amino acid motifs involved in the enzymatic functions of
the NS3 helicase and the NS5 RNA dependent RNA polymerase. The
genome is organized with 5' and 3' untranslated regions (UTRs) that
are highly conserved and that are involved both in translation and
in replication of the genome.
[0005] The Pegivirus genus, is named for the persistent (pe) GB
virus (g) infection that is not associated with a specific
pathogenicity. In 1995-1996, the first human pegivirus, GVB-C
(HPgV-1), was detected independently by two groups in sera from
patients with non-A, non-B hepatitis. Although originally
discovered in chronic hepatitis patients, HPgV-1 appears to be
lymphotropic, and not hepatotropic, and has not been associated
with hepatitis or any other clinical illness in follow-up clinical
and experimental studies. Some studies, however, have suggested
that co-infection with HPgV-1 may slow the progression of HIV
disease (Heringlake S, J Infect Dis 1998;177:1723-1726). Together
the incidence rate of HCV and HPgV-1 is estimated to be between
2-5% of the world's population (Stapleton et al., J Gen Virol 2011:
92: 233-246).
[0006] Pegiviruses infect a wide range of mammals, not limited to
chimpanzees, new world primates, bats, rodents, and horses.
Recently there have been viral discovery reports indicating the
novel hepaciviruses and pegiviruses have been identified in rodents
and that bats may be a natural reservoir for these genera of the
Flavivirdae (Quan et al., PNAS 110: 8194-8199. 2013: Drexler et
al., PLoS Pathog 9 (6) e1003438. 2013: Kapoor et al., mBlo 4(2)
e000216-13. 2013). The only pegiviruses previously known to infect
humans is HPgV-1. There is considerable sequence divergence between
pegivirus variants in the structural proteins and conservation
within the nonstructural NS3 and NS5B genes (Kapoor A, mBio. 2013
Mar-Apr; 4(2): e00216-13). Sampling of bats from different
continents shows several distinct bat-derived pegivirus lineages
suggesting bats are a natural reservoir for pegiviruses (Quan P,
Proc Natl Acad Sci U.S.A. May 14, 2013; 110(20): 8194-8199).
Characterization of HPgV-2 described in this patent shows the viral
variant is distinct from the other human-tropic virus HPgV-1.
[0007] Recently, it has been proposed that Theiler's disease, the
most common cause of acute hepatitis in horses, is likely to be
caused by TDAV (Theiler's Disease Associated Virus), a newly
described horse flavivirus, phylogenetically related to the GB
viruses (Chandriana et al., PNAS 110 (15): E 1407-1415. 2013) and
classified as a pegivirus. Thus, unlike the case for HPgV-1, where
there has been no clear association with disease, TDAV appears to
be causally related to hepatitis cases in horses.
SUMMARY OF THE INVENTION
[0008] Provided herein are compositions, methods, and kits for
detecting a human virus which has been termed "human Pegivirus 2"
(HPgV-2) based on certain homology to human Pegivirus 1. In certain
embodiments, provided herein are HPgV-2 specific nucleic acid
probes and primers, and methods for detecting HPgV-2 nucleic acid.
In other embodiments, provided herein are HPgV-2 immunogenic
compositions, methods of treating a subject with immunogenic HPgV-2
peptides, and methods of detecting HPgV-2 specific subject
antibodies in a sample.
[0009] In some embodiments, provided herein are compositions
comprising a synthetic nucleic acid molecule which comprises at
least 12 (e.g., at least 12 . . . 15 . . . 25 . . . 35 . . . 45 . .
. or 55) consecutive nucleotides from human Pegivirus 2 (HPgV-2),
and/or the encoded peptides from such nucleic acids, such as from
type UC0125.US (aka "index case"), ABT0070P.US, ABT0096P.US,
ABT0029A, ABT0239AN, ABT0055A, ABT0030P.US, ABT0041P.US,
ABT0188P.US, and/or ABT0128AUS.
[0010] In certain embodiments, provided herein are compositions
comprising a synthetic nucleic acid molecule, wherein said
synthetic nucleic acid molecule comprises a nucleotide sequence at
least 12 nucleotides in length (e.g., at least 12 . . . 15 . . . 18
. . . 27 . . . 35 . . . etc.) that hybridizes under stringent
conditions (e.g., highly stringent conditions) to region 1, region
2, region 3, or region 4 of a genomic sequence of human Pegivirus 2
(HPgV-2) or complement thereof, wherein the genomic sequence of
HPgV-2 is shown in SEQ ID NO:1, 75, and 299-303, and wherein region
1 is nucleotides 1-1401 of SEQ ID NO:1 or 75, region 2 is
nucleotides 1431-4777 of SEQ ID NO:1 or 75, region 3 is nucleotides
4818-8134 of SEQ ID NO:1 or 75, and region 4 is nucleotides
8167-9778 of SEQ ID NO: 1 or 75.
[0011] In particular embodiments, the synthetic nucleic acid
molecule is at least 15 nucleotides in length and no more than 75
nucleotides in length. In further embodiments, the synthetic
nucleic acid molecule comprises a detectable label (e.g.,
fluorescent label, chemiluminescent, enzymatic, etc.). In further
embodiments, the synthetic nucleic acid molecule comprises at least
one modified base (e.g., for nuclease resistance, higher binding
efficiency, etc.). In certain embodiments, all or nearly all of the
nucleotides are modified. In particular embodiments, at least one
modified base is selected from the group consisting of:
phosphorothioate, boranophosphate, 4'-thio-ribose, locked nucleic
acid, 2'-O-(2'-methoxyethyl), 2'-0-methyl, 2'-fluoro,
2'-deoxy-2'-fluoro-b-D-arabinonucleic acid, phosphonoacetate,
2'-3'-seco-RNA, Morpholino nucleic acid analog, Peptide nucleic
acid analog, phosphorodithioate, phosphoramidate,
methylphosphonate, 4-acetylcytosine, 8-hydroxy-N6-methyladenosine,
aziridinylcytosine, pseudoisocytosine,
5-(carboxyhydroxylmethyl)uracil, 5-fluorouracil, 5-bromouracil,
5-carboxymethylaminomethyl-2-thiouracil,
5-carboxymethylaminomethyluracil, dihydrouracil, inosine,
N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-methyladenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxy-aminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarbonylmethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,
oxybutoxosine, pseudouracil, queosine, 2-thiocytosine,
5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,
N-uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,
pseudouracil, queosine, 2-thiocytosine, 2,6-diaminopurine,
2-aminopurine, 5-amino-allyluracil, 5-hydroxymethylcytosine,
5-iodouracil, 5-nitroindole, 5-propynylcytosine, 5-propynyluracil,
hypoxanthine, N3-methyluracil, N6,N6-dimethyladenine, purine,
C-5-propynyl cytosine, C-5-propynyl uracil, and difluorotouyl.
[0012] In particular embodiments, the synthetic nucleic acid
molecule comprises DNA (e.g., the entire or nearly the entire
nucleic acid molecules is DNA). In further embodiments, the
composition further comprises a hybridization and/or amplification
buffer. In other embodiments, the synthetic nucleic acid molecule
is linked to a heterologous nucleic acid sequence (e.g., an
expression vector, a sequencing tag, a promoter, etc.).
[0013] In certain embodiments, the synthetic nucleic acid molecule
hybridizes to a portion of the HPgV-2 genome selected from the
group consisting of: the 5'UTR, the S gene, the E1 gene, the E2
gene, the X gene, and the NS2 gene. In particular embodiments, the
5' UTR is nucleotides 1-103 of SEQ ID NO:1, nucleotides 1-110 of
SEQ ID NO:75, nucleotides 1-327 of SEQ ID NOs:299-301, 419, and
430, nucleotides 12-327 of SEQ ID NO:302, and nucleotides 24-327 of
SEQ ID NO:303; said S gene is nucleotides 104-475 of SEQ ID NO:1,
nucleotides 111-482 of SEQ ID NO:75, or nucleotides 328-564 of SEQ
ID NOs: 299-303, 419, and 430; said E1 gene is nucleotides 476-1048
of SEQ ID NO:1, nucleotides 483-1055 of SEQ ID NO:75, or
nucleotides 565-1137 of SEQ ID NOs:299-303; said E2 gene is
nucleotides 1049-2110 of SEQ ID NO:1, nucleotides 1056-2117 of SEQ
ID NO:75, or nucleotides 1138-2199 of SEQ ID NOs:299-303, 419, and
430; said X gene is nucleotides 2111-2821 of SEQ ID NO:1,
nucleotides 2118-2828 of SEQ ID NO:75, or nucleotides 2200-2910 of
SEQ ID NOs:299-303, 419, and 430; and said NS2 gene is nucleotides
2822-3541 of SEQ ID NO:1, nucleotides 2829-3548 of SEQ ID NO:75, or
nucleotides 2911-3630 of SEQ ID NOs:299-303, 419, and 430. In
certain embodiments, the nucleic acid molecule hybridizes to a
portion of said HPgV-2 genome selected from the group consisting
of: the NS3 gene, the NS4A gene, the NS4B gene, the NS5A gene, the
NS5B gene, and the 3'UTR (e.g., as shown in any of the figures with
such sequences). In some embodiments, the NS3 gene is nucleotides
3542-5425 of SEQ ID NO:1, nucleotides 3549-5432 of SEQ ID NO:75, or
nucleotides 3631-5514 of SEQ ID NOs:299-303, 419, and 430; said
NS4A gene is nucleotides 5426-5548 of SEQ ID NO:1, nucleotides
5433-5555 of SEQ ID NO:75, or nucleotides 5515-5637 of SEQ ID
NOs:299-303, 419, and 430; said NS4B gene is nucleotides 5549-6334
of SEQ ID NO:1, nucleotides 5556-6341 of SEQ ID NO:75, or
nucleotides 5638-6423 of SEQ ID NOs:299-303, 419, and 430; said
NS5A gene is nucleotides 6335-7708 of SEQ ID NO:1, nucleotides
6342-7715 of SEQ ID NO:75, or nucleotides 6424-7797 of SEQ ID
NOs:299-303 and 430, or 6424-7794 of SEQ ID NO:419; said NS5B gene
is nucleotides 7709-9409 of SEQ ID NO:1, nucleotides 7716-9416 of
SEQ ID NO:75 or nucleotides 7798-9498 of SEQ ID NOs:299-303 and
430, or 7795-9495 of SEQ ID NO:419; and wherein at least a portion
of said 3'UTR is nucleotides 9410-9778 of SEQ ID NO:1,nucleotides
9417-9431 of SEQ ID NO:75, or nucleotides 9499-9867 of SEQ ID
NOs:299-303 and 430, or 9496-9864 of SEQ ID NO:419.
[0014] In certain embodiments, provided herein are compositions
comprising a synthetic nucleic acid molecule, wherein the synthetic
nucleic acid molecule comprises a nucleotide sequence at least 12
nucleotides in length (e.g., at least 12 . . . 15 . . . 25 . . . or
35 nucleotides in length) that: i) does not hybridize under
stringent conditions (e.g., highly stringent conditions) to three
regions of SEQ ID NO:1 or 75 selected from 1402-1430, 4778-4817,
and 8135-8166, and ii) does hybridize under stringent conditions
(e.g., highly stringent conditions) to: a) a 5' untranslated region
(5'UTR) of human Pegivirus 2 (HPgV-2) or complement thereof,
wherein the 5'UTR has a nucleic acid sequence as shown in
nucleotides 1-103 of SEQ ID NO:1 or nucleotides 1-110 of SEQ ID
NO:75; b) a first nucleic acid sequence, or complement thereof,
wherein the first nucleic acid sequence encodes a HPgV-2 S-protein
with the amino acid sequence shown in SEQ ID NO:2, 76, 304, 314,
324, 334, or 344; c) a second nucleic acid sequence, or complement
thereof, wherein the second nucleic acid sequence encodes a HPgV-2
E1 protein with the amino acid sequence shown in SEQ ID NO:3, 77,
305, 315, 325, 335, or 345; d) a third nucleic acid sequence, or
complement thereof, wherein the third nucleic acid sequence encodes
a HPgV-2 E2 protein with the amino acid sequence shown in SEQ ID
NO:4, 78, 306, 316, 326, 336, or 346; e) a fourth nucleic acid
sequence, or complement thereof, wherein the fourth nucleic acid
sequence encodes a HPgV-2 X-protein with the amino acid sequence
shown in SEQ ID NO:5, 79, 307, 317, 327, 337, or 347; f) a fifth
nucleic acid sequence, or complement thereof, wherein the fifth
nucleic acid sequence encodes a HPgV-2 NS2 protein with the amino
acid sequence shown in SEQ ID NO:6, 80, 308, 318, 328, 338, or 348;
g) a sixth nucleic acid sequence, or complement thereof, wherein
the sixth nucleic acid sequence encodes a HPgV-2 NS3 protein with
the amino acid sequence shown in SEQ ID NO:7, 81, 309, 319, 329,
339, or 349; h) a seventh nucleic acid sequence, or complement
thereof, wherein the seventh nucleic acid sequence encodes a HPgV-2
NS4A protein with the amino acid sequence shown in SEQ ID NO:8, 82,
310, 320, 330, 340, or 350; i) a eighth nucleic acid sequence, or
complement thereof, wherein the eighth nucleic acid sequence
encodes a HPgV-2 NS4B protein with the amino acid sequence shown in
SEQ ID NO:9, 83, 311, 321, 331, 341, or 351; j) a ninth nucleic
acid sequence, or complement thereof, wherein the ninth nucleic
acid sequence encodes a HPgV-2 NS5A protein with the amino acid
sequence shown in SEQ ID NO:10, 84, 312, 322, 332, 342, or 352; k)
a tenth nucleic acid sequence, or complement thereof, wherein the
tenth nucleic acid sequence encodes a HPgV-2 NS5B protein with the
amino acid sequence shown in SEQ ID NO:11, 85, 313, 323, 333, 343,
or 353; and 1) a 3' untranslated region (3'UTR) of Human Pegivirus
2 (HPgV-2) or complement thereof, wherein at least a portion of the
3'UTR has a nucleic acid sequence as shown in nucleotides 9410-9778
of SEQ ID NO:1, nucleotides 9417-9431 of SEQ ID NO:75, or
nucleotides 9499-9867 of SEQ ID NOs:299-303
[0015] In certain embodiments, the synthetic nucleic acid molecule
is at least 15 nucleotides in length and no more than 75
nucleotides in length. In further embodiments, the synthetic
nucleic acid molecule comprises a detectable label. In other
embodiments, the composition further comprises a hybridization
and/or amplification buffer. In some embodiments, the synthetic
nucleic acid molecule is linked to a heterologous nucleic acid
sequence. In other embodiments, the heterologous nucleic acid
sequence comprises an expression vector. In additional embodiments,
the synthetic nucleic acid molecule comprises at least one modified
base. In further embodiments, the synthetic nucleic acid molecules
comprises DNA.
[0016] In particular embodiments, provided herein are compositions
comprising a synthetic nucleic acid molecule, wherein said
synthetic nucleic acid molecule comprises a nucleotide sequence
that has at least 75% identity (e.g., at least 75% . . . 85% . . .
95% . . . 99% or 99.5%) to a portion of region 1, region 2, region
3, or region 4 of SEQ ID NO:1 or 75 or complement thereof, or to a
portion of SEQ ID NOs:299-303 or 354-356, wherein said portion is
at least 15 nucleotides in length (e.g., at least 15 . . . 25 . . .
37 . . . 48 . . . 57 . . . or 65 nucleotides in length), and
wherein region 1 is nucleotides 1-1401 of SEQ ID NO:1 or 75, region
2 is nucleotides 1431-4777 of SEQ ID NO:1 or 75, region 3 is
nucleotides 4818-8134 of SEQ ID NO:1 or 75, and region 4 is
nucleotides 8167-9778 of SEQ ID NO:1 or 75. In certain embodiments,
the portion is at least 15 nucleotides in length, but not more than
75 nucleotides in length. In other embodiments, the synthetic
nucleic acid molecule comprises a detectable label. In additional
embodiments, the composition further comprises a hybridization
and/or amplification buffer. In other embodiments, the synthetic
nucleic acid molecule is linked to a heterologous nucleic acid
sequence. In certain embodiments, the heterologous nucleic acid
sequence comprises an expression vector.
[0017] In some embodiments, provided herein are compositions
comprising a substantially purified recombinant peptide, wherein
the recombinant peptide comprises an amino acid sequence that has
at least 75% identity (e.g., at least 75% . . . 85% . . . 95% or
99% identity) to a portion of any one of SEQ ID NOs:2-11, 76-85,
304-353, 420-429, or 431-440, wherein the portion is at least 10
amino acids in length (e.g., at least 10 . . . 15 . . . 25 . . . or
35 amino acids in length). In particular embodiments, the
recombinant peptide is conjugated to a label (e.g., at detectable
label or a hapten). In other embodiments, the peptides are
glycosylated (e.g., an E2 glycosylated peptide). In further
embodiments, the compositions further comprise a physiologically
tolerable buffer suitable for injection into a mammal.
[0018] In some embodiments, described herein are methods for
detecting human Pegivirus 2 (HPgV-2) nucleic acid comprising: a)
contacting a sample suspected of containing HPgV-2 nucleic acid
with a nucleic acid molecule, wherein the nucleic acid molecule
comprises a nucleotide sequence at least 12 nucleotides in length
(e.g., at least 12 . . . 18 . . . 25 . . . 35 . . . or more) that
hybridizes under stringent conditions (e.g., highly stringent
conditions) to SEQ ID NOs:299-303, or to region 1, region 2, region
3, or region 4 of a genomic sequence of human Pegivirus 2 (HPgV-2)
or complement thereof, wherein the genomic sequence of HPgV-2 is
shown in SEQ ID NO:1 or 75, and wherein region 1 is nucleotides
1-1401 of SEQ ID NO:1 or 75, region 2 is nucleotides 1431-4777 of
SEQ ID NO:1 or 75, region 3 is nucleotides 4818-8134 of SEQ ID NO:1
or 75, and region 4 is nucleotides 8167-9778 of SEQ ID NO:1 or 75;
and b) detecting the presence or absence of hybridization of the
nucleic acid molecule to the HPgV-2 nucleic acid, wherein detecting
said presence of hybridization indicates the presence of the HPgV-2
nucleic acid in said sample.
[0019] In certain embodiments, the nucleotide sequence is at least
15 nucleotides in length and wherein the nucleic acid molecule is
no more than 75 nucleotides in length (e.g., 15 . . . 25 . . . 39 .
. . 54 . . . 68 . . . 75). In other embodiments, the nucleic acid
molecule comprises a detectable label. In further embodiments, the
nucleic acid molecule hybridizes to a portion of the HPgV-2 genome
selected from the group consisting of: the 5'UTR, the S gene, the
E1 gene, the E2 gene, the X gene, and the NS2 gene.
[0020] In additional embodiments, provided herein are methods for
detecting human Pegivirus 2 (HPgV-2) nucleic acid comprising: a)
contacting a sample suspected of containing HPgV-2 nucleic acid
with a first primer such that HPgV-2 amplification products are
produced, wherein the first primer comprises a nucleotide sequence
at least 12 nucleotides in length that hybridizes under stringent
conditions (e.g., highly stringent conditions) to SEQ ID
NOs:299-303, or to region 1, region 2, region 3, or region 4 of a
genomic sequence of human Pegivirus 2 (HPgV-2) or complement
thereof, (or, if a second primer is employed, the first primer
hybridizes under stringent conditions (e.g., highly stringent
conditions) to any portion of SEQ ID NO:1 or 75), wherein said
genomic sequence of HPgV-2 is shown, for example, in SEQ ID NO:1 or
75, and wherein region 1 is nucleotides 1-1401 of SEQ ID NO:1 or
75, region 2 is nucleotides 1431-4777 of SEQ ID NO:1 or 75, region
3 is nucleotides 4818-8134 of SEQ ID NO:1 or 75, and region 4 is
nucleotides 8167-9778 of SEQ ID NO:1 or 75; and b) detecting said
HPgV-2 amplification products, thereby detecting the presence of
the HPgV-2 nucleic acid in said sample (e.g., detecting type
UC0125.US, ABT0070P.US, ABT0096P.US, ABT0029A, ABT0239AN,
ABT0030P.US, ABT0041P.US, ABT0188P.US, and/or ABT0128A.US). In
certain embodiments, the methods further comprise contacting the
sample with a second primer that comprises a nucleotide sequence at
least 12 nucleotides in length that hybridizes to SEQ ID NOs:1, 75,
299-303, 419, 430, or complement thereof. In some embodiments, the
first and second primers together generate an amplicon that is
between 50 and 400 base pairs in length. In certain embodiments,
the amplified nucleic acid is sequenced (e.g., adapters are ligated
onto the amplified nucleic acid and it is subjected to sequencing
protocol).
[0021] In other embodiments, the detecting comprises sequencing the
HPgV-2 amplification products. In additional embodiments, the first
and/or second primer is at least 15 nucleotides in length and no
more than 75 nucleotides in length. In certain embodiments, the
first and/or second primer comprises a detectable label. In further
embodiments, the first primer hybridizes to a portion of the HPgV-2
genome selected from the group consisting of: the 5'UTR, the S
gene, the E1 gene, the E2 gene, the X gene, and the NS2 gene.
[0022] In certain embodiments, provided herein are methods for
detecting human Pegivirus 2 (HPgV-2) in a sample comprising: a)
contacting a sample suspected of containing human Pegivirus 2
(HPgV-2) with an antibody (e.g., biotin labeled antibody) that
specifically binds a portion of the HPgV-2 to form a
HPgV-2/antibody complex, wherein the antibody is a full antibody or
an antigen binding portion of a full antibody (Fab fragment or Fc
fragment); and b) detecting the presence of the HPgV-2/antibody
complex, thereby detecting the presence of the HPgV-2.
[0023] In certain embodiment, the HPgV-2 comprises the amino acid
sequences encoded by the nucleic acid sequence shown in SEQ ID
NO:1, 75, 299-303, 419, or 430. In further embodiments, the portion
of the HPgV-2 is part of a HPgV-2 protein selected from the group
consisting of: the S protein, the E1 protein, the E2 protein, the X
protein, the NS2 protein, the NS3 protein, the NS4A protein, the
NS4B protein, the NS5A protein, and the NS5B protein. In some
embodiments, the amino acid sequence of the S protein is as shown
in SEQ ID NO:2, 76, 304, 314, 324, 334, 334, 420, or 431, or a
variant having 90-99% amino acid sequence identity with SEQ ID
NO:2, 76, 304, 314, 324, 334, 344, 420, or 431. In additional
embodiments, the amino acid sequence of the E1 protein is as shown
in SEQ ID NO:3, 77, 305, 315, 325, 335, 345, 421, or 432, or a
variant having 90-99% amino acid sequence identity with SEQ ID
NO:3, 77, 305, 315, 325, 335, 345, 421, or 432. In further
embodiments, the amino acid sequence of the E2 protein is as shown
in SEQ ID NO:4, 78, 306, 316, 326, 336, 346, 422, 433, or a variant
having 90-99% amino acid sequence identity with SEQ ID NO:4, 78,
306, 316, 326, 336, 346, 422, or 433. In other embodiments, the
amino acid sequence of the X protein is as shown in SEQ ID NO:5,
79, 307, 317, 327, 337, 347, 423, 434, or a variant having 90-99%
amino acid sequence identity with SEQ ID NO:5, 79, 307, 317, 327,
337, 347, 423, or 434. In additional embodiments, the amino acid
sequence of the NS2 protein is as shown in SEQ ID NO:6, 80, 308,
318, 328, 338, 348, 424, 435, or a variant having 90-99% amino acid
sequence identity with SEQ ID NO:6,80, 308, 318, 328, 338, 348,
424, or 435. In additional embodiments, the amino acid sequence of
the NS3 protein is as shown in SEQ ID NO:7, 81, 309, 319, 329, 339,
349, 425, 436, or a variant having 90-99% amino acid sequence
identity with SEQ ID NO:7, 81, 309, 319, 329, 339, 349, 425, or
436. In additional embodiments, the amino acid sequence of the NS4A
protein is as shown in SEQ ID NO:8, 82, 310, 320, 330, 340, 350,
426, 437, or a variant having 90-99% amino acid sequence identity
with SEQ ID NOs:8, 82, 310, 320, 330, 340, 350, 426, or 437. In
some embodiments, the amino acid sequence of the NS4B protein is as
shown in SEQ ID NO:9, 83, 311, 321, 331, 341, 351, 427, 438, or a
variant having 90-99% amino acid sequence identity with SEQ ID
NO:9, 83, 311, 321, 331, 341, 351, 427, or 438. In further
embodiments, the amino acid sequence of the NS5A protein is as
shown in SEQ ID NO:10, 84, 312, 322, 332, 342, 352, 428, 439, or a
variant having 90-99% amino acid sequence identity with SEQ ID
NO:10, 84, 312, 322, 332, 342, 352, 428, or 439. In additional
embodiments, the amino acid sequence of the NS5B protein is as
shown in SEQ ID NO:11, 85, 313, 323, 333, 343, 353, 429, 440, or a
variant having 90-99% amino acid sequence identity with SEQ ID
NO:11, 85, 313, 323, 333, 343, 353, 429, or 440. In some
embodiments, the antibody is labeled. In certain embodiments, the
antigen binding portion of a full antibody comprises a Fab
fragment.
[0024] In some embodiments, provided herein are methods of
detecting human Pegivirus 2 (HPgV-2) infection in a subject
comprising: a) contacting a sample from a subject suspected of
containing a patient antibody to HPgV-2 with a peptide (or antibody
specific for the HPgV-2 antibody), wherein the peptide (or the
antibody specific for the HPgV-2 antibody) specifically binds the
patient antibody to form a complex; and b) detecting the presence
of the complex, thereby detecting the presence of past or present
HPgV-2 infection in the subject. In certain embodiments, the
peptide comprises or consists of at least part of the peptides
shown in SEQ ID NOs:86-99 or 100-218, 420-429, 431-440, or variants
thereof with 1 or 2 conservative amino acid changes, or with 1 or 2
non-conservative amino acid changes, or with 4 or more amino acid
changes. In certain embodiments, the peptide is glycosylated (e.g.,
an E2 glycosylated peptide).
[0025] In other embodiments, the peptide has at least 75% identity
(e.g., at least 75% . . . 85% . . . 95% . . . or 99% identity) to a
portion of any one of SEQ ID NOs:2-11, 76-85, 86-99, 100-218,
304-353, 420-429, or 431-440, and wherein the portion is at least
10 amino acids in length (e.g., at least 10 . . . 15 . . . 25 . . .
30 or 35 amino acids in length). In further embodiments, the
peptide is labeled. In certain embodiments, the peptide is labeled
and is free in solution to bind to the subject antibody to form a
complex. In certain embodiments, this complex is then bound, via
the label on the peptide to a solid support that has a moiety that
binds the label (e.g., streptavidin-biotin binding). In particular
embodiments, a secondary antibody is added that is able to bind to
the patient antibody in the complex. In other embodiments, the
peptide is labeled and free in solution to bind to: 1) antibodies
free solution which could then bind to an unlabeled peptide or
other antigen provided on the solid phase; 2) antibodies free in
solution which could then bind to an unlabeled peptide also free in
solid solution but containing a biotin molecule (or other moiety)
which can then be complexed to a solid phase containing a
biotin-binding molecule (e.g. streptavidin, neutravidin, antibodies
to biotin, etc.); or 3) an antibody/peptide complex present on the
solid phase.
[0026] In certain embodiments, provided herein are methods for
detecting human Pegivirus 2 (HPgV-2) infection in a subject
comprising: a) contacting a sample from a subject suspected of
containing a patient antibody to HPgV-2 with a peptide and a solid
support, wherein said peptide comprises a label, and wherein said
solid support comprises moieties that bind said label; and b)
incubating said sample under conditions such that: i) said peptide
specifically binds said patient antibody to form a complex, and ii)
said complex binds to said solid support via said label binding at
least one of said moieties; c) washing said solid support; d)
adding a detectably labeled secondary antibody capable of binding
said patient antibody in said complex; e) washing said solid
support; and f) detecting said the presence of said complex,
thereby detecting the presence of past or present HPgV-2 infection
in the subject. In certain embodiments, the label on said peptide
comprises biotin. In further embodiments, the moieties on said
solid support comprise avidin molecules. In other embodiments, the
solid support comprises beads.
[0027] In certain embodiments, the peptide comprises at least a
portion of the HPgV-2 selected from the group consisting of: the S
protein, the E1 protein, the E2 protein, the X protein, the NS2
protein, the NS3 protein, the NS4A protein, the NS4B protein, the
NS5A protein, and the NS5B protein. In some embodiments, the amino
acid sequence of the S protein is as shown in SEQ ID NO:2, 76, 304,
314, 324, 334, or 334, or a variant having 90-99% amino acid
sequence identity with SEQ ID NO:2, 76, 304, 314, 324, 334, or 344.
In additional embodiments, the amino acid sequence of the E1
protein is as shown in SEQ ID NO:3, 77, 305, 315, 325, 335, or 345,
or a variant having 90-99% amino acid sequence identity with SEQ ID
NO:3, 77, 305, 315, 325, 335, or 345. In further embodiments, the
amino acid sequence of the E2 protein is as shown in SEQ ID NO:4,
78, 306, 316, 326, 336, or 346, or a variant having 90-99% amino
acid sequence identity with SEQ ID NO:4, 78, 306, 316, 326, 336, or
346. In other embodiments, the amino acid sequence of the X protein
is as shown in SEQ ID NO:5, 79, 307, 317, 327, 337, or 347, or a
variant having 90-99% amino acid sequence identity with SEQ ID
NO:5, 79, 307, 317, 327, 337, or 347. In additional embodiments,
the amino acid sequence of the NS2 protein is as shown in SEQ ID
NO:6, 80, 308, 318, 328, 338, or 348, or a variant having 90-99%
amino acid sequence identity with SEQ ID NO:6,80, 308, 318, 328,
338, or 348. In additional embodiments, the amino acid sequence of
the NS3 protein is as shown in SEQ ID NO:7, 81, 309, 319, 329, 339,
or 349, or a variant having 90-99% amino acid sequence identity
with SEQ ID NO:7, 81, 309, 319, 329, 339, or 349. In additional
embodiments, the amino acid sequence of the NS4A protein is as
shown in SEQ ID NO:8, 82, 310, 320, 330, 340, or 350, or a variant
having 90-99% amino acid sequence identity with SEQ ID NOs:8, 82,
310, 320, 330, 340, or 350. In some embodiments, the amino acid
sequence of the NS4B protein is as shown in SEQ ID NO:9, 83, 311,
321, 331, 341, or 351, or a variant having 90-99% amino acid
sequence identity with SEQ ID NO:9, 83, 311, 321, 331, 341, or 351.
In further embodiments, the amino acid sequence of the NS5A protein
is as shown in SEQ ID NO:10, 84, 312, 322, 332, 342, or 352, or a
variant having 90-99% amino acid sequence identity with SEQ ID
NO:10, 84, 312, 322, 332, 342, or 352. In additional embodiments,
the amino acid sequence of the NS5B protein is as shown in SEQ ID
NO:11, 85, 313, 323, 333, 343, or 353, or a variant having 90-99%
amino acid sequence identity with SEQ ID NO:11, 85, 313, 323, 333,
343, or 353. In some embodiments, the antibody is labeled. In
certain embodiments, the antigen binding portion of a full antibody
comprises a Fab fragment.
[0028] In certain embodiments, provided herein are methods of
sequencing HPgV-2 nucleic acid comprising: a) treating a sample to
generate isolated HPgV-2 RNA; b) contacting said isolated HPgV-2
RNA with random primers, or primers specific to a region of said
HPgV-2 RNA, and amplifying such that a cDNA library is generated;
c) contacting said cDNA library with sequencing adapters under
conditions such that an adapter-conjugated library is generated;
and d) sequencing said adapter-conjugated cDNA library to at least
partially determine the nucleic acid sequence of said isolated
HPgV-2 RNA.
[0029] In some embodiments, provided herein are methods for
treating or preventing a human Pegivirus 2 (HPgV-2) infection in a
subject comprising: administering to a subject a composition
comprising attenuated or inactivated HPgV-2 particles, and/or an
antigenic portion of the HPgV-2, thereby generating an immune
response in the subject directed against the HPgV-2. In other
embodiments, the immune response is sufficient to prevent or treat
an infection by the HPgV-2. In some embodiments, the antigenic
portion of the HPgV-2 comprises a peptide, wherein the peptide
comprises at least a portion of the HPgV-2 selected from the group
consisting of: the S protein, the E1 protein, the E2 protein, the X
protein, the NS2 protein, the NS3 protein, the NS4A protein, the
NS4B protein, the NS5A protein, and the NS5B protein.
[0030] In other embodiments, provided herein are immunogenic
compositions suitable for administration to a subject comprising: a
composition comprising attenuated or inactivated HPgV-2 particles,
and/or an antigenic portion of the HPgV-2. In further embodiments,
the antigenic portion of the HPgV-2 comprises a peptide, wherein
the peptide wherein the peptide comprises at least a portion of the
HPgV-2 selected from the group consisting of: the S protein, the E1
protein, the E2 protein, the X protein, the NS2 protein, the NS3
protein, the NS4A protein, the NS4B protein, the NS5A protein, and
the NS5B protein.
[0031] In further embodiments, provided herein are kits or systems
for detecting human Pegivirus 2 (HPgV-2) comprising at least one of
the following components: a) a first composition comprising a first
synthetic nucleic acid molecule, wherein the first synthetic
nucleic acid molecule comprises a nucleotide sequence at least 12
nucleotides in length that hybridizes under stringent conditions to
SEQ ID NOs:209-303, 419, 430, or to region 1, region 2, region 3,
or region 4 of a genomic sequence of human Pegivirus 2 (HPgV-2) or
complement thereof, wherein the genomic sequence of HPgV-2 is shown
in SEQ ID NO:1 or 75, and wherein region 1 is nucleotides 1-1401 of
SEQ ID NO:1 or 75, region 2 is nucleotides 1431-4777 of SEQ ID NO:1
or 75, region 3 is nucleotides 4818-8134 of SEQ ID NO:1 or 75, and
region 4 is nucleotides 8167-9778 of SEQ ID NO:1 or 75; and b) a
second composition comprising a second synthetic nucleic acid
molecule, wherein the second synthetic nucleic acid molecule
comprises a nucleotide sequence that has at least 75% identity to a
portion of SEQ ID NOs:209-303, or to region 1, region 2, region 3,
or region 4 of SEQ ID NO:1 or 75 or complement thereof, wherein the
portion is at least 15 nucleotides in length (e.g., at least 15 . .
. 24 . . . 37 . . . . etc.), and wherein region 1 is nucleotides
1-1401 of SEQ ID NO:1 or 75, region 2 is nucleotides 1431-4777 of
SEQ ID NO:1 or 75, region 3 is nucleotides 4818-8134 of SEQ ID NO:1
or 75, and region 4 is nucleotides 8167-9778 of SEQ ID NO:1 or 75.
In some embodiments, the kits and systems further comprise an
additional component selected from the group consisting of: an
amplification buffer; reagents for sequencing, reagents for PCR,
written instructions for using the first or second synthetic
nucleic acid molecule; a liquid container for holding the first
and/or second composition; and a shipping container for holding the
liquid container.
[0032] In certain embodiments, provided herein are kits and systems
for detecting human Pegivirus 2 (HPgV-2) comprising a composition
comprising a substantially purified recombinant peptide, wherein
the recombinant peptide comprises an amino acid sequence that has
at least 75% identity (e.g., at least 75% . . . 85% . . . 95% . . .
99% identity) to a portion of any one of SEQ ID NOs:2-11, 76-85,
86-99, 100-218, 304-353, 420-429, and 431-440, and wherein the
portion is at least 10 amino acids in length (e.g., at least 10 . .
. 15 . . . 25 or 35 amino acids in length). In other embodiments,
the kits comprise an antibody (e.g., biotin labeled antibody)
specific to a patient's HPgV-2 antibody. In some embodiments, the
kits and systems further comprise an additional component selected
from the group consisting of: an immunoassay buffer; immunoassay
beads, chemiluminescent microparticles, a solid support (e.g., a
solid support capable of binding biotin, such as an avidin labeled
solid support), such as beads, with the recombinant peptide
attached thereto, reagents for a sandwich assay, written
instructions for using the composition to detect patient
antibodies; a liquid container for holding the composition; and a
shipping container for holding the liquid container.
[0033] In some embodiments, provided herein are methods of assaying
for an anti-HPgV-2 compound comprising: a) contacting a sample
containing a human Pegivirus 2 (HPgV-2) with a test compound; and
b) determining whether the test compound inhibits HPgV-2
replication, wherein inhibition of HPgV-2 replication indicates
that the test compound is an anti-HPgV-2 compound.
[0034] In certain embodiments, provided herein are compositions
comprising a substantially purified recombinant peptide, wherein
said recombinant peptide comprises at least one of the following:
a) a first amino acid sequence that comprises at least 17
consecutive amino acids (e.g., at least 17 . . . 24 . . . 35 . . .
or more) from the HPgV-2 NS3 protein; b) a second amino acid
sequence that comprises at least 13 consecutive amino acids from
the HPgV-2 NS5B protein (e.g., at least 13 . . . 17 . . . 25 . . .
35 or more); c) a third amino acid sequence that comprises at least
11 consecutive amino acids (e.g., at least 11 . . . 15 . . . 19 . .
. 25 . . . 35 or more) from the HPgV-2 NS2 protein; d) a fourth
amino acid sequence that comprises at least 8 consecutive amino
acids (e.g., at least 8 . . . 11 . . . 15 . . . 23 . . . 35 or
more) from the HPgV-2 NS4B protein; e) a fifth amino acid sequence
that comprises at least 5 consecutive amino acids from the HPgV-2
NS4a protein (e.g., at least 5 . . . 10 . . . 18 . . . 25 . . . 35
or more); f) a sixth amino acid sequence that comprises at least 6
consecutive amino acids (e.g., at least 6 . . . 12 . . . 18 . . .
25 . . . 35 or more) from the HPgV-2 S protein; g) a seventh amino
acid sequence that comprises at least 6 consecutive amino acids
(e.g., at least 6 . . . 12 . . . 17 . . . 25 . . . 35 or more) from
the HPgV-2 E1 protein; and h) an eighth amino acid sequence that
comprises at least 8 consecutive amino acid (e.g., at least 8 . . .
14 . . . 25 . . . 35 or more) from the HPgV-2 X protein. In
particular embodiments, the HPgV-2 NS3 protein is as shown in SEQ
ID NOs: 7, 81, 309, 319, 329, 339, and 349. In other embodiments,
the HPgV-2 NS5B protein is as shown in SEQ ID NOs: 11, 85, 313,
323, 333, 343, or 353. In additional embodiments, the HPgV-2 NS2
protein is as shown in SEQ ID NOs: 6, 80, 308, 318, 328, 338, or
348. In additional embodiments, the HPgV-2 NS4B protein is as shown
in SEQ ID NOs: 9,83, 311, 321, 331, 341, or 351. In further
embodiments, the HPgV-2 NS4a protein is as shown in SEQ ID NOs: 8,
82, 310, 320, 330, 340, or 350. In other embodiments, the HPgV-2 S
protein is as shown in SEQ ID NOs: 2, 76, 304, 314, 324, 334, or
344. In further embodiments, the HPgV-2 E1 protein is as shown in
SEQ ID NOs: 3, 77, 305, 315, 325, 335, or 345. In additional
embodiments, the HPgV-2 X protein is as shown in SEQ ID NOs: 5, 79,
307, 317, 327, 337, or 347.
[0035] In certain embodiments, the peptides and proteins described
herein are expressed recombinantly in prokaryotic cells. In other
embodiments, the peptides and proteins described herein (e.g., E1
and E1) are expressed recombinantly in mammalian cells.
[0036] In some embodiments, provided herein are methods (and
corresponding kits with recited components) for detection of HPgV-2
antigen and HPgV-2 antibody in a test sample comprising: a)
providing the following reagents: i) a solid phase capable of
binding to biotin, ii) biotinylated anti-HPgV-2 antibody for the
capture of an HPgV-2 antigen present said test sample; iii) a
biotinylated HPgV-2 antigen for the capture of anti-HPgV-2 antibody
in said test sample; and iv) a detectably labeled HPgV-2 antigen
for binding to anti-HPgV-2 antibody captured by the biotinylated
HPgV-2 antigen of (iii); and b) incubating the reagents of step (a)
under conditions to produce a reaction mixture that: (i) the
biotinylated anti-HPgV-2 antibody of (a)(ii) binds to said solid
phase through said biotin and specifically binds to HPgV-2 antigen
present in said test sample to produce an anti-HPgV-2
antibody-HPgV-2 antigen complex captured on said solid phase; (ii)
the biotinylated antigen of (a)(iii) binds to said solid phase
through said biotin and specifically binds to anti-HPgV-2
antibodies present in said test sample to produce an HPgV-2
antigen-anti-HPgV-2 antibody complex captured on said solid phase
and said detectably labeled HPgV-2 antigen of (a)(iv) specifically
binds to the anti-HPgV-2 antibody in said an HPgV-2
antigen-anti-HPgV-2 antibody complex captured on said solid phase;
c) isolating solid phase that comprises attached captured antibody,
and captured HPgV-2 antigen from unreacted test sample and
reagents, d) contacting the isolated solid phase with a detectably
labeled conjugate antibody that binds to said HPgV-2 antigen
captured in the an anti-HPgV-2-antibody-HPgV-2 antigen complex of
(b)(ii); and e) detecting the signal generated from the detectable
label moieties upon triggering of said signal, wherein presence of
said signal indicates presence of HPgV-2 in said test sample.
[0037] In certain embodiments, the methods (and corresponding kits)
further comprise providing: (v) a second biotinylated HPgV-2
antigen for the capture of anti-HPgV-2 antibody in said test sample
wherein said second HPgV-2 antigen is distinct from the HPgV-2
antigen in step (aiii); and (vi) a detectably labeled HPgV-2
antigen for binding to anti-HPgV-2 antibody captured by the
biotinylated HPgV2 antigen of (v); and (b) (iii) the biotinylated
antigen of (a)(v) binds to said solid phase through said biotin and
specifically binds to anti-HPgV-2 antibodies present in said test
sample to produce an HPgV-2 antigen-anti-HPgV-2 antibody complex
captured on said solid phase and said detectably labeled HPgV-2
antigen of (a)(vi) specifically binds to the anti-HPgV-2 antibody
in said an HPgV-2 antigen-anti-HPgV-2 antibody complex captured on
said solid phase. In certain embodiments, the methods further
comprises: (a) providing (vii) a third biotinylated HPgV-2 antigen
for the capture of anti-HPgV-2 antibody in said test sample wherein
said third HPgV-2 antigen is distinct from the HPgV-2 antigen in
step 1(a)(iii) or step 2(a)(v); and (viii) a detectably labeled
HPgV-2 antigen for binding to anti-HPgV-2 antibody captured by the
biotinylated HPgV-2 antigen of (vii); and (b) (iv) the biotinylated
antigen of (a)(vii) binds to said solid phase through said biotin
and specifically binds to anti-HPgV-2 antibodies present in said
test sample to produce an HPgV-2 antigen-anti-HPgV-2 antibody
complex captured on said solid phase and said detectably labeled
HPgV2 antigen of (a)(viii) specifically binds to the anti-HPgV-2
antibody in said an HPgV-2 antigen-anti-HPgV2 antibody complex
captured on said solid phase.
[0038] In certain embodiments, provided herein are methods for the
simultaneous detection of both HPgV-2 antigens and HPgV-2
antibodies in a test sample, wherein said combination assay
comprises: a) protein; a first capture antigen comprising a peptide
sequence of a first HPgV-2, b) a first detection antigen comprising
a peptide sequence of a first HPgV-2 protein and further comprising
a detectable label, c) a second capture antigen comprising an
antigenic sequence from a second HPgV-2 protein, d) a second
detection antigen comprising an antigenic sequence from a second
HPgV-2 protein and further comprising a detectable label, e) a
third capture antigen comprising an antigenic sequence from a third
HPgV-2 protein, f) a third detection antigen comprising an
antigenic sequence from a third HPgV-2 protein and further
comprising a detectable label, g) a first capture antibody, h) a
conjugate antibody comprising a detectable label, wherein said
capture antibody and said conjugate antibody specifically bind a
fourth HPgV-2 protein from said test sample, and said combination
assay is performed by: (i) contacting said test sample with said
capture antigen, said detection antigen, said capture antibody and
said conjugate antibody under conditions to allow: a) formation of
a sandwich complex between said first capture antigen and said
detection antigen and first anti-HPgV-2 antibody present in said
test sample; b) formation of a sandwich complex between said second
capture antigen and said second detection antigen and an
anti-HPgV-2 antibody against said second HPgV-2 protein present in
said test sample; c) formation of a sandwich complex between said
third capture antigen and said third detection antigen and an
anti-HPgV-2 antibody against said third HPgV-2 protein present in
said test sample; and d) formation of a complex between said
capture antibody, said conjugate antibody and an HPgV-2 antigen
present in said sample; and (ii) measuring a signal generated from
said detectable labels as a result of formation of said complexes,
thereby simultaneously detecting HPgV-2 antigens and HPgV-2
antibodies present in said sample.
[0039] In certain embodiments, the compositions and kits described
anywhere herein further comprise at least one reagent selected from
the group consisting of: microparticles (e.g., configured to bind a
label on the peptide), Na Pyrophosphate (e.g., pH 6.3), NaCl (e.g.,
about 0.9 M), EDTA, Sucrose, Tergitol 15-S-40, BME, Tergitol
15-S-9, Azide (e.g., 0.08%), Korasilon Antifoam (e.g., 1 ppm),
Bis-Tris buffer (e.g., pH 6.3), Sorbitol, Dextran, PVSA, BSA,
Benzethonium chloride, Heparin sodium salt, Sodium fluoride, Triton
X-100, Gentamycine, A56620, Glycine, Lauryl sulfobetaine, Palmityl
sulfobetaine, Stearyl sulfobetaine, C16TAB, C18TAB, CHAPS, Saponin,
Methyl Cellulose, Sodium Sulfite, Sodium azide, Urea, HCl, C12TAB,
Palmityl sulfobetaine, Stearyl sulfobetaine, Maltose, Citric acid,
2-Diethylaminoethanthiol, NaHCO3/Na2CO3, Laurylsulfobetaine, CHAPS,
NaOH, MES Buffer w/ Triton X-405, NaCl, BSA, Nipasept, Quinolone,
TRIS Buffer w/CKS protein, Yeast SOD, Triton X-405, Goat serum,
EDTA, Quinolone, Antifoam, Dentran sulfate, Proclin, Gentamicin
sulfate, labeled (e.g., acridinium labeled) anti-human IgG or IgM
monoclonal antibody, streptavidin labeled microparticles, NFDM,
SB3-14, PVSA (e.g., 0.8%), ACD, CPDA-1, CPD, CP2D, potassium
oxalate, sodium EDTA, potassium EDTA sodium citrate, heparin,
lithium heparin, sodium heparin, and sodium citrate.
[0040] In some embodiments, provided herein are methods of
detecting both human Pegivirus 2 (HPgV-2) and human Pegivirus 1
(HPgV-1; aka GVC-C) infection in a subject comprising: a)
contacting a sample from a subject suspected of containing a
subject antibody to HPgV-2 and a subject antibody to HPgV-1, with a
HPgV-2 derived peptide and a HPgV-1 derived peptide, wherein said
peptides specifically bind said subject antibodies to form a
complexes; and b) detecting the presence of said complexes, thereby
detecting the presence of past or present HPgV-2 and HPgV-1
infection in said subject. In certain embodiments, provided herein
are methods for detecting human Pegivirus 2 (HPgV-2) nucleic acid
and human Pegivirus 1 (HPgV-1; aka GBV-C) nucleic acid comprising:
a) contacting a sample suspected of containing HPgV-2 and HPgV-1
nucleic acid with: i) a first nucleic acid molecule at least 12
nucleotides in length that hybridizes under stringent conditions to
a nucleic acid sequence of HPgV-2, and ii) a second nucleic acid
molecule at least 12 nucleotides in length that hybridizes under
stringent conditions to a nucleic acid sequence of HPgV-1, and b)
detecting the presence or absence of hybridization of said first
and second nucleic acid molecules to said HPgV-2 and HPgV-1 nucleic
acid, wherein detecting said presence of hybridization indicates
the presence of said HPgV-2 and HPgV-1 nucleic acid in said sample.
In certain embodiments, the peptide and amino acid sequences for
detecting HPgV-1 and HPgV-1 subject antibodies are found in U.S.
Pat. No. 6,870,042 and Souza et al., J. Clin. Microbiol., 2006,
44(9):3105-3113.
[0041] In certain embodiments, provided herein are compositions
comprising an anti-HPgV-2 antibody. Such antibody can be generated
using any of the peptides described herein as an immunogen in a
host animal (e.g., mouse, rabbit, etc.), such that polyclonal or
monoclonal antibodies to HPgV-2 are generated.
DESCRIPTION OF THE FIGURES
[0042] FIGS. 1A-C provide a genomic nucleic acid sequence of an
HPgV-2 isolate called UC0125.US, which is labeled SEQ ID NO:1. It
is understood that the HPgV-2 genome is a positive strand RNA
sequence. SEQ ID NO:1 is shown with the uracils as thymine.
[0043] FIGS. 2A-C show the amino acid sequences and describes the
nucleic acid sequences of HPgV-2 index case UC0125.US. FIG. 2A
shows: 1) the amino acid sequence (SEQ ID NO:2) and describes the
nucleic acid sequence (nucleotides 104-475 of SEQ ID NO:1) of the S
protein of HPgV-2; 2) the amino acid sequence (SEQ ID NO:3) and
describes the nucleic acid sequences (nucleotides 476-1048 of SEQ
ID NO:1) of the E1 protein of HPgV-2; 3) the amino acid sequence
(SEQ ID NO:4) and describes the nucleic acid sequence (nucleotides
1049-2110 of SEQ ID NO:1) of the E2 protein of HPgV-2; and 4) the
amino acid sequence (SEQ ID NO:5) and describes the nucleic acid
sequence (nucleotides 2111-2821 of SEQ ID NO:1) of the X protein of
HPgV-2. FIG. 2B shows: 1) the amino acid sequence (SEQ ID NO:6) and
describes the nucleic acid sequences (nucleotides 2822-3541 of SEQ
ID NO:1) of the NS2 protein of HPgV-2; 2) the amino acid sequence
(SEQ ID NO:7) and describes the nucleic acid sequence (nucleotides
3542-5425 of SEQ ID NO:1) of the NS3 protein of HPgV-2; 3) the
amino acid sequence (SEQ ID NO:8) and describes the nucleic acid
sequence (nucleotides 5426-5548 of SEQ ID NO:1) of the NS4A protein
of HPgV-2; and 4) the amino acid sequence (SEQ ID NO:9) and
describes the nucleic acid sequences (nucleotides 5549-6334 of SEQ
ID NO:1) of the NS4B protein of HPgV-2. FIG. 2C shows: 1) the amino
acid sequence (SEQ ID NO:10) and describes the nucleic acid
sequence (nucleotides 6335-7708 of SEQ ID NO:1) of the NS5A protein
of HPgV-2; 2) the amino acid sequence (SEQ ID NO:11) and describes
the nucleic acid sequence (nucleotides 7709-9409 of SEQ ID NO:1) of
the NS5B protein of HPgV-2; 3) the 5' UTR nucleic acid sequence
(nucleotides 1-103 of SEQ ID NO:1) of HPgV-2; and 4) the nucleic
acid sequence (nucleotides 9410-9778 of SEQ ID NO:1) of the 3' UTR
of HPgV-2.
[0044] FIGS. 3A-N show an annotated version of the nucleic acid
sequence of SEQ ID NO:1 with the corresponding encoded amino acid
sequence below.
[0045] FIG. 4 shows a cloned portion of the HPgV-2 genome
representing nucleotides 3253-4512 of SEQ ID NO:1, along with seven
sets of primers and probes (underlined) used for qPCR in Example
3.
[0046] FIGS. 5A-B show the results of the qPCR TaqMan based
detection assays in Example 3 below. In particular, FIG. 5A shows
HPgV-2 primer/TaqMan probe sets (1-2-3-5-7; see FIG. 4 for
sequences and positions) were used to detect 10-fold serial
dilutions of the NS23Ex in vitro transcript and a 10-fold dilution
of the HPgV-2 index case (UC0125.US) RNA (highlighted in bold). The
lower right panel shows detection of 100 ng of NS23Ex and HPgV-2
RNA for each primer/probe set. FIG. 5B shows Ct values that were
normalized to set 1_100 ng results and plotted on a log scale to
estimate the amount of HPgV-2 RNA present in the index case.
Negative controls included in the experiment were: 1) water, 2)
pTRI (an irrelevant in vitro transcript), 3) CHU2725
(HIV+/GBV-C+sample), and 4) N-505 (HIV+/GBV-C-sample) indicate
there is no cross-reactivity with other infections (HIV,
GBV-C).
[0047] FIG. 6 shows the results of SYBR green qPCR assays that were
conducted using probe and primer sets 1, 2, 3, 4, 5, 7, and 15 and
44F (SEQ ID NO:12) and 342R (SEQ ID NO:13), which were used to
detect 10-fold serial dilutions of cDNA made from the NS23Ex in
vitro transcript (FIG. 6, curves A, B, C) and the HPgV-2 index
(UC0125.US) case RNAs (FIG. 6, curve D). Negative controls (FIG. 6,
curves E and F), N-505 (HIV(+)/GBV-C(-)) and water, were not
amplified. Each graph is labeled with the primer set that was
employed.
[0048] FIG. 7A shows the results of a TaqMan qPCR assay using
primer/probe set 3 (top panel) and primer/probe set 2 (bottom
panel), which detected isolates ABT0070P.US and ABT0096P.US in an
HCV(+) plasmapheresis donor plasma samples.
[0049] FIG. 7B shows results of an assay where RNA extracted from
American Red Cross blood donor plasma (HCV RNA+/antibody+) samples
were screened with TaqMan primer/probe sets 2 and 3 ABT0128A.US was
detected, but only by set 2 (bold).
[0050] FIG. 8 provides a schematic of an exemplary solution phase
capture assay that can be used to detect subject antibodies to
HPgV-2 in a sample. Both sample and biotinylated peptide (s) are
incubated together, followed by incubation with the streptavidin
coated solid phase support. Immune complexes are captured on the
solid phase support by the biotin linkage on the peptide (Step 1).
Immune complexes are detected indirectly by using a
chemiluminescent labeled human IgG (Step 2).
[0051] FIGS. 9A-C provide the genomic nucleic acid sequence of an
HPgV-2 isolate called ABT0070P.US, which is labeled SEQ ID NO:75.
It is understood that the HPgV-2 genome is a positive strand RNA
sequence. SEQ ID NO:75 is shown with the uracils as thymine.
[0052] FIGS. 10A-C show the amino acid sequences of various
proteins from HPgV-2 variant ABT0070P.US, as well as the
corresponding nucleic acid coding sequences. FIG. 10A shows: 1) the
amino acid sequence (SEQ ID NO:76) and describes the nucleic acid
sequence (nucleotides 111-482 of SEQ ID NO:75) of the S protein of
HPgV-2; 2)the amino acid sequence (SEQ ID NO:77) and describes the
nucleic acid sequences (nucleotides 483-1055 of SEQ ID NO:75) of
the E1 protein of HPgV-2; 3) the amino acid sequence (SEQ ID NO:78)
and describes the nucleic acid sequence (nucleotides 1056-2117 of
SEQ ID NO:75) of the E2 protein of HPgV-2; and 4) the amino acid
sequence (SEQ ID NO:79) and describes the nucleic acid sequence
(nucleotides 2118-2828 of SEQ ID NO:75) of the X protein of HPgV-2.
FIG. 10B shows: 1) the amino acid sequence (SEQ ID NO:80) and
describes the nucleic acid sequences (nucleotides 2829-3548 of SEQ
ID NO:75) of the NS2 protein of HPgV-2; 2) the amino acid sequence
(SEQ ID NO:81) and describes the nucleic acid sequence (nucleotides
3549-5432 of SEQ ID NO:75) of the NS3 protein of HPgV-2; 3) the
amino acid sequence (SEQ ID NO:82) and describes the nucleic acid
sequence (nucleotides 5433-5555 of SEQ ID NO:75) of the NS4A
protein of HPgV-2; and 4)the amino acid sequence (SEQ ID NO:83) and
describes the nucleic acid sequences (nucleotides 5556-6341 of SEQ
ID NO:75) of the NS4B protein of HPgV-2. FIG. 10C shows: 1) the
amino acid sequence (SEQ ID NO:84) and describes the nucleic acid
sequence (nucleotides 6342-7715 of SEQ ID NO:75) of the NS5A
protein of HPgV-2; 2) the amino acid sequence (SEQ ID NO:85) and
describes the nucleic acid sequence (nucleotides 7716-9416 of SEQ
ID NO:75) of the NS5B protein of HPgV-2; 3) describes the 5' UTR
nucleic acid sequence (nucleotides 1-110 of SEQ ID NO:75) of
HPgV-2; and 4) describes the nucleic acid sequence (nucleotides
9417-9431 of SEQ ID NO:75) of a portion of the 3' UTR of
HPgV-2.
[0053] FIGS. 11A-Y show an alignment of the genomes or partial
genomes of HPgV-2 variants UC0125.US, ABT0070P.US, ABT0096P.US (SEQ
ID NO:354), and ABT0128A.US (SEQ ID NO:355), along with a majority
consensus sequence (SEQ ID NO:356).
[0054] FIGS. 12A-C show phylogenetic trees of HPgV-2 (UC0125.US;
SEQ ID 1) and ABT0070P.US (SEQ ID 75) along with 29 representative
flaviviruses (GenBank accession numbers listed in Figure)
constructed in Geneious using the Jukes-Cantor model and neighbor
joining algorithm with 10,000 bootstrap replicates used to
calculate branch supports. These tree topologies were then refined
using a maximum likelihood Bayesian approach with MrBayes V3.2
software (1,000,000 sample trees, 10% of trees discarded as
burn-in, convergence defined at an average standard deviation of
<0.01). Each tree was rooted with dengue virus type 1 (DENV1)
and yellow fever virus (YFV) as outgroups. Analysis was performed
on entire (A) polyprotein sequences, as well as on (B) NS3 and (C)
NS5B proteins individually.
[0055] FIGS. 13A-C provide the genomic nucleic acid sequence of an
HPgV-2 consensus sequence, which is labeled SEQ ID NO:299. It is
understood that the HPgV-2 genome is a positive strand RNA
sequence. SEQ ID NO:299 is shown with the uracils as thymine.
[0056] FIGS. 14A-C show the amino acid sequences of various
proteins from a HPgV-2 consensus sequence, as well as the
corresponding nucleic acid coding sequences. FIG. 14A: 1) describes
the 5'UTR sequence (nucleotides 1-327 of SEQ ID NO:299); 2) shows
the amino acid sequence (SEQ ID NO:304) and describes the nucleic
acid sequence (nucleotides 328-564 of SEQ ID NO:299) of the S
protein of HPgV-2; 3) shows the amino acid sequence (SEQ ID NO:305)
and describes the nucleic acid sequence (nucleotides 565-1137 of
SEQ ID NO:299) of the E1 protein of HPgV-2; 4) shows the amino acid
sequence (SEQ ID NO:306) and describes the nucleic acid sequence
(nucleotides 1138-2199 of SEQ ID NO:299) of the E2 protein of
HPgV-2; and 5) shows the amino acid sequence (SEQ ID NO:307) and
describes the nucleic acid sequence (nucleotides 2200-2910 of SEQ
ID NO:299) of the X protein of HPgV-2. FIG. 14B shows: 1) the amino
acid sequence (SEQ ID NO:308) and describes the nucleic acid
sequences (nucleotides 2911-3630 of SEQ ID NO:299) of the NS2
protein of HPgV-2; 2) the amino acid sequence (SEQ ID NO:309) and
describes the nucleic acid sequence (nucleotides 3631-5514 of SEQ
ID NO:299) of the NS3 protein of HPgV-2; 3) the amino acid sequence
(SEQ ID NO:310) and describes the nucleic acid sequence
(nucleotides 5515-5637 of SEQ ID NO:299) of the NS4A protein of
HPgV-2; and 4) the amino acid sequence (SEQ ID NO:311) and
describes the nucleic acid sequences (nucleotides 5638-6423 of SEQ
ID NO:299) of the NS4B protein of HPgV-2. FIG. 14C shows: 1) the
amino acid sequence (SEQ ID NO:312) and describes the nucleic acid
sequence (nucleotides 6424-7797 of SEQ ID NO:299) of the NS5A
protein of HPgV-2; 2) the amino acid sequence (SEQ ID NO:313) and
describes the nucleic acid sequence (nucleotides 7798-9498 of SEQ
ID NO:299) of the NS5B protein of HPgV-2; and 3) describes the
nucleic acid sequence (nucleotides 9499-9867 of SEQ ID NO:299) of
at least a portion of the 3' UTR of HPgV-2.
[0057] FIGS. 15A-C provide the genomic nucleic acid sequence of an
HPgV-2 isolate called ABT0070P, which is labeled SEQ ID NO:300. It
is understood that the HPgV-2 genome is a positive strand RNA
sequence. SEQ ID NO:300 is shown with the uracils as thymine.
[0058] FIGS. 16A-C show the amino acid sequences of various
proteins from an HPgV-2 isolate called ABT0070P, as well as the
corresponding nucleic acid coding sequences. FIG. 16A: 1) describes
the 5'UTR sequence (nucleotides 1-327 of SEQ ID NO:300); 2) shows
the amino acid sequence (SEQ ID NO:314) and describes the nucleic
acid sequence (nucleotides 328-564 of SEQ ID NO:300) of the S
protein of HPgV-2; 3) shows the amino acid sequence (SEQ ID NO:315)
and describes the nucleic acid sequence (nucleotides 565-1137 of
SEQ ID NO:300) of the E1 protein of HPgV-2; 4) shows the amino acid
sequence (SEQ ID NO:316) and describes the nucleic acid sequence
(nucleotides 1138-2199 of SEQ ID NO:300) of the E2 protein of
HPgV-2; and 5) shows the amino acid sequence (SEQ ID NO:317) and
describes the nucleic acid sequence (nucleotides 2200-2910 of SEQ
ID NO:300) of the X protein of HPgV-2. FIG. 16B shows: 1) the amino
acid sequence (SEQ ID NO:318) and describes the nucleic acid
sequences (nucleotides 2911-3630 of SEQ ID NO:300) of the NS2
protein of HPgV-2; 2) the amino acid sequence (SEQ ID NO:319) and
describes the nucleic acid sequence (nucleotides 3631-5514 of SEQ
ID NO:300) of the NS3 protein of HPgV-2; 3) the amino acid sequence
(SEQ ID NO:320) and describes the nucleic acid sequence
(nucleotides 5515-5637 of SEQ ID NO:300) of the NS4A protein of
HPgV-2; and 4) the amino acid sequence (SEQ ID NO:321) and
describes the nucleic acid sequences (nucleotides 5638-6423 of SEQ
ID NO:300) of the NS4B protein of HPgV-2. FIG. 16C shows: 1) the
amino acid sequence (SEQ ID NO:322) and describes the nucleic acid
sequence (nucleotides 6424-7797 of SEQ ID NO:300) of the NS5A
protein of HPgV-2; 2) the amino acid sequence (SEQ ID NO:323) and
describes the nucleic acid sequence (nucleotides 7798-9498 of SEQ
ID NO:300) of the NS5B protein of HPgV-2; and 3) describes the
nucleic acid sequence (nucleotides 9499-9867 of SEQ ID NO:300) of
at least a portion of the 3' UTR of HPgV-2.
[0059] FIG. 17A-C provides the genomic nucleic acid sequence of an
HPgV-2 isolate called ABT0029A, which is labeled SEQ ID NO:301. It
is understood that the HPgV-2 genome is a positive strand RNA
sequence. SEQ ID NO:301 is shown with the uracils as thymine.
[0060] FIGS. 18A-C show the amino acid sequences of various
proteins from an HPgV-2 isolate called ABT0029A, as well as the
corresponding nucleic acid coding sequences. FIG. 18A: 1) describes
the 5'UTR sequence (nucleotides 1-327 of SEQ ID NO:301); 2) shows
the amino acid sequence (SEQ ID NO:324) and describes the nucleic
acid sequence (nucleotides 328-564 of SEQ ID NO:301) of the S
protein of HPgV-2; 3) shows the amino acid sequence (SEQ ID NO:325)
and describes the nucleic acid sequence (nucleotides 565-1137 of
SEQ ID NO:301) of the E1 protein of HPgV-2; 4) shows the amino acid
sequence (SEQ ID NO:326) and describes the nucleic acid sequence
(nucleotides 1138-2199 of SEQ ID NO:301) of the E2 protein of
HPgV-2; and 5) shows the amino acid sequence (SEQ ID NO:327) and
describes the nucleic acid sequence (nucleotides 2200-2910 of SEQ
ID NO:301) of the X protein of HPgV-2. FIG. 18B shows: 1) the amino
acid sequence (SEQ ID NO:328) and describes the nucleic acid
sequences (nucleotides 2911-3630 of SEQ ID NO:301) of the NS2
protein of HPgV-2; 2) the amino acid sequence (SEQ ID NO:329) and
describes the nucleic acid sequence (nucleotides 3631-5514 of SEQ
ID NO:301) of the NS3 protein of HPgV-2; 3) the amino acid sequence
(SEQ ID NO:330) and describes the nucleic acid sequence
(nucleotides 5515-5637 of SEQ ID NO:301) of the NS4A protein of
HPgV-2; and 4) the amino acid sequence (SEQ ID NO:331) and
describes the nucleic acid sequences (nucleotides 5638-6423 of SEQ
ID NO:301) of the NS4B protein of HPgV-2. FIG. 18C shows: 1) the
amino acid sequence (SEQ ID NO:332) and describes the nucleic acid
sequence (nucleotides 6424-7797 of SEQ ID NO:301) of the NS5A
protein of HPgV-2; 2) the amino acid sequence (SEQ ID NO:333) and
describes the nucleic acid sequence (nucleotides 7798-9498 of SEQ
ID NO:301) of the NS5B protein of HPgV-2; and 3) describes the
nucleic acid sequence (nucleotides 9499-9867 of SEQ ID NO:301) of
at least a portion of the 3' UTR of HPgV-2.
[0061] FIGS. 19A-C provide the genomic nucleic acid sequence of an
HPgV-2 isolate called ABT0239AN.US, which is labeled SEQ ID NO:302.
It is understood that the HPgV-2 genome is a positive strand RNA
sequence. SEQ ID NO:302 is shown with the uracils as thymine.
[0062] FIGS. 20A-C show the amino acid sequences of various
proteins from an HPgV-2 isolate called ABT0239AN.US, as well as the
corresponding nucleic acid coding sequences. FIG. 20A: 1) describes
the 5'UTR sequence (nucleotides 1-327 of SEQ ID NO:302); 2) shows
the amino acid sequence (SEQ ID NO:334) and describes the nucleic
acid sequence (nucleotides 328-564 of SEQ ID NO:302) of the S
protein of HPgV-2; 3) shows the amino acid sequence (SEQ ID NO:335)
and describes the nucleic acid sequence (nucleotides 565-1137 of
SEQ ID NO:302) of the E1 protein of HPgV-2; 4) shows the amino acid
sequence (SEQ ID NO:336) and describes the nucleic acid sequence
(nucleotides 1138-2199 of SEQ ID NO:302) of the E2 protein of
HPgV-2; and 5) shows the amino acid sequence (SEQ ID NO:337) and
describes the nucleic acid sequence (nucleotides 2200-2910 of SEQ
ID NO:302) of the X protein of HPgV-2. FIG. 20B shows: 1) the amino
acid sequence (SEQ ID NO:338) and describes the nucleic acid
sequences (nucleotides 2911-3630 of SEQ ID NO:302) of the NS2
protein of HPgV-2; 2) the amino acid sequence (SEQ ID NO:339) and
describes the nucleic acid sequence (nucleotides 3631-5514 of SEQ
ID NO:302) of the NS3 protein of HPgV-2; 3) the amino acid sequence
(SEQ ID NO:340) and describes the nucleic acid sequence
(nucleotides 5515-5637 of SEQ ID NO:302) of the NS4A protein of
HPgV-2; and 4) the amino acid sequence (SEQ ID NO:341) and
describes the nucleic acid sequences (nucleotides 5638-6423 of SEQ
ID NO:302) of the NS4B protein of HPgV-2. FIG. 20C shows: 1) the
amino acid sequence (SEQ ID NO:342) and describes the nucleic acid
sequence (nucleotides 6424-7797 of SEQ ID NO:302) of the NS5A
protein of HPgV-2; 2) the amino acid sequence (SEQ ID NO:343) and
describes the nucleic acid sequence (nucleotides 7798-9498 of SEQ
ID NO:302) of the NS5B protein of HPgV-2; and 3) describes the
nucleic acid sequence (nucleotides 9499-9867 of SEQ ID NO:302) of
at least a portion of the 3' UTR of HPgV-2.
[0063] FIG. 21A-C provide the genomic nucleic acid sequence of an
HPgV-2 isolate called UC0125.US, which is labeled SEQ ID NO:303,
and which is an extended version of UC0125 (SEQ ID NO:1) provided
in FIG. 1 (e.g., 5' UTR was extended). It is understood that the
HPgV-2 genome is a positive strand RNA sequence. SEQ ID NO:303 is
shown with the uracils as thymine.
[0064] FIGS. 22A-C show the amino acid sequences of various
proteins from an HPgV-2 isolate called UC0125.US, as well as the
corresponding nucleic acid coding sequences. FIG. 22A: 1) describes
the 5'UTR sequence (nucleotides 1-327 of SEQ ID NO:303); 2) shows
the amino acid sequence (SEQ ID NO:344) and describes the nucleic
acid sequence (nucleotides 328-564 of SEQ ID NO:303) of the S
protein of HPgV-2; 3) shows the amino acid sequence (SEQ ID NO:345)
and describes the nucleic acid sequence (nucleotides 565-1137 of
SEQ ID NO:303) of the E1 protein of HPgV-2; 4) shows the amino acid
sequence (SEQ ID NO:346) and describes the nucleic acid sequence
(nucleotides 1138-2199 of SEQ ID NO:303) of the E2 protein of
HPgV-2; and 5) shows the amino acid sequence (SEQ ID NO:347) and
describes the nucleic acid sequence (nucleotides 2200-2910 of SEQ
ID NO:303) of the X protein of HPgV-2. FIG. 22B shows: 1) the amino
acid sequence (SEQ ID NO:348) and describes the nucleic acid
sequences (nucleotides 2911-3630 of SEQ ID NO:303) of the NS2
protein of HPgV-2; 2) the amino acid sequence (SEQ ID NO:349) and
describes the nucleic acid sequence (nucleotides 3631-5514 of SEQ
ID NO:303) of the NS3 protein of HPgV-2; 3) the amino acid sequence
(SEQ ID NO:350) and describes the nucleic acid sequence
(nucleotides 5515-5637 of SEQ ID NO:303) of the NS4A protein of
HPgV-2; and 4) the amino acid sequence (SEQ ID NO:351) and
describes the nucleic acid sequences (nucleotides 5638-6423 of SEQ
ID NO:303) of the NS4B protein of HPgV-2. FIG. 22C shows: 1) the
amino acid sequence (SEQ ID NO:352) and describes the nucleic acid
sequence (nucleotides 6424-7797 of SEQ ID NO:303) of the NS5A
protein of HPgV-2; 2) the amino acid sequence (SEQ ID NO:353) and
describes the nucleic acid sequence (nucleotides 7798-9498 of SEQ
ID NO:303) of the NS5B protein of HPgV-2; and 3) describes the
nucleic acid sequence (nucleotides 9499-9867 of SEQ ID NO:303) of
at least a portion of the 3' UTR of HPgV-2.
[0065] FIGS. 23A-EE show an alignment of the genomes or partial
genomes of HPgV-2 variants UC0125, ABT0070P, ARC29v36 (ABT0029A),
ARCNAT239 (ABT0239AN), ARC128 (ABT0128A), PMDx96 (ABT0096P), and
ARC55 (ABT0055A), along with a majority consensus sequence (SEQ ID
NO:299).
[0066] FIG. 24A shows anti-His Western blot of 293F cells
transfected with HPgV-2 E-E2 expression plasmid. Supernatants were
collected and concentrated. Cells were lysed in sample buffer
(2.times. Laemmli sample buffer+2-mercaptoethanol (BioRad,
Hercules, Calif., USA). Samples were run on a 4-12% SDS-PAGE gel
and transferred to a PDVF membrane. Western blot was performed
using the Western Breeze kit and an anti-His (C-term)/Alkaline
phosphatase primary antibody (Novex by Life Technologies,Carlsbad,
Calif., USA).
[0067] FIG. 24B shows purification of HPgV-2 E2. Concentrated
HPgV-2 E2 supernatant was run over a Nickel column and eluted with
250 mM imidazole. Fraction of pre-column concentrate, flow-through,
washes, and eluted protein were diluted 1:2 with Laemelli sample
buffer containing beta-mercaptoethanol and run on a 4-12% SDS-PAGE
gel followed by visualizing with Oriole protein stain (BioRad,
Hercules, Calif., USA). Arrow indicates purified HPgV-2 E2.
[0068] FIG. 24C shows western blot of purified HPgV-2 E2. Fractions
diluted 1:2 in Laemelli sample buffer with beta-mercaptoethanol and
run on 4-12% SDS-PAGE gel as above and transferred to PDVF
membrane. Western blot was performed using the Western Breeze kit
and an anti-His (C-term)/Alkaline phosphatase primary antibody
(Novex by Life Technologies, Carlsbad, Calif., USA). Arrow
indicates purified HPgV-2 E2.
[0069] FIG. 24D shows PNGase F removal of HPgV-2 E2 glycosylation.
Purified HPgV-2 E2 was denatured and incubated with PNGase F
followed by resolution by electrophoresis on a 10% SDS-PAGE
gel.
[0070] FIG. 25 shows seroreactivity of HPgV-2 PCR+ samples for
glycoprotein E2. Purified HPgV-2 E2 was bound to nitrocellulose
membrane and probed with a 1:100 dilution of serum (samples
indicated above). Strip blots were washed and visualized using a
goat-anti-Human alkaline phosphatase secondary antibody and
BCIP/NBT chromagen substrate. All samples were reactive with human
IgG on the membrane but only the HPgV-2 PCR+ samples were reactive
with purified E2. Samples ABT0096P, ABT0070P, ABT0188P were from
ProMedDx and ABT0055A was from the American Red Cross (ARC).
[0071] FIG. 26 shows blocking immunoreactivity on slot blot with
homologous, not heterologous, proteins. 15 .mu.l of sample ABT0055A
was incubated with PBS, 10 .mu.g of HPgV-2 E2, or 10 .mu.g of
HPgV-1 E2 in a total of 100 .mu.l of sample diluent (20 mM
TRIS-HCl, 0.5M NaCl, 0.3% Tween-20 pH 8, 5% non-fat dry milk, 10%
heat inactivated newborn bovine serum) for 1 hour at room
temperature (25.degree. C.) with rotation. Samples were diluted
with 1.4 mls of sample diluent then incubated with pre-made slot
blots bound with human IgG, HPgV-2 E2 (10 .mu.g and 100 .mu.g), and
HPgV-1 E2 (10 .mu.g and 100 .mu.g). Bound antibodies were detected
with an anti-human IgG conjugated to alkaline phosphatase.
[0072] FIG. 27 shows amples ABT0055A, ABT0096P, ABT0188P were
incubated with nitrocellulose membrane containing bound NS4AB
protein. Antibodies against the indicated proteins are shown as
dark lines for each sample.
[0073] FIG. 28 shows the results of a newly designed Tri-plex
mastermix for qPCR screening described in the Materials and
Methods. A panel of 100 HIV positive specimens obtained from
ProMedDx was extracted on the m2000sp and RNA was combined with the
Tri-plex mastermix for thermocycling on the m2000rt. Six specimens
were positive for the HPgV-1 (GBV-C) RNA through detection of it
5'UTR in the VIC channel. Four identical specimens were positive
for HPgV-2 RNA and detected in the CY5 (5'UTR) and FAM (NS2)
channels. These four specimens (PMDx30, 33, 35, 41) represent two
bleeds from the same patients (e.g. 30=33, 35=41). Sanger
sequencing confirmed the presence of HPgV-2 RNA and the identical
nature of sequences from same patients.
[0074] FIGS. 29A-C provide the genomic nucleic acid sequence of an
HPgV-2 isolate called ABT0030P.US, which is labeled SEQ ID NO:419.
It is understood that the HPgV-2 genome is a positive strand RNA
sequence. SEQ ID NO:419 is shown with the uracils as thymine.
[0075] FIGS. 30A-C show the amino acid sequences of various
proteins from an HPgV-2 isolate called ABT0030P.US, as well as the
corresponding nucleic acid coding sequences. FIG. 30A: 1) describes
the 5'UTR sequence (nucleotides 1-327 of SEQ ID NO:419); 2) shows
the amino acid sequence (SEQ ID NO:420) and describes the nucleic
acid sequence (nucleotides 328-564 of SEQ ID NO:419) of the S
protein of HPgV-2; 3) shows the amino acid sequence (SEQ ID NO:421)
and describes the nucleic acid sequence (nucleotides 565-1137 of
SEQ ID NO:419) of the E1 protein of HPgV-2; 4) shows the amino acid
sequence (SEQ ID NO:422) and describes the nucleic acid sequence
(nucleotides 1138-2199 of SEQ ID NO:419) of the E2 protein of
HPgV-2; and 5) shows the amino acid sequence (SEQ ID NO:423) and
describes the nucleic acid sequence (nucleotides 2200-2910 of SEQ
ID NO:419) of the X protein of HPgV-2. FIG. 30B shows: 1) the amino
acid sequence (SEQ ID NO:424) and describes the nucleic acid
sequences (nucleotides 2911-3630 of SEQ ID NO:419) of the NS2
protein of HPgV-2; 2) the amino acid sequence (SEQ ID NO:425) and
describes the nucleic acid sequence (nucleotides 3631-5514 of SEQ
ID NO:419) of the NS3 protein of HPgV-2; 3) the amino acid sequence
(SEQ ID NO:426) and describes the nucleic acid sequence
(nucleotides 5515-5637 of SEQ ID NO:419) of the NS4A protein of
HPgV-2; and 4) the amino acid sequence (SEQ ID NO:427) and
describes the nucleic acid sequences (nucleotides 5638-6423 of SEQ
ID NO:419) of the NS4B protein of HPgV-2. FIG. 30C shows: 1) the
amino acid sequence (SEQ ID NO:428) and describes the nucleic acid
sequence (nucleotides 6424-7794 of SEQ ID NO:419) of the NS5A
protein of HPgV-2; 2) the amino acid sequence (SEQ ID NO:429) and
describes the nucleic acid sequence (nucleotides 7795-9495 of SEQ
ID NO:419) of the NS5B protein of HPgV-2; and 3) describes the
nucleic acid sequence (nucleotides 9496-9864 of SEQ ID NO:419) of
at least a portion of the 3' UTR of HPgV-2.
[0076] FIGS. 31A-C provide the genomic nucleic acid sequence of an
HPgV-2 isolate called ABT0030P.US, which is labeled SEQ ID NO:430.
It is understood that the HPgV-2 genome is a positive strand RNA
sequence. SEQ ID NO:430 is shown with the uracils as thymine.
[0077] FIGS. 32A-C show the amino acid sequences of various
proteins from an HPgV-2 isolate called ABT0041P.US, as well as the
corresponding nucleic acid coding sequences. FIG. 32A: 1) describes
the 5'UTR sequence (nucleotides 1-327 of SEQ ID NO:430); 2) shows
the amino acid sequence (SEQ ID NO:431) and describes the nucleic
acid sequence (nucleotides 328-564 of SEQ ID NO:430) of the S
protein of HPgV-2; 3) shows the amino acid sequence (SEQ ID NO:432)
and describes the nucleic acid sequence (nucleotides 565-1137 of
SEQ ID NO:430) of the E1 protein of HPgV-2; 4) shows the amino acid
sequence (SEQ ID NO:433) and describes the nucleic acid sequence
(nucleotides 1138-2199 of SEQ ID NO:430) of the E2 protein of
HPgV-2; and 5) shows the amino acid sequence (SEQ ID NO:434) and
describes the nucleic acid sequence (nucleotides 2200-2910 of SEQ
ID NO:430) of the X protein of HPgV-2. FIG. 32B shows: 1) the amino
acid sequence (SEQ ID NO:435) and describes the nucleic acid
sequences (nucleotides 2911-3630 of SEQ ID NO:430) of the NS2
protein of HPgV-2; 2) the amino acid sequence (SEQ ID NO:436) and
describes the nucleic acid sequence (nucleotides 3631-5514 of SEQ
ID NO:430) of the NS3 protein of HPgV-2; 3) the amino acid sequence
(SEQ ID NO:437) and describes the nucleic acid sequence
(nucleotides 5515-5637 of SEQ ID NO:430) of the NS4A protein of
HPgV-2; and 4) the amino acid sequence (SEQ ID NO:438) and
describes the nucleic acid sequences (nucleotides 5638-6423 of SEQ
ID NO:430) of the NS4B protein of HPgV-2. FIG. 32C shows: 1) the
amino acid sequence (SEQ ID NO:439) and describes the nucleic acid
sequence (nucleotides 6424-7797 of SEQ ID NO:430) of the NS5A
protein of HPgV-2; 2) the amino acid sequence (SEQ ID NO:440) and
describes the nucleic acid sequence (nucleotides 7798-9498 of SEQ
ID NO:430) of the NS5B protein of HPgV-2; and 3) describes the
nucleic acid sequence (nucleotides 9499-9867 of SEQ ID NO:430) of
at least a portion of the 3' UTR of HPgV-2.
DEFINITIONS
[0078] The terms "sample" and "specimen" are used in their broadest
sense and encompass samples or specimens obtained from any source,
including a human patient. In some embodiments of this invention,
biological samples include tissue or cells, cerebrospinal fluid
(CSF), serous fluid, urine, saliva, blood, and blood products such
as plasma, serum and the like. However, these examples are not to
be construed as limiting the types of samples that find use with
the methods and compositions described herein. In some embodiments,
the sample is a blood, serum, or plasma sample from a patient known
to be infected with HCV and/or HIV.
[0079] As used herein, the terms "host," "subject" and "patient"
refer to any animal, including but not limited to, human and
non-human animals (e.g., dogs, cats, cows, horses, sheep, poultry,
fish, crustaceans, etc.) that is studied, analyzed, tested,
diagnosed or treated. As used herein, the terms "host," "subject"
and "patient" are used interchangeably, unless indicated
otherwise.
[0080] As used herein, the terms "administration" and
"administering" refer to the act of giving therapeutic treatment
(e.g., a immunogenic composition) to a subject (e.g., a subject or
in vivo, in vitro, or ex vivo cells, tissues, and organs).
Exemplary routes of administration to the human body can be through
space under the arachnoid membrane of the brain or spinal cord
(intrathecal), the eyes (ophthalmic), mouth (oral), skin (topical
or transdermal), nose (nasal), lungs (inhalant), oral mucosa
(buccal), ear, rectal, vaginal, by injection (e.g., intravenously,
subcutaneously, intratumorally, intraperitoneally, etc.) and the
like.
[0081] As used herein, the term "nucleic acid molecule" refers to
any nucleic acid containing molecule, including but not limited to,
DNA or RNA. The term encompasses sequences that include any of the
known base analogs of DNA and RNA including, but not limited to,
4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine,
pseudoisocytosine, 5-(carboxyhydroxylmethyl)uracil, 5-fluorouracil,
5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil,
5-carboxymethylaminomethyluracil, dihydrouracil, inosine,
N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-methyladenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxy-aminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarbonylmethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,
oxybutoxosine, pseudouracil, queosine, 2-thiocytosine,
5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,
N-uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,
pseudouracil, queosine, 2-thiocytosine, 2,6-diaminopurine,
2-aminopurine, 5-amino-allyluracil, 5-hydroxymethylcytosine,
5-iodouracil, 5-nitroindole, 5-propynylcytosine, 5-propynyluracil,
hypoxanthine, N3-methyluracil, N6,N6-dimethyladenine, purine,
C-5-propynyl cytosine, C-5-propynyl uracil, and difluorotouyl.
[0082] The term "gene" refers to a nucleic acid (e.g., DNA)
sequence that comprises coding sequences necessary for the
production of a polypeptide, precursor, or RNA (e.g., rRNA, tRNA).
The polypeptide can be encoded by a full length coding sequence or
by any portion of the coding sequence so long as the desired
activity or functional properties (e.g., enzymatic activity, ligand
binding, signal transduction, immunogenicity, etc.) of the
full-length or fragment are retained. The term also encompasses the
coding region of a structural gene and the sequences located
adjacent to the coding region on both the 5' and 3' ends for a
distance of about 1 kb or more on either end such that the gene
corresponds to the length of the full-length mRNA. Sequences
located 5' of the coding region and present on the mRNA are
referred to as 5' non-translated sequences. Sequences located 3' or
downstream of the coding region and present on the mRNA are
referred to as 3' non-translated sequences. The term "gene"
encompasses both cDNA and genomic forms of a gene.
[0083] As used herein, the terms "gene expression" and "expression"
refer to the process of converting genetic information encoded in a
gene into RNA (e.g., mRNA, rRNA, tRNA, or snRNA) through
"transcription" of the gene (i.e., via the enzymatic action of an
RNA polymerase), and for protein encoding genes, into protein
through "translation" of mRNA. Gene expression can be regulated at
many stages in the process. "Up-regulation" or "activation" refer
to regulation that increases and/or enhances the production of gene
expression products (e.g., RNA or protein), while "down-regulation"
or "repression" refer to regulation that decrease production.
Molecules (e.g., transcription factors) that are involved in
up-regulation or down-regulation are often called "activators" and
"repressors," respectively.
[0084] The term "isolated" when used in relation to a nucleic acid,
as in "an isolated oligonucleotide" or "isolated polynucleotide"
refers to a nucleic acid sequence that is identified and separated
from at least one component or contaminant with which it is
ordinarily associated in its natural source. Isolated nucleic acid
is present in a form or setting that is different from that in
which it is found in nature. In contrast, non-isolated nucleic
acids are nucleic acids such as DNA and RNA found in the state they
exist in nature. For example, a given DNA sequence (e.g., a gene)
is found on the host cell chromosome in proximity to neighboring
genes; RNA sequences, such as a specific viral RNA sequences
encoding a specific protein, are generally found in the cell as a
mixture with numerous other host mRNAs that encode a multitude of
proteins. The isolated nucleic acid, oligonucleotide, or
polynucleotide may be present in single-stranded or double-stranded
form.
[0085] The term "synthetic" when used in reference to nucleic acid
molecules (e.g., primers or probes of HPgV-2) refers to non-natural
molecules made directly (e.g., in a laboratory) or indirectly
(e.g., from expression in a cell of a construct made in a
laboratory) by mankind.
[0086] As used herein, the terms "complementary" or
"complementarity" are used in reference to polynucleotides (i.e., a
sequence of nucleotides) related by the base-pairing rules. For
example, for the sequence "A-G-T," is complementary to the sequence
"T-C-A." Complementarity may be "partial," in which only some of
the nucleic acids' bases are matched according to the base pairing
rules. Or, there may be "complete" or "total" complementarity
between the nucleic acids. The degree of complementarity between
nucleic acid strands has significant effects on the efficiency and
strength of hybridization between nucleic acid strands. This is of
particular importance in amplification reactions, as well as
detection methods that depend upon binding between nucleic
acids.
[0087] The term "homology" refers to a degree of complementarity.
There may be partial homology or complete homology (i.e.,
identity). A partially complementary sequence is one that at least
partially inhibits a completely complementary sequence from
hybridizing to a target nucleic acid and is referred to using the
functional term "substantially homologous." The term "inhibition of
binding," when used in reference to nucleic acid binding, refers to
inhibition of binding caused by competition of homologous sequences
for binding to a target sequence. The inhibition of hybridization
of the completely complementary sequence to the target sequence may
be examined using a hybridization assay (e.g., Southern or Northern
blot, solution hybridization and the like) under conditions of low
stringency. A substantially homologous sequence or probe will
compete for and inhibit the binding (i.e., the hybridization) of a
completely homologous to a target under conditions of low
stringency. This is not to say that conditions of low stringency
are such that non-specific binding is permitted; low stringency
conditions require that the binding of two sequences to one another
be a specific (i.e., selective) interaction. The absence of
non-specific binding may be tested by the use of a second target
that lacks even a partial degree of complementarity (e.g., less
than about 30% identity); in the absence of non-specific binding
the probe will not hybridize to the second non-complementary
target.
[0088] The art knows well that numerous equivalent conditions may
be employed to comprise low stringency conditions; factors such as
the length and nature (DNA, RNA, base composition) of the probe and
nature of the target (DNA, RNA, base composition, present in
solution or immobilized, etc.) and the concentration of the salts
and other components (e.g., the presence or absence of formamide,
dextran sulfate, polyethylene glycol) are considered and the
hybridization solution may be varied to generate conditions of low
stringency hybridization different from, but equivalent to, the
above listed conditions. In addition, the art knows conditions that
promote hybridization under conditions of high stringency (e.g.,
increasing the temperature of the hybridization and/or wash steps,
the use of formamide in the hybridization solution, etc.).
[0089] As used herein, the term "hybridization" is used in
reference to the pairing of complementary nucleic acids.
Hybridization and the strength of hybridization (i.e., the strength
of the association between the nucleic acids) is impacted by such
factors as the degree of complementary between the nucleic acids,
stringency of the conditions involved, the Tm of the formed hybrid,
and the G:C ratio within the nucleic acids.
[0090] As used herein, the term "Tm" is used in reference to the
"melting temperature." The melting temperature is the temperature
at which a population of double-stranded nucleic acid molecules
becomes half dissociated into single strands. The equation for
calculating the Tm of nucleic acids is well known in the art. As
indicated by standard references, a simple estimate of the Tm value
may be calculated by the equation: Tm=81.5+0.41(% G+C), when a
nucleic acid is in aqueous solution at 1 M NaCl (See e.g., Anderson
and Young, Quantitative Filter Hybridization, in Nucleic Acid
Hybridization [1985]). Other references include more sophisticated
computations that take structural as well as sequence
characteristics into account for the calculation of Tm.
[0091] As used herein the term "stringency" is used in reference to
the conditions of temperature, ionic strength, and the presence of
other compounds such as organic solvents, under which nucleic acid
hybridizations are conducted. Those skilled in the art will
recognize that "stringency" conditions may be altered by varying
the parameters just described either individually or in concert.
With "high stringency" conditions, nucleic acid base pairing will
occur only between nucleic acid fragments that have a high
frequency of complementary base sequences (e.g., hybridization
under "high stringency" conditions may occur between homologs with
about 85-100% identity, preferably about 70-100% identity). With
medium stringency conditions, nucleic acid base pairing will occur
between nucleic acids with an intermediate frequency of
complementary base sequences (e.g., hybridization under "medium
stringency" conditions may occur between homologs with about 50-70%
identity). Thus, conditions of "weak" or "low" stringency are often
required with nucleic acids that are derived from organisms that
are genetically diverse, as the frequency of complementary
sequences is usually less.
[0092] "High stringency conditions" when used in reference to
nucleic acid hybridization comprise conditions equivalent to
binding or hybridization at 42 degrees Celsius in a solution
consisting of 5.times. SSPE (43.8 g/l NaCl, 6.9 g/l NaH2PO4.H2O and
1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5.times.
Denhardt's reagent and 100 .mu.g/ml denatured salmon sperm DNA
followed by washing in a solution comprising 0.1.times. SSPE, 1.0%
SDS at 42 degrees Celsius when a probe of about 500 nucleotides in
length is employed.
[0093] "Medium stringency conditions" when used in reference to
nucleic acid hybridization comprise conditions equivalent to
binding or hybridization at 42 degrees Celsiuis in a solution
consisting of 5.times. SSPE (43.8 g/l NaCl, 6.9 g/l NaH2PO4.H2O and
1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5.times.
Denhardt's reagent and 100 .mu.g/ml denatured salmon sperm DNA
followed by washing in a solution comprising 1.0.times. SSPE, 1.0%
SDS at 42 degrees C. when a probe of about 500 nucleotides in
length is employed.
[0094] "Low stringency conditions" comprise conditions equivalent
to binding or hybridization at 42 degrees Celsius in a solution
consisting of 5.times. SSPE (43.8 g/l NaCl, 6.9 g/l NaH2PO4.H2O and
1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS, 5.times.
Denhardt's reagent [50X Denhardt's contains per 500 ml: 5 g Ficoll
(Type 400, Pharamcia), 5 g BSA (Fraction V; Sigma)] and 100
.mu.g/ml denatured salmon sperm DNA followed by washing in a
solution comprising 5.times. SSPE, 0.1% SDS at 42 degrees Celsius
when a probe of about 500 nucleotides in length is employed.
[0095] The following terms are used to describe the sequence
relationships between two or more polynucleotides: "reference
sequence," "sequence identity," "percentage of sequence identity,"
and "substantial identity." A "reference sequence" is a defined
sequence used as a basis for a sequence comparison; a reference
sequence may be a subset of a larger sequence, for example, as a
segment of a full-length cDNA sequence given in a sequence listing
(e.g., SEQ ID NO:1 or 75) or may comprise a gene sequence.
Generally, a reference sequence is at least 20 nucleotides in
length, frequently at least 25 nucleotides in length, and often at
least 50 nucleotides in length. Since two polynucleotides may each
(1) comprise a sequence (i.e., a portion of the complete
polynucleotide sequence) that is similar between the two
polynucleotides, and (2) may further comprise a sequence that is
divergent between the two polynucleotides, sequence comparisons
between two (or more) polynucleotides are typically performed by
comparing sequences of the two polynucleotides over a "comparison
window" to identify and compare local regions of sequence
similarity. A "comparison window," as used herein, refers to a
conceptual segment of at least 20 contiguous nucleotide positions
wherein a polynucleotide sequence may be compared to a reference
sequence of at least 20 contiguous nucleotides and wherein the
portion of the polynucleotide sequence in the comparison window may
comprise additions or deletions (i.e., gaps) of 20 percent or less
as compared to the reference sequence (which does not comprise
additions or deletions) for optimal alignment of the two sequences.
Optimal alignment of sequences for aligning a comparison window may
be conducted by the local homology algorithm of Smith and Waterman
(Smith and Waterman, Adv. Appl. Math. 2: 482 (1981)) by the
homology alignment algorithm of Needleman and Wunsch (Needleman and
Wunsch, J. Mol. Biol. 48:443 (1970)), by the search for similarity
method of Pearson and Lipman (Pearson and Lipman, Proc. Natl. Acad.
Sci. (U.S.A.) 85:2444 (1988)), by computerized implementations of
these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin
Genetics Software Package Release 7.0, Genetics Computer Group, 575
Science Dr., Madison, Wis.), or by inspection, and the best
alignment (i.e., resulting in the highest percentage of homology
over the comparison window) generated by the various methods is
selected. The term "sequence identity" means that two
polynucleotide sequences are identical (i.e., on a
nucleotide-by-nucleotide basis) over the window of comparison. The
term "percentage of sequence identity" is calculated by comparing
two optimally aligned sequences over the window of comparison,
determining the number of positions at which the identical nucleic
acid base (e.g., A, T, C, G, U, or I) occurs in both sequences to
yield the number of matched positions, dividing the number of
matched positions by the total number of positions in the window of
comparison (i.e., the window size), and multiplying the result by
100 to yield the percentage of sequence identity. The terms
"substantial identity" as used herein denotes a characteristic of a
polynucleotide sequence, wherein the polynucleotide comprises a
sequence that has at least 85 percent sequence identity, preferably
at least 90 to 95 percent sequence identity, more usually at least
99 percent sequence identity as compared to a reference sequence
over a comparison window of at least 20 nucleotide positions,
frequently over a window of at least 25-50 nucleotides, wherein the
percentage of sequence identity is calculated by comparing the
reference sequence to the polynucleotide sequence which may include
deletions or additions which total 20 percent or less of the
reference sequence over the window of comparison.
[0096] As applied to polypeptides, the term "substantial identity"
means that two peptide sequences, when optimally aligned, such as
by the programs GAP or BESTFIT using default gap weights, share at
least 80 percent sequence identity, preferably at least 90 percent
sequence identity, more preferably at least 95 percent sequence
identity or more (e.g., 99 percent sequence identity). Preferably,
residue positions which are not identical differ by conservative
amino acid substitutions. Conservative amino acid substitutions
refer to the interchangeability of residues having similar side
chains. For example, a group of amino acids having aliphatic side
chains is glycine, alanine, valine, leucine, and isoleucine; a
group of amino acids having aliphatic-hydroxyl side chains is
serine and threonine; a group of amino acids having
amide-containing side chains is asparagine and glutamine; a group
of amino acids having aromatic side chains is phenylalanine,
tyrosine, and tryptophan; a group of amino acids having basic side
chains is lysine, arginine, and histidine; and a group of amino
acids having sulfur-containing side chains is cysteine and
methionine. Preferred conservative amino acids substitution groups
are: valine-leucine-isoleucine, phenylalanine-tyrosine,
lysine-arginine, alanine-valine, and asparagine-glutamine. In
certain embodiments, provided herein are peptides that have
substantial identity to at least a portion of the amino acid
sequences shown in SEQ ID NOs:2-11 or 304-353.
[0097] The term "fragment" or "portion" as used herein refers to a
polypeptide that has an amino-terminal and/or carboxy-terminal
deletion as compared to the native protein, but where the remaining
amino acid sequence is identical to the corresponding positions in
the amino acid sequence deduced from a full-length cDNA sequence.
Fragments typically are at least 4 amino acids long, preferably at
least 20 amino acids long, usually at least 50 amino acids long or
longer.
[0098] As used herein, the term "primer" refers to an
oligonucleotide, whether occurring naturally as in a purified
restriction digest or produced synthetically, which is capable of
acting as a point of initiation of synthesis when placed under
conditions in which synthesis of a primer extension product which
is complementary to a nucleic acid strand is induced, (i.e., in the
presence of nucleotides and an inducing agent such as DNA
polymerase and at a suitable temperature and pH). The primer is
preferably single stranded for maximum efficiency in amplification,
but may alternatively be double stranded. If double stranded, the
primer is first treated to separate its strands before being used
to prepare extension products. Preferably, the primer is an
oligodeoxyribonucleotide. The primer must be sufficiently long to
prime the synthesis of extension products in the presence of the
inducing agent. The exact lengths of the primers will depend on
many factors, including temperature, source of primer and the use
of the method.
[0099] As used herein, the term "probe" refers to an
oligonucleotide (i.e., a sequence of nucleotides), whether
occurring naturally as in a purified restriction digest or produced
synthetically, recombinantly or by PCR amplification, that is
capable of hybridizing to another oligonucleotide of interest. A
probe may be single-stranded or double-stranded. Probes are useful
in the detection, identification and isolation of particular
nucleic acid sequences. It is contemplated that, in certain
embodiments, a probe used in the present methods and compositions
will be labeled with any "reporter molecule," so that is detectable
in any detection system, including, but not limited to enzyme
(e.g., ELISA, as well as enzyme-based histochemical assays),
fluorescent, radioactive, and luminescent systems. It is not
intended that the methods and compositions be limited to any
particular detection system or label.
[0100] As used herein, the term "recombinant nucleic acid molecule"
as used herein refers to a nucleic molecule that is comprised of
segments of nucleic acid joined together by means of molecular
biological techniques.
[0101] As used herein, "peptide" refers to a linear polymer of
amino acids joined by peptide bonds in a specific sequence. As used
herein, "peptide" also encompasses polypeptide, oligopeptide and
protein. A peptide may be, for example, a short amino acid stretch
from the HPgV-2 virus (e.g., 25 amino acids), but may also be a
long sequence, such as the amino acid sequence of the entire NS3
protein (e.g., SEQ ID NO:7).
[0102] The term "recombinant protein" or "recombinant polypeptide"
as used herein refers to a protein molecule that is expressed from
a recombinant nucleic acid molecule.
[0103] The term "antigenic determinant" or "epitope" as used herein
refers to that portion of an antigen that makes contact with a
particular antibody. When a protein or fragment of a protein is
used to immunize a host animal, numerous regions of the protein may
induce the production of antibodies that bind specifically to a
given region or three-dimensional structure on the protein; these
regions or structures are referred to as antigenic determinants. An
antigenic determinant may compete with the intact antigen (i.e.,
the "immunogen" used to elicit the immune response) for binding to
an antibody.
[0104] As used herein, a "solid support" is any surface or material
to which a biological molecules, such as a HPgV-2 nucleic acid,
protein, or antibody may be attached and employed in a biological
assay. Examples of solid supports which may be utilized in the
assays described herein are well-known in the art and included, but
are not limited to, a magnetic particles, a beads, microparticle, a
test tube, a microtiter plate, a cuvette, a membrane, a scaffolding
molecule, a film, a filter paper, a disc and a chip.
DETAILED DESCRIPTION
[0105] Provided herein are compositions, methods, and kits for
detecting human Pegivirus 2 (HPgV-2). In certain embodiments,
provided herein are HPgV-2 specific nucleic acid probes and
primers, and methods for detecting HPgV-2 nucleic acid. In other
embodiments, provided herein are HPgV-2 immunogenic compositions,
methods of treating a subject with immunogenic peptides to develop
resistance to infection, and methods of detecting HPgV-2 subject
antibodies in a sample.
I. Human Pegivirus 2
[0106] Complete genomic nucleic acid sequences of human Pegivirus 2
(HPgV-2), also called GBV-E, are shown in SEQ ID NOs:1 and 75
(FIGS. 1 and 8) and SEQ ID NOs:299-303. HPgV-2 is a positive single
stranded RNA virus and encodes the following proteins: S protein
(e.g., SEQ ID NO:2, 76, 304, 314, 324, 334, and 344), E1 protein
(e.g., SEQ ID NO:3, 77, 305, 315, 325, 335, and 345), E2 protein
(e.g., SEQ ID NO:4, 78, 306, 316, 326, 336, and 346), X protein
(e.g., SEQ ID NO:5,79, 307, 317, 327, 337, and 347), NS2 protein
(e.g., SEQ ID NO:6, 80, 308, 318, 328, 338, and 348), NS3 protein
(e.g., SEQ ID NO:7, 81, 309, 319, 329, 339, and 349), NS4A protein
(e.g., SEQ ID NO:8, 82, 310, 320, 330, 340, and 350), NS4B protein
(e.g., SEQ ID NO:9, 83, 311, 321, 331, 341, and 351), NS5A protein
(e.g., SEQ ID NO:10, 84, 312, 322, 332, 342, and 352), and NS5B
protein (e.g., SEQ ID NO:11, 85, 313, 323, 333, 343, and 353).
Certain determined portions of the 5' UTR include nucleotides 1-103
of SEQ ID NO:1, nucleotides 1-110 of SEQ ID NO:75, nucleotides
1-327 of SEQ ID NOs:299-301, nucleotides 12-327 of SEQ ID NO:302,
and nucleotides 24-327 of SEQ ID NO:303; and exemplary certain
determined portions of the 3' UTR include nucleotides 9410-9778 of
SEQ ID NO:1, nucleotides 9417-9431 of SEQ ID NO:75, and nucleotides
9499-9867 of SEQ ID NOS:299-303. Based on sequence relatedness, the
organization of the HPgV-2 genome is most similar to both the
Pegiviruses and the Hepaciviruses, both currently classified as
members of the Flaviviridae family. It has recently been proposed
that HCV and GBV-B are to be included within the Hepacivirus genus
of the Flaviviridae family and that GBV-A and GBV-C (HPgV-1) are to
be included within the Pegivirus genus of the Flaviviridae family
(Stapleton et al., J Gen Virol 2011: 92: 233-246).
[0107] All members of the Flaviviridae family of viruses have a
positive sense, single stranded RNA genome of about 10 kb, that
that contains a single long open reading frame (ORF) encoding a
polyprotein of about 3,000 amino acids (Lindenbach et al.,
Flaviviridae: The Viruses and Their Replication. Chapter 33. In
Fields Virology Fifth Edition, (Knipe et al., Eds.) Wolters
Kluwer/Lippincott Williams and Williams, Philadelphia Pa. Pages
1101-1152). For Flaviviridae, the polyproteins are cleaved into
smaller functional nonstructural and structural components by a
combination of host and viral proteases. The viral structural
proteins are located at the amino terminal portion of the genome
and include two envelope glycoproteins, E1 and E2 for both HCV and
the GB viruses. While HCV and GBV-B contain a capsid protein, GBV-A
and GBV-C (HPgV-1) lack a typical capsid-like protein. For all
Flaviviruses, the nonstructural proteins are located downstream of
the structural proteins, and include an NS3 protein that contains
an N-terminus serine protease and a C-terminal RNA helicase protein
and an NS5 protein that is a multifunctional protein with
methyltransferase and RNA-dependent RNA replication activities. The
ORF is flanked at both the 5' end and the 3' by untranslated
regions that are highly conserved and that are involved both in
translation and in replication of the genome.
[0108] In certain embodiments, patient samples that are known to be
HCV positive are tested for the presence of HPgV-2 (e.g., for
patient antibodies or by PCR). In particular embodiments, HCV
infected blood donors who are anti-HCV negative but HCV RNA
positive are tested. Such samples are termed as "preseroconversion
window period" samples. In other embodiments, samples are tested
from HCV infected patients who are anti-HCV positive and HCV RNA
positive. In certain embodiments, samples that are HIV positive are
tested. In particular embodiments, high risk groups are tested for
the presence of HPgV-2 infection. High risk groups include multiply
transfused individuals, plasmapheresis donors (some of whom may be
positive for HBV, HIV or HCV), intravenous drug users, and
individuals with sexually transmitted diseases.
[0109] FIG. 10 shows an alignment of the genomes or partial genomes
of HPgV-2 variants UC0125.US, ABT0070P.US, ABT0096P.US, and
ABT0128A.US, along with a majority consensus sequence. The amino
acid similarities between UC0125.US and ABT0070P.US are shown in
Table 16 below:
TABLE-US-00001 TABLE 16 protein amino acids mismatches % identity
polyprotein 3102 157 94.94 S 124 4 96.77 E1 191 6 96.86 E2 354 24
93.22 X 237 13 94.51 NS2 240 8 96.67 NS3 628 22 96.50 NS4A 41 3
92.68 NS4B 262 10 96.18 NS5A 458 43 90.61 NS5B 567 24 95.77 3102
157
II. Amplification
[0110] In some embodiments, provided herein are compositions and
methods for the amplification of HPgV-2 nucleic acids (e.g. DNA,
RNA, etc.). In some embodiments, amplification is performed on a
bulk sample of nucleic acids. In some embodiments, amplification is
performed on a single nucleic acid target molecule. In some
embodiments, provided herein are compositions (e.g. primers,
buffers, salts, nucleic acid targets, etc.) and methods for the
amplification of nucleic acid (e.g. digital droplet amplification,
PCR amplification, combinations thereof, etc.). In some
embodiments, an amplification reaction is any reaction in which
nucleic acid replication occurs repeatedly over time to form
multiple copies of at least one segment of a template or target
nucleic acid molecule (e.g., HPgV-2 nucleic acid). In some
embodiments, amplification generates an exponential or linear
increase in the number of copies of the template nucleic acid.
Amplifications may produce in excess of a 1,000-fold increase in
template copy-number and/or target-detection signal. Exemplary
amplification reactions include, but are not limited to the
polymerase chain reaction (PCR) or ligase chain reaction (LCR),
each of which is driven by thermal cycling.
[0111] Amplification may be performed with any suitable reagents
(e.g. template nucleic acid (e.g. DNA or RNA), primers, probes,
buffers, replication catalyzing enzyme (e.g. DNA polymerase, RNA
polymerase), nucleotides, salts (e.g. MgCl.sub.2), etc. In some
embodiments, an amplification mixture includes any combination of
at least one primer or primer pair, at least one probe, at least
one replication enzyme (e.g., at least one polymerase, such as at
least one DNA and/or RNA polymerase), and deoxynucleotide (and/or
nucleotide) triphosphates (dNTPs and/or NTPs), etc.
[0112] In some embodiments, the systems, devices, and methods
utilize nucleic acid amplification that relies on alternating
cycles of heating and cooling (i.e., thermal cycling) to achieve
successive rounds of replication (e.g., PCR). In some embodiments,
PCR is used to amplify target nucleic acids (e.g. HPgV-2 nucleic
acid). PCR may be performed by thermal cycling between two or more
temperature set points, such as a higher melting (denaturation)
temperature and a lower annealing/extension temperature, or among
three or more temperature set points, such as a higher melting
temperature, a lower annealing temperature, and an intermediate
extension temperature, among others. PCR may be performed with a
thermostable polymerase, such as Taq DNA polymerase (e.g.,
wild-type enzyme, a Stoffel fragment, FastStart polymerase, etc.),
Pfu DNA polymerase, S-Tbr polymerase, Tth polymerase, Vent
polymerase, or a combination thereof, among others. Typical PCR
methods produce an exponential increase in the amount of a product
amplicon over successive cycles, although linear PCR methods also
find use.
[0113] Any suitable PCR methodology, combination of PCR
methodologies, or combination of amplification techniques may be
utilized, such as allele-specific PCR, assembly PCR, asymmetric
PCR, digital PCR, endpoint PCR, hot-start PCR, in situ PCR,
intersequence-specific PCR, inverse PCR, linear after exponential
PCR, ligation-mediated PCR, methylation-specific PCR, miniprimer
PCR, multiplex ligation-dependent probe amplification, multiplex
PCR, nested PCR, overlap-extension PCR, polymerase cycling
assembly, qualitative PCR, quantitative PCR, real-time PCR, RT-PCR,
single-cell PCR, solid-phase PCR, thermal asymmetric interlaced
PCR, touchdown PCR, or universal fast walking PCR, etc.
[0114] In some embodiments, the systems, devices, and methods
employ RT-PCR (reverse transcription-PCR). In some embodiments, the
systems, devices, and methods employ real-time PCR. In some
embodiments, the systems, devices, and methods employ endpoint
PCR.
[0115] In some embodiments, the systems, devices, and methods
utilize isothermal nucleic acid amplification methods. Any suitable
isothermal amplification methodology or combination of
amplification techniques may be utilized, such as transcription
mediated amplification, nucleic acid sequence-based amplification,
signal mediated amplification of RNA technology, strand
displacement amplification, rolling circle amplification,
loop-mediated isothermal amplification of DNA, isothermal multiple
displacement amplification, helicase-dependent amplification,
single primer isothermal amplification, and circular
helicase-dependent amplification.
III. Nucleic Acid Detection
[0116] In some embodiments, provided herein are systems, devices,
methods, and compositions to identify the presence of HPgV-2
nucleic acids (e.g. amplicons, labeled nucleic acids). In some
embodiments, detection involves measurement or detection of a
characteristic of a non-amplified nucleic acid, amplified nucleic
acid, a component comprising amplified nucleic acid, or a byproduct
of the amplification process, such as a physical, chemical,
luminescence, or electrical aspect, which correlates with
amplification (e.g. fluorescence, pH change, heat change,
etc.).
[0117] In some embodiments, fluorescence detection methods are
provided for detection of amplified HPgV-2 nucleic acid, and/or
identification of amplified nucleic acids. In addition to the
reagents already discussed, and those known to those of skill in
the art of nucleic acid amplification and detection, various
detection reagents, such as fluorescent and non-fluorescent dyes
and probes are provided. For example, the protocols may employ
reagents suitable for use in a TaqMan reaction, such as a TaqMan
probe; reagents suitable for use in a SYBR Green fluorescence
detection; reagents suitable for use in a molecular beacon
reaction, such as molecular beacon probes; reagents suitable for
use in a scorpion reaction, such as a scorpion probe; reagents
suitable for use in a fluorescent DNA-binding dye-type reaction,
such as a fluorescent probe; and/or reagents for use in a LightUp
protocol, such as a LightUp probe. In some embodiments, provided
herein are methods and compositions for detecting and/or
quantifying a detectable signal (e.g. fluorescence) from amplified
target nucleic acid. Thus, for example, methods may employ labeling
(e.g. during amplification, post-amplification) amplified nucleic
acids with a detectable label, exposing partitions to a light
source at a wavelength selected to cause the detectable label to
fluoresce, and detecting and/or measuring the resulting
fluorescence. Fluorescence emitted from label can be tracked during
amplification reaction to permit monitoring of the reaction (e.g.,
using a SYBR Green-type compound), or fluorescence can be measure
post-amplification.
[0118] In some embodiments, detection of amplified nucleic acids
employs one or more of fluorescent labeling, fluorescent
intercalation dyes, FRET-based detection methods (U.S. Pat. No.
5,945,283; PCT Publication WO 97/22719; both of which are
incorporated by reference in their entireties), quantitative PCR,
real-time fluorogenic methods (U.S. Pat. Nos. 5,210,015 to Gelfand,
5,538,848 to Livak, et al., and 5,863,736 to Haaland, as well as
Heid, C. A., et al., Genome Research, 6:986-994 (1996); Gibson, U.
E. M, et al., Genome Research 6:995-1001 (1996); Holland, P. M., et
al., Proc. Natl. Acad. Sci. USA 88:7276-7280, (1991); and Livak, K.
J., et al., PCR Methods and Applications 357-362 (1995), each of
which is incorporated by reference in its entirety), molecular
beacons (Piatek, A. S., et al., Nat. Biotechnol. 16:359-63 (1998);
Tyagi, S. and Kramer, F. R., Nature Biotechnology 14:303-308
(1996); and Tyagi, S. et al., Nat. Biotechnol. 16:49-53 (1998);
herein incorporated by reference in their entireties), Invader
assays (Third Wave Technologies, (Madison, Wis.)) (Neri, B. P., et
al., Advances in Nucleic Acid and Protein Analysis 3826:117-125,
2000; herein incorporated by reference in its entirety), nucleic
acid sequence-based amplification (NASBA; (See, e.g., Compton, J.
Nucleic Acid Sequence-based Amplification, Nature 350: 91-91,
1991.; herein incorporated by reference in its entirety), Scorpion
probes (Thelwell, et al. Nucleic Acids Research, 28:3752-3761,
2000; herein incorporated by reference in its entirety), partially
double-stranded linear probes (Luk, K.-C., et al, J. Virological
Methods 144:1-11, 2007; herein incorporated by reference in its
entirety), capacitive DNA detection (See, e.g., Sohn, et al. (2000)
Proc. Natl. Acad. Sci. U.S.A. 97:10687-10690; herein incorporated
by reference in its entirety), etc.
IV. Nucleic Acid Analysis
[0119] Nucleic acid molecules (e.g., amplified HPgV-2 nucleic acid)
may be analyzed by any number of techniques to determine the
presence of, amount of, or identity of the molecule. Non-limiting
examples include sequencing, mass determination, and base
composition determination. The analysis may identify the sequence
of all or a part of the amplified nucleic acid or one or more of
its properties or characteristics to reveal the desired
information. For example, in some embodiments, the presence of a
polymorphism or of a particular HPgV-2 strain or isolate is
determined. In some embodiments, the methylation status of a
nucleic acid is determined.
[0120] Illustrative non-limiting examples of nucleic acid
sequencing techniques include, but are not limited to, chain
terminator (Sanger) sequencing and dye terminator sequencing, as
well as "next generation" sequencing techniques. Those of ordinary
skill in the art will recognize that because RNA is less stable in
the cell and more prone to nuclease attack experimentally RNA is
usually, although not necessarily, reverse transcribed to DNA
before sequencing.
[0121] A number of DNA sequencing techniques are known in the art,
including fluorescence-based sequencing methodologies (See, e.g.,
Birren et al., Genome Analysis: Analyzing DNA, 1, Cold Spring
Harbor, N.Y.; herein incorporated by reference in its entirety). In
some embodiments, automated sequencing techniques understood in
that art are utilized. In some embodiments, the systems, devices,
and methods employ parallel sequencing of partitioned amplicons
(PCT Publication No: W02006084132 to Kevin McKernan et al., herein
incorporated by reference in its entirety). In some embodiments,
DNA sequencing is achieved by parallel oligonucleotide extension
(See, e.g., U.S. Pat. No. 5,750,341 to Macevicz et al., and U.S.
Pat. No. 6,306,597 to Macevicz et al., both of which are herein
incorporated by reference in their entireties). Additional examples
of sequencing techniques include the Church polony technology
(Mitra et al., 2003, Analytical Biochemistry 320, 55-65; Shendure
et al., 2005 Science 309, 1728-1732; U.S. Pat. No. 6,432,360, U.S.
Pat. No. 6,485,944, U.S. Pat. No. 6,511,803; herein incorporated by
reference in their entireties) the 454 picotiter pyrosequencing
technology (Margulies et al., 2005 Nature 437, 376-380; US
20050130173; herein incorporated by reference in their entireties),
the Solexa single base addition technology (Bennett et al., 2005,
Pharmacogenomics, 6, 373-382; U.S. Pat. No. 6,787,308; U.S. Pat.
No. 6,833,246; herein incorporated by reference in their
entireties), Illumina Single base sequencing technology, the Lynx
massively parallel signature sequencing technology (Brenner et al.
(2000). Nat. Biotechnol. 18:630-634; U.S. Pat. No. 5,695,934; U.S.
Pat. No. 5,714,330; herein incorporated by reference in their
entireties) and the Adessi PCR colony technology (Adessi et al.
(2000). Nucleic Acid Res. 28, E87; WO 00018957; herein incorporated
by reference in its entirety).
[0122] In some embodiments, chain terminator sequencing is
utilized. Chain terminator sequencing uses sequence-specific
termination of a DNA synthesis reaction using modified nucleotide
substrates. Extension is initiated at a specific site on the
template DNA by using a short radioactive, or other labeled,
oligonucleotide primer complementary to the template at that
region. The oligonucleotide primer is extended using a DNA
polymerase, standard four deoxynucleotide bases, and a low
concentration of one chain terminating nucleotide, most commonly a
di-deoxynucleotide. This reaction is repeated in four separate
tubes with each of the bases taking turns as the
di-deoxynucleotide. Limited incorporation of the chain terminating
nucleotide by the DNA polymerase results in a series of related DNA
fragments that are terminated only at positions where that
particular di-deoxynucleotide is used. For each reaction tube, the
fragments are size-separated by electrophoresis in a slab
polyacrylamide gel or a capillary tube filled with a viscous
polymer. The sequence is determined by reading which lane produces
a visualized mark from the labeled primer as you scan from the top
of the gel to the bottom.
[0123] Dye terminator sequencing alternatively labels the
terminators. Complete sequencing can be performed in a single
reaction by labeling each of the di-deoxynucleotide
chain-terminators with a separate fluorescent dye, which fluoresces
at a different wavelength.
[0124] A set of methods referred to as "next-generation sequencing"
techniques have emerged as alternatives to Sanger and
dye-terminator sequencing methods (Voelkerding et al., Clinical
Chem., 55: 641-658, 2009; MacLean et al., Nature Rev. Microbiol.,
7: 287-296; each herein incorporated by reference in their
entirety). These techniques may be used to sequence portions of
HPgV-2 nucleic acid. Next-generation sequencing (NGS) methods share
the common feature of massively parallel, high-throughput
strategies, with the goal of lower costs in comparison to older
sequencing methods. NGS methods can be broadly divided into those
that require template amplification and those that do not.
Amplification-requiring methods include pyrosequencing
commercialized by Roche as the 454 technology platforms (e.g., GS
20 and GS FLX), the Solexa platform commercialized by Illumina, and
the Supported Oligonucleotide Ligation and Detection (SOLiD)
platform commercialized by Applied Biosystems. Non-amplification
approaches, also known as single-molecule sequencing, are
exemplified by the HeliScope platform commercialized by Helicos
BioSciences, and emerging platforms commercialized by VisiGen,
Oxford Nanopore Technologies Ltd., and Pacific Biosciences,
respectively.
[0125] In pyrosequencing (Voelkerding et al., Clinical Chem., 55:
641-658, 2009; MacLean et al., Nature Rev. Microbiol., 7: 287-296;
U.S. Pat. No. 6,210,891; U.S. Pat. No. 6,258,568; each herein
incorporated by reference in its entirety), template DNA is
fragmented, end-repaired, ligated to adaptors, and clonally
amplified in-situ by capturing single template molecules with beads
bearing oligonucleotides complementary to the adaptors. Each bead
bearing a single template type is compartmentalized into a
water-in-oil microvesicle, and the template is clonally amplified
using a technique referred to as emulsion PCR. The emulsion is
disrupted after amplification and beads are deposited into
individual wells of a picotiter plate functioning as a flow cell
during the sequencing reactions. Ordered, iterative introduction of
each of the four dNTP reagents occurs in the flow cell in the
presence of sequencing enzymes and luminescent reporter such as
luciferase. In the event that an appropriate dNTP is added to the
3' end of the sequencing primer, the resulting production of ATP
causes a burst of luminescence within the well, which is recorded
using a CCD camera. It is possible to achieve read lengths greater
than or equal to 400 bases, and 1.times.10.sup.6 sequence reads can
be achieved, resulting in up to 500 million base pairs (Mb) of
sequence.
[0126] In the Solexa/Illumina platform (Voelkerding et al.,
Clinical Chem., 55: 641-658, 2009; MacLean et al., Nature Rev.
Microbiol., 7: 287-296; U.S. Pat. No. 6,833,246; U.S. Pat. No.
7,115,400; U.S. Pat. No. 6,969,488; each herein incorporated by
reference in its entirety), sequencing data are produced in the
form of shorter-length reads. In this method, single-stranded
fragmented DNA is end-repaired to generate 5'-phosphorylated blunt
ends, followed by Klenow-mediated addition of a single A base to
the 3' end of the fragments. A-addition facilitates addition of
T-overhang adaptor oligonucleotides, which are subsequently used to
capture the template-adaptor molecules on the surface of a flow
cell that is studded with oligonucleotide anchors. The anchor is
used as a PCR primer, but because of the length of the template and
its proximity to other nearby anchor oligonucleotides, extension by
PCR results in the "arching over" of the molecule to hybridize with
an adjacent anchor oligonucleotide to form a bridge structure on
the surface of the flow cell. These loops of DNA are denatured and
cleaved. Forward strands are then sequenced with reversible dye
terminators. The sequence of incorporated nucleotides is determined
by detection of post-incorporation fluorescence, with each fluor
and block removed prior to the next cycle of dNTP addition.
Sequence read length ranges from 36 nucleotides to over 600
nucleotides, with overall output exceeding 1 billion nucleotide
pairs per analytical run.
[0127] Sequencing nucleic acid molecules using SOLiD technology
(Voelkerding et al., Clinical Chem., 55: 641-658, 2009; MacLean et
al., Nature Rev. Microbiol., 7: 287-296; U.S. Pat. No. 5,912,148;
U.S. Pat. No. 6,130,073; each herein incorporated by reference in
their entirety) also involves fragmentation of the template,
ligation to oligonucleotide adaptors, attachment to beads, and
clonal amplification by emulsion PCR. Following this, beads bearing
template are immobilized on a derivatized surface of a glass
flow-cell, and a primer complementary to the adaptor
oligonucleotide is annealed. However, rather than utilizing this
primer for 3' extension, it is instead used to provide a 5'
phosphate group for ligation to interrogation probes containing two
probe-specific bases followed by 6 degenerate bases and one of four
fluorescent labels. In the SOLiD system, interrogation probes have
16 possible combinations of the two bases at the 3' end of each
probe, and one of four fluors at the 5' end. Fluor color and thus
identity of each probe corresponds to specified color-space coding
schemes. Multiple rounds (usually 7) of probe annealing, ligation,
and fluor detection are followed by denaturation, and then a second
round of sequencing using a primer that is offset by one base
relative to the initial primer. In this manner, the template
sequence can be computationally re-constructed, and template bases
are interrogated twice, resulting in increased accuracy. Sequence
read length averages 35 nucleotides, and overall output exceeds 4
billion bases per sequencing run.
[0128] In certain embodiments, nanopore sequencing in employed
(see, e.g., Astier et al., J Am Chem Soc. 2006 Feb
8;128(5):1705-10, herein incorporated by reference). The theory
behind nanopore sequencing has to do with what occurs when the
nanopore is immersed in a conducting fluid and a potential
(voltage) is applied across it: under these conditions a slight
electric current due to conduction of ions through the nanopore can
be observed, and the amount of current is exceedingly sensitive to
the size of the nanopore. If DNA molecules pass (or part of the DNA
molecule passes) through the nanopore, this can create a change in
the magnitude of the current through the nanopore, thereby allowing
the sequences of the DNA molecule to be determined.
[0129] Another exemplary nucleic acid sequencing approach that may
be adapted for use with the systems, devices, and methods was
developed by Stratos Genomics, Inc. and involves the use of
Xpandomers. This sequencing process typically includes providing a
daughter strand produced by a template-directed synthesis. The
daughter strand generally includes a plurality of subunits coupled
in a sequence corresponding to a contiguous nucleotide sequence of
all or a portion of a target nucleic acid in which the individual
subunits comprise a tether, at least one probe or nucleobase
residue, and at least one selectively cleavable bond. The
selectively cleavable bond(s) is/are cleaved to yield an Xpandomer
of a length longer than the plurality of the subunits of the
daughter strand. The Xpandomer typically includes the tethers and
reporter elements for parsing genetic information in a sequence
corresponding to the contiguous nucleotide sequence of all or a
portion of the target nucleic acid. Reporter elements of the
Xpandomer are then detected. Additional details relating to
Xpandomer-based approaches are described in, for example, U.S.
Patent Publication No. 20090035777, entitled "HIGH THROUGHPUT
NUCLEIC ACID SEQUENCING BY EXPANSION," that was filed Jun. 19,
2008, which is incorporated herein in its entirety.
[0130] Other emerging single molecule sequencing methods include
real-time sequencing by synthesis using a VisiGen platform
(Voelkerding et al., Clinical Chem., 55: 641-658, 2009; U.S. Pat.
No. 7,329,492; U.S. Pat. App. Ser. No. 11/671956; U.S. Pat. App.
Ser. No. 11/781166; each herein incorporated by reference in their
entirety) in which immobilized, primed DNA template is subjected to
strand extension using a fluorescently-modified polymerase and
florescent acceptor molecules, resulting in detectible fluorescence
resonance energy transfer (FRET) upon nucleotide addition.
[0131] Processes and systems for such real time sequencing that may
be adapted for use with the invention are described in, for
example, U.S. Pat. No. 7,405,281, entitled "Fluorescent nucleotide
analogs and uses therefor", issued Jul. 29, 2008 to Xu et al., U.S.
Pat. No. 7,315,019, entitled "Arrays of optical confinements and
uses thereof, issued Jan. 1, 2008 to Turner et al., U.S. Pat. No.
7,313,308, entitled "Optical analysis of molecules", issued
December 25, 2007 to Turner et al., U.S. Pat. No. 7,302,146,
entitled "Apparatus and method for analysis of molecules", issued
Nov. 27, 2007 to Turner et al., and U.S. Pat. No. 7,170,050,
entitled "Apparatus and methods for optical analysis of molecules",
issued Jan. 30, 2007 to Turner et al., U.S. Patent Publications
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for simultaneous real-time monitoring of optical signals from
multiple sources", filed Feb. 9, 2007 by Lundquist et al.,
20070196846, entitled "Polymerases for nucleotide analogue
incorporation", filed Dec. 21, 2006 by Hanzel et al., 20070188750,
entitled "Methods and systems for simultaneous real-time monitoring
of optical signals from multiple sources", filed Jul. 7, 2006 by
Lundquist et al., 20070161017, entitled "MITIGATION OF PHOTODAMAGE
IN ANALYTICAL REACTIONS", filed Dec. 1,2006 by Eid et al.,
20070141598, entitled "Nucleotide Compositions and Uses Thereof,
filed Nov. 3, 2006 by Turner et al., 20070134128, entitled "Uniform
surfaces for hybrid material substrate and methods for making and
using same", filed Nov. 27, 2006 by Korlach, 20070128133, entitled
"Mitigation of photodamage in analytical reactions", filed Dec. 2,
2005 by Eid et al., 20070077564, entitled "Reactive surfaces,
substrates and methods of producing same", filed Sep. 30, 2005 by
Roitman et al., 20070072196, entitled "Fluorescent nucleotide
analogs and uses therefore", filed Sep. 29, 2005 by Xu et al., and
20070036511, entitled "Methods and systems for monitoring multiple
optical signals from a single source", filed Aug. 11, 2005 by
Lundquist et al., and Korlach et aI. (2008) "Selective aluminum
passivation for targeted immobilization of single DNA polymerase
molecules in zero-mode waveguide nanostructures" Proc. Nat'I. Acad.
Sci. U.S.A. 105(4): 11761181--all of which are herein incorporated
by reference in their entireties.
[0132] In some embodiments, nucleic acids are analyzed by
determination of their mass and/or base composition. For example,
in some embodiments, nucleic acids are detected and characterized
by the identification of a unique base composition signature (BCS)
using mass spectrometry (e.g., Abbott PLEX-ID system, Abbot Ibis
Biosciences, Abbott Park, Ill.) described in U.S. Pat. Nos.
7,108,974, 8,017,743, and 8,017,322; each of which is herein
incorporated by reference in its entirety. In some embodiments, a
MassARRAY system (Sequenom, San Diego, Calif.) is used to detect or
analyze sequences (See e.g., U.S. Pat. Nos. 6,043,031; 5,777,324;
and 5,605,798; each of which is herein incorporated by
reference).
[0133] In certain embodiments, the Ion Torrent sequencing
technology is employed to sequence HPgV-2 nucleic acid. The Ion
Torrent technology is a method of DNA sequencing based on the
detection of hydrogen ions that are released during the
polymerization of DNA (see, e.g., Science 327(5970): 1190 (2010);
U.S. Pat. Appl. Pub. Nos. 20090026082, 20090127589, 20100301398,
20100197507, 20100188073, and 20100137143, incorporated by
reference in their entireties for all purposes). A microwell
contains a fragment of the NGS fragment library to be sequenced.
Beneath the layer of microwells is a hypersensitive ISFET ion
sensor. All layers are contained within a CMOS semiconductor chip,
similar to that used in the electronics industry. When a dNTP is
incorporated into the growing complementary strand a hydrogen ion
is released, which triggers a hypersensitive ion sensor. If
homopolymer repeats are present in the template sequence, multiple
dNTP molecules will be incorporated in a single cycle. This leads
to a corresponding number of released hydrogens and a
proportionally higher electronic signal. This technology differs
from other sequencing technologies in that no modified nucleotides
or optics are used. The per-base accuracy of the Ion Torrent
sequencer is .about.99.6% for 50 base reads, with .about.100 Mb
generated per run. The read-length is 100 base pairs. The accuracy
for homopolymer repeats of 5 repeats in length is .about.98%. The
benefits of ion semiconductor sequencing are rapid sequencing speed
and low upfront and operating costs.
[0134] In certain embodiments, HPgV-2 nucleic acid, including
polymorphisms or bases that identify particular types, strains, or
isolates are detected a hybridization assay. In a hybridization
assay, the presence of absence of a given sequence, SNP, or
mutation is determined based on the ability of the nucleic acid
from the sample to hybridize to a complementary nucleic acid
molecule (e.g., a oligonucleotide probe). A variety of
hybridization assays using a variety of technologies for
hybridization and detection are available and known in the art.
V. HPgV-2 Peptides
[0135] In other embodiments, provided herein are HPgV-2
polynucleotide sequences that encode HPgV-2 polypeptide sequences.
HPgV-2 polypeptides (e.g., SEQ ID NOs:2-11, 76-85, and 304-353) are
described in FIGS. 2, 9, 14, 16, 18, 20, and 22. Other embodiments
provide fragments, fusion proteins or functional equivalents of
these HPgV-2 proteins. In still other embodiments, nucleic acid
sequences corresponding to various HPgV-2 homologs and mutants may
be used to generate recombinant DNA molecules that direct the
expression of HPgV-2 homologs and mutants in appropriate host
cells. In some embodiments, the polypeptide may be a purified
product, in other embodiments it may be a product of chemical
synthetic procedures, and in still other embodiments it may be
produced by recombinant techniques using a prokaryotic or
eukaryotic host (e.g., by bacterial, yeast, higher plant, insect
and mammalian cells in culture).
[0136] In certain embodiments, due to the inherent degeneracy of
the genetic code, DNA sequences other than the polynucleotide
sequences of SEQ ID NO:1, 75, or 299-303 which encode substantially
the same or a functionally equivalent amino acid sequence, may be
used to clone and express HPgV-2 peptides. In general, such
polynucleotide sequences hybridize to SEQ ID NO:1, SEQ ID NO:75, or
SEQ ID NOS:299-303, under conditions of high to medium stringency
as described above. As will be understood by those of skill in the
art, it may be advantageous to produce HPgV-2 encoding nucleotide
sequences possessing non-naturally occurring codons. Therefore, in
some preferred embodiments, codons preferred by a particular
prokaryotic or eukaryotic host (Murray et al., Nucl. Acids Res., 17
[1989]) are selected, for example, to increase the rate of HPgV-2
expression or to produce recombinant RNA transcripts having
desirable properties, such as a longer half-life, than transcripts
produced from naturally occurring sequence.
[0137] In certain embodiments, the peptides comprises or consist of
the peptides in Table 15 below, or variants thereof with 1, 2, 3,
or 4 amino acid changes. Such peptides, for example, may be used as
capture peptides and/or immunogens to generate antibodies.
TABLE-US-00002 TABLE 15 Isolate/ Peptide Protein Amino Acid
Sequence SEQ ID NO: Peptide 1 S
GGSCRSPSRVQVARRVLQLSAFLALIGSGMSSIRSKTEGRIESGQ 100 Peptide 1 S
GGSCRSPSRVQVARRVLQLSAFL 101 Peptide 1 S SAFLALIGSGMSSIRSKTEGRIESGQ
102 Peptide 1 S ARRVLQLSAFLALIGSGMSS 103 UC0125.US S
GGSCRSPSRVQVARRVLQLCAFLALIGSGMCSIRSKTEGRIESGQ 104 UC0125.US S
GGSCRSPSRVQVARRVLQLCAFL 105 UC0125.US S CAFLALIGSGMCSIRSKTEGRIESGQ
106 UC0125.US S ARRVLQLCAFLALIGSGMCS 107 ABT0070P.US S
GGSCRSPSRVQVAGRVLRLCAFLALIGSGMCSIRSKNEGRIESGQ 108 ABT0070P.US S
GGSCRSPSRVQVAGRVLRLCAFL 109 ABT0070P.US S MCSIRSKNEGRIESGQ 110
ABT0070P.US S VAGRVLRLCAFLALIGSGMC 111 Peptide 2 S
RDGSLHWSHARHHSVQPDRVAAGPPSVTSVERNMGSSTDQT 112 Peptide 2 S
RDGSLHWSHARHHSVQPDRVAAG 113 UC0125.US S
RDGSLHWCHARHHSVQPDRVAAGPPSVTSVERNMGSSTDQT 114 UC0125.US S
RDGSLHWCHARHHSVQPDRVAAG 115 UC0125.US S VAAGPPSVTSVERNMGSSTDQT 116
UC0125.US S RHHSVQPDRVAAGPPSVTSVE 117 Peptide 3 E2
SMNSDSPFGTFTRNTESRFSIPRFSPVKINS 118 Peptide 3 E2
SMNCDCPFGTFTRNTESRFSIPRFCPVKINS 119 UC0125.US E2
SMNSDSPFGTFTRNTESRF 120 UC0125.US E2 SMNCDCPFGTFTRNTESRF 121
UC0125.US E2 SRFSIPRFSPVKINS 122 UC0125.US E2 FGTFTRNTESRFSIPR 123
ABT0070P.US E2 AMNCDCPFGTFTRNTESGFTIPRFCPVKLNS 124 ABT0070P.US E2
AMNCDCPFGTFTRNTESGF 125 ABT0070P.US E2 SGFTIPRFCPVKLNS 126
ABT0070P.US E2 FGTFTRNTESGFTIPR 127 ABT0096P.US
AMNCDCPFGTFTRNTESGFSISIDSVLLKSI 128 UC0125.US NS3
QAPAVTPTYSEITYYAPTGSGKSTKYPVDLVKQGHKVLVL 129 UC0125.US NS3
QAPAVTPTYSEITYYAPTGSGKST 130 UC0125.US NS3 GKSTKYPVDLVKQGHKVLVL 131
UC0125.US NS3 ITYYAPTGSGKSTKYPVDLVKQG 132 UC0125.US NS3
VKSMAPYIKETYKIRPEIRAGTGPDGVTVITG 133 UC0125.US NS3
VKSMAPYIKETYKIRPEI 134 UC0125.US NS3 PEIRAGTGPDGVTVITG 135
UC0125.US NS3 IKETYKIRPEIRAGTGPDG 136 ABT0070P.US NS3
VKSMAPYIKEKYKIRPEIRAGTGPDGVTVITG 137 ABT0070P.US NS3
VKSMAPYIKEKYKIRPEI 138 ABT0070P.US NS3 IKEKYKIRPEIRAGTGPDG 139
UC0125.US NS3 LVDPETNLRGYAVVICDECHDTSSTTLLGIGAVRMYAEKA 140
UC0125.US NS3 LVDPETNLRGYAVVICDECHDTSS 141 UC0125.US NS3
TNLRGYAVVICDECHDTSSTTLLGI 142 UC0125.U5 NS3 DTSSTTLLGIGAVRMYAEKA
143 UC0125.US NS3 PETNLRGYAVVISDESHDTSS 144 UC0125.US NS3
PETNLRGYAVVISD 145 UC0125.US NS3 VISDESHDTSS 146 ABT0070P.US NS3
PETNLRGYAVVICDECHDTSS 147 ABT0070P.US NS3 PETNLRGYAVVICD 148
ABT0096P.US NS3 RRGFAVVICVECHEHIT 149 UC0125.US NS3
PCTAALRMQRRGRTGRGRRGAYYTTSPGAAPCVS 150 UC0125.US NS3
PCTAALRMQRRGRTGRGRRG 151 UC0125.US NS3 GRRGAYYTTSPGAAPCVS 152
UC0125.US NS3 RRGRTGRGRRGAYYTTSPG 153 ABT0070P.US NS3
PCTAALRMQRRGRTGRGRRGAYYTTTPGAAPCV 154 ABT0070P.US NS3
GRRGAYYTTTPGAAPCV 155 ABT0070P.US NS3 RRGRTGRGRRGAYYTTTPG 156
UC0125.US NS4B LSERFGQQLSKLSLWRSVYHWAQAREGYTQCG 157 UC0125.US NS4B
LSERFGQQLSKLSLWRSV 158 UC0125.US NS4B RSVYHWAQAREGYTQCG 159
UC0125.US NS4B LSKLSLWRSVYHWAQAREG 160 ABT0070P.US NS4B
LTEKFGQQLSKLSLWRSVYHWAQAREGYTQCG 161 ABT0070P.US NS4B
LTEKFGQQLSKLSLWRSV 162 UC0125.US NS5A
NPTTTGTGTLRPDISDANKLGFRYGVADIVELERRGDKWH 163 UC0125.US NS5A
FNPTTTGTGTLRPDISDANKLGFR 164 UC0125.US NS5A GFRYGVADIVELERRGDKWH
165 UC0125.US NS5A RPDISDANKLGFRYGVADI 166 ABT0070P.US NS5A
NPTTTATGTLRPDISDATKLGFRYGVAEIVELERRGNKWH 167 ABT0070P.US NS5A
NPTTTATGTLRPDISDATKLGFR 168 ABT0070P.US NS5A GFRYGVAEIVELERRGNKWH
169 ABT0070P.US NS5A RPDISDATKLGFRYGVAEI 170 UC0125.US NS5A
QNLAARRRAEYDAWQVRQAVGDEYTRLADEDVD 171 UC0125.US NS5A
QNLAARRRAEYDAWQVRQAV 172 UC0125.US NS5A RQAVGDEYTRLADEDVD 173
UC0125.US NS5A RAEYDAWQVRQAVGDEYTR 174 ABT0070P.US NS5A
QNLEARRRAEFDAWQVREAIRDEYTRLADEDVD 175 ABT0070P.US NS5A
QNLEARRRAEFDAWQVREAI 176 ABT0070P.US NS5A REAIRDEYTRLADEDVD 177
ABT0070P.US NS5A RAEFDAWQVREAIRDEYTR 178 ABT0096P.US NS5A
FEAWQVREAIRDEYTRLADEDVD 179 UC0125.US NS5A
RFVPPVPKPRTRVSGVLERVRMCMRTPPIKF 180 UC0125.US NS5A RFVPPVPKPRTRVSGV
181 UC0125.US NS5A SGVLERVRMCMRTPPIKF 182 UC0125.US NS5A
KPRTRVSGVLERVRM 183 ABT0096P.US NS5A RTRVSGVLERVRMCMTT 184
UC0125.US NS5B NTTRDHNNGITYTDLVSGRAKP 185 UC0125.US NS5B
NTTRDHNNGITYTD 186 UC0125.US NS5B YTDLVSGRAKP 187 ABT0070P.US NS5B
NTTRDHNNGITYSDLVSGRAKP 188 ABT0070P.US NS5B NTTRDHNNGITYSD 189
ABT0070P.US NS5B YSDLVSGRAKP 190 UC0125.US NS5B
DAPMRIIPKPEVFPRDKSTRKPPRFIVFPGCAARV 191 UC0125.US NS5B
DAPMRIIPKPEVFPRDKSTRKPPR 192 UC0125.US NS5B DKSTRKPPRFIVFPGCAARV
193 UC0125.US NS5B IPKPEVFPRDKSTRKPPRFI 194 ABT0070P.US NS5B
DAPMRIIPKPEVFPRDKTTRKPPRFIVFPGCAARV 195 ABT0070P.US NS5B
DAPMRIIPKPEVFPRDKTTRKPPR 196 ABT0070P.US NS5B DKTTRKPPRFIVFPGCAARV
197 ABT0070P.US NS5B IPKPEVFPRDKTTRKPPRFI 198 UC0125.US NS5B
MPLLCMLIRNEPSQTGTLVT 199 UC0125.US NS5B MPLLCMLIRNEPSQT 200
UC0125.US NS5B MLIRNEPSQTGTLVT 201 ABT0070P.US NS5B
LPLLCMLIRNEPSQTGTLVT 202 ABT0070P.US NS5B LPLLCMLIRNEPSQT 203
ABT0096P.US NS5B LPLLCMLIRNEPSQTGTLVT 204 UC0125.US S
AEAAPKSGELDSQCDHLAWSFMEGMPTGTLIVQRDGSLH 205 UC0125.US S
AEAAPKSGELDSQCDHLAWSFME 206 UC0125.US S FMEGMPTGTLIVQRDGSLH 207
UC0125.US S QCDHLAWSFMEGMPTGT 208 UC0125.US NS4A-
SVEVRPAGVTRPDATDETAAYAQRLYQACADSGIFASLQGTASAA 209 B LGKLA UC0125.US
NS4A- SVEVRPAGVTRPDATDETAAYAQRLYQACAD 210 B UC0125.US NS4A-
ACADSGIFASLQGTASAALGKLA 211 B UC0125.US NS4A-
VTRPDATDETAAYAQRLYQACADSGIFASLQG 212 B ABT0070P.US NS4A-
SVENGLAGVTRPDATDETAAYAQRLYQACADSGILASLQGTASAA 213 B LSRLA
ABT0070P.US NS4A- SVENGLAGVTRPDATDETAAYAQRLYQACAD 214 B ABT0070P.US
NS4A- ACADSGILASLQGTASAALSRLA 215 B ABT0070P.US NS4A-
VTRPDATDETAAYAQRLYQACADSGILASLQG 216 B
[0138] The polynucleotides described herein may be employed for
producing HPgV-2 polypeptides by recombinant techniques. Thus, for
example, the polynucleotide may be included in any one of a variety
of expression vectors for expressing a polypeptide. In some
embodiments, vectors include, but are not limited to, chromosomal,
nonchromosomal and synthetic DNA sequences (e.g., derivatives of
SV40, bacterial plasmids, phage DNA; baculovirus, yeast plasmids,
vectors derived from combinations of plasmids and phage DNA, and
viral DNA such as vaccinia, adenovirus, fowl pox virus, and
pseudorabies). It is contemplated that any vector may be used as
long as it is replicable and viable in the host.
[0139] In particular, some embodiments provided herein are
recombinant constructs comprising one or more of the sequences as
broadly described above (e.g., SEQ ID NO:1, 75, 299-303, or
sub-portion thereof). In some embodiments, the constructs comprise
a vector, such as a plasmid or viral vector, into which a sequence
of the invention has been inserted, in a forward or reverse
orientation. In still other embodiments, the heterologous
structural sequence is assembled in appropriate phase with
translation initiation and termination sequences. In certain
embodiments, the appropriate DNA sequence is inserted into the
vector using any of a variety of procedures. In general, the DNA
sequence is inserted into an appropriate restriction endonuclease
site(s) by procedures known in the art.
[0140] Large numbers of suitable vectors are known to those of
skill in the art, and are commercially available. Such vectors
include, but are not limited to, the following vectors: 1)
Bacterial--pQE70, pQE60, pQE 9 (Qiagen), pBS, pD10, phagescript,
psiX174, pbluescript SK, pBSKS, pNH8A, pNH16a, pNH18A, pNH46A
(Stratagene); ptrc99a, pKK223 3, pKK233 3, pDR540, pRIT5
(Pharmacia); pET vectors (Novagen); and 2) Eukaryotic--pWLNEO,
pSV2CAT, pOG44, PXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL
(Pharmacia). Any other plasmid or vector may be used as long as
they are replicable and viable in the host.
[0141] In certain embodiments, the DNA sequence in the expression
vector is operatively linked to an appropriate expression control
sequence(s) (promoter) to direct mRNA synthesis. Useful promoters
include, but are not limited to, the LTR or SV40 promoter, the E.
coli lac or trp, the phage lambda PL and PR, T3 and T7 promoters,
and the cytomegalovirus (CMV) immediate early, herpes simplex virus
(HSV) thymidine kinase, and mouse metallothionein I promoters and
other promoters known to control expression of gene in prokaryotic
or eukaryotic cells or their viruses. In other embodiments,
recombinant expression vectors include origins of replication and
selectable markers permitting transformation of the host cell
(e.g., dihydrofolate reductase or neomycin resistance for
eukaryotic cell culture, or tetracycline or ampicillin resistance
in E. coli).
[0142] In a further embodiment, provided herein are host cells
containing the above described constructs. In some embodiments, the
host cell is a higher eukaryotic cell (e.g., a mammalian or insect
cell). In other embodiments, the host cell is a lower eukaryotic
cell (e.g., a yeast cell). In still other embodiments, the host
cell can be a prokaryotic cell (e.g., a bacterial cell). Specific
examples of host cells include, but are not limited to, Escherichia
coli, Salmonella typhimurium, Bacillus subtilis, and various
species within the genera Pseudomonas, Streptomyces, and
Staphylococcus, as well as Saccharomycees cerivisiae,
Schizosaccharomycees pombe, Drosophila S2 cells, Spodoptera Sf9
cells, Chinese hamster ovary (CHO) cells, COS 7 lines of monkey
kidney fibroblasts, (Gluzman, Cell 23:175 [1981]), C127, 3T3, 293,
293T, HeLa and BHK cell lines.
[0143] HPgV-2 proteins can be expressed in mammalian cells, yeast,
bacteria, or other cells under the control of appropriate
promoters. Cell free translation systems can also be employed to
produce such proteins using RNAs derived from the DNA constructs
described herein. Appropriate cloning and expression vectors for
use with prokaryotic and eukaryotic hosts are described by
Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second
Edition, Cold Spring Harbor, N.Y., (1989).
[0144] The present disclosure also provides methods for recovering
and purifying HPgV-2 proteins from recombinant cell cultures
including, but not limited to, ammonium sulfate or ethanol
precipitation, acid extraction, anion or cation exchange
chromatography, phosphocellulose chromatography, hydrophobic
interaction chromatography, affinity chromatography,
hydroxylapatite chromatography and lectin chromatography. In other
embodiments, protein refolding steps can be used as necessary, in
completing configuration of the mature protein. In still other
embodiments, high performance liquid chromatography (HPLC) can be
employed for final purification steps.
[0145] The present disclosure further provides polynucleotides
having the coding sequence (e.g., portions of SEQ ID NOs:1, 75, or
299-303) fused in frame to a marker sequence which allows for
purification polypeptides. A non-limiting example of a marker
sequence is a hexahistidine tag which may be supplied by a vector,
preferably a pQE 9 vector, which provides for purification of the
polypeptide fused to the marker in the case of a bacterial host,
or, for example, the marker sequence may be a hemagglutinin (HA)
tag when a mammalian host (e.g., COS 7 cells) is used. The HA tag
corresponds to an epitope derived from the influenza hemagglutinin
protein (Wilson et al., Cell, 37:767 [1984]).
[0146] In certain embodiments, the HPgV-2 peptides (e.g., SEQ ID
NOs: 86-218 and 304-353) are conservatively modified.
Conservatively modified variants applies to both amino acid and
nucleic acid sequences. With respect to particular nucleic acid
sequences, conservatively modified variants refers to those nucleic
acids which encode identical or essentially identical amino acid
sequences, or where the nucleic acid does not encode an amino acid
sequence, to essentially identical sequences. Because of the
degeneracy of the genetic code, a large number of functionally
identical nucleic acids encode any given protein. For instance, the
codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
Thus, at every position where an alanine is specified by a codon,
the codon can be altered to any of the corresponding codons
described without altering the encoded polypeptide. Every nucleic
acid sequence herein which encodes a polypeptide also describes
every possible silent variation of the nucleic acid. One of skill
will recognize that each codon in a nucleic acid (except AUG, which
is ordinarily the only codon for methionine, and TGG, which is
ordinarily the only codon for tryptophan) can be modified to yield
a functionally identical molecule. Accordingly, each silent
variation of a nucleic acid which encodes a polypeptide is implicit
in each described sequence with respect to the expression product,
but not with respect to actual probe sequences. As to amino acid
sequences, one of skill will recognize that individual
substitutions, deletions or additions to a nucleic acid, peptide,
polypeptide, or protein sequence which alters, adds or deletes a
single amino acid or a small percentage of amino acids in the
encoded sequence is a conservatively modified variant where the
alteration results in the substitution of an amino acid with a
chemically similar amino acid. Conservative substitution tables
providing functionally similar amino acids are well known in the
art. Such conservatively modified variants are in addition to and
do not exclude polymorphic variants, interspecies homologs, and
alleles of the invention. The following eight groups each contain
amino acids that are conservative substitutions for one another: 1)
Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5)
Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6)
Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S),
Threonine (T); and 8) Cysteine (C), Methionine (M).
VI. Antibody Generation and Immunoassays
[0147] In some embodiments, antibodies are used for the detection
of HPgV-2 protein. The antibodies may be prepared using various
immunogens. In one embodiment, the immunogen is a HPgV-2 peptide
(e.g., as shown in SEQ ID NOs:2-11, 76-218, and 304-353), or
portions thereof, to generate antibodies that recognize HPgV-2.
Such antibodies include, but are not limited to polyclonal,
monoclonal, chimeric, single chain, Fab fragments, and Fab
expression libraries.
[0148] Various procedures known in the art may be used for the
production of polyclonal antibodies directed against HPgV-2. For
the production of antibody, various host animals can be immunized
by injection with the peptide corresponding to an HPgV-2 epitope
including but not limited to rabbits, mice, rats, sheep, goats,
etc. In certain embodiments, the peptide is conjugated to an
immunogenic carrier (e.g., diphtheria toxoid, bovine serum albumin
(BSA), or keyhole limpet hemocyanin (KLH)). Various adjuvants may
be used to increase the immunological response, depending on the
host species, including but not limited to Freund's (complete and
incomplete), mineral gels (e.g., aluminum hydroxide), surface
active substances (e.g., lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, keyhole limpet hemocyanins,
dinitrophenol, and potentially useful human adjuvants such as BCG
(Bacille Calmette Guerin) and Corynebacterium parvum).
[0149] For preparation of monoclonal antibodies directed toward
HPgV-2, it is contemplated, in certain embodiments, that any
technique that provides for the production of antibody molecules by
continuous cell lines in culture will find use with the present
disclosure (See e.g., Harlow and Lane, Antibodies: A Laboratory
Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y.). These include but are not limited to the hybridoma technique
originally developed by Kohler and Milstein (Kohler and Milstein,
Nature 256:495 497 [1975]), as well as the trioma technique, the
human B cell hybridoma technique (See e.g., Kozbor et al., Immunol.
Tod., 4:72 [1983]), and the EBV hybridoma technique to produce
human monoclonal antibodies (Cole et al., in Monoclonal Antibodies
and Cancer Therapy, Alan R. Liss, Inc., pp. 77 96 [1985]).
[0150] In certain embodiments, monoclonal antibodies are produced
in germ free animals utilizing technology such as that described in
PCT/US90/02545. Furthermore, it is contemplated that human
antibodies may be generated by human hybridomas (Cote et al., Proc.
Natl. Acad. Sci. USA 80:2026 2030 [1983]) or by transforming human
B cells with EBV virus in vitro (Cole et al., in Monoclonal
Antibodies and Cancer Therapy, Alan R. Liss, pp. 77 96 [1985]).
[0151] In addition, it is contemplated that techniques described
for the production of single chain antibodies (U.S. Pat. No.
4,946,778; herein incorporated by reference) will find use in
producing HPgV-2 specific single chain antibodies. An additional
embodiment of the invention utilizes the techniques described for
the construction of Fab expression libraries (Huse et al., Science
246:1275 1281 [1989]) to allow rapid and easy identification of
monoclonal Fab fragments with the desired specificity for
HPgV-2.
[0152] It is contemplated that any technique suitable for producing
antibody fragments will find use in generating antibody fragments
that contain the idiotype (antigen binding region) of the antibody
molecule. For example, such fragments include but are not limited
to: F(ab')2 fragment that can be produced by pepsin digestion of
the antibody molecule; Fab' fragments that can be generated by
reducing the disulfide bridges of the F(ab')2 fragment, and Fab
fragments that can be generated by treating the antibody molecule
with papain and a reducing agent.
[0153] In the production of antibodies, it is contemplated that
screening for the desired antibody will be accomplished by
techniques known in the art (e.g., radioimmunoassay, ELISA (enzyme
linked immunosorbant assay), "sandwich" immunoassays,
immunoradiometric assays, gel diffusion precipitation reactions,
immunodiffusion assays, in situ immunoassays (e.g., using colloidal
gold, enzyme or radioisotope labels, for example), Western blots,
precipitation reactions, agglutination assays (e.g., gel
agglutination assays, hemagglutination assays, etc.), complement
fixation assays, immunofluorescence assays, protein A assays, and
immunoelectrophoresis assays, etc.
[0154] In one embodiment, antibody binding is detected by detecting
a label on the primary antibody. In another embodiment, the primary
antibody is detected by detecting binding of a secondary antibody
or reagent to the primary antibody. In a further embodiment, the
secondary antibody is labeled. As is well known in the art, the
immunogenic peptide should generally be provided free of the
carrier molecule used in any immunization protocol. For example, if
the peptide was conjugated to KLH, it may be conjugated to BSA, or
used directly, in a screening assay to detect HPgV-2 in a sample.
The foregoing antibodies can be used to detect HPgV-2 in a
biological sample from an individual (e.g., suspected of being
infected with HPgV-2). The biological sample can be a biological
fluid, such as, but not limited to, blood, serum, plasma,
interstitial fluid, urine, cerebrospinal fluid, and the like,
containing cells.
[0155] The biological samples can then be tested directly for the
presence of HPgV-2 using an appropriate strategy (e.g., ELISA or
radioimmunoassay) and format (e.g., microwells, dipstick (e.g., as
described in International Patent Publication WO 93/03367), etc.
Alternatively, proteins in the sample can be size separated (e.g.,
by polyacrylamide gel electrophoresis (PAGE), in the presence or
not of sodium dodecyl sulfate (SDS), and the presence of HPgV-2
detected by immunoblotting (Western blotting). Immunoblotting
techniques are generally more effective with antibodies generated
against a peptide corresponding to an epitope of a protein, and
hence, are particularly suited to the methods and compositions
disclosed herein.
[0156] In some embodiments, HPgV-2 is detected with an immunoassay
such as: 1) a sandwich immunoassay (e.g., monoclonal, polyclonal
and/or DVD-Ig sandwich immunoassays or any variation thereof (e.g.,
monoclonal/DVD-Ig or DVD-Ig/polyclonal), including
chemiluminescence detection, radioisotope detection (e.g.,
radioimmunoassay (MA)) and enzyme detection (e.g., enzyme
immunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA)
(e.g., Quantikine ELISA assays, R&D Systems, Minneapolis,
Minn.))), 2) a competitive inhibition immunoassay (e.g., forward
and reverse), 3) a fluorescence polarization immunoassay (FPIA), 4)
an enzyme multiplied immunoassay technique (EMIT), 5) a
bioluminescence resonance energy transfer (BRET), 6) a homogeneous
chemiluminescent assay, 7) a SELDI-based immunoassay, 8)
chemiluminescent microparticle immunoassay (CMIA) and 9) a clinical
chemistry colorimetric assay (e.g., IMA, creatinine for eGFR
determination and LC-MS/MS). (See, e.g., Tietz Textbook of Clinical
Chemistry and Molecular Diagnostics. 4th Edition, edited by C A
Burtis, E R Ashwood and D E Bruns, Elsevier Saunders, St. Louis,
Mo., 2006.).
[0157] Further, if an immunoassay is being utilized, any suitable
detectable label as is known in the art can be used. For example,
the detectable label can be a radioactive label (such as 3H, 1251,
35S, 14C, 32P, and 33P), an enzymatic label (such as horseradish
peroxidase, alkaline peroxidase, glucose 6-phosphate dehydrogenase,
and the like), a chemiluminescent label (such as acridinium esters,
thioesters, or sulfonamides; luminol, isoluminol, phenanthridinium
esters, and the like), a fluorescent label (such as fluorescein
(e.g., 5-fluorescein, 6-carboxyfluorescein, 3'6-carboxyfluorescein,
5(6)-carboxyfluorescein, 6-hexachloro-fluorescein,
6-tetrachlorofluorescein, fluorescein isothiocyanate, and the
like)), rhodamine, phycobiliproteins, R-phycoerythrin, quantum dots
(e.g., zinc sulfide-capped cadmium selenide), a thermometric label,
or an immuno-polymerase chain reaction label. An introduction to
labels, labeling procedures and detection of labels is found in
Polak and Van Noorden, Introduction to Immunocytochemistry, 2nd
ed., Springer Verlag, N.Y. (1997), and in Haugland, Handbook of
Fluorescent Probes and Research Chemicals (1996), which is a
combined handbook and catalogue published by Molecular Probes,
Inc., Eugene, Oreg. A fluorescent label can be used in FPIA (see,
e.g., U.S. Pat. Nos. 5,593,896, 5,573,904, 5,496,925, 5,359,093,
and 5,352,803, which are hereby incorporated by reference in their
entireties). An acridinium compound can be used as a detectable
label in a homogeneous or heterogeneous chemiluminescent assay
(see, e.g., Adamczyk et al., Bioorg. Med. Chem. Lett. 16: 1324-1328
(2006); Adamczyk et al., Bioorg. Med. Chem. Lett. 4: 2313-2317
(2004); Adamczyk et al., Biorg. Med. Chem. Lett. 14: 3917-3921
(2004); and Adamczyk et al., Org. Lett. 5: 3779-3782 (2003)).
[0158] A preferred acridinium compound is an
acridinium-9-carboxamide. Methods for preparing acridinium
9-carboxamides are described in Mattingly, J. Biolumin. Chemilumin.
6: 107-114 (1991); Adamczyk et al., J. Org. Chem. 63: 5636-5639
(1998); Adamczyk et al., Tetrahedron 55: 10899-10914 (1999);
Adamczyk et al., Org. Lett. 1: 779-781 (1999); Adamczyk et al.,
Bioconjugate Chem. 11: 714-724 (2000); Mattingly et al., In
Luminescence Biotechnology: Instruments and Applications; Dyke, K.
V. Ed.; CRC Press: Boca Raton, pp. 77-105 (2002); Adamczyk et al.,
Org. Lett. 5: 3779-3782 (2003); and U.S. Pat. Nos. 5,468,646,
5,543,524 and 5,783,699 (each of which is incorporated herein by
reference in its entirety for its teachings regarding same).
Another preferred acridinium compound is an
acridinium-9-carboxylate aryl ester. An example of an
acridinium-9-carboxylate aryl ester is
10-methyl-9-(phenoxycarbonyl)acridinium fluorosulfonate (available
from Cayman Chemical, Ann Arbor, Mich.). Methods for preparing
acridinium 9-carboxylate aryl esters are described in McCapra et
al., Photochem. Photobiol. 4: 1111-21 (1965); Razavi et al.,
Luminescence 15: 245-249 (2000); Razavi et al., Luminescence 15:
239-244 (2000); and U.S. Pat. No. 5,241,070 (each of which is
incorporated herein by reference in its entirety for its teachings
regarding same). Further details regarding acridinium-9-carboxylate
aryl ester and its use are set forth in U.S. published application
no. 2008-0248493. Chemiluminescent assays (e.g., using acridinium
as described above or other chemiluminescent agents) can be
performed in accordance with the methods described in Adamczyk et
al., Anal. Chim. Acta 579(1): 61-67 (2006). While any suitable
assay format can be used, a microplate chemiluminometer (Mithras
LB-940, Berthold Technologies U.S.A., LLC, Oak Ridge, Tenn.)
enables the assay of multiple samples of small volumes rapidly.
Upon the simultaneous or subsequent addition of at least one basic
solution to the sample, a detectable signal, namely, a
chemiluminescent signal, indicative of the presence of analyte is
generated. The basic solution contains at least one base and has a
pH greater than or equal to 10, preferably, greater than or equal
to 12. Examples of basic solutions include, but are not limited to,
sodium hydroxide, potassium hydroxide, calcium hydroxide, ammonium
hydroxide, magnesium hydroxide, sodium carbonate, sodium
bicarbonate, calcium hydroxide, calcium carbonate, and calcium
bicarbonate. The amount of basic solution added to the sample
depends on the concentration of the basic solution. Based on the
concentration of the basic solution used, one skilled in the art
can easily determine the amount of basic solution to add to the
sample.
[0159] The chemiluminescent signal that is generated can be
detected using routine techniques known to those skilled in the
art. Based on the intensity of the signal generated, the amount of
analyte in the sample can be quantified. Specifically, the amount
of analyte in the sample is proportional to the intensity of the
signal generated. The amount of analyte present can be quantified
by comparing the amount of light generated to a standard curve for
the analyte or by comparison to a reference standard. The standard
curve can be generated using serial dilutions or solutions of known
concentrations of analyte by mass spectroscopy, gravimetric
methods, and other techniques known in the art. While the above is
described with emphasis on use of an acridinium compound as the
chemiluminescent agent, one of ordinary skill in the art can
readily adapt this description for use of other chemiluminescent
agents.
[0160] Immunoassays can generally be conducted using any format
known in the art, such as, but not limited to, a sandwich format.
Specifically, in one immunoassay format, at least two antibodies
are employed to separate and quantify HPgV-2 or a fragment thereof
in a sample. More specifically, the at least two antibodies bind to
different epitopes on HPgV-2 (or a fragment thereof) forming an
immune complex, which is referred to as a "sandwich." Generally, in
the immunoassays, one or more antibodies can be used to capture the
HPgV-2 (or a fragment thereof) in the test sample (i.e., these
antibodies are frequently referred to as a "capture" antibody or
"capture" antibodies) and one or more antibodies can be used to
bind a detectable (namely, quantifiable) label to the sandwich
(i.e., these antibodies are frequently referred to as the
"detection antibody," the "detection antibodies," the "conjugate,"
or the "conjugates"). Thus, in the context of a sandwich
immunoassay format, an antibody (or a fragment, a variant, or a
fragment of a variant thereof) can be used as a capture antibody, a
detection antibody, or both. For example, one DVD-Ig having a
domain that can bind a first epitope on HPgV-2 (or a fragment
thereof) can be used as a capture antibody and/or another DVD-Ig
having a domain that can bind a second epitope on HPgV-2 (or a
fragment thereof) can be used as a detection antibody. In this
regard, a DVD-Ig having a first domain that can bind a first
epitope on an analyte (or a fragment thereof) and a second domain
that can bind a second epitope on an analyte (or a fragment
thereof) can be used as a capture antibody and/or a detection
antibody. Alternatively, one DVD-Ig having a first domain that can
bind an epitope on a first analyte (or a fragment thereof) and a
second domain that can bind an epitope on a second analyte (or a
fragment thereof) can be used as a capture antibody and/or a
detection antibody to detect, and optionally quantify, two or more
analytes.
[0161] Generally speaking, in an immunoassay, a sample being tested
for (for example, suspected of containing) HPgV-2 (or a fragment
thereof) can be contacted with at least one capture antibody (or
antibodies) and at least one detection antibody (which can be a
second detection antibody or a third detection antibody or even a
successively numbered antibody, e.g., as where the capture and/or
detection antibody comprise multiple antibodies) either
simultaneously or sequentially and in any order. For example, the
test sample can be first contacted with at least one capture
antibody and then (sequentially) with at least one detection
antibody. Alternatively, the test sample can be first contacted
with at least one detection antibody and then (sequentially) with
at least one capture antibody. In yet another alternative, the test
sample can be contacted simultaneously with a capture antibody and
a detection antibody.
[0162] In the sandwich assay format, described above, a sample
suspected of containing HPgV-2 (or a fragment thereof) is first
brought into contact with at least one first capture antibody under
conditions that allow the formation of a first antibody/HPgV-2
complex. If more than one capture antibody is used, a first capture
antibody/HPgV-2 complex comprising two or more capture antibodies
is formed. In a sandwich assay, the antibodies, i.e., preferably,
the at least one capture antibody, are used in molar excess amounts
of the maximum amount of HPgV-2 (or a fragment thereof) expected in
the test sample. For example, from about 5.mu.g to about 1 mg of
antibody per mL of buffer (e.g., microparticle coating buffer) can
be used.
[0163] In contrast, competitive inhibition immunoassays, which are
often used to measure small analytes because binding by only one
antibody is required, comprise sequential and classic formats. In a
sequential competitive inhibition immunoassay, a capture antibody
to an analyte of interest (e.g., HPgV-2 protein) is coated onto a
well of a microtiter plate or other solid support. When the sample
containing the analyte of interest is added to the well, the
analyte of interest binds to the capture antibody. After washing, a
known amount of labeled analyte (e.g., acridinium, biotin or
horseradish peroxidase (HRP)) is added to the well. A substrate for
an enzymatic label is necessary to generate a signal. An example of
a suitable substrate for HRP is 3,3',5,5'-tetramethylbenzidine
(TMB). After washing, the signal generated by the labeled analyte
is measured and is inversely proportional to the amount of analyte
in the sample. In a classic competitive inhibition immunoassay, an
antibody to an analyte of interest is coated onto a solid support
(e.g., a well of a microtiter plate). However, unlike the
sequential competitive inhibition immunoassay, the sample and the
labeled analyte are added to the well at the same time. Any analyte
in the sample competes with labeled analyte for binding to the
capture antibody. After washing, the signal generated by the
labeled analyte is measured and is inversely proportional to the
amount of analyte in the sample.
[0164] The concentration of HPgV-2 or a fragment thereof in the
test sample is determined by appropriate means, such as by use of a
standard curve that has been generated using serial dilutions of
analyte or a fragment thereof of known concentration. Other than
using serial dilutions of analyte or a fragment thereof, the
standard curve can be generated gravimetrically, by mass
spectroscopy and by other techniques known in the art.
[0165] In a chemiluminescent microparticle assay employing the
ARCHITECT analyzer, the conjugate diluent pH may be about
6.0+/-0.2, the microparticle coating buffer may be maintained at
about room temperature (i.e., at from about 17 to about 27.degree.
C.), the microparticle coating buffer pH may be about 6.5+/-0.2,
and the microparticle diluent pH may be about 7.8+/-0.2. Solids
preferably are less than about 0.2%, such as less than about 0.15%,
less than about 0.14%, less than about 0.13%, less than about
0.12%, or less than about 0.11%, such as about 0.10%. Of course,
these ranges or numbers may be altered in order to enhance such
properties of the assay including, for example, reduction in
background interference, increased sensitivity, increased
specificity, etc.
[0166] FPIAs are based on competitive binding immunoassay
principles. A fluorescently labeled compound, when excited by a
linearly polarized light, will emit fluorescence having a degree of
polarization inversely proportional to its rate of rotation. When a
fluorescently labeled tracer-antibody complex is excited by a
linearly polarized light, the emitted light remains highly
polarized because the fluorophore is constrained from rotating
between the time light is absorbed and the time light is emitted.
When a "free" tracer compound (i.e., a compound that is not bound
to an antibody) is excited by linearly polarized light, its
rotation is much faster than the corresponding tracer-antibody
conjugate produced in a competitive binding immunoassay. FPIAs are
advantageous over RIAs inasmuch as there are no radioactive
substances requiring special handling and disposal. In addition,
FPIAs are homogeneous assays that can be easily and rapidly
performed.
[0167] The present description is not limited by the type of
immunoassay employed to detect patient antibodies in a sample. A
number of exemplary formats are as follows. In an indirect assay,
HPgV-2 peptide or protein is coated on solid phase (e.g., beads)
and then contacted with a sample (e.g. 18 minutes), followed by a
wash step. Then, in a second step, patient antibodies to HPgV-2 are
detected by contacting the immune complex with labeled "second"
antibody to detect human IgG (or IgM) bound to the solid phase
(e.g. for 4 minutes). Another assay is a two step direct (sandwich)
assay. In this assay, HPgV-2 peptide or protein is coated on solid
phase (e.g., beads) and contacted with sample (e.g. for about 18
minutes) and then washed. In a second step, antibodies to HPgV-2
are detected with a labeled HPgV-2 peptide/protein that binds to
human IgG (or IgM) bound to the solid phase containing the HPgV-2
protein (e.g. for 4 minutes). A one-step direct (sandwich) assay
could also be employed. In such an assay, HPgV-2 peptide or protein
is coated on solid phase and contacted with sample (e.g., for about
18 minutes) and with labeled HPgV-2 peptide/protein at the same
time or about the same time (e.g., for 18 minutes). Another type of
assay is a solution phase capture. In such an assay, the sample is
contacted with both protein tagged HPgV-2 peptide or protein (e.g.,
biotin tag, FLAG-tag, HA-tag, etc.) and labeled HPgV-2 peptide or
protein in the presence of a solid phase coated with an affinity
molecule (e.g., streptavidin or protein tag antibody). If the
patient antibodies are present in the sample, the tagged peptide or
protein and labeled HPgV-2 peptides or proteins can bind to patient
antibodies in a complex that can be captured by the associated
protein tag to a solid phase support. In all of these assay
formats, the solid phase is further processed to elicit a signal
from labeled HPgV-2 associated with patient antibodies and with the
solid phase.
[0168] In particular embodiments, the antigens and antibodies
described herein are contemplated for use as immunodiagnostic
reagents in combination immunoassays designed for the detection of
multiple HPgV-2 components found in a test sample suspected of
having been infected with HPgV-2. Immunodiagnostic reagents may
used in a combination assay that detects both peptides and patient
antibodies. For purposes of capture, the antigens and/or antibodies
of which the immunodiagnostic reagent is comprised can be coated on
a solid support such as for example, a microparticle, (e.g.,
magnetic particle), bead, test tube, microtiter plate, cuvette,
membrane, scaffolding molecule, film, filter paper, disc or chip.
In this regard, where the immunodiagnostic reagent comprises a
combination of antigens (e.g., directed at different HPgV-2
proteins or different fragments of the same HPgV-2 protein), the
antigens can be co-coated on the same solid support or can be on
separate solid supports. Likewise, where the immunodiagnostic
reagent comprises one or more antibodies that will be used to
capture one or more antigens from the test sample, such-antibodies
can be co-coated on the same solid support or can be on separate
solid supports.
[0169] Notably, the immunodiagnostic reagent may include the
antigens and antibodies labeled with a detectable label or labeled
with a specific partner that allows capture or detection. For
example, the labels may be a detectable label, such as a
fluorophore, radioactive moiety, enzyme, biotin/avidin label,
chromophore, chemiluminescent label, or the like. Still further the
invention contemplates the preparation of HPgV-2 diagnostic kits
comprising the immunodiagnostic reagents described herein and
instructions for the use of the immunodiagnostic reagents in
immunoassays for determining the presence of HPgV-2 in a test
sample by detecting the presence of two or more HPgV-2 proteins
and/or anti-HPgV-2 antibodies in such a sample. For example, the
kit can comprise instructions for assaying the test sample for
anti-HPgV-2 antibody (e.g., an anti Core antibody in the test
sample) by immunoassay. While certain embodiments employ
chemiluminescent microparticle immunoassay for assaying the test
sample, it should be understood that the antigens and antibodies
used in the immunoassays of the present invention may be used in
any other immunoassay formats known to those of skill in the art
for determining the presence of HPgV-2 in a test sample. The
instructions can be in paper form or computer-readable form, such
as a disk, CD, DVD, or the like. Alternatively or additionally, the
kit can comprise a calibrator or control, e.g., purified, and
optionally lyophilized, anti-HPgV-2 antibody or antigen, and/or at
least one container (e.g., tube, microtiter plates or strips, which
can be already coated with one or more of the capture components
(antigens and/or antibodies) of the immunoassay) for conducting the
assay, and/or a buffer, such as an assay buffer or awash buffer,
either one of which can be provided as a concentrated solution, a
substrate solution for the detectable label (e.g., an enzymatic
label), or a stop solution. In certain embodiments, the kit
comprises all components, i.e., reagents, standards, buffers,
diluents, etc., which are necessary to perform the assay. In
specific embodiments, the components are individually presented in
the kit such that the immunoassay may be performed as a
capture-on-the-fly type combination immunoassay in which the solid
support is coated with an agent that allows binding of the
capturing moiety (e.g., a-33-biotinylated antigen or a biotinylated
antibody) and the kit further comprises each of the individual
capture and detection antigen pairs and the biotinylated capture
antibodies in one container and a second container provides the
detection antibody conjugate. The instructions for conducting the
assay also can include instructions for generating a standard curve
or a reference standard for purposes of quantifying anti-HPgV-2
antibody.
[0170] Any antibodies, which are provided in the kit, such as
anti-IgG antibodies and anti-IgM antibodies, can also incorporate a
detectable label, such as a fluorophore, radioactive moiety,
enzyme, biotin/avidin label, chromophore, chemiluminescent label,
or the like, or the kit can include reagents for labeling the
antibodies or reagents for detecting the antibodies (e.g.,
detection antibodies) and/or for labeling the analytes or reagents
for detecting the analyte. The antibodies, calibrators and/or
controls can be provided in separate containers or pre-dispensed
into an appropriate assay format, for example, into microtiter
plates. In certain immunoassays, there are two containers provided.
In the first container is provided at least a first, second and
third pair of antigens, wherein the first antigen in each pair is a
capture antigen from a given HPgV-2 protein that is biotinylated
and the second antigen in each pair is a detection antigen from the
same protein as the first antigen but is labeled with a detectable
label(e.g., it is acridinylated) as well as one or more
biotinylated antibodies designed for detecting one or more HPgV-2
antigens from a test sample; and in the second container is
provided the antibody that forms the conjugation partner for
detection of the antigen that is captured by the biotinylated
antibodies from the first container. It is contemplated that where
there are multiple biotinylated antibodies in the first container,
the multiple antibodies that form the conjugation partners may be
present in a single container or individual containers for each
different antigen detecting conjugate antibody.
[0171] Optionally, the kit includes quality control components (for
example, sensitivity panels, calibrators, and positive controls).
Preparation of quality control reagents is well-known in the art
and is described on insert sheets for a variety of immunodiagnostic
products. Sensitivity panel members optionally are used to
establish assay performance characteristics, and further optionally
are useful indicators of the integrity of the immunoassay kit
reagents, and the standardization of assays.
[0172] The kit can also optionally include other reagents required
to conduct a diagnostic assay or facilitate quality control
evaluations, such as buffers, salts, enzymes, enzyme co-factors,
substrates, detection reagents, and the like. Other components,
such as buffers and solutions for the isolation and/or treatment of
a test sample (e.g., pretreatment reagents), also can be included
in the kit. The kit can additionally include one or more other
controls. One or more of the components of the kit can be
lyophilized, in which case the kit can further comprise reagents
suitable for the reconstitution of the lyophilized components.
[0173] The various components of the kit optionally are provided in
suitable containers as necessary, e.g., a microtiter plate. The kit
can further include containers for holding or storing a sample
(e.g., a container or cartridge for a sample). Where appropriate,
the kit optionally also can contain reaction vessels, mixing
vessels, and other components that facilitate the preparation of
reagents or the test sample. The kit can also include one or more
instrument for assisting with obtaining a test sample, such as a
syringe, pipette, forceps, measured spoon, or the like.
[0174] In preferred embodiments, the detectable label is at least
one acridinium compound. In such embodiments, the kit can comprise
at least one acridinium-9carboxamide,at least one
acridinium-9-carboxylate aryl ester, or any combination thereof. If
the detectable label is at least one acridinium compound, the kit
also can comprise a source of hydrogen peroxide, such as a buffer,
solution, and/or at least one basic solution. It should be
understood that in the immunodiagnostic reagent the antigens for
antibody detection may be detectably labeled, and any antibodies
provided in kit for use along with such reagents also may be
detectably labeled. If desired, the kit can contain a solid support
phase, such as a magnetic particle, bead, test tube, microtiter
plate, cuvette, membrane, scaffolding molecule, film, filter paper,
disc or chip.
[0175] The present disclosure provides immunoassays and combination
immunoassays method for determining the presence, amount or
concentration of anti-HPgV-2 antibodies and HPgV-2 antigens in a
test sample. Any suitable assay known in the art can be used in
such methods. Examples of such assays include, but are not limited
to, immunoassay, such as sandwich immunoassay (e.g.,
monoclonal-polyclonal sandwich immunoassays, including radioisotope
detection (radioimmunoassay (RIA)) and enzyme detection (enzyme
immunoassay (EIA) or enzyme-linked immunosorbent assay
(ELISA)(e.g., Quantikine ELISA assays, R&D Systems,
Minneapolis, Minn.)), competitive inhibition immunoassay (e.g.,
forward and reverse), fluorescence polarization immunoassay (FPIA),
enzyme multiplied immunoassay technique (EMIT),bioluminescence
resonance energy transfer (BRET), and homogeneous chemiluminescent
assay, etc.
[0176] In specific embodiments of the immunoassays, the recombinant
antigens (e.g., core, NS3 and NS4 antigens) may be used as capture
reagents (e.g., by using such antigens in which the amino- or
carboxy-terminal of the antigen comprises a biotin tag) or as a
detection (conjugate) reagents in which the antigens are either
directly or indirectly labeled with acridinium. Indirect labeling
may employ the use of acridinylated BSA covalently coupled to the
free thiol of unpaired cysteine residues within a protein via
SMCC-type linker. To facilitate such indirect labeling certain of
the antigens used in the immunoassays of the present invention may
readily be further modified to include additional cysteine residues
at the C-terminus.
[0177] Typically, immunoassays are performed in 1-step or 2-step
format. Solid phase reagents for capture of immune complexes formed
in solution in the 1-step assay include anti-biotin monoclonal
antibody, streptavidin or neutravidin to capture the biotinylated
moiety (be it a biotinylated antigen for capture of an HPgV-2
antibody or a biotinylated antibody for the capture of an HPgV-2
protein/antigen in the test sample).
[0178] In a SELDI-based immunoassay, a capture reagent that
specifically binds anti-HPgV-2-antibody or an HPgV-2 antigen is
attached to the surface of a mass spectrometry probe, such as a
pre-activated protein chip array. The anti-HPgV-2 antibody or the
antigen is then specifically captured on the biochip, and the
captured moiety is detected by mass spectrometry. Alternatively,
the anti-HPgV-2 antibody can be eluted from the capture reagent and
detected by traditional MALDI (matrix-assisted laser
desorption/ionization) or by SELOI. A chemiluminescent
microparticle immunoassay, in particular one employing the
ARCHITECT.RTM. automated analyzer (Abbott Laboratories, Abbott
Park), is an example of an immunoassay in which a combination of
multiple antigens (e.g., antigens from two or more HPgV-2 proteins)
as well as multiple anti-HPgV-2 antibodies may readily be employed.
An agglutination assay, such as a passive hemaglutination assay,
also can be used. In an agglutination assay an antigen antibody
reaction is detected by agglutination or clumping. In a passive
hemaglutination assay, erythrocytes are coated with the antigen and
the coated erythrocytes are used in the agglutination assay. A
second embodiment of the measurement of HPgV-2 neutralizing
antibodies is the traditional virus neutralization test which
employs cell lines susceptible to infection with HPgV-2, and
measuring inhibition by one or more methods (e.g.
immunofluorescence, plaque assay methods, etc.) (see, Temperton et
al., Virol. J., 10:266-213, 2013, herein incorporated by
reference).
[0179] Methods well-known in the art for collecting, handling and
processing urine, blood, serum and plasma, and other body fluids,
are used in the practice of the present disclosure, for instance,
when the immunodiagnostic reagents comprise multiple antigens
and/or in an anti-HPgV-2 antibody immunoassay kit. The test sample
can comprise further moieties in addition to the polypeptide of
interest, such as antibodies, antigens, haptens, hormones, drugs,
enzymes, receptors, proteins, peptides, polypeptides,
oligonucleotides or polynucleotides. For example, the sample can be
a whole blood sample obtained from a subject. It can be necessary
or desired that a test sample, particularly whole blood, be treated
prior to immunoassay as described herein, e.g., with a pretreatment
reagent. Even in cases where pretreatment is not necessary (e.g.,
most urine samples), pretreatment optionally can be done for mere
convenience (e.g., as part of a regimen on a commercial
platform).
[0180] The pretreatment reagent can be any reagent appropriate for
use with the immunoassays and kits of the invention. The
pretreatment optionally comprises: (a) one or more solvents (e.g.,
methanol and ethylene glycol) and salt, (b) one or more solvents,
salt and detergent, (c) detergent, or (d) detergent and salt.
Pretreatment reagents are known in the art, and such pretreatment
can be employed, e.g., as used for assays on Abbott TOx,
AxSYM.RTM., and ARCHITECT.RTM. analyzers (Abbott Laboratories,
Abbott Park, IL), as described in the literature (see, e.g.,
Yatscoff et ai.,-37-Abbott TDx Monoclonal Antibody Assay Evaluated
for Measuring Cyclosporine in WholeBlood, Clin. Chem. 36: 1969-1973
(1990), and Wallemacq et aI., Evaluation of the New AxSYM
Cyclosporine Assay: Comparison with TDx Monoclonal Whole Blood and
EMITCyclosporine Assays, Clin. Chem. 45: 432-435 (1999)), and/or as
commercially available. Additionally, pretreatment can be done as
described in Abbott's U.S. Pat. No. 5,135,875, European Pat. Pub.
No. 0 471 293, U.S. Provisional Pat. App. 60/878,017,filed Dec. 29,
2006, and U.S. Pat. App. Pub. No. 2008/0020401 (incorporated by
reference in its entirety for its teachings regarding
pretreatment). The pretreatment reagent can be a heterogeneous
agent or a homogeneous agent.
[0181] With use of a heterogeneous pretreatment reagent, the
pretreatment reagent precipitates analyte binding protein (e.g.,
protein that can bind to anti-HPgV-2 antibody or an antigen that
can bind to an anti-HPgV-2 antibody form the present in the sample.
Such a pretreatment step comprises removing any analyte binding
protein by separating from the precipitated analyte binding protein
the supernatant of the mixture formed by addition of the
pretreatment agent to sample. In such an assay, the supernatant of
the mixture absent any binding protein is used in the assay,
proceeding directly to the antibody capture step.
[0182] With use of a homogeneous pretreatment reagent there is no
such separation step. The entire mixture of test sample and
pretreatment reagent are contacted with a labeled specific binding
partner for anti-HPgV-2 antibody (i.e., an antigen) or the labeled
specific binding partner for the HPgV-2 antigen (i.e., an
antibody). The pretreatment reagent employed for such an assay
typically is diluted in the pretreated test sample mixture, either
before or during capture by the first specific binding partner.
Despite such dilution, a certain amount of the pretreatment reagent
(for example, 5 M methanol and/or 0.6 methylene glycol) is still
present (or remains) in the test sample mixture during capture.
[0183] In a heterogeneous format, after the test sample is obtained
from a subject, a first mixture is prepared. The mixture contains
the test sample being assessed for anti-HPgV-2 antibodies and a
first specific capture binding partner, wherein the first specific
capture binding partner and any anti-HPgV-2 antibodies contained in
the test sample form a first specific capture binding
partner-anti-HPgV-2 antibody complex. The first specific capture
binding partner may be any of a core antigen, an NS3 antigen or an
NS3, or other HPgV-2 protein. Likewise, in certain embodiments, in
the combination assays of the invention the mixture also contains a
second and third specific capture binding partner and these second
and third specific capture binding partners form second and third
specific capture binding partner-anti-HPgV-2 antibody complexes
with anti-HPgV-2 antibodies that are present in the test
sample.
[0184] In addition the combination immunoassay may include at least
one anti-HPgV-2 capture antibody that will form a specific complex
with a fourth specific binding partner that is found in the test
sample (i.e., an antigen or HPgV-2 protein that is found in the
test sample) so as to form an anti-HPgV-2 antibody-four the
specific binding partner complex with the fourth antigen that is
present in the test sample.
[0185] In the combination immunoassays, the order in which the test
sample and the various specific binding partners are added to form
the mixture is not critical. In some embodiments, the first,
second, and third specific capture binding partners (i.e.,
antigens) and the anti-HPgV-2 capture antibody are immobilized on a
solid phase. In still other embodiments, none of these four
components are immobilized but are instead all added at the same
time to the test sample to effect capture onto the solid phase. The
solid phase used in the combination immunoassay can be any solid
phase known in the art, such as, but not limited to, a magnetic
particle, a bead, a test tube, a microtiter plate, a cuvette, a
membrane, a scaffolding molecule, a film, a filter paper, a disc
and a chip.
[0186] After the immunocomplexes are formed between the first,
second and third specific capture binding partners and their
respective anti-HPgV-2 antibodies found in the test sample, and the
first anti-HPgV-2 capture antibodies (e.g., anti-Core) and their
respective HPgV-2 antigens or HPgV-2 proteins found in the test
sample, any unbound antiHPgV-2 antibody or HPgV-2 antigen/protein
is removed from the complex using any technique known in the art.
For example, the unbound anti-HPgV-2 antibody or antigen can be
removed by washing. Desirably, however, the first, second and third
specific binding partners and the anti-HPgV-2 antibodies are
present in excess of any anti-HPgV-2 antibody and antigens,
respectively present in the test sample, such that all anti-HPgV-2
antibody and antigens that are present in the test sample become
bound by the first, second, and third specific binding partner and
anti-HPgV-2 capture antibodies respectively.
[0187] After any unbound anti-HPgV-2 antibody and antigen is
removed, detection is achieved by addition of a first specific
detection binding partner to the mixture to form a first specific
capture binding partner-anti-HPgV-2 antibody-first specific
detection binding partner complex. The first specific detection
binding partner is preferably a combination of an anti-IgG antibody
and an anti-IgM antibody. Moreover, also preferably, the first
specific detection binding partner is labeled with or contains a
detectable label. In specific embodiments, the first specific
detection partner may instead or in addition be an antigen that
binds the captured antibody. Likewise, in the combination assays of
the invention the mixture also contains a second and third specific
detection binding partner and these second and third specific
detection binding partners form second or third specific capture
binding partner-anti-HPgV-2 antibody second or third specific
detection binding partner complexes with the captured anti-HPgV-2
antibodies that are present in the test sample. Again, the second
and third specific detection binding partners may be a combination
of an anti-IgG antibody and an anti-IgM antibody. In specific
embodiments, the second and third specific detection partners may
instead or in addition be an antigen that binds the captured
antibody. Moreover, the second and third specific detection binding
partners, be they anti IgM or IgG antibodies or antigens, are
labeled with or contains a detectable label. In addition the
combination immunoassay may include at least one anti-HPgV-2
conjugate antibody that will form a specific complex with the
captured antigen or HPgV-2 protein that is found in the test sample
so as to form an anti-HPgV-2 antibody-fourth specific binding
partner-anti-HPgV-2 conjugate antibody complex with the fourth
antigen that captured from the test sample.
[0188] Any suitable detectable label as is known in the art can be
used as anyone or more of the detectable labels. For example, the
detectable label can be a radioactive label (such as 3H, 1251, 35S,
14C, 32p, and 33p), an enzymatic label (such as horseradish
peroxidase, alkaline peroxidase, glucose 6-phosphate dehydrogenase,
and the like), a chemiluminescent label (such as acridinium esters,
thioesters, orsulfonamides; luminol, isoluminol, phenanthridinium
esters, and the like), a fluorescentlabel (such as fluorescein
(e.g., 5-fluorescein, 6-carboxyfluorescein, 3'6-carboxyfluorescein,
5(6)-carboxyfluorescein, 6-hexachloro-fluorescein,
6-tetrachlorofluorescein, fluorescein isothiocyanate, and the
like)), rhodamine,phycobiliproteins, R-phycoerythrin, quantum dots
(e.g., zinc sulfide-capped cadmiumselenide), a thermometric label,
or an immuno-polymerase chain reaction label. An introduction to
labels, labeling procedures and detection of labels is found in
Polak andVan Noorden, Introduction to Immunocytochemistry, 2nd ed.,
Springer Verlag, N.Y.(1997), and in Haugland, Handbook of
Fluorescent Probes and Research Chemicals(1996), which is a
combined handbook and catalogue published by Molecular Probes,Inc.,
Eugene, Oreg. A fluorescent label can be used in FPIA (see, e.g.,
U.S. Pat. Nos. 5,593,896, 5,573,904, 5,496,925, 5,359,093, and
5,352,803, which are hereby incorporated by reference in their
entireties). An acridinium compound can be used as a detectable
label in a homogeneous chemiluminescent assay (see, e.g., Adamczyk
etaI., Bioorg. Med. Chem. Lett. 16: 1324-1328 (2006); Adamczyk et
aI, Bioorg. Med.Chem. Lett. 4: 2313-2317 (2004); Adamczyk et aI.,
Biorg. Med. Chem. Lett. 14: 39173921(2004); and Adamczyk et aI.,
Org. Lett. 5: 3779-3782 (2003)).
[0189] An exemplary acridinium compound is an
acridinium-9-carboxamide.Methods for preparing acridinium
9-carboxamides are described in Mattingly, J.Biolumin. Chemilumin.
6: 107-114 (1991); Adamczyk et aI., J. Org. Chem. 63:
56365639(1998); Adamczyk et aI., Tetrahedron 55: 10899-10914
(1999); Adamczyk et aI., Org. Lett. 1: 779-781 (1999); Adamczyk et
aI., Bioconjugate Chem. 11: 714-724 (2000);Mattingly et aI., In
Luminescence Biotechnology: Instruments and Applications; Dyke, K.
V. Ed.; CRC Press: Boca Raton, pp. 77-105 (2002); Adamczyk et aI.,
Org. Lett. 5: 37793782(2003); and U.S. Pat. Nos. 5,468,646,
5,543,524 and 5,783,699 (each of which isincorporated herein by
reference in its entirety for its teachings regarding same).
[0190] Another exemplary acridinium compound is an
acridinium-9-carboxylatearyl ester. An example of an
acridinium-9-carboxylate aryl ester of formula II is
10methyl-9-(phenoxycarbonyl) acridinium fluorosulfonate (available
from Cayman Chemical, Ann Arbor, Mich.). Methods for preparing
acridinium 9-carboxylate aryl esters are described in McCapra et
aI., Photochem. Photobiol. 4: 1111-21 (1965); Razavi etaI.,
Luminescence 15: 245-249 (2000); Razavi et aI., Luminescence 15:
239-244 (2000);and U.S. Pat. No. 5,241,070 (each of which is
incorporated herein by reference in its entirety for its teachings
regarding same). Such acridinium-9-carboxylate aryl esters are
efficient chemiluminescent indicators for hydrogen peroxide
produced in the oxidation of an analyte by at least one oxidase in
terms of the intensity of the signal and/or the rapidity of the
signal. The course of the chemiluminescent emission for the
acridinium-9carboxylatearyl ester is completed rapidly, i.e., in
under 1 second, while the acridinium9-carboxamide chemiluminescent
emission extends over 2 seconds. Acridinium-9carboxylatearyl ester,
however, loses its chemiluminescent properties in the presence of
protein. Therefore, its use requires the absence of protein during
signal generation and detection. Methods for separating or removing
proteins in the sample are well known to those skilled in the art
and include, but are not limited to, ultrafiltration, extraction,
precipitation, dialysis, chromatography, and/or digestion (see,
e.g., Wells, High Throughput Bioanalytical Sample Preparation.
Methods and AutomationStrategies, Elsevier (2003)). The amount of
protein removed or separated from the test sample can be about 40%,
about 45%, about 50%, about 55%, about 60%, about 65%,about 70%,
about 75%, about 80%, about 85%, about 90%, or about 95%. Further
details regarding acridinium-9-carboxylate aryl ester and its use
are set forth in U.S. patent application Ser. No. 11/697,835, filed
Apr. 9, 2007, and published on Oct. 9, 2008, as U.S. Pat. App. Pub.
No. 2008/0248493. Acridinium-9-carboxylate aryl esters can be
dissolved in any suitable solvent, such as degassed anhydrous N,N
dimethylformamide (DMF) or aqueous sodium cholate.
[0191] Chemiluminescent assays can be performed, for example, in
accordance with the methods described in Adamczyk et aI., Anal.
Chim. Acta 579(1): 61-67 (2006). While any suitable assay format
can be used, a microplate chemiluminometer (Mithras LB940, Berthold
Technologies U.S.A., LLC, Oak Ridge, Tenn.) enables the assay of
multiple samples of small volumes rapidly. The chemiluminometer can
be equipped with multiple reagent injectors using 96-well black
polystyrene microplates (Costar #3792). Each sample can be added
into a separate well, followed by the simultaneous/sequential
addition of other reagents as determined by the type of assay
employed. Desirably, the formation of pseudobases in neutral or
basic solutions employing an acridinium aryl ester is avoided, such
as by acidification. The chemiluminescent response is then recorded
well-by-well. In this regard, the time for recording the
chemiluminescent response will depend, in part, on the delay
between the addition of the reagents and the particular acridinium
employed.
[0192] Hydrogen peroxide can be generated in situ in the mixture or
provided or supplied to the mixture before, simultaneously with, or
after the addition of an above described acridinium compound.
Hydrogen peroxide can be generated in situ in a number of ways such
as would be apparent to one skilled in the art. Alternatively, a
source of hydrogen peroxide can be simply added to the mixture. For
example, the source of the hydrogen peroxide can be one or more
buffers or other solutions that are known to contain hydrogen
peroxide. In this regard, a solution of hydrogen peroxide can
simply be added.
[0193] Upon the simultaneous or subsequent addition of at least one
basic solution to the sample, a detectable signal, namely, a
chemiluminescent signal, indicative of the presence of anti-HPgV-2
antibody (where capture is with an antigen) or antigen (where
capture is with an antibody) is generated. In certain embodiments,
the basic solution contains at least one base and has a pH greater
than or equal to 10, preferably, greater than or equal to 12.
Examples of basic solutions include, but are not limited to, sodium
hydroxide, potassium hydroxide, calcium hydroxide, ammonium
hydroxide, magnesium hydroxide, sodium carbonate, sodium
bicarbonate, calcium hydroxide, calcium carbonate, and calcium
bicarbonate. The amount of basic solution added to the sample
depends on the concentration of the basic solution. Based on the
concentration of the basic solution used, one skilled in the art
can easily determine the amount of basic solution to add to the
sample.
[0194] The chemiluminescent signal that is generated can be
detected using routine techniques known to those skilled in the
art. Based on the intensity of the signal generated, the amount of
anti-HPgV-2 antibody and/or antigen in the sample can be
quantified. Specifically, the amount of anti-HPgV-2 antibody and/or
in the sample is proportional to the intensity of the signal
generated. The amount of anti-HPgV-2 antibody and/or antigen
present can be quantified by comparing the amount of light
generated to a standard curve for anti-HPgV-2 antibody and/or
antigen or by comparison to a reference standard. The standard
curve can be generated using serial dilutions or solutions of known
concentrations of anti-HPgV-2 antibody by mass spectroscopy,
gravimetric methods, and other techniques known in the art.
[0195] Anti-HPgV-2 antibody and/or antigen immunoassays can be
conducted using any suitable format known in the art. In certain
embodiments, a sample being tested for (for example, suspected of
containing) anti-HPgV-2 antibodies can be contacted with a capture
antigen and at least one detection antibody (which can be a second
detection antibody or a third detection antibody), such as labeled
anti-IgG and anti-IgM antibodies, either simultaneously or
sequentially and in any order. Similarly, the test for presence of
an antigen can be contacted with a captured antibody which binds
the antigen in the test sample and the bound antigen may then be
detected by a detection antibody.
[0196] For example, the test sample can be first contacted with at
least one capture antigen and then (sequentially) with at least one
detection antibody. Alternatively, the test sample can be first
contacted with at least one detection antibody and then
(sequentially) with at least one capture antibody. In yet another
alternative, the test sample can be contacted simultaneously with a
capture antigen and a detection antibody.
[0197] In the sandwich assay format, in certain embodiments, a
sample suspected of containing anti-HPgV-2 antibodies (or a
fragment thereof) is first brought into contact with an at least
one first capture antigen under conditions that allow the formation
of a first capture antigen/antiHPgV-2 antibody complex. In the
combination assay, the same is repeated or simultaneously conducted
with a second, third or more capture antigens. If more than one
capture antigen is used, multiple first capture antigen/anti-HPgV-2
antibody complexes are formed. In a sandwich assay, the antigen(s),
in certain embodiments, the at least one capture antigen, is/are
used in molar excess amounts of the maximum amount of anti-HPgV-2
antibodies expected in the test sample. For example, from about 5
ug to about 1 mg of antigen per mL of buffer (e.g., microparticle
coating buffer) can be used.
[0198] Competitive inhibition immunoassays, which are often used to
measure small analytes, comprise sequential and classic formats. In
a sequential competitive inhibition immunoassay the one or more
capture antigen(s) (i.e., a polypeptide, and a pair of
polypeptides, as described herein) to an antibody of interest
(i.e., an anti-HPgV-2 antibody) is/are coated onto a well of a
microtiter plate. When the sample containing the
antibody/antibodies of interest is added to the well, the antibody
of interest binds to the capture antigen(s). After washing, a known
amount of labeled (e.g., biotin or horseradish peroxidase (HRP))
antibody is added to the well. A substrate for an enzymatic label
is necessary to generate a signal. An example of a suitable
substrate for HRP is 3,3',5,5'-tetramethylbenzidine (TMB). After
washing, the signal generated by the labeled antibody is measured
and is inversely proportional to the amount of antibody in the
sample. In a classic competitive inhibition immunoassay antigen for
an antibody of interest is coated onto a well of a microtiter
plate. However, unlike the sequential competitive inhibition
immunoassay, the sample containing the antibody of interest (i.e.,
an anti-HPgV-2 antibody) and the labeled antibody are added to the
well at the same. Any antibody in the sample competes with labeled
antibody for binding to the capture antigen. After washing, the
signal generated by the labeled analyte is measured and is
inversely proportional to the amount of analyte in the sample.
[0199] Optionally, prior to contacting the test sample with the at
least one capture antigen (for example, the first capture antigen),
the at least one capture antigen can be bound to a solid support,
which facilitates the separation of the first antigen/anti-HPgV-2
antibody complex from the test sample. The substrate to which the
capture antigen is bound can be any suitable solid support or solid
phase that facilitates separation of the capture
antigen-anti-HPgV-2 antibody complex from the sample. Examples
include a well of a plate, such as a microtiter plate, a test tube,
a porous gel (e.g., silica gel, agarose, dextran, or gelatin), a
polymeric film (e.g., polyacrylamide), beads (e.g., polystyrene
beads or magnetic beads), a strip of a filter/membrane (e.g.,
nitrocellulose or nylon), microparticles (e.g., latex particles,
magnetizable microparticles (e.g., microparticles having ferric
oxide or chromium oxide cores and homo- or hetero-polymeric coats
and radii of about 1-10 microns). The substrate can comprise a
suitable porous material with a suitable surface affinity to bind
antigens and sufficient porosity to allow access by detection
antibodies. A microporous material is generally preferred, although
a gelatinous material in a hydrated state can be used. Such porous
substrates may be in the form of sheets having a thickness of about
0.01 to about 0.5 mm, or about 0.1 mm. While the pore size may vary
quite a bit, preferably the pore size is from about 0.025 to about
15 microns, or from about 0.15 to aboutl5 microns. The surface of
such substrates can be activated by chemical processes that cause
covalent linkage of an antibody to the substrate. Irreversible
binding, generally by adsorption through hydrophobic forces, of the
antigen to the substrate results; alternatively, a chemical
coupling agent or other means can be used to bind covalently the
antigen to the substrate, provided that such binding does not
interfere with the ability of the antigen to bind to anti-HPgV-2
antibodies.
[0200] Alternatively, the anti-HPgV-2 antibody from the test sample
can be bound with microparticles, which have been previously coated
with antigen. If desired, one or more capture reagents, such as a
pair of polypeptides as described herein, each of which can be
bound by an anti-HPgV-2 antibody, can be attached to solid phases
indifferent physical or addressable locations (e.g., such as in a
biochip configuration (see,-46-e.g., U.S. Pat. No. 6,225,047, Int'l
Pat. App. Pub. No. WO 99/51773; U.S. Pat. No. 6,329,209; Int'l Pat.
App. Pub. No. WO 00/56934, and U.S. Pat. No. 5,242,828). If the
capture reagent is attached to a mass spectrometry probe as the
solid support, the amount of anti-HPgV-2 antibodies bound to the
probe can be detected by laser desorption ionization mass
spectrometry. Alternatively, a single column can be packed with
different beads, which are derivatized with the one or more capture
reagents, thereby capturing the anti-HPgV-2 antibody in a single
place (see, antibody derivatized, bead based technologies, e.g.,
the xMAP technology of Luminex (Austin, Tex.)).
[0201] After the test sample being assayed for anti-HPgV-2
antibodies is brought into contact with at least one capture
antigen (for example, the first capture antigen), the mixture is
incubated in order to allow for the formation of a first antigen
(or multiple antigen)-anti-HPgV-2 antibody (or a fragment thereof)
complex. The incubation can be carried out at a pH of from about
4.5 to about 10.0, at a temperature of from about 20 C. to about 45
0 C., and for a period from at least about one (1) minute to about
eighteen(18) hours, or from about 1 to about 24 minutes, or for
about 4 to about 18 minutes. The immunoassay described herein can
be conducted in one step (meaning the test sample, at least one
capture antibody and at least one detection antibody are all added
sequentially or simultaneously to a reaction vessel) or in more
than one step, such as two steps, three steps, etc.
[0202] After or simultaneously with formation of the (first or
multiple) capture antigen/anti-HPgV-2 antibody complex, the complex
is then contacted with at least one detection antibody (under
conditions which allow for the formation of a (first or multiple)
capture antigen/anti-HPgV-2 antibody/first antibody detection
complex). The at least one detection antibody can be the second,
third, fourth, etc. antibodies used in the immunoassay. If the
capture antigen/anti-HPgV-2 antibody complex is contacted with more
than one detection antibody, then a (first or multiple) capture
antigen/anti-HPgV-2 antibody/(multiple) detection antibody complex
is formed. As with the capture antigen (e.g., the first capture
antigen), when the at least second (and subsequent) detection
antibody is brought into contact with the capture
antigen/anti-HPgV-2 antibody complex, a period of incubation under
conditions similar to those described above is required for the
formation of the (first or multiple) capture antigen/anti-HPgV-2
antibody/(second or multiple) detection antibody complex.
Preferably, at least one detection antibody contains a detectable
label. The detectable label can be bound to the at least one
detection antibody (e.g., the second detection antibody) prior to,
simultaneously with, or after the formation of the (first or
multiple) capture antigen/anti-HPgV-2 antibody/(second or multiple)
detection antibody complex. Any detectable label known in the art
can be used (see discussion above, including Polak and Van Noorden
(1997) and Haugland (1996)).
[0203] The detectable label can be bound to the antibodies (or
antigens which may comprise detectable labels) either directly or
through a coupling agent. An example of a coupling agent that can
be used is EDAC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide,
hydrochloride), which is commercially available from Sigma-Aldrich,
St. Louis, Mo. Other coupling agents that can be used are known in
the art. Methods for binding a detectable label to an antibody are
known in the art. Additionally, many detectable labels can be
purchased or synthesized that already contain end groups that
facilitate the coupling of the detectable label to the antibody,
such as CPSP-Acridinium Ester (i.e.,
9-[N-tosyl-N-(3-carboxypropyl)]-1 O-(3-sulfopropyl) acridinium
carboxamide) or SPSP-Acridinium Ester (i.e.,
N10-(3-sulfopropyl)-N-(3-sulfopropyl)-acridinium-9carboxamide).
[0204] The (first or multiple) capture antigen/anti-HPgV-2
antibody/(second or multiple) detection antibody complex can be,
but does not have to be, separated from the remainder of the test
sample prior to quantification of the label. For example, if the at
least one capture antigen (e.g., the first capture antigen) is
bound to a solid support, such as a well or a bead, separation can
be accomplished by removing the fluid (of the test sample) from
contact with the solid support. Alternatively, if the at least
first capture antigen is bound to a solid support, it can be
simultaneously contacted with the anti-HPgV-2 antibody-containing
sample and the at least one second detection antibody (or the
labeled detection antigen) to form a first (multiple)
antigen/anti-HPgV-2 antibody/second(multiple) antibody (and/or
labeled detection antigen) complex, followed by removal of the
fluid (test sample) from contact with the solid support. If the at
least one first capture antigen is not bound to a solid support,
then the (first or multiple) capture antigen/anti-HPgV-2
antibody/(second or multiple) detection antibody (and/or detection
antigen for the-48-captured antibody) complex does not have to be
removed from the test sample for quantification of the amount of
the label.
[0205] After formation of the labeled capture antigen/anti-HPgV-2
antibody/detection antigen (and/or detection antibody) complex
(e.g., the first capture antigen/anti-HPgV-2 antibody/first
detection antigen complex optionally also with a second detection
antibody), the amount of label in the complex is quantified using
techniques known in the art. For example, if an enzymatic label is
used, the labeled complex is reacted with a substrate for the label
that gives a quantifiable reaction such as the development of
color. If the label is a radioactive label, the label is quantified
using a scintillation counter. If the label is a fluorescent label,
the label is quantified by stimulating the label with a light of
one color (which is known as the "excitation wavelength") and
detecting another color (which is known as the "emission
wavelength") that is emitted by the label in response to the
stimulation. If the label is a chemiluminescent label, the label is
quantified by detecting the light emitted either visually or by
using luminometers, x-ray film, high speed photographic film, a CCO
camera, etc. Once the amount of the label in the complex has been
quantified, the concentration of anti-HPgV-2 antibody or antigen in
the test sample is determined by use of a standard curve that has
been generated using serial dilutions of anti-HPgV-2 antibody or
antigens of known concentration. Other than using serial dilutions
of anti-HPgV-2 antibodies or HPgV-2 antigens, the standard curve
can be generated gravimetrically, by mass spectroscopy and by other
techniques known in the art.
[0206] In a chemiluminescent microparticle assay employing the
ARCHITECT.RTM. analyzer, the conjugate diluent pH may be about
6.0+/-0.2, the microparticle coating buffer should be maintained at
room temperature (i.e., at about 17 to about 27.degree. C.), the
microparticle coating buffer pH should be about 6.5+/-0.2, and the
microparticle diluent pH should be about 6.5+/-0.2. Solids
preferably are less than about 0.2%, such as less than about 0.15%,
less than about 0.14%, less than about 0.13%, less than about0.12%,
or less than about 0.11%, such as about 0.10%.
[0207] FPIAs are based on competitive binding immunoassay
principles. A fluorescently labeled compound, when excited by a
linearly polarized light, will emit fluorescence having a degree of
polarization inversely proportional to its rate of rotation. When a
fluorescently labeled tracer-antibody complex is excited by a
linearly polarized light, the emitted light remains highly
polarized because the fluorophore is constrained from rotating
between the time light is absorbed and the time light is emitted.
When a "free" tracer compound (i.e., a compound that is not bound
to an antibody) is excited by linearly polarized light, its
rotation is much faster than the corresponding tracer-antibody
conjugate produced in a competitive binding immunoassay. FPIAs are
advantageous over RIAs inasmuch as there are no radioactive
substances requiring special handling and disposal. In addition,
FPIAs are homogeneous assays that can be easily and rapidly
performed.
[0208] In certain embodiments, the present disclosure provides
methods of determining the presence, amount, or concentration of
anti-HPgV-2 antibodies or antigens in a test sample. In some
embodiments, the methods comprise assaying the test sample for
anti-HPgV-2 antibodies or antigens by an assay:(i) employing an
immunodiagnostic reagent comprising at least an isolated or
purified polypeptide comprising HPgV-2 antigens, and at least one
detectable label, and comparing a signal generated by the
detectable label as a direct or indirect indication of the
presence, amount or concentration of anti-HPgV-2 antibodies in the
test sample to a signal generated as a direct or indirect
indication of the presence, amount or concentration of anti-HPgV-2
antibodies in a control or calibrator, which is optionally part of
a series of calibrators in which each of the calibrators differs
from the other calibrators in the series by the concentration of
anti-HPgV-2 antibodies. The method can comprise the following
steps:(i) contacting the test sample with the immunodiagnostic
reagent comprising one of more recombinant HPgV-2 antigens so as to
form first, second and third specific capture binding
partner/anti-HPgV-2 antibody complexes with HPgV-2 antibodies that
may be present in the test sample, (ii) contacting the first,
second and third specific capture binding partner/first, second and
third anti-HPgV-2 antibody complexes with at least one detectably
labeled second specific binding partner for anti-HPgV-2 antibody
(e.g., anti-IgG antibody and anti-IgM antibody or polypeptides as
described herein) so as to form first specific binding
partner/first, second and third anti-HPgV-2 antibody,
respectively/second specific binding partner complexes, and (iii)
determining the presence, amount or concentration of anti-HPgV-2
antibodies in the test sample by detecting or measuring the signal
generated by the detectable label in the first specific binding
partner/anti-HPgV-2 antibody/second specific binding partner
complexes formed in (ii).
[0209] In certain embodiments, in addition to, or instead of, use
of the anti-IgG and IgM antibodies, the second step comprises
addition of first, second and third detection antigens that will
specifically bind the anti-HPgV-2 antibodies that have been
specifically captured by the first, second and third capture
antigens, respectively so as to form first specific binding
partner/anti-HPgV-2 antibody/second specific binding partner
complexes, and the third step comprises:(iii) determining the
presence, amount or concentration of anti-HPgV-2 antibodies in the
test sample by detecting or measuring the signal generated by the
detectable label in the first, second and third specific capture
binding partner/first, second and third anti-HPgV-2
antibodies/first, second and third specific detection binding
partner complexes formed in (ii).
[0210] In some embodiments, the methods can comprise the following
steps: (i) contacting the test sample with the immunodiagnostic
reagent comprising one of more recombinant antigens and
simultaneously or sequentially, in either order, contacting the
test sample with at least one detectably labeled second specific
binding partner, which can compete with anti-HPgV-2 antibody for
binding to the at least one pair of first specific binding partners
and which comprises detectably labeled anti-HPgV-2 antibodies,
wherein any anti-HPgV-2 antibody present in the test sample and the
at least one detectably labeled second specific binding partner
compete with each other to form first specific binding
partner/anti-HPgV-2 antibody complexes and first specific binding
partner/second specific binding partner complexes, respectively,
and (ii) determining the presence, amount or concentration of
anti-HPgV-2 antibodies in the test sample by detecting or measuring
the signal generated by the detectable label in the first specific
binding partner/second specific binding partner complex formed in
(ii), wherein the signal generated by the detectable label in the
first specific binding partner/second specific binding partner
complex is inversely proportional to the amount or concentration of
anti-HPgV-2 antibodies in the test sample. The recombinant antigens
of which the immunodiagnostic reagent is comprised can be coated on
microparticles. In this regard, the antigens of which the
immunodiagnostic reagent is comprised can be co-coated on the same
microparticles as additional HPgV-2 antigens. When the polypeptides
of which the immunodiagnostic reagent is comprised are co-coated on
the same microparticles (e.g., a microparticle suspension
containing 4% solids (4% weight/volume microparticles or 4 g
microparticles/100 mL microparticle suspension)), preferably the
polypeptides are co-coated on the same microparticles in a ratio of
about 1:2 to about 1:6, wherein, when the polypeptides are
co-coated on the same microparticles in a ratio of about 1:2, the
concentration of an isolated or purified antigen of the present
invention is at least about 40 .mu.g/mL and the concentration of
the other isolated or purified polypeptide is at least about 80
.mu.g/mL. If the test sample was obtained from a patient, the
method may further comprise diagnosing, prognosticating, or
assessing the efficacy of a therapeutic/prophylactic treatment of
the patient. If the method further comprises assessing the efficacy
of a therapeutic/prophylactic treatment of the patient, the method
optionally can further comprise modifying the
therapeutic/prophylactic treatment of the patient as needed to
improve efficacy. The method can be adapted for use in an automated
system or a semi-automated system.
[0211] In certain embodiments, provided here are methods of
determining the presence, amount, and/or concentration of
anti-HPgV-2 antibodies or HPgV-2 antigens or proteins in a test
sample. In some embodiments, the methods comprise assaying the test
sample by an assay:(i) employing: an immunodiagnostic reagent
comprising at least one HPgV-2 antigen (and preferably two, three
or more antigens) at least one detectable label (preferably each
antigen being detectably labeled), and (ii) comparing a signal
generated by the detectable label as a direct or indirect
indication of the presence, amount or concentration of anti-HPgV-2
antibodies in the test sample to a signal generated as a direct or
indirect indication of the presence, amount or concentration of
anti-HPgV-2 antibodies in a control or calibrator, which is
optionally part of a series of calibrators in which each of the
calibrators differs from the other calibrators in the series by the
concentration of anti-HPgV-2 antibodies. The method can comprise
the following steps:(i) contacting the test sample with the
immunodiagnostic reagent comprising at least one, two, three or
more recombinant HPgV-2 antigens invention so as to form first
specific capture binding partner/anti-HPgV-2 antibody complexes,
(ii) contacting the first specific capture binding
partner/anti-HPgV-2 antibody complexes with at least one detectably
labeled second specific binding partner for anti-HPgV-2 antibody
(e.g., anti-IgG antibody and anti-IgM antibody or labeled antigens
that bind the anti-HPgV-2 antibodies) so as to form first specific
binding partner/anti-HPgV-2antibody/second specific binding partner
complexes, and (iii) determining the presence, amount or
concentration of anti-HPgV-2 antibodies in the test sample by
detecting or measuring the signal generated by the detectable label
in the first specific binding partner/anti-HPgV-2 antibody/second
specific binding partner complexes formed in (ii). Alternatively,
the method can comprise the following steps: (i) contacting the
test sample with the immunodiagnostic reagent comprising at least
one, two, three or more different HPgV-2 antigens and
simultaneously or sequentially, in either order, contacting the
test sample with at least one detectably labeled second specific
binding partner, which can compete with anti-HPgV-2 antibody for
binding to the at least one pair of first specific binding partners
and which comprises detectably labeled anti-HPgV-2 antibodies,
wherein any anti-HPgV-2 antibody present in the test sample and the
at least one second specific binding partner compete with each
other to form first specific binding partner/anti-HPgV-2 antibody
complexes and a first specific binding partner/second specific
binding partner complexes, respectively, and (ii) determining the
presence, amount or concentration of anti-HPgV-2 antibodies in the
test sample by detecting or measuring the signal generated by the
detectable label in the first specific binding partner/second
specific binding partner complex formed in (ii), wherein the signal
generated by the detectable label in the first specific binding
partner/second specific binding partner complex is inversely
proportional to the amount or concentration of anti-HPgV-2
antibodies in the test sample. The polypeptides of which the
immunodiagnostic reagent is comprised can be coated on
microparticles. In this regard, the polypeptides of which the
immunodiagnostic reagent is comprised can be co-coated on the same
microparticles. When the polypeptides of which the immunodiagnostic
reagent is comprised are co-coated on the same microparticles
(e.g., a microparticle suspension containing 4% solids
(4%weight/volume microparticles or 4 g microparticles/100 mL
microparticle suspension)), preferably the polypeptides are
co-coated on the same microparticles in a ratio of about 1:2 to
about 1:6, wherein, when the polypeptides are co-coated on the same
microparticles in a ratio of about 1:2, the concentration of an
isolated or purified polypeptide comprising the recombinant HPgV-2
antigen is at least about 40 .mu.g/mL and the concentration of the
other isolated or purified polypeptide is at least about 80
.mu.g/mL. If the test sample was obtained from a patient, the
method can further comprise diagnosing, prognosticating, or
assessing the efficacy of a therapeutic/prophylactic treatment of
the patient. If the method further comprises assessing the efficacy
of a therapeutic/prophylactic treatment of the patient, the method
optionally can further comprise modifying the
therapeutic/prophylactic treatment of the patient as needed to
improve efficacy. The method can be adapted for use in an automated
system or a semi-automated system.
[0212] In certain embodiments, the kits (or components thereof), as
well as the methods of determining the concentration of anti-HPgV-2
antibodies and/or HPgV-2 antigens in a test sample by an
immunoassay as described herein, can be adapted for use in a
variety of automated and semi-automated systems (including those
wherein the solid phase comprises a microparticle), as described,
e.g., in U.S. Pat. Nos. 5,089,424 and 5,006,309, and as
commercially marketed, e.g., by Abbott Laboratories (Abbott Park,
Ill.) as ARCHITECT.RTM..
[0213] In particular embodiments, some of the differences between
an automated or semi-automated system as compared to a
non-automated system (e.g., ELISA) include the substrate to which
the first specific binding partner (e.g., antigen) is attached
(which can impact sandwich formation and analyte reactivity), and
the length and timing of the capture, detection and/or any optional
wash steps. Whereas a non-automated format such as an ELISA may, in
certain embodiments, require a relatively longer incubation time
with sample and capture reagent (e.g., about 2 hours), an automated
or semi-automated format (e.g., ARCHITECT.RTM., Abbott
Laboratories) may have a relatively shorter incubation time (e.g.,
approximately 18 minutes for ARCHITECT.RTM.). Similarly, whereas a
non-automated format such as an ELISA may incubate a detection
antibody such as the conjugate reagent for a relatively longer
incubation time (e.g., about 2 hours), an automated or
semi-automated format (e.g., ARCHITECT.RTM.) may have a relatively
shorter incubation time (e.g., approximately 4 minutes for the
ARCHITECT.RTM.).
[0214] Other platforms available from Abbott Laboratories include,
but are not limited to, AxSYM.RTM., IMx.RTM. (see, e.g., U.S. Pat.
No. 5,294,404, which is hereby incorporated by reference in its
entirety), PRISM.RTM., EIA (bead), and Quantum.TM. II, as well as
other platforms. Additionally, the assays, kits and kit components
can be employed in other formats, for example, on electrochemical
or other hand-held or point of-care assay systems. The present
disclosure is, for example, applicable to the commercial Abbott
Point of Care (i-STAT.RTM., Abbott Laboratories) electrochemical
immunoassay system that performs sandwich immunoassays.
Immunosensors and their methods of manufacture and operation in
single-use test devices are described, for-63-example in, U.S. Pat.
No. 5,063,081, U.S. Pat. App. Pub. No. 2003/0170881, U.S. Pat. App.
Pub. No. 2004/0018577, U.S. Pat. App. Pub. No. 2005/0054078, and
U.S. Pat. App. Pub. No. 2006/0160164, which are incorporated in
their entireties by reference for their teachings regarding
same.
[0215] In particular, with regard to the adaptation of an assay to
the I-STAT.RTM.system, the following configuration is exemplary. A
microfabricated silicon chip is manufactured with a pair of gold
amperometric working electrodes and a silver-silver chloride
reference electrode. On one of the working electrodes, polystyrene
beads (0.2 mm diameter) with immobilized capture antibody are
adhered to a polymer coating of patterned polyvinyl alcohol over
the electrode. This chip is assembled into an I-STAT.RTM. cartridge
with a fluidics format suitable for immunoassay. On a portion of
the wall of the sample-holding chamber of the cartridge there is a
layer comprising the detection antibody labeled with alkaline
phosphatase (or other label). Within the fluid pouch of the
cartridge is an aqueous reagent that includes p-aminophenol
phosphate.
[0216] In certain embodiments, a sample suspected of containing
anti-HPgV-2 antibody and/or HPgV-2 antigens is added to the holding
chamber of the test cartridge and the cartridge is inserted into
the I-STAT.RTM. reader. After the detection antibody or detectably
labeled detection antigen has dissolved into the sample, a pump
element within the cartridge forces the sample into a conduit
containing the chip. Here it is oscillated to promote formation of
the sandwich between the capture antigen (or capture antibody),
anti-HPgV-2 antibody (or HPgV-2 antigen), and the labeled detection
antibody (and/or detection antigen). In the penultimate step of the
assay, fluid is forced out of the pouch and into the conduit to
wash the sample off the chip and into a waste chamber. In the final
step of the assay, the alkaline phosphatase label reacts with
p-aminophenol phosphate to cleave the phosphate group and permit
the liberated p-aminophenol to be electrochemically oxidized at the
working electrode. Based on the measured current, the reader is
able to calculate the amount of anti-HPgV-2 antibody or HPgV-2
antigen in the sample by means of an embedded algorithm and
factory-determined calibration curve.
[0217] The methods and kits as described herein encompass other
reagents and methods for carrying out the immunoassay. For
instance, encompassed are various buffers such as are known in the
art and/or which can be readily prepared or optimized to be
employed, e.g., for washing, as a conjugate diluent, and/or as a
calibrator diluent. An exemplary conjugate diluent is
ARCHITECT.RTM. conjugate diluent employed in certain kits (Abbott
Laboratories, Abbott Park, Ill.) and containing 2-(N morpholino)
ethanesulfonic acid (IVIES), a salt, a protein blocker, an
antimicrobial agent, and a detergent. An exemplary calibrator
diluent is ARCHITECT.RTM. human calibrator diluent employed in
certain kits (Abbott Laboratories, Abbott Park, Ill.), which
comprises a buffer containing MES, other salt, a protein blocker,
and an antimicrobial agent. Additionally, as described in U.S.
Patent Application No. 61/142,048 filed Dec. 31, 2008,and U.S.
patent application Ser. No. 12/650,241, improved signal generation
may be obtained, e.g., in an I-STAT.RTM. cartridge format, using a
nucleic acid sequence linked to the signal antibody as a signal
amplifier.
VII. HPgV-2 Immunogenic Compositions
[0218] In certain embodiments, provided herein are immunogenic
composition compositions for treating or preventing HPgV-2
infection. In certain embodiments, provided herein are
pharmaceutical compositions comprise one or more such immunogenic
composition compounds (e.g., portion of proteins shown in SEQ ID
NOs:2-11,76-218, and 304-353) and a physiologically acceptable
carrier. Immunogenic compositions may comprise one or more such
compounds and a non-specific immune response enhancer. A
non-specific immune response enhancer may be any substance that
enhances an immune response to an exogenous antigen. Examples of
non-specific immune response enhancers include adjuvants,
biodegradable microspheres (e.g., polylactic galactide) and
liposomes (into which the compound is incorporated; see, e.g., U.S.
Pat. No. 4,235,877). Most adjuvants contain a substance designed to
protect the antigen from rapid catabolism, such as aluminum
hydroxide or mineral oil, and a stimulator of immune responses,
such as lipid A, Bortadella pertussis or Mycobacterium tuberculosis
derived proteins. Suitable adjuvants are commercially available as,
for example, Freund's Incomplete Adjuvant and Complete Adjuvant
(Difco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and
Company, Inc., Rahway, NT); AS-2 (Smith line Beecham); aluminum
salts such as aluminum hydroxide gel (alum) or aluminum phosphate;
salts of calcium, iron or zinc; an insoluble suspension of acylated
tyrosine; acylated sugars; cationically or anionically derivatized
polysaccharides; polyphosphazenes; biodegradable microspheres;
monophosphoryl lipid A and quil A. Cytokines, such as GM-CSF or
interleukin -2, -7, or -12, may also be used as adjuvants.
[0219] Pharmaceutical compositions and immunogenic compositions
within the scope of the present disclosure may also contain other
compounds, which may be biologically active or inactive. For
example, one or more immunogenic portions of other antigens may be
present, either incorporated into a fusion polypeptide or as a
separate compound, within the composition or immunogenic
composition. Polypeptides may, but need not, be conjugated to other
macromolecules as described, for example, within U.S. Pat. Nos.
4,372,945 and 4,474,757. Pharmaceutical compositions and
immunogenic compositions may generally be used for prophylactic and
therapeutic purposes.
[0220] Nucleic acid immunogenic compositions encoding a genome,
structural protein or non-structural protein or a fragment thereof
of HPgV-2 can also be used to elicit an immune response to treat or
prevent HPgV-2 infection. Numerous gene delivery techniques are
well known in the art, such as those described by Rolland (1998)
Crit. Rev. Therap. Drug Carrier Systems 75: 143-198, and references
cited therein. Appropriate nucleic acid expression systems contain
the necessary DNA sequences for expression in the patient (such as
a suitable promoter and terminating signal). In certain
embodiments, the DNA may be introduced using a viral expression
system (e.g., vaccinia, pox virus, retrovirus, or adenovirus),
which may involve the use of a non-pathogenic (defective),
replication competent virus. Suitable systems are disclosed, for
example, in Fisher-Hoch et al. (1989) Proc. Natl. Acad. Sci. USA
55:317-321; Flexner et al. (1989) Ann. N. Y. Acad. Sci. 569:86-103;
Flexner et al. (1990) Immunogenic composition 5: 17-21; U.S. Pat.
Nos. 4,603,112, 4,769,330, 4,777,127 and 5,017,487; WO
89/01973.
[0221] Pharmaceutically acceptable carriers are determined in part
by the particular composition being administered (e.g., nucleic
acid, protein, modulatory compounds or transduced cell), as well as
by the particular method used to administer the composition.
Accordingly, there are a wide variety of suitable formulations of
pharmaceutical compositions of the present disclosure (see, e.g.,
Remington's Pharmaceutical Sciences, 17th ed., 1989).
Administration can be in any convenient manner, e.g., by injection,
oral administration, inhalation, transdermal application, or rectal
administration. Formulations suitable for oral administration can
be composed of (a) liquid solutions, such as an effective amount of
the packaged nucleic acid suspended in diluents, such as water,
saline or PEG 400; (b) capsules, sachets or tablets, each
containing a predetermined amount of the active ingredient, as
liquids, solids, granules or gelatin; (c) suspensions in an
appropriate liquid; and (d) suitable emulsions. Tablet forms can
include one or more of lactose, sucrose, mannitol, sorbitol,
calcium phosphates, corn starch, potato starch, microcrystalline
cellulose, gelatin, colloidal silicon dioxide, talc, magnesium
stearate, stearic acid, and other excipients, colorants, fillers,
binders, diluents, buffering agents, moistening agents,
preservatives, flavoring agents, dyes, disintegrating agents, and
pharmaceutically compatible carriers. Lozenge forms can comprise
the active ingredient in a flavor, e.g., sucrose, as well as
pastilles comprising the active ingredient in an inert base, such
as gelatin and glycerin or sucrose and acacia emulsions, gels, and
the like containing, in addition to the active ingredient, carriers
known in the art.
VIII. Compound Screening
[0222] In certain embodiments, the HPgV-2 virus, nucleic acids and
proteins of the present disclosure are used to assay for antiviral
compounds, including compounds that inhibit (1) viral interactions
at the cell surface, e.g., viral transduction (e.g., block viral
cell receptor binding or internalization); (2) viral replication
and gene expression, e.g., viral replication (e.g., by inhibiting
non-structural protein activity, origin activity, or primer
binding), viral transcription (promoter or splicing inhibition,
nonstructural protein inhibition), viral protein translation,
protein processing (e.g., cleavage or phosphorylation); and (3)
viral assembly and egress, e.g., viral packaging, and virus
release. Assays to identify compounds with HPgV-2 modulating
activity can be performed in vitro. Such assays can use full length
HPgV-2 or a variant thereof, or a mutant thereof, or a fragment
thereof. Purified recombinant or naturally occurring protein can be
used in the in vitro methods of the invention. In addition to
purified HPgV-2, the recombinant or naturally occurring protein can
be part of a cellular lysate or a cell membrane. In certain
embodiments, the binding assay is either solid state or soluble. In
certain embodiments, the protein or membrane is bound to a solid
support, either covalently or non-covalently. In particular
embodiments, the in vitro assays of the invention are substrate or
ligand binding or affinity assays, either non-competitive or
competitive. Other in vitro assays include measuring changes in
spectroscopic (e.g., fluorescence, absorbance, refractive index),
hydrodynamic (e.g., shape), chromatographic, or solubility
properties for the protein.
[0223] In certain embodiments, a high throughput binding assay is
performed in which the protein or a fragment thereof is contacted
with a potential modulator and incubated for a suitable amount of
time. In one embodiment, the potential modulator is bound to a
solid support, and the protein is added. In another embodiment, the
protein is bound to a solid support. A wide variety of modulators
can be used, as described below, including small organic molecules,
peptides, antibodies, etc. A wide variety of assays can be used to
identify HPgV-2-modulator binding, including labeled
protein-protein binding assays, electrophoretic mobility shifts,
immunoassays, enzymatic assays, and the like. In some cases, the
binding of the candidate modulator is determined through the use of
competitive binding assays, where interference with binding of a
known ligand or substrate is measured in the presence of a
potential modulator. Either the modulator or the known ligand or
substrate is bound first, and then the competitor is added. After
the protein is washed, interference with binding, either of the
potential modulator or of the known ligand or substrate, is
determined. Often, either the potential modulator or the known
ligand or substrate is labeled.
[0224] Many different chemical compounds can be used as a potential
modulator or ligand in the screening assays of the invention. In
certain embodiments, the assays are designed to screen large
chemical libraries by automating the assay steps and providing
compounds from any convenient source to assays, which are typically
run in parallel (e.g., in microtiter formats on microtiter plates
in robotic assays). It will be appreciated that there are many
suppliers of chemical compounds, including Sigma (St. Louis, Mo.),
Aldrich (St. Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.), and Fluka
Chemika-Biochemica Analytika (Buchs Switzerland). In certain
embodiments, high throughput screening methods involve providing a
combinatorial small organic molecule or peptide library containing
a large number of potential therapeutic compounds (potential
modulator or ligand compounds). Such libraries are then screened in
one or more assays, as described herein, to identify those library
members (particular chemical species or subclasses) that display a
desired characteristic activity. The compounds thus identified can
serve as lead compounds or can themselves be used as potential or
actual therapeutics.
IX. Kits and Systems
[0225] In certain embodiments, provided herein are kits and systems
comprising one or more reagents for use in a variety of diagnostic
assays, including for example, immunoassays such as ELISA and
"sandwich"-type immunoassays, as well as nucleic acid assay, e.g.,
PCR assays. Such kits and systems may preferably include at least a
first peptide, or a first antibody or antigen binding fragment of
the invention, a functional fragment thereof, or a cocktail
thereof, or a first oligonucleotide or oligonucleotide pair, and
means for signal generation. The kit's components may be
pre-attached to a solid support, or may be applied to the surface
of a solid support when the kit is used. The signal generating
means may come pre-associated with an antibody or nucleic acid of
the invention or may require combination with one or more
components, e.g., buffers, nucleic acids, antibody-enzyme
conjugates, enzyme substrates, or the like, prior to use. Kits and
systems may also include additional reagents, e.g., blocking
reagents for reducing nonspecific binding to the solid phase
surface, washing reagents, enzyme substrates, enzymes, and the
like. The solid phase surface may be in the form of microtiter
plates, microspheres, or other materials suitable for immobilizing
nucleic acids, proteins, peptides, or polypeptides. An enzyme that
catalyzes the formation of a chemiluminescent or chromogenic
product or the reduction of a chemiluminescent or chromogenic
substrate is one such component of the signal generating means.
Such enzymes are well known in the art. Where a radiolabel,
chromogenic, fluorigenic, or other type of detectable label or
detecting means is included within the kit or system, the labeling
agent may be provided either in the same container as the
diagnostic or therapeutic composition itself, or may alternatively
be placed in a second distinct container means into which this
second composition may be placed and suitably aliquoted.
Alternatively, the detection reagent and the label may be prepared
in a single container means, and in most cases, the kit or system
will also typically include a means for containing the vial(s) in
close confinement for commercial sale and/or convenient packaging
and delivery (e.g., a box or other container).
[0226] In certain embodiments, the kits disclosed herein comprise
at least one component for assaying the test sample for HPgV-2 (or
a fragment thereof) and instructions for assaying the test sample
for the HPgV-2 (or a fragment thereof). The at least one component
for assaying the test sample for the HPgV-2 (or a fragment thereof)
can include a composition comprising, for example, an antibody or
antibodies against HPgV-2 (or a fragment, a variant, or a fragment
of a variant thereof), which is optionally immobilized on a solid
phase. In some embodiments, the kit can comprise at least one
component for assaying the test sample for HPgV-2 by assay, e.g.,
chemiluminescent microparticle immunoassay, and instructions for
assaying the test sample for an analyte by immunoassay, e.g.,
chemiluminescent microparticle immunoassay. For example, the kit
can comprise at least one specific binding partner for HPgV-2 such
as an anti-analyte, monoclonal/polyclonal antibody (or a fragment
thereof that can bind to HPgV-2, a variant thereof that can bind to
HPgV-2, or a fragment of a variant that can bind to HPgV-2), either
of which can be detectably labeled. Alternatively or additionally,
the kit can comprise detectably labeled HPgV-2 protein (or a
fragment thereof that can bind to an anti-HPgV-2,
monoclonal/polyclonal antibody or an anti-HPgV-2 DVD-Ig (or a
fragment, a variant, or a fragment of a variant thereof)), which
can compete with any HPgV-2 proteins in a test sample for binding
to an anti-HPgV-2, monoclonal/polyclonal antibody (or a fragment
thereof that can bind to HPgV-2, a variant thereof that can bind to
HPgV-2, or a fragment of a variant that can bind to the HPgV-2) or
an anti-HPgV-2 DVD-Ig (or a fragment, a variant, or a fragment of a
variant thereof), either of which can be immobilized on a solid
support. The kit can comprise one or more calibrators or controls,
e.g., isolated or purified HPgV-2. The kit can comprise at least
one container (e.g., tube, microtiter plates or strips, which can
be already coated with a first specific binding partner, for
example) for conducting the assay, and/or a buffer, such as an
assay buffer or a wash buffer, either one of which can be provided
as a concentrated solution, a substrate solution for the detectable
label (e.g., an enzymatic label) or a stop solution. Preferably,
the kit comprises all components, i.e., reagents, standards,
buffers, diluents, etc. which are necessary to perform the assay.
The instructions can be in paper form or computer-readable form
such as a disk, CD, DVD or the like.
[0227] Any antibodies, such as an anti-biomarker antibody or an
anti-HPgV-2 DVD-Ig, or tracer can incorporate a detectable label as
described herein such as a fluorophore, a radioactive moiety, an
enzyme, a biotin/avidin label, a chromophore, a chemiluminescent
label or the like, or the kit can include reagents for carrying out
detectable labeling. The antibodies, calibrators and/or controls
can be provided in separate containers or pre-dispensed into an
appropriate assay format, for example, into microtiter plates.
[0228] Optionally, the kit includes quality control components (for
example, sensitivity panels, calibrators, and positive controls).
Preparation of quality control reagents is well-known in the art
and is described on insert sheets for a variety of immunodiagnostic
products. Sensitivity panel members optionally are used to
establish assay performance characteristics, and further optionally
are useful indicators of the integrity of the assay kit reagents,
and the standardization of assays.
[0229] The kit can also optionally include other reagents required
to conduct a diagnostic assay or facilitate quality control
evaluations such as buffers, salts, enzymes, enzyme co-factors,
enzyme substrates, detection reagents, and the like. Other
components, such as buffers and solutions for the isolation and/or
treatment of a test sample (e.g., pretreatment reagents) also can
be included in the kit. The kit can additionally include one or
more other controls. One or more of the components of the kit can
be lyophilized, in which case the kit can further comprise reagents
suitable for the reconstitution of the lyophilized components.
[0230] The various components of the kit optionally are provided in
suitable containers as necessary, e.g., a microtiter plate. The kit
can further include containers for holding or storing a sample
(e.g., a container or cartridge for a urine sample). Where
appropriate, the kit optionally also can contain reaction vessels,
mixing vessels, and other components that facilitate the
preparation of reagents or the test sample. The kit can also
include one or more instruments for assisting with obtaining a test
sample, such as a syringe, pipette, forceps, measured spoon, or the
like.
[0231] If the detectable label is at least one acridinium compound,
the kit can comprise at least one acridinium-9-carboxamide, at
least one acridinium-9-carboxylate aryl ester, or any combination
thereof. Further, if the detectable label is at least one
acridinium compound, the kit also can comprise a source of hydrogen
peroxide, such as a buffer, a solution, and/or at least one basic
solution. If desired, the kit can contain a solid phase, such as a
magnetic particle, bead, test tube, microtiter plate, cuvette,
membrane, scaffolding molecule, film, filter paper, disc or
chip.
[0232] The kit (or components thereof), as well as the method of
determining the presence, amount or concentration of HPgV-2 in a
test sample by an assay, such as the assays described herein, can
be adapted for use in a variety of automated and semi-automated
systems (including those wherein the solid phase comprises a
microparticle), as described, e.g., in U.S. Pat. Nos. 5,089,424 and
5,006,309, and as commercially marketed, e.g., by Abbott
Laboratories (Abbott Park, Ill.) as ARCHITECT. Some of the
differences between an automated or semi-automated system as
compared to a non-automated system (e.g., ELISA) include the
substrate to which the first specific binding partner (e.g., an
anti-HPgV-2, monoclonal/polyclonal antibody (or a fragment thereof,
a variant thereof, or a fragment of a variant thereof) or an
anti-HPgV-2 DVD-Ig (or a fragment thereof, a variant thereof, or a
fragment of a variant thereof) is attached; either way, sandwich
formation and HPgV-2 reactivity can be impacted), and the length
and timing of the capture, detection and/or any optional wash
steps. Whereas a non-automated format, such as an ELISA, may
require a relatively longer incubation time with sample and capture
reagent (e.g., about 2 hours), an automated or semi-automated
format (e.g., ARCHITECT, Abbott Laboratories) may have a relatively
shorter incubation time (e.g., approximately 18 minutes for
ARCHITECT). Similarly, whereas a non-automated format, such as an
ELISA, may incubate a detection antibody, such as the conjugate
reagent, for a relatively longer incubation time (e.g., about 2
hours), an automated or semi-automated format (e.g., ARCHITECT) may
have a relatively shorter incubation time (e.g., approximately 4
minutes for the ARCHITECT).
[0233] Other platforms available from Abbott Laboratories include,
but are not limited to, AxSYM, IMx (see, e.g., U.S. Pat. No.
5,294,404, which is hereby incorporated by reference in its
entirety), PRISM, EIA (bead), and Quantum.TM. II, as well as other
platforms. Additionally, the assays, kits and kit components can be
employed in other formats, for example, on electrochemical or other
hand-held or point-of-care assay systems. The present disclosure
is, for example, applicable to the commercial Abbott Point of Care
(i-STATED, Abbott Laboratories) electrochemical immunoassay system
that performs sandwich immunoassays. Immunosensors and their
methods of manufacture and operation in single-use test devices are
described, for example in, U.S. Pat. No. 5,063,081, U.S. Pat. App.
Pub. No. 2003/0170881, U.S. Pat. App. Pub. No. 2004/0018577, U.S.
Pat. App. Pub. No. 2005/0054078, and U.S. Pat. App. Pub. No.
2006/0160164, which are incorporated in their entireties by
reference for their teachings regarding same.
[0234] In particular, with regard to the adaptation of an HPgV-2
assay to the I-STAT system, the following configuration may be
employed. A microfabricated silicon chip is manufactured with a
pair of gold amperometric working electrodes and a silver-silver
chloride reference electrode. On one of the working electrodes,
polystyrene beads (0.2 mm diameter) with immobilized anti-HPgV-2,
monoclonal/polyclonal antibody (or a fragment thereof, a variant
thereof, or a fragment of a variant thereof) or anti-HPgV-2 DVD-Ig
(or a fragment thereof, a variant thereof, or a fragment of a
variant thereof), are adhered to a polymer coating of patterned
polyvinyl alcohol over the electrode. This chip is assembled into
an I-STAT cartridge with a fluidics format suitable for
immunoassay. On a portion of the wall of the sample-holding chamber
of the cartridge, there is a layer comprising a specific binding
partner for an analyte, such as an anti-HPgV-2,
monoclonal/polyclonal antibody (or a fragment thereof, a variant
thereof, or a fragment of a variant thereof that can bind the
HPgV-2) or an anti-HPgV-2 DVD-Ig (or a fragment thereof, a variant
thereof, or a fragment of a variant thereof that can bind the
HPgV-2), either of which can be detectably labeled. Within the
fluid pouch of the cartridge is an aqueous reagent that includes
p-aminophenol phosphate.
[0235] In operation, a sample suspected of containing an HPgV-2 is
added to the holding chamber of the test cartridge, and the
cartridge is inserted into the I-STAT reader. After the specific
binding partner for HPgV-2 has dissolved into the sample, a pump
element within the cartridge forces the sample into a conduit
containing the chip. Here it is oscillated to promote formation of
the sandwich. In the penultimate step of the assay, fluid is forced
out of the pouch and into the conduit to wash the sample off the
chip and into a waste chamber. In the final step of the assay, the
alkaline phosphatase label reacts with p-aminophenol phosphate to
cleave the phosphate group and permit the liberated p-aminophenol
to be electrochemically oxidized at the working electrode. Based on
the measured current, the reader is able to calculate the amount of
HPgV-2 in the sample by means of an embedded algorithm and
factory-determined calibration curve. In certain embodiments, the
methods and kits as described herein may include other reagents and
methods for carrying out the assays. For instance, encompassed are
various buffers such as are known in the art and/or which can be
readily prepared or optimized to be employed, e.g., for washing, as
a conjugate diluent, microparticle diluent, and/or as a calibrator
diluent. An exemplary conjugate diluent is ARCHITECT conjugate
diluent employed in certain kits (Abbott Laboratories, Abbott Park,
Ill.) and containing 2-(N-morpholino) ethanesulfonic acid (MES), a
salt, a protein blocker, an antimicrobial agent, and a detergent.
An exemplary calibrator diluent is ARCHITECT human calibrator
diluent employed in certain kits (Abbott Laboratories, Abbott Park,
Ill.), which comprises a buffer containing MES, other salt, a
protein blocker, and an antimicrobial agent. Additionally, as
described in U.S. Patent Application No. 61/142,048 filed Dec. 31,
2008, improved signal generation may be obtained, e.g., in an
I-Stat cartridge format, using a nucleic acid sequence linked to
the signal antibody as a signal amplifier.
EXAMPLES
Example 1
Discovery of a Novel Virus Tentatively Named HPgV-2
[0236] A cohort of 105 serum samples collected from patients in the
United States with chronic hepatitis were pooled into groups of 6
samples each (50 .mu.l.times.6=300 .mu.l serum per pool) and
treated with a mix of DNases (Turbo DNase, Ambion, Foster City,
Calif. and Baseline DNase, Epicentre, San Diego, Calif.) at room
temperature for 2.5 hours, followed by bead-based extraction using
Qiagen's EZ1 viral extraction kit according to the manufacturer's
protocol. Extracted nucleic acid from individual pools were grouped
into two pools (N=12 samples) then prepared for next-generation
sequencing library construction and indexing using a modified
TruSeq (Illumina, San Diego, Calif.) protocol as previously
described (Grard, et al. 2012; PLoS Pathogens 8(9): e1002924). The
libraries were sequenced by Elim Biopharmaceuticals (Elim
Biopharmaceuticals, Hayward, Calif.) in 2 lanes using Illumina's
HiSeq2000 instrument. A total of 255 million raw paired-end reads
were generated. These reads were processed using a customized
bioinformatics pipeline titled sequence-based ultra-rapid pathogen
identification (SURPI) (Naccache et al. 2014; Genome Research in
press). By protein alignment, 3 paired-end reads were identified
with remote homology to simian pegivirus, GB virus A in a single
pool consisting of 32.7 million raw reads. PCR using primers (SEQ
IDs:219 and 220) targeting one of the paired-end reads followed by
confirmatory Sanger sequencing was used to pinpoint the individual
sample containing HPgV-2.
[0237] To recover additional sequences from the viral genome,
libraries were prepared from the remaining pooled nucleic acids
(N=6 samples) using two library methods:
[0238] Illumina TruSeq and Rubicon Genomics ThruPLEX (Ann Arbor,
Mich.). These libraries were indexed and sequenced using Illumina
MiSeq, generating a total of 13.4 million raw paired-end reads. The
genome of the novel pegivirus was then reconstructed using (1)
identification of overlapping reads by BLASTn (nucleotide)
alignment to existing contigs at an e-value cutoff of
1.times.10.sup.-8, (2) identification of additional reads by BLASTx
(translated nucleotide) alignment at an e-value cutoff of
1.times.10.sup.-2 to the published sequence of GBV-A (now Pegivirus
A, NC_001837) as a reference, and (3) iterative de novo assembly
and reference-based assembly to existing contigs using the PRICE
aligner (Ruby, et al., 2013; G3 3:865-880) and Geneious
(Biomatters, Auckland, New Zealand), respectively. This resulted in
the generation of six contiguous sequences (contigs) spanning
.about.60% of the estimated pegivirus genome. These contigs were
verified and gaps were closed using bridging PCR (primers shown in
Table 18) and confirmation of resulting amplicons by Sanger
sequencing. To recover the 5' and 3' ends, we ran a Rapid
Amplification of cDNA Ends (RACE) procedure according to the
manufacturer's instructions (FirstChoice RLM-RACE Kit, Ambion,
Foster City, Calif.). The 3' RACE protocol required the addition of
a poly(A)-tail to the 3' terminus with poly-A polymerase (New
England BioLabs, Ipswich, Mass.) prior to RACE. The final 9,778-nt
pegivirus draft genome included 604 sequence reads from the
Illumina HiSeq run and 2,571 reads from the Illumina MiSeq run,
with 87.8% of the assembled genome confirmed by PCR and Sanger
sequencing
[0239] All confirmatory Sanger sequencing was performed by
purifying amplicons on a 2% agarose gel, cloning them into plasmid
vectors using TOPO TA (Invitrogen, Carlsbad, Calif.), and sending
them to an outside company (Elim Biopharmaceuticals, Hayward,
Calif.), for Sanger sequencing in both directions using vector
primers M13F and M13R.
TABLE-US-00003 TABLE 18 Sequence_Name Sequence Tm SEQ ID NO:
HPgV-2_set_1F GAG TCA CGC GGG GTG CTT 60.9 219 HPgV-2_set_1R CTT
AAT ATA AGG GGC CAT ACT TTT GA 52.9 220 HPgV-2_set_2F TGG AAC CAG
TCG TGT TGG AG 60.5 221 HPgV-2_set_2R GAA CAG CAG CAG GGG TCT AG
62.5 222 HPgV-2_set_3F CTA GAC CCC TGC TGC TGT TC 62.5 223
HPgV-2_set_3R TGA CTA CAG CCA CAC TTG GT 58.4 224 HPgV-2_set_4F ATA
TGG GAG CTA CCA CTG CGG T 64.2 225 HPgV-2_set_4R TAA CAG GAC AGA
ATC TAG GTA TGG AG 64.6 226 HPgV-2_set_5F TGT CTA TTG CTC TAC CTC
CAG GTG 65.2 227 HPgV-2_set_5R TTC CAA AGC AAC GTA ACA CGG CG 64.6
228 HPgV-2_set_6F CGC CGT GTT ACG TTG CTT TGG AA 64.6 229
HPgV-2_set_6R TTA CCA GAA CCA GTA GGG GCA TAG 65.2 230
HPgV-2_set_7F ACA GTC ACA TTC CAA CAT TGA TGA ATA C 64.4 231
HPgV-2_set_7R TGC TCC CCT TCT ACC ACG ACC 65.3 232 HPgV-2_set_8F
TAG GCG TGT GGT TTT CCG GTC T 64.2 233 HPgV-2_set_8R CCA ATC CCA
CAC AGC GCG TAG A 65.8 234 HPgV-2_set_9F TCT ACG CGC TGT GTG GGA
TTG G 65.8 235 HPgV-2_set_9R GGG TCT CAA ACT TGA TTG GAG GC 64.6
236 HPgV-2_set_10F CTA TGC CCC TAC TGG TTC TGG TAA 65.2 237
HPgV-2_set_10R GTA TTC ATC AAT GTT GGA ATG TGA CTG T 64.4 238
HPgV-2_set_11F ACT TAT TGA CTG ACA CAG GCG ACG 65.2 239
HPgV-2_set_11R TGC ATG CGC AAT GCA GCA GTA CAT 65.2 240
HPgV-2_set_12F GTG CCC ATA AGT GGC TAT TAG CTA T 64.1 241
HPgV-2_set_12R TGC TTA ATT GTT GGC CAA ATC TTT CAC 63.7 242
HPgV-2_set_13F ACG ATT CCG TGT GCC TAT GAT TGG 65.2 243
HPgV-2_set_13R CTG GGT TAC ATA AGT TAG TAG ACA TGC 65.3 244
HPgV-2_set_14F TCT CCG CCT CCA GCA GTT CAA 63.2 245 HPgV-2_set_14R
AGC CGC AGT AGG ATA CAT GAC AAT A 64.1 246 HPgV-2_set_15F TGT GAT
ATC ACA GGA AAA GTT GTC GG 64.6 247 HPgV-2_set_15R ACA GTC ACA GCC
GCA GTA GGA TA 64.6 248 HPgV-2_set_16F GAG AAA ATG ATC CTG GGC GAT
CCT G 67.4 249 HPgV-2_set_16R GCC GTG ATG GTG CTA TCA AAG CA 64.6
250 HPgV-2_set_17F GGG ACA CCT CAA CCC TGA AG 62.5 251
HPgV-2_set_17R TCA CTG CGG TAC CCA TTG AC 60.5 252 HPgV-2_set_18F
ATA TGG GAG CTA CCA CTG CGG T 64.2 253 HPgV-2_set_18R GGT ACA GTA
TTT GAG GTA GCT TTC AG 64.6 254 HPgV-2_set_19F CTT TTT GGT GCG CAG
TGT TTG CCT 65.2 255 HPgV-2_set_19R TGT CAG GGA AGA CAA CAC CAC GAT
65.2 256 HPgV-2_set_20F ACA CTC ACA GGG CGT GCT GAA A 64.2 257
HPgV-2_set_20R ACG CCA AGT TCT CAC CAG TGA TG 64.6 258
HPgV-2_set_21F GTG TGG CTG TAG TCA AAA GTA TGG C 65.8 259
HPgV-2_set_21R CAG CAG TAC ATG GCA CCA CTC G 65.8 260
HPgV-2_set_22F TTC GGT CAT CGA CTG CGG GTG T 65.8 261
HPgV-2_set_22R CCA GCC AAG TTC CTG CAA TAG CTA A 65.8 262
HPgV-2_set_23F TTC ACT GCG CTT GCT GGC TTG G 65.8 263
HPgV-2_set_23R CCG TAA GGT GCC AGT GCC TGT 65.3 264 HPgV-2_set_24F
AAG CAT CAA TCT GAA AGC TAC CTC AAA 63.7 265 HPgV-2_set_24R TGA ATC
TTA TAG TGT CGT CCA AGT G 62.5 266 HPgV-2_set_25F
CTGTGACTGCCCCTTTGGAA 60.5 267 HPgV-2_set_25R CATGCCAACGTCCGTGTATG
60.5 268 HPgV-2_set_26F AAC CCT TGC AAT TCT GGC CGA TGA 65.2 269
HPgV-2_set_26R AGA TCC CTG ACT GCT TGC GCC A 65.8 270
HPgV-2_set_27F CCC ACG GTC CTG ATG ATA GCA T 64.2 271
HPgV-2_set_27R GCT AAC CAG CCA AGT TCC TGC AA 64.6 272
HPgV-2_set_28F AAG TGA AAG ATT TGG CCA ACA ATT AAG CAA 65.1 273
HPgV-2_set_28R CCA CGC AGG TGA GCA GCC AA 64.6 274 HPgV-2_set_29F
ACA ATT ATT ACA AAA GAA GCC CCA T 59.2 275 HPgV-2_set_29R AGT CAC
AGC CGC AGT AGG A 59.5 276 HPgV-2_set_30F TTG ACA TGA CAG CGT CGG
TG 60.5 277 HPgV-2_set_30R AGCGCGCATCTGATCTACAA 58.5 278
HPgV-2_set_31F AAC AGC GGT TGA TGT CTC C 57.5 279 HPgV-2_set_31R
AAA GAT GCG CGC AAA CAC C 57.5 280 HPgV-2_set_32F
AGCGCGCATCTGATCTACAA 58.5 281 HPgV-2_set_32R GCT AGT GCT ATA CTC
GCT CTG CTT 65.2 282 HPgV-2_set_33F ATG ATG CAT GGC AGG TTC GCC AA
64.6 283 HPgV-2_set_33R GAT CAA TCG TGA CCT TAG CCT GC 64.6 284
HPgV-2_set_34F AAC TCG GCG ACC AGT GCC AAA AT 64.6 285
HPgV-2_set_34R TGT CTG CGC GCA AAA TGC CAG C 65.8 286
HPgV-2_set_35F GGC AAA GAC CTT CAG ACA ATC TGG 65.2 287
HPgV-2_set_35R CAC CCC GAC AAC TTT TCC TGT GA 64.5 288
HPgV-2_set_36F TTG ATC GTG CAA AGG GAT GGG TC 64.6 289
HPgV-2_set_36R CTA ACA GTC CAA GCC AAC CTG CA 64.5 290
HPgV-2_set_37F GCC ATG AGG GAT CAT GAC ACT G 64.5 291
HPgV-2_set_37R TTT GCA CGA TCA GCG TTC CCG T 65.2 292
HPgV-2_set_38F CTG TCC TGT TAC TCC ATA CCT AGA TT 64.6 293
HPgV-2_set_38R CTG ACA CCT GGA GGT AGA GCA A 63.2 294
HPgV-2_set_39F CCT CCA ATC AAG TTT GAG ACC C 62.1 295
HPgV-2_set_39R GTA CAC TCC AGC GCG CAT CT 62.5 296 HPgV-2_set_40F
ACC AAG TGT GGC TGT AGT CA 58.4 297 HPgV-2_set_40R CCC CTG TTG TAT
GCC TAG CC 62.5 298
Example 2
Confirmation of the Novel Virus, HPgV-2
[0240] This example describes methods used to confirm the presence
of HPgV-2 virus in biological samples.
A. Sample Pre-Treatment and RNA Extraction
[0241] To independently verify and confirm the novel virus, plasma
collected from the index patient (harboring UC0125.US) was
evaluated at Abbott laboratories. The plasma sample (130 p.1)
containing HPgV-2 was thawed at room temperature. The sample was
spun at 2650g for 5 min at room temperature and the supernatant was
transferred to a fresh tube. The sample was pre-treated with
benzonase for 2 hrs at 37.degree. C. to degrade free DNA and RNA.
The 10.times. benzonase buffer was as follows: 200 mM Tris-Cl pH
7.5, and 100 mM NaCl, 20 mM MgCl.sub.2. The benzonase reaction was
as follows: 14 .mu.l 10.times. benzonase buffer, 130 .mu.l plasma,
and 0.5 .mu.l (250 U/.mu.l benzonase: 892 U/ml final) (Sigma,
E8263-25KU). The sample was then filtered with 0.22 .mu.M spin
filters (Millipore, UFC306V00) by spinning at 2650 g for 3 min.
[0242] The Qiagen Viral Mini extraction spin protocol was used for
viral RNA purification. Alternatively, the Total Nucleic Acid prep
can be used and samples processed on an Abbott m2000 according to
manufacturer instructions (not described here). This protocol is
suitable for purification of viral RNA from 140 .mu.l plasma,
serum, urine, cell culture media, or cell-free body fluids using a
microcentrifuge. Larger starting volumes, up to 560 .mu.l (in
multiples of 140 .mu.l), can be processed by increasing the initial
volumes proportionally and loading the QIAamp Mini column multiple
times, as described below in the protocol.
[0243] Before starting the purification protocol: i) equilibrate
samples to room temperature (15-25.degree. C.); ii) equilibrate
Buffer AVE to room temperature for elution later; iii) check that
Buffer AW1 and Buffer AW2 have been prepared according to the
instructions; and iv) add carrier RNA reconstituted in Buffer AVE
to Buffer AVL according to instructions.
[0244] The purification procedure is as follows. Step 1, pipet 560
.mu.l of prepared Buffer AVL containing 2 .mu.l of tRNA-MagMax
carrier RNA into a 1.5 ml microcentrifuge tube. Step 2, add 140
.mu.l plasma, serum, urine, cell-culture supernatant, or cell-free
body fluid to the Buffer AVL-carrier RNA in the microcentrifuge
tube. Mix by pulse-vortexing for 15 seconds. Step 3, incubate at
room temperature (15-25.degree. C.) for 10 min. Viral particle
lysis is complete after lysis for 10 min at room temperature.
Longer incubation times have no effect on the yield or quality of
the purified RNA. Potentially infectious agents and RNases are
inactivated in Buffer AVL.
[0245] Step 4, briefly centrifuge the tube to remove drops from the
inside of the lid. Step 5, add 560 .mu.l of ethanol (96-100%) to
the sample, and mix by pulse-vortexing for 15 seconds. After
mixing, briefly centrifuge the tube to remove drops from inside the
lid. Step 6, carefully apply 630 .mu.l of the solution from step 5
to the QIAamp Mini column (in a 2 ml collection tube) without
wetting the rim. Close the cap, and centrifuge at 6000.times.g
(8000 rpm) for 1 minute. Place the QIAmp Mini column into a clean 2
ml collection tube, and discard the tube containing the filtrate.
Close each spin column to avoid cross-contamination during
centrifugation. If the solution has not completely passed through
the membrane, centrifuge again at a higher speed until all of the
solution has passed through.
[0246] Step 7, carefully open the QIAmp Mini column, and repeat
step 6. If the sample volume was greater than 140 repeat this step
until all of the lysate has been loaded onto the spin column. Step
8, carefully open the QIAmp Mini column, and add 500 .mu.l of
Buffer AW1. Close the cap, and centrifuge at 6000.times.g (8000
rpm) for 1 min. Place the QIAmp Mini column in a clean 2 ml
collection tube (provided), and discard the tube containing the
filtrate. It is not necessary to increase the volume of Buffer AW1
even if the original sample volume was larger than 140 .mu.l.
[0247] Step 9, carefully open the QIAmp Mini column, and add 500
.mu.l of Buffer AW2. Close the cap and centrifuge at full speed
(20,000.times.g; 14,000 rpm) for 3 min. Continue directly with step
11, or to eliminate any chance of possible Buffer AW2 carryover,
perform step 10, and then continue with step 11. Step 10,
optionally place the QIAmp Mini column in a new 2 ml collection
tube, and discard the old collection tube with the filtrate.
Centrifuge at full speed for 1 min.
[0248] Step 11, place the QIAmp Mini column in a clean 1.5 ml
microcentrifuge tube. Discard the old collection tube containing
the filtrate. Carefully open the QIAmp Mini column and add 60 .mu.l
of Buffer AVE equilibrated to room temperature. Close the cap, and
incubate at room temperature for 1 min. Centrifuge at 6000.times.g
(8000 rpm) for 1 min. A first elution with 60 .mu.l of Buffer AVE
was performed and aliquots of 8 .mu.l or 5 .mu.l were stored at
-20.degree. C. or -70.degree. C. A second elution of 60 .mu.l was
performed and frozen in one tube.
[0249] Step 12, RNA stocks are concentrated with RNA Clean &
Concentrator-5 columns (Zymo Research) by adding 2 volumes RNA
Binding Buffer to each sample RNA, mixing, then adding an equal
volume of ethanol (95-100%). Samples are transferred to the
Zymo-Spin.TM. IC Column in a 2 ml collection tube, centrifuged for
30 seconds and the flow-through is discarded. 400 .mu.l of RNA Prep
Buffer is added to the column, centrifuged for 30 seconds and the
flow through discarded, followed by 700 .mu.l of RNA Wash Buffer,
centrifuging for 30 seconds, and discarding of the flow-through. A
final 400 .mu.l volume of RNA Wash Buffer is added to the column
and centrifuged for 2 minutes to ensure complete removal of the
wash buffer, then the column is transferred carefully to an
RNase-free tube. 7 .mu.lof DNase/RNase-Free water is added directly
to the column matrix and centrifuged for 30 seconds to elute the
RNA.
B. Reverse Transcription
[0250] cDNA was generated by random hexamer priming using SS RTIII
(Invitrogen: 18080-051) with the HPgV-2 sample RNA as template.
This was to allow detection by PCR using the primers listed in
below (section C.) in Table 1.
[0251] The following volumes were employed: 7.6 .mu.l sample RNA, 1
.mu.l of random hexamer (50 ng/.mu.l), 0.4 .mu.l of oligo dT (50
.mu.M), 1 .mu.l dNTP mix (10 mM). This was heated at 65.degree. C.
for 5 min and returned to ice to add 10 .mu.l of master mix. The
mastermix was as follows: 2 .mu.l 10 RT buffer, 4 .mu.l 25 mM
MgCl.sub.2, 2 .mu.l 0.1 M DTT, 1 .mu.l RNAseOUT (40 U/.mu.l), 1
.mu.l Superscript III RT (200 U/.mu.l). Incubate for 10 min at
25.degree. C., then 80 min at 50.degree. C. Terminate at 85.degree.
C. for 5 min then 4.degree. C. Add 1 .mu.l of RNAseH (Invitrogen,
Y01220) to reactions and incubate for 20 min at 37.degree. C. and
then to 4.degree. C. cDNA was aliquoted into 2.times.10 .mu.l
samples and frozen at -80C.
C. HPgV-2 Primer Design and Testing
[0252] The following primers, in Table 1, were shown to work for
amplifying portions of the recited region of HPgV-2.
TABLE-US-00004 TABLE 1 Forward Primers Region v35 SEQ ID detected
Name of Primer coordinates Sequence (5'.fwdarw.3') NO: S 35F 24
TATTGCTACTTCGGTACGCCTAAT 12 S 81F 76 AAGGGCCTAGTAGGACGTGTGACA 13 S
1F 119 CACTGGG GTGAGCGGAG GCAGCAC 14 S 15F 133 GAG GCAGCACCGA
AGTCGGGTGA A 15 E1 307F 702 TGCCACCCATCCTATCTGCT 16 E1 487F 882
TATTGCTTGGTATGGCTGGGGTAT 17 E1 804F 893 ATGGCTGGGGTATACCTAARACA 18
E2 1147F 1133 TGGCGTACAAGCATCAATC 19 E2 1392F 1374
ACCGATTTCCGCTTTGTGCTAT 20 E2 1840F 1819 CCTGGGCTTGGGAAATGG 21 X
2283F 2372 CATG GGTGATTTCG CGGACTACT 22 X 2368F 2487 CCTCGGGGA
CATCACGGGC ATCTA 23 X 2584F 2702 CTGTTAATGCTGCGCTCAATAGAA 24 NS2
2801F 2919 GCGGGTATTTGGTCTTGAGGTTTG 25 NS2 2978F 3372
TTTGGATCACGCGGCACATACATA 26 NS2 3028F 3423 ACGGGTGGCGCAAGCAGTCAGG
27 NS2 NS23_F5 3478 GAGGAGCCCACCTTTACTGA 28 NS3 Primer Set 2 3601
GAAGATCTGCCACCTGGTTT 29 NS3 Primer Set 7 3668 TCCTTCCTTAAGGCGACACT
30 NS3 3535F 3929 GAGCCGGGTTTGGGTGATGAATAA 31 NS3 Primer Set 4 3940
TGGGTGATGAATAACAACGG 32 NS3 Primer Set 3 4005 AATGACGACGTCTGTTTGGA
33 NS3 3681 4076 ACGCGTTGATGCTCGGTGGTTACT 34 NS3 Primer Set 15 4189
CCAGCTGTGACACCAACATA 35 NS3 Primer Set 1 4508 AGTCATTTGCGACGAGTGTC
36 NS3 5353F 5348 GGC CCA CGG TCC TGA TGA TA 37 NS3 5353Fv2 5348
GGCCCAYGGTCCAGACGATR 38 NS3 5353Fv128 5348 GGCCCATGGTCCGGATGATG 39
NS3 5605Fv2 5600 CCGTTTGGAGYGTTGAYAAC 40 NS4B 5605F 5600
CTGTTTGGAGCGTTGAGGTC 41 NS5A 6466F 6555 TACYGGCACCTTGTTGACCACCTG 42
NS5A 6383F 6778 CTAGAGCGGCGGGGCGACAAA 43 NS5A 6608F 7003
GAGGCGGTTGAGCTGCTGGAAGAG 44 NS5A 7284F 7279 TAG TTC AGG CGG CTT CAC
GGT TTG 45 NS5A 7499F 7588 TGCGCCGT ACCAACAAAG 46 NS5A 7661F 7606
TGT CAC CCC TTG CAA ACT CCT ATT 47 NS5B 7783F 8178
AGTGTACGACGCTCCAATG 48 NS5B 8285F 8280 GCA CGA GTC GCG GAG AAA ATG
A 49 NS5B 7886F 8283 ACG AGTCGCGGAG AAAATGA 50 NS5B 8385F 8380 CGC
GCC TAC TGG AAC AAT 51 NS5B 8781F 8776 ACG CGC TTGATG ACT ATG GGT
TTA 52 NS5B 9080F 9075 TTG ACG GTC CAC GGT AAC AG 53 NS5B/3'UTR
3raceo_9186F 9275 TGATCAAGTYGGGCGGGTGGAAT 357 NS5B/3'UTR
3raceo2_9249F 9338 GAACACCACARCCCGAACCAA 358 NS5B/3'UTR 9276F 9394
CGTCCGTACGAAAATTTGCACTTGAG 359 NS5B/3'UTR 9312F 9429
CGCAATCGTGGTGCTAGTCGCTTACG 360 NS5B/3'UTR 3racei_9380F 9469
GCTAGTGCTATACTCGCTCTGCTT 361 Reverse Primers Region Name on v35
Antiparallel Sequence SEQ. ID detected Primer coordinates
(5'.fwdarw.3') NO: S 208R 297 TGATAGGGTG GCGGCGGGC 362 S
E5RACEin311R 400 ATACCTCCTC GGGCTGCC 363 S E313R 430 ATGGGAGCTA
CCACTGCGGT G 364 S 345R 462 GCCGGTCACC AAGTCGTRTG CAG 365 S
E5RACEo448R 537 GGTATGTGTT CSATCCGGTC CAAA 366 E2 1042R 1131
ATCCTTCT GGCTAGTCCT ACGGTT 367 E2 1227R 1316 TT TATTTGTTCA
TGGGGGTCGTG 368 E2 957R 1352 GAAAACATCA CGCGTCCATA CAC 369 E2 990R
1385 CACCAGCACC GATTTCCGC 370 E2 1558R 1539 TTGTATTCTT GACCGCCGG
371 E2 2009R 1993 GTAATCCGAC GCCTGGCCG 372 X 2331R 2302 TGTGTCTGCC
GGTTGCGA 373 X 2628R 2717 TCTCACCCTGT TAATGCTGCG CT 374 NS2 3015R
3133 CTGTCGGTTG TGGTCCTCTC GGTC 375 NS2 3317R 3435 AAGTCTCGGA
ACGGGTGGCG CAA 376 NS3 Primer Set 5 3548 TGGACAATTGCTTGGAGGTA 377
NS3 3553R 3690 TCCTTCCTTA AGGCGACACT 378 NS3 3607R 3744 ATCGTGGTGT
TGTCTTCCCT 379 NS3 3402R 3797 CACTGTATGC GACCGGCCA 380 NS3 3536R
3922 TAGACCCCTG CTGCTGTTCG CCGA 381 NS3 Primer Set 4 4025
CTGTCCCACGCACATAGATC 382 NS3 Primer Set 3 4073 TTTGTGTGATCACGGTCATG
383 NS3 Primer Set 15 4372 CCAAGTGTGGCTGTAGTCAAA 384 NS3 4092R 4487
GACGAATCTG CGGGGCTATG CTGT 385 NS3 4151R 4446 GCTACTCGGC ATTGGCGCAG
386 NS3 Primer Set 1 4584 AAAGCTGGAGTGAAGACCGT 387 NS4 5325R 5414
AACAACAGTAACAA AACACCCCT 388 NS4B 6109R 6104 CTTCCTGTTTGGGTGCCTTAC
389 NS4B 6129R 6124 TTACAGGTTGGGAAGCCGTGGTCG 390 NS4B 6129Rv2 6124
TTACAGGTTG GGAGGCCGTG GTYG 391 NS4B 6129Rv128 6124 TTACGGGTTG
GGAAGCCGTG GTCG 392 NS5A 6701R 6789 WTCGTGGAKY TAGAGCGSCG G 393
NS5A 6907R 7293 GTAGTTCAGG CGGCTTCACG GTTT 394 NS5A 7012R 7407
AGTTTGAGGC CACCGCAGTA CCA 395 NS5B 7850R 7939 TTGAYGTCYC CGAGCGGCAG
G 396 NS5B 8211R 8606 GTTGTCGGGG TGCGTAGCTG TCG 397 NS5B 8278R 8666
CTCAAGGTTC GCGCAGCT 398 NS5B 8080R 8076 AGGATGCTGTGTCAAAGATGCGCG
399 NS5B 9012R 9007 TATCCTACTGCGGCTGTGACTGTC 400 NS5B 9227R 9222
CCTCAGCGTTGGCCTTCTTTG 401 3'UTR 9616R 9611 CCTATCCGAGTTGGGCAAG 402
NS5B 9363R 9358 GTAAGAACACCACAGCCCGAACCA 403 3'UTR 9690R 9808
ACCACTTAAT GGTCGTAACT CGACC 404 3'UTR 9749R 9867(END) GTCAACGGCC
CCTTTCATT 405
[0253] The forward and reverse primers listed in Table 1 form
primer pairs in the order listed in the table. For example, the
following are primer pairs based on SEQ ID NOs are provided:
12:362, 13:363, 14:364, 15:365, 16:366, 17:367, 18:368, 19:369,
20:370, 21:371, 22:372, 23:373, 24:374, 25:375, 26:376, 27:377,
28:378, 29:379, 30:380, 31:381, 32:382, 33:383, 34:384, 35:385,
36:386, 37:387, 38:388, 39:389, 40:390, 41:391, 42:392, 43:393,
44:394, 45:395, 46:396, 47:397, 48:398, 49:399, 50:400, 51:401,
52:402, 53:403, 357:404, and 358:405. Other combinations of these
primers can be used to generate other primer pairs.
Results
[0254] The presence of HPgV-2 in the UC0125.US sample was initially
confirmed using the following primer pairs found in NS3: 3841F
& 4460R (SEQ IDs: 38 & 39), 3987F & 4398R (SEQ IDs: 40
& 41); in NS5A: 6689F & 7318R (SEQ IDs: 42 & 43), 6914F
and 7213R (SEQ IDs: 44 & 45); in NS2-NS3: 3334F & 3708R
(SEQ IDs: 34 & 35); and in E1-E2: 793F & 1263R (SEQ IDs: 14
& 15). Bands of the expected sizes were detected after one
round of 40 cycle RT-PCR and sequenced by Sanger, providing
conclusive evidence of the existence of the HPgV-2 virus.
D. Next Generation Sequencing Library Prep
[0255] A randomly primed library was prepared from the sample
discussed above for next generating sequencing (NGS) using the
Ovation.RTM. RNA-SeqV2 kit (NuGen, Part No. 7102) according to
manufacturer recommendations. Use 5 .mu.l of each RNA (pre-treated
and extracted as above in (A) as starting material. The thermal
cycler programs were as follows.
[0256] Program 1: First Strand Primer Annealing (For RNA inputs
>1 ng) 65.degree. C.--5 min, hold at 4.degree. C.
[0257] Program 2: First Strand Synthesis 4.degree. C.--1 min,
25.degree. C.--10 min, 42.degree. C.--10 min, 70.degree. C.--15
min, hold at 4.degree. C. Second strand cDNA Synthesis
[0258] Program 3: Second Strand Synthesis 4.degree. C.--1 min,
25.degree. C.--10 min, 50.degree. C.--30 min, 80.degree. C.--20
min, hold at 4.degree. C.
[0259] Program 4: SPIA.RTM. Amplification 4.degree. C.--1 min,
47.degree. C.--60 min, 80.degree. C.--20 min, hold at 4.degree.
C.
[0260] First Strand cDNA Synthesis--Thaw the First Strand cDNA
Synthesis reagents (blue) and nuclease-free Water (green). Spin A3
ver 1 briefly and place on ice. Vortex A1 ver 4 and A2 ver 3, spin
and place on ice. Leave nuclease-free water at room temperature. On
ice, mix 2 .mu.l of A1 and 5 .mu.l of total RNA sample (500 pg to
100 ng) in a 0.2 ml PCR tube. Place the tubes in a thermal cycler
running Program 1 (65.degree. C.--2 min, hold at 4.degree. C. or
65.degree. C.--5 min, hold at 4.degree. C.). Once the thermal
cycler reaches 4.degree. C., remove tubes and place on ice. Prepare
First Strand Master Mix. Be sure to pipet A3 enzyme slowly and
rinse out tip at least five times into buffer.
[0261] Per sample combine 2.5 .mu.l Buffer Mix A2+0.5 .mu.l Enzyme
Mix A3. Add 3 .mu.l of First Strand Master Mix to each tube, mix by
pipetting, spin and place on ice. Place the tubes in a thermal
cycler running Program 2 (4.degree. C.--1 min, 25.degree. C.--10
min, 42.degree. C.--10 min, 70.degree. C.--15 min, hold at
4.degree. C.). Once the thermal cycler reaches 4.degree. C., remove
tubes, spin and place on ice. Continue immediately with Second
Strand cDNA Synthesis.
[0262] Second Strand cDNA Synthesis--Resuspend the Agencourt.RTM.
RNA Clean.RTM. XP beads provided with the Ovation RNA-Seq System V2
and leave at room temperature for use in the next step. Thaw the
Second Strand cDNA Synthesis reagents (yellow). Spin B2 ver 2
briefly and place on ice. Vortex B1 ver 3, spin and place on ice.
Prepare Second Strand Master Mix. Be sure to pipet B2 enzyme
slowly.
[0263] Per sample combine: 9.7 .mu.l Buffer Mix B1+0.3 .mu.l Enzyme
Mix B2. Mix well. Add 10 .mu.l of Second Strand Master Mix to each
reaction tube, mix by pipetting, spin and place on ice. Place the
tubes in a thermal cycler running Program 3 (4.degree. C.--1 min,
25.degree. C.--10 min, 50.degree. C.--30 min, 80.degree. C.--20
min, hold at 4.degree. C.). Once the thermal cycler reaches
4.degree. C., remove tubes, spin and place on bench top. Continue
immediately with Purification of cDNA.
[0264] Purification of cDNA--Ensure the RNAClean XP beads have
reached room temperature. Mix the beads by inverting several times.
At room temperature, add 32 .mu.l of RNAClean XP beads to each
reaction tube and mix by pipetting 10 times. Incubate at room
temperature for 10 minutes. Transfer the tubes to the magnet and
let stand for an additional 5 minutes. Remove only 45 .mu.l of the
binding buffer. Add 200 .mu.l of freshly prepared 70% ethanol and
let stand for 30 seconds. Remove the ethanol using a pipette.
Repeat the ethanol wash 2 more times. Remove all excess ethanol
after the final wash and let beads air dry for 15 to 20 minutes.
Ensure the tubes have completely dried and no residual ethanol is
left. Continue immediately with SPIA Amplification, with the cDNA
bound to the dry beads.
[0265] SPIA Amplification--SPIA is an isothermal
strand-displacement amplification process that uses a DNA/RNA
chimeric SPIA primer, DNA polymerase and RNAse H to amplify DNA.
Thaw the SPIA Amplification reagents (red). Invert C3 ver 7 to mix,
spin and place on ice. Vortex C1 ver 9 and C2 ver 11, spin and
place on ice. Prepare SPIA Master Mix. Per sample combine 20 .mu.l
Buffer Mix C2+10 .mu.l Primer Mix C1+10 .mu.l Enzyme Mix C3. Add 40
.mu.l of SPIA Master Mix to each reaction tube and resuspend beads
thoroughly by pipetting. Place on ice. Place the tubes in a thermal
cycler running Program 4 (4.degree. C.--1 min, 47.degree. C.--60
min, 80.degree. C.--20 min, hold at 4.degree. C.). Once the thermal
cycler reaches 4.degree. C., remove tubes, spin and place on ice.
[0266] Remove beads with magnet and transfer to new tube; store
SPIA cDNA at -20.degree. C.
[0267] SPIA amplified cDNA for sample UC0125.US was then purified
with AMP Pure magnetic beads (Beckman Coutler, A63880) (1.8.times.
volume=72 .mu.l of beads) and eluted in 30 .mu.l of EB buffer. SPIA
amplified cDNA for samples ABT0070P.US and ABT0096P.US were
purified using the Qiagen MinElute protocol as follows: (note: this
is the preferred method of purification): Add 300 .mu.l of buffer
ERC (Enzymatic reaction clean-up) to 40 .mu.l of sample. Vortex 5
sec then load entire sample to column (stored at 4.degree. C.).
Spin at 14K rpm for 1 min. Discard flow through and add 700 .mu.l
of buffer PE (EtOH added) to column. Spin at 14K rpm for 1 min then
discard flow through. Replace column in same collection tube and
spin again at 14K rpm for 2 min. Discard collection tube and blot
column tip to paper towel to remove any residual EtOH from PE
buffer. Finally, add 25 .mu.l of EB buffer to the center of column
filter placed in fresh Eppendorf collection tube. Let stand for 1
min then spin at 11K rpm for 1 min. Store eluted library on ice or
at -20.degree. C.
E. Determine cDNA Yield and Size Range
[0268] Run 1-2 .mu.l of each cDNA on an agarose gel to estimate
library size. With Qiagen Viral Mini extractions they are generally
150-250 bp in length. Measure the concentrations on a Qubit
Fluorometer using dsDNA_BR (broad range) reagents (Molecular
Probes/Life Technologies). Alternatively, libraries can be
evaluated on a BioAnalyzer 2200 TapeStation.
F. Confirmatory PCR
[0269] Perform confirmatory PCRs to evaluate the virus-specific
content of libraries Select primers for regions/genes of interest
(see Table 1 of Section C). Use 2 .mu.l of Ovation cDNA as template
in PCR reaction. Determine appropriate controls and use Applied
Biosystems Taq reagents. To 2 .mu.l of cDNA template, add 2.5 .mu.l
10.times. PCR buffer with 15 mM MgCl.sub.2, 0.5 .mu.l dNTP mix, 0.5
.mu.l 10 uM fwd primer, 0.5 .mu.l 10 .mu.M rev primer, 0.2 .mu.l
Taq DNA polymerase, and 18.8 .mu.l water. Run for 30-50 cycles
depending on expectations and resolve products on an agarose gel to
determine library quality/content.
G. Nextera Tagmentation
[0270] Nextera XT was used to incorporate Illumina adaptors and
indexes into libraries. When multiplexing samples, it is essential
to select compatible barcodes to achieve color balance. The
Illumina protocol described below and was adapted from the Nextera
XT Sample Prep kit (Illumina, #15032350) and used in conjuction
with the Nextera Index kit: ref# 15032353. Reactions can be
assembled in 96 well plates, however, the samples were done here in
0.2 ml PCR tubes.
[0271] Step 1. Preparation--Remove the ATM, TD, and input DNA from
-15.degree. to -25.degree. C. storage and thaw on ice. Ensure that
NT is at room temperature. Visually inspect NT to ensure there is
no precipitate. If there is precipitate, vortex until all
particulates are resuspended. After thawing, ensure all reagents
are adequately mixed by gently inverting the tubes 3-5 times,
followed by a brief spin in a microcentrifuge. Reagent volumes are
aliquoted as described and placed on ice (except NT) until added to
reactions: TD Buffer: (sample # +1) * 10 .mu.l, ATM: (sample # +1)
* 5 .mu.l, NT: (sample # +1) * 5 NPM: (sample # +1) * 15 .mu.l,
Index 2: (sample# +1) * 5 .mu.l, Index 1: (sample# +1) * 5
.mu.l.
[0272] Step 2. Tagmentation of Input DNA--Ensure the reaction is
assembled in the order described for optimal kit performance. The
reaction does not need to be assembled on ice. Label a new 96-well
TCY plate NTA (Nextera XT Tagment Amplicon Plate) or 0.2 ml tubes.
Dilute cDNA in water to 0.2 ng/.mu.l. This is typically
.about.30-300X dilution of starting cDNA. 1 ng of input (5 .mu.l of
0.2 ng/.mu.l) is required for optimal results. Use .about.2 .mu.l
of stock in the appropriate volume of water to most accurately
reach the final concentration of 0.2 ng/.mu.l. Add 10 .mu.l of TD
Buffer to each well to be used in this assay. Change tips between
samples. Add 5 .mu.l of input DNA at 0.2 ng/.mu.l (1 ng total) to
each sample well of the NTA plate/tube. Add 5 .mu.l of ATM to the
wells/tubes containing input DNA and TD Buffer. Change tips between
samples. Gently pipette up and down 5 times to mix. Change tips
between samples. Cover the NTA plate with Microseal `B`. Centrifuge
at 280.times.g at 20.degree. C. for 1 minute. Place the NTA
plate/tubes in a thermocycler and run the following program:
55.degree. C. for 5 minutes; Hold at 10.degree. C. Once the sample
reaches 10.degree. C. proceed immediately to Neutralize NTA.
[0273] Step 3. Neutralize NTA--Carefully remove the Microseal "B"
seal and add 5 .mu.l of NT Buffer to each well/tube of the NTA
plate. Change tips between samples. Gently pipette up and down 5
times to mix. Change tips between samples. Cover the NTA plate with
Microseal `B`/close tube. Centrifuge at 280.times.g at 20.degree.
C. for 1 minute. Place the NTA plate/tubes at room temperature for
5 minutes. After the tagmentation step, set up cycling conditions
as described below.
[0274] Step 4. Library amplification and barcode addition--Place
the NTA plate in the TruSeq Index Plate Fixture/tubes on ice. Add
15 .mu.l of NPM (Nextera PCR Mix) to each well of the NTA
plate/tube containing index primers. Add 5 .mu.l of index 2 primers
(white caps) to each column of the NTA plate/tube. Changing tips
between columns is important to avoid cross-contamination. Add 5
.mu.l of index 1 primers (orange caps) to each row of the NTA
plate/tube. Tips should be changed after each row to avoid index
cross-contamination.
[0275] Cover the plate with Microseal `A` and seal with a rubber
roller/close tubes. Centrifuge at 280.times.g at 20.degree. C. for
1 minute. Perform PCR using the following program on a thermal
cycler: Ensure that the thermocycler lid is heated during the
incubation; 72.degree. C. for 3 minutes; 95.degree. C. for 30
seconds; set for 16 cycles of: 95.degree. C. for 10 seconds;
55.degree. C. for 30 seconds; 72.degree. C. for 30 seconds; and
72.degree. C. for 5 minutes. Hold at 10.degree. C.
[0276] Library Purification--Samples were then purified using 1.8X
AMP-PURE XP beads (Beckman Coulter, A63880) and eluted in 40 .mu.l
of RSB buffer (Illumina provided).
H. Library Quantification and Visualization
[0277] Visualize samples (1 .mu.l) on the BioAnalyzer 2200
TapeStation using the following reagents from Agilent: D1K Screen
Tape: ref# 00-S019-120707-02-000084D1K ladder: ref# 52715 90-240
Sample Buffer: ref# 52907 98-221 Add 3 .mu.l of loading buffer to 1
.mu.l of library sample. Cap tube strips and vortex thenspin to
collect. Remove caps and place in appropriate slot of TapeStation.
Enter sample information and run electrophoresis. Adjust window
limits for integration measurement of peak to determine
concentration.
I. Prepare for MiSeq Run
[0278] The following procedure was followed to run
HPgV-2-containing libraries on a MiSeq. Step 1. Thaw -20.degree. C.
reagent cartridge (e.g. 300 or 500 cycle V2 reagent kit; Illumina,
15033625) in water container filled to designated line. Put HT
buffer on bench at RT. Let both thaw for 1 hr, invert cartridge
several times then hit gently on bench to dislodge to any bubbles
from the bottom. Put cartridge and HT1 buffer in refrigerator until
ready to dilute/load sample. Place 4.degree. C. reagent box at room
temperature (buffer and flow cell). Shake/invert buffer and allow
bubbles to dissipate for >1 hour. Wash flow cell with ddH.sub.20
thoroughly to remove salts thedry completely with a Kimwipe.
[0279] Step 2. Create a new sample sheet in Illumina Experiment
Manager (IEM). Select MiSeq.fwdarw.small genome.fwdarw.resequencing
workflow. Record experiment name, reagent IDs, sample IDs and
barcode information.
[0280] Step 3. Combine libraries in equimolar amounts (or as
desired) for multiplexing of sequencing library. Using
concentrations (nM) determined on BioAnalyzer, calculate the
dilutions to be made with water to bring each library to a 1.1 nM
final concentration in 18 .mu.l. Add 2 .mu.l of 1N NaOH and
denature for 5 min at room temperature.
[0281] Step 4. Dilute 1 nM library (20 .mu.l) with 980 .mu.l of HT1
for a final library concentration of 20 pM. Vortex and place on
ice.
[0282] Step 5. Dilute a second time with HT1 buffer at 1:1 for a 10
pM library, adding in 1% PhiX control, vortex then place on ice.
The library should contain 500 .mu.l 20 pM library, 10 .mu.l PhiX
(diluted and denatured), and 490 .mu.l of HT1 buffer.
[0283] Step 6. Denature the 10 pM library (+1% PhiX) by heat for 2
min at 96.degree. C. then place on ice water bath for >5
min.
[0284] Step 7. Go to MiSeq Control Software and hit "Sequence" to
set up the instrument. Follow instructions as provided and load
experiment sample sheet.
[0285] Step 8. Dispense 600 .mu.l of 10 pM library into reagent
cartridge and hit "Start".
Results
[0286] The MiSeq run was completed and the data was aligned to the
consensus genome sequence generated in Example 1. From a total of
16,306,796 reads assigned to the HPg-V2 sample barcode, 249,693
(1.53%) of these mapped to HPgV-2. Sequences aligned uniformly and
without gaps, covering 98.4% of the genome with an average depth of
3314X.+-.426 reads/nt. In the region of overlap, this independent
NGS dataset had 99.73% identity (9290/9315) to the draft genome
produced in Example 1, with every mismatch either conserved (e.g.
A.fwdarw.G, C.fwdarw.T) or resolving an ambiguous base in the
latter (e.g. R.fwdarw.4A, G). 156 bp of the 3' end previously
determined by 3'RACE was absent in this NGS run, however, the data
was mined by de novo assembly to extend the 5' end by an additional
306 nucleotides, thereby adding to the putative core protein (S)
and/or 5'UTR sequence. The combined data from Examples 1 and 2
yielded the 9778 bp genomic sequence (SEQ ID 1) found in FIG. 1.
Subsequent NGS runs for additional strains of HPgV-2 extended the
5'UTR such that the total length of the genome is now 9867 nt.
Example 3
Generating an In Vitro HPgV-2 Template Control and qPCR Assay
[0287] This example describes methods of generating a HPgV-2
template positive control and qPCR assays carried out with this
template. The NS2-NS3 region in HPgV-2 was selected to probe
specimens by qPCR. Based on our NGS data, this region exhibited the
least amount of sequence heterogeneity. The region we cloned is
approximately 1260 bp long and was inserted into pGEM-11Zf(+)
(Promega, Madison Wis.) to enable in vitro transcription of this
HPgV-2 template. This cloned sequence represents bases 3224 to 4483
of SEQ ID NO:1 and is shown in FIG. 4. As described further below,
5 sets of primers and probes were designed to detect the HPgV-2
RNA.
A. Linearize NS23EX Plasmid for Use as In Vitro Transcription
Template.
[0288] Step 1--Resuspend the 4 .mu.g of lyophilized plasmid in 20
.mu.l of elution buffer (EB) to bring concentration to 200
ng/.mu.l.
[0289] Step 2--Digest with restriction enzyme(s) XbaI and HindIII
found at the end of the 1260 bp insert. The pGEM.RTM.-11Zf(+)
Vector can be used as a standard cloning vector and as a template
for in vitro transcription and the production of ssDNA. The plasmid
contains T7 and SP6 RNA polymerase promoters flanking a multiple
cloning region within the alpha-peptide coding region of
beta-galactosidase. The following reaction mixture was generated: 5
.mu.l DNA (1 .mu.g), 2 .mu.l 10.times. M buffer, 2 .mu.l 0.1% BSA,
1 .mu.l XbaI, 1 .mu.l HindIII, and 9 .mu.l water. This was
incubated at 37.degree. C. for 2.25 hrs.
[0290] Step 3--Precipitate linearized plasmid DNA in a reaction
mixture of: 20 .mu.l DNA, 2 .mu.l NH4 stop solution (Ambion), and
40 .mu.l EtOH. Incubate at -20.degree. C. for 15 min; spin at top
speed for 15 minutes; wash with 80% EtOH and respin 5 min. Dry for
5 min and resuspend in 10 .mu.l of EB to an estimated concentration
of 100 ng/.mu.l.
[0291] Step 4--Measure DNA concentration on a NanoDrop.
[0292] Step 5--Visualize plasmid digestion on agarose gel. Run
uncut and cut vector side by side to compare.
B. In Vitro Transcribe NS23EX Insert
[0293] Follow the Ambion Megascript T7 kit (AM1334) protocol as
recommended then purify RNA. Step 1: Assemble 20 .mu.l in vitro
transcription reactions. Template DNA input volumes were as
follows: 6.3 .mu.l of linearized NS23Ex_pGEM11Zf (1 .mu.g)+1.7
.mu.l water; 2.0 .mu.l of control pTRI-Xef vector+6 .mu.l of water.
Incubate for 4 hr at 37.degree. C. in a plate incubator (dry). Add
1 .mu.l of DNAse and digest for 15 min at 37.degree. C.
[0294] Step 2: Trizol purification--Resuspend reaction in 230 .mu.l
of water then add 750 .mu.l of Trizol. Suspended pTRI pellet in 25
.mu.l of waterand NS23Ex pellet in 50 .mu.l of water.
[0295] Step 3: Quantify RNA by Nanodrop and on the BioAnalyzer as
described above using the R6K screen tape.
C. Reverse Transcription
[0296] cDNA was generated using SS RTIII (Invitrogen: 18080-051)
for random hexamer priming. The HPgV-2 RNA in vitro transcribed
from NS23EX/pGEM11-Zf(+) plasmid and the pTRI-Xef control RNA from
Ambion were used as templates. Reactions were assembled as follows:
2.0 .mu.l RNA (.about.400 ng), (used the 1/10 dilution of NS23EX
RNA; straight for pTRI), 5.6 .mu.l water, 1 .mu.l of random hexamer
(50 ng/.mu.l), 0.4 .mu.l of oligo dT (50 .mu.M), And 1 .mu.l dNTP
mix (10 mM). Heat at 65.degree. C. for 5 min and return to ice to
add 10 .mu.l of master mix. Prepare the mastermix as follows: 2
.mu.l 10 RT buffer, 4 .mu.l 25 mM MgCl.sub.2, 2 .mu.l 0.1 M DTT, 1
.mu.l RNAseOUT (40 U/.mu.l), and 1 .mu.l Superscript III RT (200
U/.mu.l).
[0297] Incubate for 10 min at 25.degree. C., then 20 min at
50.degree. C. Terminate at 85.degree. C. for 5 min then 4.degree.
C. Add 1 .mu.l of RNAse H (Invitrogen, Y01220) to reactions and
incubate for 20 min at 37.degree. C. and then to 4.degree. C. cDNA
was aliquoted into 2.times.10 .mu.l samples and frozen at
-80.degree. C.
E. HPgV-2 PCR
[0298] The following primers were used to confirm the HPgV-2 RNA.
These primers were named with an older version of the HPgV-2
genome. Refer to their sequence ID numbers.
TABLE-US-00005 PCR 1_617 bp product (SEQ ID NO: 38)
GAGCCGGGTTTGGGTGATGAATAA 3841F (SEQ ID NO: 39) CTGCGCCAATGCCGAGTAGC
4460R PCR 2_412 bp product (SEQ ID NO: 40) ACGCGTTGATGCTCGGTGGTTACT
3987F (SEQ ID NO: 41) ACAGCATAGCCCCGCAGATTCGTC 4398R
[0299] The following volumes of reagents were used for PCR. 2 .mu.l
of diluted cDNA as template (NS23EX, pTRI; add 1.5 .mu.l of cDNA
stock+8 .mu.l of water); 2.5 .mu.l 10.times. PCR buffer with 15 mM
MgCl.sub.2; 0.5 .mu.l dNTP mix; 0.5 .mu.l 10 .mu.M forward primer ;
0.5 .mu.l 10 .mu.M reverse primer ; 0.2 .mu.l Taq DNA polymerase;
and 18.8 .mu.l water. Use the following PCR conditions: 1 cycle
@94.degree. C.: 2 min ; 35 cycles @94.degree. C.: 20 sec,
55.degree. C.: 30 sec, 72.degree. C.: 40 sec; 1 cycle72.degree. C.:
7 min; 1 cycle 4.degree. C.: hold. 5 .mu.l of each sample was
resolved on an ethidium bromide gel and photographed under UV
light. F. qPCR with 7 Primer Sets
[0300] The following was performed to establish a TaqMan-based qPCR
assay for the detection of HPgV-2 RNA in patient specimens. In
vitro transcribed RNA (NS23Ex=2000 ng/.mu.l), start the first
dilution at 1/100 (20 ng/.mu.l) 4 load 5 .mu.l for 100 ng total.
Repeat 10 fold dilutions using 2 .mu.l in 18 .mu.l of water for a
total of 6 dilutions. For pTRI(200 ng/.mu.l),dilute at
1/10.fwdarw.load 5 .mu.l for 100 ng total._For total RNA, Take 3
.mu.l of each RNA and dilute it with 27 .mu.l of water ( 1/10).
Included in this experiment were UC0125.US, CHU2725 [an
HIV(+)/GBV-C(+) sample], and N-505 [HIV(+)/GBV-C(-) sample] all
extracted in the same manner.
[0301] Make dilutions of primers & probes at the concentrations
listed and combine reactions as follows for qPCR on an ABI
light-cycler (Abbott m2000rt): [0302] 12.5 .mu.l 2.times. RT-PCR
Buffer [0303] 1 .mu.l Forward primer, 10 uM [0304] 1 .mu.l Reverse
primer, 10 uM [0305] 1 .mu.l Taq man probe, 3 uM [0306] 1 .mu.l
25.times. RT-PCR enzyme mix [0307] 5 .mu.l RNA sample [0308] 3.5
.mu.l Nuclease-free water [0309] 25 .mu.l total 20 ul of master-mix
per well
[0310] The 7 sets of probes and primer pairs employed are shown in
Table 2 below. The binding locations for these sequences are shown
in FIG. 4.
TABLE-US-00006 TABLE 2 Primer Set 5: Amplicon Size = 70 NS23Ex_F5
GAGGAGCCCACCTTTACTGA (SEQ ID NO: 54) NS23Ex_R5 TACCTCCAAGCAATTGTCCA
(SEQ ID NO: 55) NS23Ex_Prb5 CACCAAACTCATTGTGTCATCCACGA (SEQ ID NO:
56) Primer Set 2: Amplicon Size = 89 NS23Ex_F2 GAAGATCTGCCACCTGGTTT
(SEQ ID NO: 57) NS23Ex_R2 AGTGTCGCCTTAAGGAAGGA (SEQ ID NO: 58)
NS23Ex_Prb2 CCACCGGAGCACTCAGCTGG (SEQ ID NO: 59) Primer Set 7:
Amplicon Size = 74 NS23Ex_F7 TCCTTCCTTAAGGCGACACT (SEQ ID NO: 60)
NS23Ex_R7 AGGGAAGACAACACCACGAT (SEQ ID NO: 61) NS23Ex_Prb7
AAACACCAGGGTCCGGCCAG (SEQ ID NO: 62) Primer Set 4: Amplicon Size =
85 NS23Ex_F4 TGGGTGATGAATAACAACGG (SEQ ID NO: 63) NS23Ex_R4
GATCTATGTGCGTGGGACAG (SEQ ID NO: 64) NS23Ex_Prb4
CCACTCTGCCACACACCAACCC (SEQ ID NO: 65) Primer Set 3: Amplicon Size
= 88 NS23Ex_F3 AATGACGACGTCTGTTTGGA (SEQ ID NO: 66) NS23Ex_R3
CATGACCGTGATCACACAAA (SEQ ID NO: 67) NS23Ex_Prb3
CTGGTGAGCCCGAAGCACCC (SEQ ID NO: 68) Primer Set 15: Amplicon Size =
138 NS23Ex_F15 CCAGCTGTGACACCAACATA (SEQ ID NO: 69) NS23Ex_R15
TTTGACTACAGCCACACTTGG (SEQ ID NO: 70) NS23Ex_Prb15
CCAGTGGACCTAGTCAAACAGGGACA (SEQ ID NO: 71) Primer Set 1: Amplicon
Size = 96 NS23Ex_F1 AGTCATTTGCGACGAGTGTC (SEQ ID NO: 72) NS23Ex_R1
ACGGTCTTCACTCCAGCTTT (SEQ ID NO: 73) NS23Ex_Prb1
TCGGCATACATGCGCACTGC (SEQ ID NO: 74)
G. qPCR Assay A--TaqMan Based Detection of HPgV2
[0311] TaqMan assays using probes and primer pairs 1, 2, 3, 5, and
7 were performed to detect 10-fold serial dilutions of the NS23Ex
in vitro transcript and a 10-fold dilution of the UC0125.US RNA.
Primers were ordered from a Applied Biosystems and HPLC purified.
Probes have 5'6FAM and 3'TAMRA modifications. Ag Path-ID One
StepRT-PCR Kit (Applied Biosystems, 438724) was used for buffer and
enzyme mixes. Primer (10 .mu.M) and probe (3 .mu.M) concentrations
were used as recommended.
[0312] Realtime qPCR cycling conditions were as shown below:
TABLE-US-00007 Step Stage Reps Temp Time Reverse transcription 1 1
45.degree. C. 10 min RT inact/initial denaturation 2 1 95.degree.
C. 10 min Amplification 3 40 (45) 95.degree. C. 15 sec 60.degree.
C. 45 sec
[0313] Dispense 5 .mu.l of sample (NS23Ex RNA dilutions, controls
or sample RNA) to each well. The optional detection enhancer was
not added. Dispense 20 .mu.l of mastermix to each well. Spin to
collect and seal.
[0314] The results are shown in FIG. 5. FIG. 5A shows HPgV-2
primer/TaqMan probe sets (1-2-3-5-7; see FIG. 4 for sequences and
positions) were used to detect 10-fold serial dilutions of the
NS23Ex in vitro transcript and a 10-fold dilution of the HPgV-2
index case (UC0125.US) RNA (highlighted in bold). The lower right
panel shows detection of 100 ng of NS23Ex and HPgV-2 RNA for each
primer/probe set. FIG. 5B shows Ct values that were normalized to
set 1_100 ng results and plotted on a log scale to estimate the
amount of HPgV-2 RNA present in the index case. Negative controls
included in the experiment were: 1) water, 2) pTRI (an irrelevant
in vitro transcript), 3) CHU2725 (HIV+/GBV-C+ sample), and 4) N-505
(HIV+/GBV-C- sample) indicate there is no cross-reactivity with
other infections (HIV, GBV-C). HCV purified virus (1000 plaque
forming units) was tested at a later time and also shown to NOT
cross-react with these primers (data not shown). The NS23Ex
template was detected in a dose-dependent fashion by all
primer/probe sets. None of the negative control RNAs were amplified
by these primer, suggesting no cross-reactivity. The index case
(UC0125.US) viral load is estimated at 1.5.times.10.sup.6 copies/ml
based on these results.
H. qPCR Assay B--SYBR Green qPCR
[0315] SYBR green qPCR assays were conducted using probe and primer
sets 1, 2, 3, 4, 5, 7, and 15 and 44F (SEQ ID NO:12) and 342R (SEQ
ID NO:13), which were used to detect 10-fold serial dilutions of
cDNA made from the NS23Ex in vitro transcript (FIG. 6, curves A, B,
C) and the HPgV-2 index case (UC0125.US) RNAs (FIG. 6, curve D).
Negative controls (FIG. 6, curves E and F), N-505 (HIV(+)/GBV-C(-))
and water, were not amplified. The following samples were in each
row: 1) NS23Ex 1:1000 cDNA; 2) NS23Ex 1:10,000 cDNA; 3) NS23Ex
1:100,000 cDNA; 4) GBV-E cDNA; 5) N505 cDNA; and 6) Water. The qPCR
reactions were set up as follows:_2 .mu.l cDNA (diluted at 1:5 from
20 .mu.l SSRTIII reactions); 7.2 .mu.l water; 0.4 .mu.l Fwd primer
(10 .mu.M); 0.4 .mu.l Rev primer (10 .mu.M); and 10 .mu.l SYBR
green mix (Applied Biosystems).
[0316] The results are shown in FIG. 6. Each primer set showed a
dose-dependent detection of the NS23Ex transcript and all detected
UC0125.US cDNA at essentially the same Ct. None produced a signal
for N505 or water.
I. qPCR Assay C
[0317] Primer/probe sets 2 and 3 were used in TaqMan qPCR assays
and revealed three cases of HPgV-2 infection in donor plasma. RNA
extracted from ProMedDx HCV(+) plasmapheresis donor plasma samples
48-96 was screened with TaqMan primer/probe sets 2 (top panel) and
3 (bottom panel) as described in FIG. 7A. Samples#70 and #96
(ABT0070P.US and ABT0096P.US) were detected by both sets. 10 ng and
10 fg of the NS23Ex in vitro transcript positive control are also
shown. FIG. 7B shows results of an assay where RNA extracted from
American Red Cross blood donor plasma [HCV RNA(+)/antibody(+)]
samples were screened with TaqMan primer/probe sets 2 and 3 as
above. Sample #128 (ABT0128A.US) was detected, but only by set 2
(bold). 10 ng and 10 fg of the NS23Ex in vitro transcript positive
control are shown in green (probe 2) and purple (probe 3).
J. Tri-Plex Mastermix qPCR Assay
[0318] A new qPCR assay was developed based off current HPgV-2
strain sequence alignments (see FIG. 23 alignment) to
simultaneously screen for HPgV-2 and GBV-C (HPgV-1)-infected
specimens. RNA was extracted from donor samples using the automated
Abbott m2000sp system followed by qPCR on the m2000rt instrument.
An example of results which led to the identification of strains
ABT0030P.US and ABT0041P.US is shown in FIG. 28. Detection of HPgV1
was achieved by targeting the 5' UTR using FP
5'-TGTTGGCCCTACCGGTGTTA-3' (SEQ ID NO: 408) and RP
5'-CCGTACGTGGGCGTCGTT-3' (SEQ ID NO: 409) and three fluorescently
labeled probes (5'-VIC-CTCGTCGTTAAACCGAGCCCGTCA-BHQ1-3'(SEQ ID NO:
410), 5'-VIC-CTCGTCGTTAAACCGAGACCGTCA-BHQ1-3'(SEQ ID NO: 411),
5'-VIC-CACGCCGTTAAACCGAGACCGTTA-BHQ1-3'(SEQ ID NO: 412)) to account
for mismatched sequences. HPgV-2 primer/probe sequences are adapted
from Keys, et al (Keys, JR. et al 2014 J Med Virol 86: 473-477)
Detection of HPgV2 relied on amplification of the NS2-3 region (FP
5'-GTGGGACACCTCAACCCTGAAG-3'(SEQ ID NO: 413), RP
5'-GGGAAGACAACACCACGATCTGGC-3'(SEQ ID NO: 414), probe
5'-FAM-CCTGGTTTCCAGCTGAGTGCTCC-BHQ1-3'(SEQ ID NO: 415)) and the 5'
UTR (FP 5'-CGCTGATCGTGCAAAGGGATG-3'(SEQ ID NO: 416), RP
5'-GCTCCACGGACGTCACACTGG-3'(SEQ ID NO: 417), probe
5'-Quasar670-GCACCACTCCGTACAGCCTGAT-BHQ2-3'(SEQ ID NO: 418)). All
primers were synthesized by IDT (Coralville, Iowa) and probes
synthesized at Abbott Molecular (Des Plaines, Ill.). Cycling
conditions were the following: 50.degree. C., 4 minutes (1.times.);
75.degree. C., 5 minutes (1.times.); 60.degree. C., 30 minutes
(1.times.); 91.degree. C., 30 seconds; 58.degree. C., 45 seconds
(6.times.); 91.degree. C., 30 second; 60.degree. C., 45 seconds (+2
sec/cycle)(4.times.); 91.degree. C., 30 second; 60.degree. C., 45
seconds (+2 sec/cycle)(43X-Read). Reverse transcription and DNA
polymerase activity was performed using the enzyme, rTth (Roche).
In FIG. 28, a total of 4 specimens among the HIV-positive set were
reactive for HPgV-2 RNA (CY5 and FAM channels: 5'UTR and NS2,
respectively), however, two of each were from the same donor. A
total of six specimens were GBV-C positive (VIC channel:
5'UTR).
Example 4
Detection of Antibodies Directed Against HPgV-2
Peptide/Proteins
[0319] This example describes the selection and evaluation of
potential antigenic peptides and recombinant proteins from the
HPgV-2 genome. A series of peptides, shown in Table 3, ranging in
length from 20 amino acid residues to 45 amino acids residues were
designed taking into account the predicted surface exposure
(hydrophillicity profile and surface probability) and antigenic
index scores.
[0320] Briefly, HPgV-2 proteins (S, E1, E2, NS3, NS3, NS4A, NS4B,
NS5A, and NS5B) were analyzed for predicted antigenic regions using
DNASTAR Lasergene 11 version 11.2.1 (29) Protean software (DNAStar,
3801 Regent Street Madison, Wis. 53705, USA). Potential antigenic
regions were determined by a combination of hydropathy plot,
antigenicity index, and predicted surface probability for each
amino acid sequence. Hydropathy profiles for each open reading
frame were generated and assessed for areas that had the potential
to be exposed when in aqueous solution (hydrophilic). Surface
probability determination refers to the bias a sequence has to
being on the surface of the molecule. The Emini surface probability
method was employed where the predicted probability of a given
amino acid to be water solvent exposed is determined for a sequence
of amino acids (Emini et al., J Virol. 1985 Sep: 55(3):836-9).
Predicted antigenic regions would have the property of surface
exposure. Antigenic index was determined using the Jameson-Wolf
algorithm where surface accessibility of residues is combined with
predicted backbone flexibility and secondary structure (Jameson B.
A and Wolf H. Comput Appl Biosci (1988) 4 (1): 181-186). Peptides
were designed to sequences found in HPgV-2 proteins (S, E1, E2,
NS3, NS3, NS4A, NS4B, NS5A, and NS5B) with consideration of
predicted surface exposure (hydrophilicity profile and surface
probability) and antigenic index scores (Table 3).
[0321] Peptides (Table 3) were generated with an amino terminal
biotinylation modification (Genscript USA Inc, Piscataway, N.J.).
Peptides were biotinylated to allow for a strong covalent
interaction with avidin and streptavidin coated microparticles
which can be used to immobilize the peptides on a solid surface
(e.g. microparticles), that are used for solid-phase based
immunoassays.
TABLE-US-00008 TABLE 3 Peptide SEQ ID # Protein Sequence NO: 1 S
GGSCRSPSRVQVARRVLQLSAFLALIGSGMSSIRSKTEGRIESGQ 86 2
RDGSLHWSHARHHSVQPDRVAAGPPSVTSVERNMGSSTDQT 87 3 E2
SMNSDSPFGTFTRNTESRFSIPRFSPVKINS 88 4 NS3
QAPAVTPTYSEITYYAPTGSGKSTKYPVDLVKQGHKVLVL 89 5
VKSMAPYIKETYKIRPEIRAGTGPDGVTVITG 90 6 PETNLRGYAVVISDESHDTSS 91 7
PCTAALRMQRRGRTGRGRRGAYYTTSPGAAPCVS 92 8 NS4B
LSERFGQQLSKLSLWRSVYHWAQAREGYTQCG 93 9 NS5A
NPTTTGTGTLRPDISDANKLGFRYGVADIVELERRGDKWH 94 10
QNLAARRRAEYDAWQVRQAVGDEYTRLADEDVD 95 11
RFVPPVPKPRTRVSGVLERVRMCMRTPPIKF 96 12 NS5B NTTRDHNNGITYTDLVSGRAKP
97 13 DAPMRIIPKPEVFPRDKSTRKPPRFIVFPGCAARV 98 14
MPLLCMLIRNEPSQTGTLVT 99 15 S
AEAAPKSGELDSQCDHLAWSFMEGMPTGTLIVQRDGSLH 217 16 NS4A-B
SVEVRPAGVTRPDATDETAAYAQRLYQACADSGIFASLQGTASAALGKLA 218
Populations to be Tested
[0322] Previous studies indicate that GBV-C, the human virus most
closely related to HPgV-2, is frequently detected in HCV infected
individuals and in commercial blood donors (e.g. paid
plasmapheresis donors). It was postulated that HPgV-2 may likewise
be found in higher prevalence rates in commercial donors than in
the general population. Thus a population of samples from
plasmapheresis donors was selected for study. Further, since the
index case (UC0125.US)) was also HCV infected, it was reasoned that
additional HPgV-2 cases might be detected among samples that are
antibody positive for HCV. Thus, one of the first populations
selected for study was plasmapheresis donors whose plasma were
previously tested as positive both for antibodies to HCV and for
HCV RNA.
Testing Procedures
[0323] Three pools of peptides were generated: Pool 1 contained the
following peptides: P 1, 2, 4, 5, 7, 8; Pool 2 contained the
following peptides: P 9, 10, 11, 13, 14; and Pool 3 contained the
following peptides: P 3, 6, 12.
[0324] In the first step of the assay, a sample (e.g. human serum
or plasma) is added to a reaction vessel along with a specimen
diluent buffer (containing buffering salts and detergents)
containing one of the pools of the biotinylated peptides (800ng/ml
each) and the solid phase coated with streptavidin (Dynabeads M-270
Streptavidin, Life Technologies 3175 Staley Road Grand Island, N.Y.
14072). This sample is incubated for 18 minutes. During this time,
the solid phase captures both the biotinylated peptide and the
antibody complexed to the peptide (immune complex). Following the
18 minute incubation step, unreacted sample is removed, and the
second step of the assay is initiated by adding a signal generating
conjugate to the reaction vessel. The conjugate (in this case, 16
ng/mL of mouse-anti-human IgG conjugated to a chemiluminescent
enzyme (acridinium)) recognizes the human immunoglobulins that have
bound to the peptide and are now affixed to the solid phase. After
a washing step to remove unreacted material, the microparticles are
washed, and then incubated with a substrate capable of triggering
chemiluminescence. The amount of luminescence was then measured in
relative light units (RLU) using a bioluminescence imager. The
steps of this assay are shown in FIG. 8.
[0325] Each sample is tested using three pools of peptides as
described above. Samples are considered to be reactive (or above
the cutoff value) if the signal for a given sample is 10-fold or
more fold higher than the signal obtained with a negative control
sample. It is noted that the negative control sample is prepared by
pooling a series of samples from individuals at low risk for viral
infection and who have tested negative for several common viruses
including HIV, HBV and HCV. A second type of control is used
wherein the samples are reacted in a vessel that contains the
diluents and streptavidin coated microparticles but does not
contain any of the 14 peptides listed in Table 3. It is expected
that sample results should be negative when peptides are not
present. When samples are reactive in the absence of the peptides,
the sample is considered to be non-specifically reactive with the
solid phase containing streptavidin, and were not included in the
various lists of antibody positive samples provided in the
following tables.
Results:
[0326] A panel of samples from first-time plasmapheresis donors,
testing positive for, both by an antibody test for HCV (Abbott
Laboratories, Abbott Park Ill. 60064) and a HCV RNA test (Bayer
Versant HCV RNA 3.0 assay (bDNA) was obtained from ProMedDx
(Norton, Mass.). The samples were tested against the three pools of
peptides as described above. A total of 19 of the 200 samples were
reactive with one or more of the three peptide pools. One sample
(S188) was reactive with all three peptides pools, while two
samples (S 80 and S 96) were reactive with two peptides pools
(Table 4).
TABLE-US-00009 TABLE 4 List of ProMedDx Samples that are antibody
positive for HPgV-2 Peptide Pools ProMedDx Samples (anti-HCV
positive, HCV RNA positive Peptide plasmapheresis donors) Pool 1
Peptide Pool 2 Peptide Pool 3 Sample ID S/CO* S/CO* S/CO* S 188
3.01 2.75 1.52 S 80 2.05 12.16 NR** S 96 NR 9.93 1.42 S 164 1.42 NR
5.25 S 66 1.20 NR NR S 147 1.04 NR NR S 182 2.07 NR NR S 70 NR 7.61
NR S 27 NR 3.11 NR S 89 NR 2.75 BR S 192 NR NR 5.74 S059 NR NR 2.31
S115 NR NR 3.21 S 5 NR NR 4.70 S 3 NR NR 2.22 S 33 NR NR 1.70 S 109
NR NR 1.48 S 148 NR NR 1.45 S 45 NR NR 1.37 *S/CO = sample to
cutoff value >1.0 is considered reactive for antibodies to that
peptide. The cutoff was determined as 10 times the signal for the
negative control value. **Non-reactive
[0327] Samples reactive with pooled peptides were retested with
individual peptides from the reactive peptide pool as shown below.
All 200 samples were also tested with individual Peptide 15 and
Peptide 16. Data below indicate that 8 samples were reactive with
two or more individual peptides and 16 samples were reactive only
with single peptides (Table 5). A total of 24 samples were reactive
with one or more of the synthetic peptides that were evaluated: 176
of the 200 samples were non-reactive for the peptides used in the
assay.
TABLE-US-00010 [0327] TABLE 5 Individual peptide results on
ProMedDx samples reactive with peptide pools. Abbott ID 1 2 3 4 5 6
7 8 9 10 11 12 13 14 15 16 PCR result 200 HCV RNA and Ab S188 1.7
5.2 1.9 4.8 1.3 positive positive samples from S182 4.2 negative
PreMedDx S080 4.3 9.6 16.5 negative S164 1.5 1.2 1.9 3.9 2.6 1.1
3.2 5.5 negative S066 4.9 negative S147 2 1.5 negative S096 1.6 3.5
1.1 1 20.7 positive S070 5.3 1.1 positive S027 4.3 4.6 negative
S089 5.4 2.5 negative "S192" 4.6 negative "S005" 4.3 negative
"S115" 1.5 negative "S059" 2.5 negative "S003" 1.3 negative "S033"
1.2 negative "S109" 1.3 negative "S148" 2.1 negative "S045" 4.7
negative S093 16.6 negative S133 1.4 negative S020 1.1 negative
S123 7.8 negative S044 2.4 negative S065 negative S114 negative
*S/CO = sample to cutoff value >1.0 is considered reactive for
antibodies to that peptide. The cutoff was determined as 10 times
the signal for the negative control value.
[0328] An antibody positive result for any HPgV-2 peptide is
consistent with previous or current infection with HPgV-2; the
prevalence in this population may be as high as 12% (24 reactive
samples among 200 samples).
[0329] It can be uncertain as to which results indicate past or
current infection with HPgV-2. Confirmatory tests are often
utilized to provide supportive evidence that a given serologic
result correlates to infection with the agent being studied. In
many cases, RT-PCR testing is utilized determine if an antibody
positive individual is actively infected with a given agent. A
positive RT-PCR result supports that idea that the serologic test
is correct. However, a negative RT-PCR result however, does not
over rule the serologic test, as some seropositive individuals may
have cleared the infection, thus producing a negative RT-PCR
result.
[0330] RT-PCR testing was performed on all 200 samples from
ProMedDx using the primer and probe sets described in Example 3.
Three samples were determined to be positive by RT-PCR (ABT0070P,
ABT0096P, ABT0188P) and have subsequently been confirmed by next
generation sequencing (Example 5).
Serology Tests with UC0125.US Case
[0331] An aliquot of the index case (UC0125.US) was tested for
antibodies to peptides 4 and 9, as both of these peptides, as noted
above having shown some positive predictive value for HPgV-2 RNA
positivity. It was found that this case was positive for antibodies
to both peptides 4 and 9, with S/N values of 12.5 and 94.7,
respectively. Thus, providing further support that detection of
antibodies to selected peptides may predict active HPgV-2 infection
as detected by RT-PCR.
Correlation Between Antibody Reactivity and RT-PCR Results
[0332] There appears to be a correlation between antibody
reactivity to certain peptides, and a positive HPgV-2 RT-PCR result
(Table 6). Reactivity to peptides 3, 4, 9, and 16 occurred in at
least 2 of the 4 HPgV-2 RNA positives including the index case.
Whereas reactivity to other peptides was absent or only occurred in
1 of the HPgV-2 RNA positive samples.
TABLE-US-00011 TABLE 6 Correlation of Antibody Test results and
RT-PCR (qPCR) results for ProMedDx panel of plasmapheresis donors.
200 HCV HPgV-2 HPgV-2 Postive RNA and Ab positive samples from PCR
PCR predicitve ProMedDx positive negative value* Antibody Positive
for any HPgV-2 3 21 12.5% peptide (N = 24) Antibody Negative for
all HPgV-2 0 176 0.0% peptides (N = 176) Antibody Positive for
HPgV-2 Peptide 2 6 25.0% 3 (N = 8) Antibody Positive for HPgV-2
Peptide 4 2 3 40.0% (N = 5) Antibody Positive for HPgV-2 Peptide 9
2 4 33.3% (N = 6) Antibody Positive for HPgV-2 Peptide 16 2 5 28.6%
(N = 7) *Positive predictive value was determined as the number of
samples that were HPgV-2 RNA positive and antibody positive for
HPgV-2 peptides divided by the total number of antibody positive
samples for each peptide.
Strategy to Identify RT-PCR Positive Samples
[0333] The discussion above indicates that antibody reactivity has
a positive predictive value for identifying RT-PCR positive
samples. Thus, several populations of samples, as described below,
are tested with the three peptide pools described below, followed
by individual peptide testing. Samples that are reactive with
individual peptides are selected for RT-PCR testing. For RT-PCR
positive samples, sequencing can be performed across the
genome.
Peptide Pools for Continued Testing:
[0334] The peptide pool composition has been modified as shown
below:
[0335] Pool 1 contained the following peptides: P 1, 5, 7, 8,
10
[0336] Pool 2 contained the following peptides: P 2, 3, 4, 6, 9
[0337] Pool 3 contained the following peptides: P 11, 12, 13,
14
HCV RNA Positive/Anti-HCV Negative Blood Donor Samples.
[0338] A panel of 240 blood donor samples were obtained from the
American Red Cross (Gaithersburg, Md.) having been identified as
NAT yield samples (i.e., samples that tested as antibody negative
for HCV, but were RT-PCR positive via minipool nucleic acid
testing). When tested with the three peptide pools, a total of 11
samples were detected as reactive (Table 7).
TABLE-US-00012 TABLE 7 240 ARC Samples that were HCV RNA positive
but anti-HCV antibody negative: List of sample ID numbers with
positive result. HCV NAT Yield Peptide Peptide Samples (ARC) Pool 1
Pool 2 Peptide Pool 3 Sample ID S/CO* S/CO* S/CO* S0217 3.6 NR** NR
S0159 1.8 NR NR S049 1.5 NR NR S0078 1.5 NR NR S0226 1.4 NR NR
S0111 NR 4.3 NR S0079 NR 1.8 NR S0061 NR 1.7 NR S0177 NR 1.3 NR
S0108 NR NR 1.3 S0145 NR NR 1.0 *S/CO = sample to cutoff value
>1.0 is considered reactive for antibodies to that peptide. The
cutoff was determined as 10 times the signal for the negative
control value. **Non-reactive
[0339] Samples reactive with pooled peptides were retested with
individual peptides from the reactive peptide pool as shown below.
Data below indicate that 1 sample was reactive with two or more
individual peptides and 11 samples were reactive only with single
peptides (Table 8).
TABLE-US-00013 [0339] TABLE 8 Individual peptide results on HCV NAT
yield samples reactive with peptide pools. Shown are signal to
noise (S/N) values compared to the negative control for each
peptide tested individually. RT-PCR was performed and results are
indicated. Abbott ID 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 PCR
result 240 HCV RNA positive S0111 28 negative samples from ARC
S0079 45 negative S0061 21 negative S0177 14 15 20 negative S0217
38 negative S0159 19 negative S0049 21 negative S0078 20 negative
S0226 18 negative S0108 19 negative S0162 55 negative S0145 26
negative
HCV RNA Positive/Anti-HCV Positive Blood Donor Samples.
[0340] A panel of 240 blood donor samples obtained from the
American Red Cross (Gaithersburg Md.) were identified as HCV
infected (being both antibody positive for HCV and RT-PCR positive
via minipool nucleic acid testing) were tested via peptide pools. A
total of 12 samples were reactive with one or more peptide pools as
listed in Table 9.
TABLE-US-00014 TABLE 9 List of sample ID numbers with positive
results from HCV RNA positive/ anti-HCV positive blood donors from
ARC. HCV Antibody Positive HCV RT-PCR Peptide Peptide Positive
Samples (ARC) Pool 1 Pool 2 Peptide Pool 3 Sample ID S/CO* S/CO*
S/CO* S0127 6.8 NR NR S0104 4.4 NR NR S0046 1.5 NR NR S0128 NR 3.6
NR S0045 NR 2.6 NR S0178 NR 2.3 NR S0044 NR 2.2 NR S0220 NR 1.9 NR
S0238 NR 1.2 NR S0141 NR 1.1 NR S0077 NR NR 1.5 S0202 NR NR 1.5
*S/CO = sample to cutoff value >1.0 is considered reactive for
antibodies to that peptide. The cutoff was determined as 10 times
the signal for the negative control value. **Non-reactive
[0341] Samples reactive with pooled peptides were re-tested with
individual peptides from the reactive peptide pool as shown below.
Data below indicate that 5 samples were reactive with two or more
individual peptides and 9 samples were reactive with single
peptides (Table 10).
TABLE-US-00015 [0341] TABLE 10 Individual peptide results on HCV
RNA positive/anti-HCV positive on ARC blood donor samples reactive
with peptide pools. Shown are signal to noise (S/N) values compared
to the negative control for each peptide tested individually.
RT-PCR was performed and results are indicated. Abbott ID 1 2 3 4 5
6 7 8 9 10 11 12 13 14 15 16 PCR result 240 HCV RNA and Ab S0029 7
19 11 12 25 46 16 15 12 30 35 23 28 positive positive samples from
S0055 15 positive ARC S0127 164 negative S0104 152 negative S0046
136 negative S0128 69 24 positive S0045 30 negative S0178 21
negative S0044 12 18 71 negative S0220 59 negative S0238 30 46
negative S0141 30 negative S0077 15 negative S0202 14 21
negative
[0342] RT-PCR testing was performed on the 14 samples. Three
samples (S029, ABT0029A.US; S0055, ABT0055A.US; and
S128,ABT0128A.US) were found to be HPgV-2 RNA positive. The three
HPgV-2 RNA postive samples showed reactivity to peptide 16, 2 out
of 3 were reactive for peptide 9, 1 was reactive for peptide 4, and
1 was reactive for peptide 3. Whereas sample ABT0029A.US was
reactive to 13 of the peptides, sample ABT0055A.US was only
reactive for petpide 16, and sample ABT0128A.US was reactive to
both peptide 9 and 16. The peptide reactivity data supports the
utility of peptides 3, 4, 9, and 16 in detecting HPgV-2 RNA
positives. Seventeen additional samples that were antibody negative
for these 14 peptides were also tested by RT-PCR: all 17 samples
were negative by RT-PCR.
Blood Donor Samples from Thailand
[0343] A total of 145 HIV positive blood donor samples (obtained
from Thailand) were tested via the three peptide pools. A total of
6 samples were detected as reactive with one or more peptide pools
as listed in Table 11.
TABLE-US-00016 TABLE 11 List of sample ID numbers with positive
results from 145 samples collected in Thailand Thailand Samples
Peptide Pool 1 Peptide Pool 2 Peptide Pool 3 Sample ID S/CO* S/CO*
S/CO* S0116 17.6 NR NR S0068 NR 18.4 NR S0145 NR 17.0 NR S0027 NR
NR 38.1 S0136 NR NR 16.1 S0016 NR NR 10.1 *S/CO = sample to cutoff
value >1.0 is considered reactive for antibodies to that
peptide. The cutoff was determined as 10 times the signal for the
negative control value. **Non-reactive
[0344] Samples reactive with pooled peptides were retested with
individual peptides from the reactive peptide pool as shown below.
Data below indicate that all samples were reactive only single
peptides (Table 12).
TABLE-US-00017 [0344] TABLE 12 Individual peptide results on
Thailand samples reactive with peptide pools. For 145 Thailand
sample: reactive to (S/CO) Run ID P1 p2 p3 p4 p5 p6 p7 p8 p9 p10
p11 p12 p13 14 S0116 2.8 S0068 1.0 S0145 1.6 S0027 9.4 S0136 2.1
S0016 1.1
HIV Positive Blood Donor Samples from South Africa.
[0345] A total of 73 HIV donor samples (obtained from South Africa)
that were tested with the three peptide pools. Most of the samples
were from HIV-infected blood donors. A total of 4 samples were
detected as reactive with one or more peptide pools as listed in
Table 13.
TABLE-US-00018 TABLE 13 List of sample ID numbers with positive
results from HIV positive blood donors from South Africa. South
African Blood Peptide Donors (anti-HCV positive) Pool 1 Peptide
Pool 2 Peptide Pool 3 Sample ID S/CO* S/CO* S/CO* S0027 1.3 NR NR
S0009 NR 2.2 NR S0004 NR 1.6 NR S0008 NR NR 3.4 *S/CO = sample to
cutoff value >1.0 is considered reactive for antibodies to that
peptide. The cutoff was determined as 10 times the signal for the
negative control value. **Non-reactive
[0346] Samples reactive with pooled peptides were re-tested with
individual peptides from the reactive peptide pool as shown below.
Data below indicate that all four samples were reactive with only
single peptides (Table 14).
TABLE-US-00019 [0346] TABLE 14 List of ID numbers with positive
results on individual peptides. For 73 S. Africa sample: reactive
to (S/CO) Run ID P1 p2 p3 p4 p5 p6 p7 p8 p9 p10 p11 p12 p13 14
S0027 2.1 S0009 17.0 S0004 3.2 S0008 14.9
Expanded HPgV-2 Prevalence Studies
[0347] The first seven isolates (ABT0096P, ABT0070P, ABT0188P,
ABT0029A, ABT0055A, and ABT0128A), besides index case, showed a
positive correlation between having an antibody response to
peptides 3, 4, 9, or 16 and the presence of HPgV-2 RNA. Continued
studies to investigate the presence of HPgV-2 in different
populations was performed using a combination of antibody testing
with a pool of peptides 3, 4, 9, 16 and the RT-PCR assay described
in Example 3.
[0348] A total of 542 samples were positive for both HCV RNA and
antibodies to HCV (including 200 ProMedDx samples, 240 HCV samples
from ARC and a second set of 100 samples from ProMedDx). Eight of
these 540 samples (1.5%) were positive for HPgV-2 RNA when screened
with the KEYS qPCR multiplex assay. Six of the 8 HPgV-2 RNA
positives samples from this group were reactive with one or more
HPgV-2 peptides. (Two HPgV-2 RNA positive samples were obtained
from individuals co-infected with HIV and HCV and were tested as
negative for antibodies toHPgV-2 peptides). Overall 24 of the 540
(4.4%) samples were reactive with one or more HPgV-2 peptides: six
of these 24 (25.0%) were HPgV-2 RNA positive, indicating a positive
predictive value of 25.0%. The negative predictive was calculated
to be 99.6%. A total of 13 of the 542 (2.4%) samples were reactive
with two or more peptides and 5 of the 13 (38.5%) samples,
indicating a positive predictive value for HPgV-2 RNA positivity as
being 38.5%, and a negative predictive value of 99.6%.
[0349] There was a fourth set of 240 HCV RNA samples obtained from
the American Red Cross (Gaithersburg, Md.) that were anti-HCV
negative, and thus, were recent infections, within the
preseroconversion window period. There was 1 sample among 240
(0.4%) samples that was HPgV-2 RNA positive. This sample was not
reactive with any of the HPgV-2 peptides. Overall, a total of 5 of
240 (2.1%) samples were reactive with one or more HPgV-2 peptides,
but none of the samples were reactive with two or more
peptides.
[0350] A total of 188 samples were positive for both HBsAg and HBV
DNA. None of these samples were positive for HPgV-2 RNA and a total
of 3 of 188 (1.6%) samples were reactive with one or more HPgV-2
peptides. One sample was reactive with two or more peptides.
[0351] A total of 298 samples were positive for both HIV RNA and
antibodies to HIV. A total of 4 of 298 (1.3%) were reactive to one
or more HPgV-2 peptides and one sample was reactive for two or more
peptides. One sample was positive for HPgV-2 RNA, and was reactive
with on HPgV-2 peptide. Two additional samples were identified
byRT-PCR screening that not detected with the peptides. The 3
RT-PCR positive samples were confirmed by RT-PCR assay but not next
generation sequencing. Upon further testing it was noted that the
two HPgV-2 RNA samples that were negative for HPgV-2 peptides were
positive for anti-HCV (Abbott ARCHITECT anti-HCV) and HCV antigen
(Abbott ARCHITECT HCV Ag). Therefore these samples are categorized
as co-infected with HCV and were removed from this sample group and
included in the HCV antibody positive HCV RNA positive group (Table
15 below).
[0352] A total of 463 samples were obtained from volunteer blood
donors, considered to be at low risk for parenterally transmitted
viruses like HCV. A total of 13 of 463 (2.8%) of samples were
reactive with one or more HPgV-2 peptides. Two of the 463 (0.4%)
samples were reactive with two or more peptides. None of the
samples were positive for HPgV-2 RNA. (However, not all of the
samples were tested using the same methodology. All 450 of the
samples that were negative for antibody detection using HPgV-2
peptides were tested as negative via the multiplex RT-PCR assay.
Among the 13 samples that were reactive for peptides only two were
tested with the multiplex assay, both being negative. The remaining
11 samples were all tested for HPgV-2 RNA using the NS2/3 primer
set and 7 of the 11 were also tested with other primer sets (E1,
5'UTR): all samples were negative for HPgV-2 RNA, as described in
example 3)
[0353] A summary of the testing performed on various sample groups
is found below. The frequency of HPgV-1 RNA positivity was higher
than HPgV-2 RNA positivity in all of the groups tested. HPgV-2 RNA
was found more frequently in HCV infected individuals than in
volunteer blood donors, HBV positive individuals, and HIV infected
individuals not co-infected with HCV. The frequency of HPgV-2
infection (as noted by HPgV-2 RNA or antibody detection to HPgV-2
peptides) was higher among HCV seropositive individuals, than in
HCV infected individuals who are seronegative. Reactivity to two or
more of peptides 4, 9, or 16 had a positive predictive value of
having HPgV-2 RNA. The negative predictive value for reactivity to
peptides was high in all of the groups tested.
TABLE-US-00020 TABLE 15 Number of samples reactive to at least one
peptide from the peptide pool (3, 4, 9, 16) for the indicated
sample groups. Antibody Reactive to Antibody reactive to one or
more peptides (%) two or more peptides (%) Antibody HPgV-2 Positive
Negative Positive Negative Reactive/ HPgV-1 RNA RNA Positive
Predictive Predictive Positive Predictive Predictive PCR Group
positive (%) positive (%) (%) value* Value** (%) value* Value**
negative - HCV Ab+/PCR+ 40 (7.4%) 8 (1.5%) 24 (4.4%) 6/24 (25%)
518/520 13 (2.4%) 5/13 (38.5%) 529/531 18/534 (n = 542*) (99.6%)
(99.6%) (3.4%) HCV Ab-/PCR+ nt 1 (0.4%) 5 (2.1%) 0/5 (0%) 239/240 0
(0%) N/A 239/240 5/239 (n = 240) (99.6%) (99.6%) (2.1%) HIV (n =
296) 29/275 (10.5%) 1 (0.3%) 4 (1.3%) 1/4 (25.0%) 288/292 1 (0.3%)
0/1 294/295 1/295 (98.6%) (99.%) (0.3%) HBV (n = 188) 6 (3.2%) 0/0
(0%) 3 (1.6%) N/A 185/188 0/0 (0.0%) N/A 188/188 3/188 (98.4%)
.sup. (100%)-- (1.6%) Blood donors 19/452 (4.2%) 0/0 (0%) 13 (2.8%)
N/A 450/463 2 (0.4%) N/A 461/463 13/463 (n = 463) (97.2%) .sup.
(99.4%)-- (2.8%) Grand total 94/1457 (6.4%) 10 (0.6%) 40 (2.3%)
7/10 (70%) 40/1708 (n = 1729) (2.3%) *Positive predictive value was
determined as the number of samples that were HPgV-2 RNA positive
and antibody positive for HPgV-2 peptides divided by the total
number of antibody positive samples **Negative predictive value was
determine as the number of samples that were negative for
antibodies to HPgV-2 peptides minus the number of HPgV-2 RNA
positive samples divided by the total number samples negative for
antibodies to HPgV-2 peptides ***Includes co-infected HCV/HIV
samples (n = 2), not confirmed by NGS.
[0354] A total of 11 HPgV-2 RNA positive samples (including the
index case) have been identified among 1729 samples tested (Table
16). A total of 782 of the 1729 (45.2%) of samples were obtained
from HCV infected individuals. Ten of the 11 HPgV-2 RNA positive
samples were found among individuals infected with HCV, suggesting
that this virus may share a similar transmission pattern as HCV
(parenteral exposure). For HPgV-1, the prevalence of HPgV-1 RNA was
highest in the HIV population (10.5%), was relatively high in HCV
infected persons (7.4%), and was detected in volunteer blood donors
(4.2%), indicating that active infection with HPgV-1 is much more
common than HPgV-2, for the populations studied.
[0355] As noted above, there were 40 samples among 1729 samples
that were reactive to antibodies to HPgV-2 peptides. Seven of these
40 samples were HPgV-2 RNA positive. A total of 7 of the 10 (70%)
HPgV-2 RNA positives were antibody reactive. The remaining 33
antibody reactive results were noted among 1719 tested samples,
resulting in a frequency of 1.9%. As noted below, several of the
HPgV-2 RNA positive samples are reactive to 2 or more peptides.
TABLE-US-00021 TABLE 22 HPgV-2 HCV Peptide reactivity Viral load
Ab/ RNA GBV-C HIV (S/CO) Log RNA HPgV-2 isolate RNA only RNA RNA 3
4 9 16 copies/ml UC0125.US + - nt - nt 1.3 9.7 nt 6.2 ABT0096P.US +
- - - 1.6 3.5 9.0 20.7 3.5 ABT0070P.US + - - - -- -- 5.3 1.1 5.2
ABT0188P.US + - - - 1.7 5.2 -- -- 2.5 ABT0055A.US + - - - 1.0 -- --
1.5 3.8 ABT0029A.US + - + - -- 1.0 1.2 2.8 4.6 ABT0128A.US + - - -
-- -- 6.9 2.4 4.5 ABT0239.AN.US - + - - -- -- -- -- 5.8 ABT0100P.US
- - - + -- -- 16.3 -- nt ABT0030P.US + - - + -- -- -- -- nt
ABT0035P.US + - + + -- -- -- -- nt
Example 5
Next Generation Sequencing of ABT0070P.US, ABT0096P.US and
ABT0128A.US
[0356] The qPCR positive samples in Example 3.1 were probed by
conventional RT-PCR as described in Example 2. Only ABT0070P.US was
reactive with primers in NS5A: 6914F and 7213R (SEQ IDs 44 &
45) and in NS2-NS3: 3334F & 3708R (SEQ IDs 34 & 35) and
products subsequently shown by Sanger sequencing to align to
UC0125.US. All three samples were extracted and prepared for NGS as
described in Example 2. Read mappings from multiple MiSeq runs were
extracted and combined.
[0357] A total of 98,017 NGS reads from sample ABT0070P.US mapped
to SEQ ID NO:1, covering 93% of the HPg-V2 genome. Coverage depth
was 1133X.+-.1364 reads/nucleotide, with a short gap (>20 nt)
present at the 5' end, four internal gaps within the region of nt
2600-3400 in SEQ ID 1 totaling approximately 300 missing bases, and
the final 354 nt lacking at the 3'end. The missing 5' end was
filled in by PCR and Sanger sequencing of products from 44F-342R
(SEQ ID NO:12 & 13) reactions. The internal gaps were covered
by 10 overlapping amplicons located in the X-NS3 region using the
primers listed in Table 1 (SEQ ID NO: 16-35). The resulting
consensus sequence (SEQ ID NO: 75) combining the NGS and Sanger
data is shown in FIG. 9 and annotated in FIG. 10. Table 16 reports
the number of amino acid mismatches and the percentage identity
compared to UC0125.US, which ranged from 90-97%, depending on the
protein.
[0358] A total of 5,099 NGS reads from sample ABT0096P.US mapped to
SEQ ID NO:1, covering 57% of the HPg-V2 genome. Coverage depth was
33X.+-.112 reads/nucleotide, with gaps seen throughout the length
of the genome. Sample 128 only had 116 NGS reads, primarily
concentrated into 2 regions: 740-975 and 2130-2270. A nucleotide
alignment of these 3 additional cases to UC0125.US is shown in FIG.
11.
[0359] Four additional strains of HPgV-2 in HCV co-infected
patients have been uncovered through our screening efforts and
sequenced by NGS (see below). Using both gene-specific and
random-primed NGS approaches, in concert with traditional RT-PCR
and Sanger sequencing, the current genome sequence coverage of each
strain is reported. The 5' ends of viruses were obtained by SMARTer
PCR cDNA synthesis (see FIG. 23A). The 3' end of ABT0070P.US was
determined by 3'RACE and for ABT0029A.US and ABT0239AN.US by using
RT-PCR, supplementing reactions with 2% DMSO (see FIG.
23DD-EE).
TABLE-US-00022 TABLE 17 HCV positive specimens % Genome Coverage
Length UC0125.US 99.8 9847 nt ABT0070P.US 100.0 9867 nt ABT0029A.US
100.0 9867 nt ABT0239AN.US 100.0 9867 nt ABT0128A.US 92.3 9109 nt
ABT0055A.US 78.3 7724 nt ABT0096P.US 58.0 5726 nt ABT0188P.US 4.0
394 nt ABT0030P.US 99.4 9812 nt ABT0041P.US 97.7 9645 nt
[0360] A total of eight HPgV-2 isolates have now been identified in
patients co-infected with HCV (above). Recent screening in a US
population of HIV+ patients from ProMedDx has revealed 6 additional
strains that have been confirmed by RT-PCR and Sanger sequencing: ,
ABT0084H.US, ABT0086H.US, ABT0100H.US, ABT0198H.US, ABT0030P.US,
and ABT0041P.US (sequences not shown).
[0361] A total of ten HPgV-2 isolates have now been identified in
patients co-infected with HCV (above). Recent screening in a US
population of HIV+patients from ProMedDx has revealed 4 additional
strains that have been confirmed by RT-PCR and Sanger sequencing:
ABT0084H.US, ABT0086H.US, ABT0100H.US, and ABT0198H.US(sequences
not shown).
TABLE-US-00023 TABLE 21 HIV positive specimens % Genome Coverage
Length ABT0084H.US 3.3 325 nt ABT0086H.US 1.6 163 nt ABT0100H.US
4.3 421 nt ABT0198H.US 1.7 168 nt
[0362] This brings the total to 14 isolates of HPgV-2 and
demonstrates that infection with this virus, for example, is not
restricted to individuals co-infected with HCV as originally
believed. An alignment of some of the full and nearly complete
genomes is shown in FIG. 23.
[0363] A multiple sequence alignment of HPgV-2 (UC0125.US) and
ABT0070P.US along with 29 representative flaviviruses was performed
in Geneious v6.1 (Kearse, et al., 2012, Bioinformatics), using
MAFFT v7.0 with the E-INS-I algorithm and at default parameters
Katoh, et al., Mol Biol Evol, 2013), followed by refinement using
MUSCLE v3.8 with 10 maximum iterative cycles (Edgar, Nuc Acids Res,
2004). Phylogenetic trees were constructed in Geneious using the
Jukes-Cantor model and neighbor joining algorithm with 10,000
bootstrap replicates used to calculate branch supports. These tree
topologies were then refined using a maximum likelihood Bayesian
approach with MrBayes V3.2 software (1,000,000 sample trees, 10% of
trees discarded as burn-in, convergence defined at an average
standard deviation of <0.01). Each tree was rooted with dengue
virus type 1 (DENV1) and yellow fever virus (YFV) as outgroups . .
. Analysis was performed on entire polyprotein sequences, as well
as on NS3 and NS5B proteins individually (FIG. 12). Two major
branches distinguish pegiviruses from hepaciviruses in the
Flavivirus family. Within the pegivirus branch, ABT0070P.US
clusters tightly with UC0125.US, with the branch supported by a
bootstrap value of 100%. Both strains share a common, albeit
distant, ancestor with bat and rodent pegiviruses, with a bootstrap
value of 99.95%. This demonstrates that HPgV-2 is a human pegivirus
distinct from pegiviruses previously identified in mammals.
Example 6
Purification of HPgV-2 E2 Glycoprotein and Serology with HPgV-2
PCR+/Ab+ Samples
Expression, Purification, Characterization of HPgV-2 E2 in
Mammalian Cells.
[0364] This example describes the design, expression, and
purification of the HPgV-2 glycoprotein E2 from mammalian cells. An
expression plasmid encoding the E2 ORF sequence up to the predicted
transmembrane region (SEQ ID NO:406) was constructed using a
pcDNA3.1 derived vector containing a mouse Ig kappa light chain
leader sequence for protein secretion.
TABLE-US-00024 SEQ ID 406: Coding sequence of E2-cassette
atgagagttcctgcacaattattaggattattattattatggtttcctgg
atctaggtgctacaagcaccagagcgagagctacctgaagtattgtacaa
ttacaaatacatctacaagcatgaactgcgattgcccttttggcaccttc
accaggaatacagagtctagattttctattcctagattttgtccagtgaa
gatcaatagcagcaccttcatctgctcttggggatcttggtggtggtttg
ctgaaaatattacaagaccttatacagatgtgggaatgcctccagctcca
atttctgctctgtgttacatctacagcaataatgatcctcctccttggta
tcataataccaccatcattcctcagaactgcagaaatagcaccgttgatc
ctacaacagctccttgtagagataaatggggaaatgctacagcttgtatt
cttgacagaagaagcagattttgcggcgattgttatggaggatgctttta
cacaaatggaagccatgatagatcttgggatagatgtggaatcggctaca
gagatggactgattgaatttgttcagttaggccagattagacccaatatc
agcaatacaaccatcgaactgcttgctggagcttctttagttattgcttc
tggattaagacctggatttggatgttctagagctcatggagttgtgcact
gctatagatgtccttcttacagagatttagagcaatttggacctggactt
ggaaaatgggtgcctttacctggagaacctgttcctgaattatgtattaa
tcctcaatgggctagaagaggattcagaatgagcaataaccctctgtctc
tgctgcagacatttgttgaagatatctttcttgcccattctgtaatccta
cacctggaagagttagagtgtgcaacaatacagccttttatcctagagga
ggaggatttgttcaacttattggcgatgttcaggttctgacccctaatac
aggatctggatctggacatcatcatcatcatcatcatcactaa
[0365] An 8.times. Histidine tag was cloned in frame at the carboxy
terminus of the E2 ORF for purification. The plasmid encoding
HPgV-2 E2 was transiently transfected into HEK293-6E (human
embryonic kidney--6E) suspension cells using Lipofectamine 2000
transfection reagent (Life Technologies, Carlsbad, Calif., USA).
After 6 days the cell cultures were centrifuged (1200 rpm 10 min)
and the supernatant was collected. The supernatant was concentrated
using an Amicon Ultra filter (EMD Millipore, Billerica, Mass.,
USA). Cells were lysed on ice for 30 minutes using phospho-buffered
saline (PBS) with 1% triton X-100. Cell debris was spun down by
centrifugation for 10 minutes at 15000 rpm, the supernatant was
collected. Cell lysates and concentrated supernatants were run on a
4-20% SDS PAGE gradient gel (Novex by Life Technologies Carlsbad,
Calif., USA) and Western blot was performed using the WesternBreeze
kit (Novex by Life Technologies) in conjunction with an anti-His
alkaline phosphatase (AP) conjugated primary antibody (Novex by
Life Technologies, Carlsbad, Calif., USA). Protein was visualized
using the BCIP/NBT chromagen staining (Novex by Life Technologies)
and the Bio-Rad Imager (BioRad GelDoc EZ Imager using Image Lab
v4.0 software). The predicted molecular weight of the expressed
HPgV-2 E2 construct is 39.6 kDa. HPgV-2 E2 from the cell lysate ran
at approximately 50 kDa and the concentrated HPgV-2 E2 from the
supernatant ran a range of molecular weights between 50-75 kDa
suggesting glycosylation of the secreted form of the protein (FIG.
24A), which may cause the protein to migrate more slowly during
electrophoresis.
[0366] HPgV-2 E2 from concentrated supernatant was purified under
native conditions using a nickel (Ni+) agarose packed affinity
column (His Bind resin, Novagen, EMD Millipore, Billerica, Mass.,
USA). Unbound material (flow-through) and eluted bound material was
run on a 4-20% SDS-PAGE gel followed by staining with Oriole
protein stain (Bio-Rad) or Western blotted using an anti-His-AP
antibody (WesternBreeze, Novex by Life Technologies, Carlsbad,
Calif., USA). Protein staining showed multiple molecule weights of
bound and eluted material between 50-70 kDa (FIG. 24B), consistent
with size detected by initial Western blot of HPgV-2 E2 transfected
cell supernatants (FIG. 24A). Western blot showed multiple
molecular weight bands between 60-70 kDa (FIG. 24C).
[0367] PNGase F (Peptide-N-Glycosidase F) treatment of purified
HPgV-2 E2 was performed to confirm the larger than estimated
molecular weight of the purified protein was due to
post-translational glycosylation. HPgV-2 has 10 potential
asparagine-linked glycosylation sites which can shift
electrophoretic mobility of the 39.6 kDa predicted molecular
weight. PNGase F is an enzyme that specifically cleaves between the
innermost GlcNAc and asparagine residues of N-linked glycoproteins.
Purified HPgV-2 E2 was denatured and incubated with PNGase F (New
England Biolabs (NEB), Ipswich, Mass., USA) for 1 hour at
37.degree. C. followed by SDS-PAGE analysis of treated and
untreated proteins (FIG. 24D). Purified HPgV-2 E2 untreated with
PNGase F had a molecular weight 60-70 kDa, PNGase F treated HPgV-2
E2 had a molecular weight closer to the 37 kDa marker by SDS-PAGE
gel. The PNGase F treated E2 ran closer to the predicted molecular
weight suggesting the majority of the protein was
deglycosylated.
[0368] Additionally, a plasmid encoding HPgV-1 (GBV-C) E2 without
the carboxy terminal transmembrane domain was also expressed in a
mammalian expression vector as described above. Upon expression in
mammalian cells the protein is secreted as a fusion with a 8X
hisitidine tag at the carboxy terminus.
Serology Testing of Purified HpGV-2 E2 with PCR+/Ab+Samples.
[0369] Antibodies against the purified HPgV-2 E2 in PCR+/Ab+samples
were assessed by performing a slot blot containing dilutions of
HPgV-2 purified E2 protein. Purified protein was bound to
nitrocellulose membrane at a titration of 0.1 ug/ml, 1 ug/ml, 10
ug/ml, and 100 ug/ml. Unbound protein was washed away and membranes
were air-dried. 1:100 dilutions of PCR+/Ab+ samples ABT0096P,
ABT0070P, ABT0188P, and ABT0055A were incubated with the slot
blots. These samples were reactive to a pool of HPgV-2 peptides (3,
4, 9, and 16). Negative controls were normal human plasma and
samples negative for HPgV-2 RNA and negative for antibodies to the
pool of HPgV-2 peptides 3, 4, 9, and 16. Antibodies were visualized
using a goat anti-human secondary antibody conjugated to alkaline
phosphatase and BCIP/NBT chromogen substrate (FIG. 25). The HPgV-2
PCR+/Ab+ samples tested had strongly detectable antibodies to the
100 ug band and were faintly reactive to the bug E2 band. This data
shows that PCR+ individuals make antibodies to the native form of
HPgV-2 E2. Further studies need to be done to determine if E2
antibodies are present following viremia. Also this data indicates
that HPgV-2 is a serologic marker of infection. Additionally a
panel of samples from first-time plasmapheresis donors, testing
positive for, both by an antibody test for HCV (Abbott
Laboratories, Abbott Park Ill. 60064) and a HCV RNA test (Bayer
Versant HCV RNA 3.0 assay (from ProMedDx, Norton, Mass.)) were used
to probe the slot blots (FIG. 25--samples ABT0045P, ABT0141P,
ABT0178P, ABT0065P).
[0370] Samples which were HPgV-2 RNA positive and positive for
antibodies to the peptides were used to validate the utility of
purified HPgV-2 E2 when coated onto a solid phase microparticle and
used in an ARCHITECT assay (See Example 4). Briefly, Spherotech
magnetic microparticles (Lake Forest, Ill., USA) were coated with
100 ug/ml of purified E2 protein using
N-(3-Dimethylaminopropyl)-N'-ethylcarbodimide hydrochloride (EDAC,
Sigma-Aldrich, St. Louis, Mo. 63103) to crosslink the purified
protein to the magnetic microparticle. The E2 coupled
microparticles were run with selected HPgV-2 RNA positive samples
in an indirect immunoassay using the ABBOTT ARCHITECT instrument
(described above, Example 3) where bound antibodies from the sample
were detected with a mouse anti-human acridinium conjugated
secondary antibody (Table 18). Additionally samples negative for
HPgV-2 RNA and antibody response were tested with the E2 coated
microparticles. Samples reactive on the slot blot were also
reactive when HPgV-2 E2 was coated onto microparticles and used in
an ARCHITECT assay (Table 18).
TABLE-US-00025 TABLE 18 ARCHITECT assay using HPgV-2 coated
microparticles. Signal to cut-off (S/CO) was set at 10 times NC. A
sample was considered positive (reactive) for antibodies to HPgV-2
E2 when the S/CO >1. Samples ABT0029A and ABT0128A had elevated
signals but not over the cut-off. Abs to HPgV-2 peptide pool Abs to
purified sample HPgV-2 RNA + 3, 4, 9, 16 S/CO HPgV-2 E2 S/CO NC Neg
Neg -- ABT0029A Pos Pos 0.6 ABT0055A Pos Pos 6.6 ABT0070P Pos Pos
4.1 ABT0096P Pos Pos 6.4 ABT0128A Pos Pos 0.9 ABT0188P Pos Pos 15.7
ABT0239A Pos Neg .05 HPgV-2 RNA negative/Antibody negative samples
ABT0045P Neg Neg .07 ABT0141P Neg Neg .004 ABT0178P Neg Neg .02
ABT0065P Neg Neg .007
[0371] Within the HPgV-2 RNA positive samples, the correlation
between having antibodies to the peptides and antibodies to the E2
protein was high. 4 out of 6 HPgV-2 RNA positive samples had a
detectable antibody response which was over 10 times the negative
control signal to the E2 recombinant protein coated microparticles.
The remaining 2 samples had elevated signals over background
suggesting there is a mild antibody response, which may be detected
upon further optimization of the assay A population of normal human
donor plasma (n=100) was also screened for reactivity to HPgV-2 E2
to indicate current or past infection. No samples were detected
with a signal to cutoff (S/CO) greater than 1 suggesting HPgV-2 is
a low frequency endemic virus in the US population.
[0372] Because many of the HPgV-2 RNA+samples also have antibodies
to HPgV-1 E2, cross-reactivity to either HPgV-1 or -2 E2 was
evaluated. A blocking experiment was performed where purified
HPgV-1 or -2 E2 was prebound to a sample that was HPgV-1 E2 Ab+ and
HPgV-2 RNA+/ E2Ab+. The sample prebound to with either HPgV-1 or
HPgV-2 was used to probe a slot blot containing both purified
HPgV-1 and HPgV-2 E2 glycoproteins. Pre-binding with HPgV-2 E2
reduced binding to the purified HPgV-2 E2 on the slot blot and
pre-binding with HPgV-1 E2 reduced binding of the sample to HPgV-1
E2 (FIG. 26). There was a minimal decrease in binding to the
heterologous proteins not used in the pre-binding suggesting the
immunoreactivity detected by slot blot analysis is specific.
Example 7
Expression, Purification, and Serology Using HPgV-2 NS4A/4B Fusion
Protein.
[0373] Design, Expression, and Purification of Plasmid Containing
NS4A/4B Fusion in Esherichia coli (E. coli).
[0374] This example describes the design of a maltose binding
protein fusion to domains of the HPgV-2 proteins NS4A and NS4B and
the subsequent expression and purification from E. coli. Briefly,
using the Protean 3D program (DNASTAR, Madison, Wis., USA) a 81
amino acid segment of HPgV-2 NS4A-4B was predicted to have
cytoplasmic localization (SEQ ID NO:407).
TABLE-US-00026 Nucleic acid sequence of encoded 81 amino acid
HPgV-2 NS4A-4B peptide (SEQ: 407): ATG GCC CTT GTT CCC AGC GCT GTG
TGG AGT GTT GAA GTC CGC CCC GCA GGC GTG ACG CGC CCT GAT GCC ACC GAT
GAA ACC GCT GCG TAC GCT CAA CGC TTG TAT CAG GCC TGC GCC GAT TCA GGT
ATC TTT GCG TCA CTT CAA GGA ACC GCG AGT GCG GCG TTG GGC AAG CTG GCG
GAT GCC TCG CGT GGC GCG AGT CAA TAC CTG GCA GCC GCC CCA CCA TCA CCT
GCC CCA CTG GTG CAG GTA TTA
[0375] This region was chosen for expression in the pMAL-05X vector
that allows for an amino-terminal maltose binding fusion and a 6X
histidine tag was designed at the C-terminal end of the construct,
all under an IPTG inducible promoter (Genscript, Piscataway, N.J.,
USA). The construct was expressed in the BL21 E. coli strain and
following IPTG induction for four hours at 37C the cells were lysed
and the soluble protein was purified using Probond nickel
purification system (Invitrogen, Grand Island, N.Y., USA). A
Western blot was performed (WesternBreeze, Invitrogen, Grand
Island, N.Y., USA) using an anti-His antibody (Invitrogen, Grand
Island, N.Y., USA) and a single band at 50 kDa was detected (42 kDa
MBP+8 kDa NS4AB) in the eluted material (FIG. 27A).
[0376] To validate the purified material both slot blot and an
indirect ARCHITECT immunoassay were performed using known HPgV-2
RNA positive samples. For the slot blot, 10 and 100 ug of purified
material was passively bound to a nitrocellulose membrane
(Invitrogen, Grand Island, N.Y., USA) followed by washing away
unbound protein and blocking of the membrane. Antibodies to the
purified NS4AB were detected using slot blots containing bound
NS4AB for the samples ABT0055A, ABT0096P, ABT0188P and not for the
negative control plasma (FIG. 27B). Bound antibodies were detected
using a goat anti-human alkaline phosphatase conjugated secondary
antibody (SouthernBiotech, Birmingham, Al., USA) conjugated to
alkaline phosphatase, colormeric detection was provided by BCIP/NBT
substrate (SigmaFAST, Sigma-Aldrich, St. Louis, Mo. 63103).
[0377] To test the purified NS4AB protein in an automated
immunoassay, Spherotech magnetic microparticles (Lake Forest, Ill.,
USA) were coated with 50 ug/ml of purified NS4AB protein using
N-(3-Dimethylaminopropyl)-N'-ethylcarbodimide hydrochloride (EDAC,
Sigma-Aldrich, St. Louis, Mo. 63103) to crosslink the purified
protein to the magnetic microparticle. The NS4AB coupled
microparticles were run with selected HPgV-2 RNA positive samples
in an indirect immunoassay using the ABBOTT ARCHITECT instrument
(described above, Example 3) where bound antibodies from the sample
were detected with a mouse anti-human acridinium conjugated
secondary antibody (Table 19).
TABLE-US-00027 TABLE 19 Indirect assay of antibodies to NS4AB in
HPgV-2 RNA positive samples. sample S/CO* ABT0029A 1.4 ABT0055A 0.4
ABT0070P 14.8 ABT0096P 9.8 ABT0128A 0.8 ABT0188P 1.9 *S/CO =
signal/cut-off. S/CO>1 is considered reactive for antibodies to
NS4AB protein.
[0378] The cut-off was determined as 10 times the signal for the
negative control. A population of normal human donor plasma (n=100)
was also screened for reactivity to HPgV-2 NS4AB to indicate
current or past infection. No samples were detected with a signal
to cutoff (S/CO) greater than 1 suggesting HPgV-2 is a low
frequency endemic virus in the US population.
Example 8
Detection of HPgV-2 Antigens and Generation of Hyperimmune Serum in
Rabbits
[0379] New Zealand Rabbits approximately 1 year of age were
selected for the generation of antibodies to HPgV-2 peptides. For
the first immunization, each rabbit was inoculated with 1.0 mg of
peptides (peptides 4, 5, and 9 from Table 3, Example 4) solubilized
in 0.9% saline mixed with 0.5 ml of adjuvant. Approximately 10
weeks after the first immunization serum was obtained from the
rabbits and tested for antibodies to the immugens. The rabbits were
immunized five additional times over the next 8 months.
[0380] Antibody production was determined for each rabbit by using
an indirect assay on the ARCHITECT. The assay format utilized 800
ng/ml of each respective biotinylated peptide diluted into a
buffered solution along with 10 ul of rabbit serum and 0.05% of
microparticles coated with streptavidin for capturing
peptide/antibody complexes via the biotin tag on each peptide.
After an18 minute incubation, the magnetic microparticles were
washed and reacted with a conjugate diluent containing 10 ng/ml of
acridinylated goat anti-rabbit IgG to detect rabbit antibodies
bound to the solid phased via immunocomplex. Samples were diluted
1:100 in ARCHITECT wash buffer solution prior to testing. The
signal (S) expressed in relative light units (rlu) for the negative
control (NC) rabbit (not inoculated with any of the HPgV-2
peptides) ran between 197 and 251. The S/N values were obtained for
the five rabbits immunized with HPgV-2 peptides are found in Table
20. The S/N values for rabbits 13212 and 13213 were 1195 and 1131.1
for reactivity to peptide 4, rabbits 13214 and 13215 were 1056 and
1160 for peptide 5, and rabbit 13216 was 849 for peptide 9, all
rabbits were negative for peptides they were not immunized against.
Rabbits showed specific antibodies for the immunogen peptides
(Table 20) and antibodies were not cross reactive to the other
HPgV-2 peptides. Thus these hyperimmune sera can be utilized to
determine the presence of HPgV-2 antigens in various tissues
(blood) or organs (liver, etc) and may be useful in determining the
cell tropism of this virus.
TABLE-US-00028 TABLE 20 Serum from rabbits immunized with HPgV-2
peptides show specific reactivity to the immunogen sequence.
Testing with Immunogen peptide 800 ng/ml BT-peptides Rabbit Number
(Genscript) peptide 4 peptide 5 peptide 9 1st bleed 13212 PV4A
(peptide 4) 1195.0 0.7 0.5 13213 PV4A (peptide 4) 1131.1 1.5 1.0
13214 PV5A (peptide 5) 1.2 1055.9 0.6 13215 PV5A (peptide 5) 1.6
1160.0 0.8 13216 PV9A (peptide 9) 1.1 1.4 849.4
[0381] Bleeds from HPgV-2 peptide immunized, or non-immunized
rabbit serum (NC) were assayed for peptide specific antibodies by
ARCHITECT indirect assay. Shown are the relative light units (rlus)
for the negative control (non-immunized rabbit serum) and the
signal to noise (S/N) for each rabbit immunized with the indicated
peptides.
[0382] All publications and patents mentioned in the present
application are herein incorporated by reference. Various
modification and variation of the described methods and
compositions of the invention will be apparent to those skilled in
the art without departing from the scope and spirit of the
invention. Although the invention has been described in connection
with specific preferred embodiments, it should be understood that
the invention as claimed should not be unduly limited to such
specific embodiments. Indeed, various modifications of the
described modes for carrying out the invention that are obvious to
those skilled in the relevant fields are intended to be within the
scope of the following claims.
Sequence CWU 1
1
44019778DNAHuman Pegivirus 2 1gtgtttgaca atgccatgag ggatcatgac
actggggtga gcggaggcag caccgaagtc 60gggtgaactc gactcccagt gcgaccacct
ggcttggtcg ttcatggagg gcatgcccac 120gggaacgctg atcgtgcaaa
gggatgggtc cctgcactgg tgccatgcgc ggcaccactc 180cgtacagcct
gatagggtgg cggcgggccc ccccagtgtg acgtccgtgg agcgcaacat
240ggggtgttca actgatcaaa ccatttgttc tccagtcgtg ggggccgact
ataatacctc 300ctcgggctgc cgggccttaa atgggagcta ccactgcggt
ggtggctctt gccggtcacc 360aagtcgtgtg caggttgcga gacgagtctt
gcagctgtgc gcattccttg cgctgatcgg 420atccggtatg tgttcgatcc
ggtccaaaac tgaagggcgc attgagtcag ggcaaatatt 480gcagtctcag
cgcgcatgtt ggactggtga gggttttgct ttcttttcta actgttgcaa
540tcaatctgat attatgtggt gtttgcaccg ttggtgtgtg acaagacctg
gctgtttagt 600gtgcacgggc aatgccaccc atcctatctg ctgggactat
cttggatccg gtgtaagtcg 660gcggcctgca cgtcgaatgg gtgagggagc
tgaagcgctt cttcgcttga tcggcattgc 720aggttggctt ggactgttag
ctgagtccct tggtatgtcc gaagtctatg cagctattct 780ttgctttgga
tttattgctt ggtatggctg gggtatacct aaaacactgg tgtgcaccgt
840ctgccctgca gtgaacattt ctccctatag cttcttatct ccagatacta
tcgcatttgg 900tacgtggata ctacaactac ctggtctttt gtggcaaatg
tttgttagct tccctatact 960ctacagcact tggattcttt ggttgttgct
cagcggcaag actgttgctg tgatagcaat 1020ccttctggct agtcctacgg
ttatggcgta caagcatcaa tctgaaagct acctcaaata 1080ctgtaccata
accaatactt caacttctat gaactgtgac tgcccctttg gaacctttac
1140tcgcaatact gagtctcgtt tctccatacc tagattctgt cctgttaaaa
tcaatagctc 1200tacatttatt tgttcatggg ggtcgtggtg gtggtttgct
gaaaacatca cgcgtccata 1260cacggacgtt ggcatgccac cagcaccgat
ttccgctttg tgctatatct attctaacaa 1320tgacccacct ccttggtatc
ataacacaac tatcatacct cagaactgtc gcaactctac 1380ggtggatcct
accacagctc catgccgtga caagtggggc aacgcaactg cttgtattct
1440tgaccgccgg tcgcggttct gcggggactg ctatggcggt tgtttctata
ctaatggtag 1500tcatgatcga tcctgggatc gatgcgggat tggttaccgt
gatggactca tagagttcgt 1560gcagctcggt cagattcgac ctaacatctc
gaatacgacc attgagctcc tcgctggcgc 1620ctcgcttgtg atcgcatccg
gtcttcggcc tgggtttggt tgcagccgag cgcatggcgt 1680ggtgcactgc
tataggtgtc cttcataccg tgaccttgaa cagtttggtc ctgggcttgg
1740gaaatgggtg ccattgcccg gcgagcctgt cccggagttg tgtatcaacc
ctcagtgggc 1800gaggcgcggc ttccggatgt ctaataatcc tctgagcttg
ctacagacct tcgttgagga 1860cattttccta gcgccttttt gtaatccgac
gcctggccgt gtacgtgtgt gtaacaatac 1920cgctttctat ccaagaggag
gcggctttgt gcagctcatc ggggacgtcc aggtgctaac 1980ccctaacact
gcatctttac actctctgct gactttgata tctcttatct tgttggtgtg
2040tgttgtttct ggtgcgcgat tcgttccact aataatcata tttttctgga
gcgcgcgcca 2100tgtatatgct tcttgttact taagctgtga ttgggctgtt
tgcaacgatg cgttctgttt 2160cacatctggc acttgtgcca ccttcaatga
cgtcttgtgt ctgccggttg cgacgcgcat 2220atcgtcctgt ggtcatgctg
tgccacctcc cgaccgtggt tgggaggtgc ctgcggcgat 2280gtcatgggtg
atttcgcgga ctactggcct gacgttcgat gtcttttcct tcattcagta
2340ccttcctact gtgcctggca acaacaccaa tatcatttac tgtggtgaac
caaccttcct 2400cggggacatc acgggcatct attggcctta ctttttgcct
ggcgcaatcc tcttgtactt 2460gactcccttc ctaggtttaa ggttaatgct
tgccggcttc aatatagatg gcttgtttcc 2520catacggcat gccacggctg
cactgaggtt ttcgacttct cgtgtgacct tgtgtgtcgt 2580agttgctttc
ctaatctata tattatctca ccctgttaat gctgcgctca atagaatgtt
2640cttagcatct gcaaatttag agatgatctt atcttttgat acctatcatg
agactgttct 2700ttatatcctt tgtctattgc tctacctcca ggtgtcgccc
cgtgcgggct tggccgctat 2760ggtggccatc aagctatctc gaggcctgtt
attcgctgtg gtgttggcgc acggtgtgtg 2820ccgacctggg cgggtatttg
gtcttgaggt ttgcgcggac atctcttggt tggtggagtt 2880tactggcaat
tgcacttggt acatgtcctg tgtcttctct ttttggtgcg cagtgtttgc
2940cttcaccagt ccacttggac gacactataa gattcagatc tatcggtact
gggcgcaggt 3000ctatgccaga ctcgtcctcg ctgtcggttg tggtcctctc
ggtcgagagt tccatttccg 3060tgcaagtgtg ggcgtgctgt ggtgtggagc
ttgcatgctc tggccccgtg agtgctctga 3120aatcagcctg gtcttcattc
tgtgtgctct gacagtggac accatagaca catggttagt 3180agcgtgcttg
tccgcagggc cgagtgcgcg aacccttgca attctggccg atgacatggc
3240gcgcattggt gaccaccggg cgttgcgcgc cgtgttacgt tgctttggat
cacgcggcac 3300atacatatac aaccacatgg gccaagtctc ggaacgggtg
gcgcaagcag tcagggatct 3360cggcggttgc ttggaaccag tcgtgttgga
ggagcccacc tttactgaga tcgtggatga 3420cacaatgagt ttggtgtgtg
gacaattgct tggaggtaaa cctgtggtgg cccgctgcgg 3480cacgcgtgtc
ttggtgggac acctcaaccc tgaagatctg ccacctggtt tccagctgag
3540tgctccggtg gttattacca ggccaagcat tggtacgtgg tccttcctta
aggcgacact 3600cacagggcgt gctgaaacac cagggtccgg ccagatcgtg
gtgttgtctt ccctgacagg 3660tcggtcaatg ggtaccgcag tgaatggcac
actgtatgcg accggccatg gtgccggcgc 3720gcgcggccta gccacgtgcg
ctggtttgag gacgccactt tacacggcat tatctgatga 3780tgtcgtggcc
tattcttgcc ttccgggcat gagttcccta gacccctgct gctgttcgcc
3840gagccgggtt tgggtgatga ataacaacgg agggttggtg tgtggcagag
tggagaatga 3900cgacgtctgt ttggactgtc ccacgcacat agatcaactg
cggggtgctt cgggctcacc 3960agttttgtgt gatcacggtc atgcatacgc
gttgatgctc ggtggttact ctaccagtgg 4020tatttgtgca cgcgtccgga
cggtccggcc atggcataac gcctattcct cctcgggggg 4080gcaaggcgga
atgcaggcgc cagctgtgac accaacatac tctgaaatca cctactatgc
4140ccctactggt tctggtaagt caacaaaata tccagtggac ctagtcaaac
agggacacaa 4200agtattggtc cttttaccaa gtgtggctgt agtcaaaagt
atggcccctt atattaagga 4260gacatataag atcagacccg aaattagagc
tggcacaggt cctgacggtg tgacggtcat 4320cactggtgag aacttggcgt
acatgaccta tggccgcttc cttgtggatc cggagacgaa 4380tctgcggggc
tatgctgtag tcatttgcga cgagtgtcac gacacatcat ccaccacgct
4440actcggcatt ggcgcagtgc gcatgtatgc cgagaaagct ggagtgaaga
ccgttgtatt 4500cgccacagcc acccctgctg gcattcaagt acagtcacat
tccaacattg atgaatactt 4560attgactgac acaggcgacg tggaatttta
cggcgccaaa atcaaaatgg acaacatcag 4620aactggtaga catgttatct
tttgccactc gaaggccagg tgtgcggaac taacgcagca 4680gctctccggc
cttggcattc gtgcagtgag tttttggcgc ggctgtgaca tcaaaaccat
4740tcccgcctca gactccattg ttgtggtggc aactgatgca ttgtccacgg
gctacacagg 4800aaactttgat tcggtcatcg actgcgggtg ttgcgtggag
caaactgtga caattgacat 4860ggaccctacg ttctccatct cggcccgagt
ggtgccatgt actgctgcat tgcgcatgca 4920gcggcgcgga cgtaccggtc
gtggtagaag gggagcgtac tacacaactt ctccaggagc 4980agcaccctgc
gtcagcgttc ccgatgctaa cgtctggcaa gcagtggaga gcgccatggt
5040cttttatgat tggagtgcta ccaggataca acagtgcctg gcggcatacc
atgatttggg 5100gtgcacacca cgcatcagct gtgacccaca cactccagtg
cgggtgatgg acacactgag 5160ggcgtacctg cgcagacctg aggtgacgac
tgcagctctc gcaggagagc agtggccgct 5220gctttatggt gcgcagttgt
gcatctgcaa agagaccgag gcccacggtc ctgatgatag 5280catcaagtgg
aagtgcttac tcaacaacag taacaaaaca cccctgttgt atgccttaga
5340caatcctaca ctggaattca caacccaaca tgacttgact cgccgtatag
ccggcgctct 5400atcgagcaca gtgttcgtgg agacaggcta cggccccatc
ctccttgctg gcgccgcttt 5460ggctgcctcc ttcgcctttg cgggcgccac
tggagcttta gtgccgtcgg ctgtttggag 5520cgttgaggtc aggcctgctg
gcgtgacccg tcccgacgcg acagacgaga ccgcggccta 5580cgcacagcgc
ttgtaccaag cctgtgcaga ttcaggaatt ttcgccagct tgcagggtac
5640ggcgagtgcg gcgctgggca aactggccga cgccagtagg ggtgctagtc
aatatctggc 5700agccgcgcct ccttcacccg cccccctggt acaggtgttg
cagttcctcg agaccaactt 5760tagctccatt gcatctttcg gcctgctctg
tgctggctgc caggctggcg agtgcttcac 5820tgcgcttgct ggcttggtgt
ccggtgctac agctggcttg gggggtgccc ataagtggct 5880attagctatt
gcaggaactt ggctggttag cttgcagacc gggtcccgtg gcggcatggt
5940tgcgggcctc tcgattctag cgggctgttg catcggtagt gtcaccgggc
ttgacttcct 6000gtttgggtgc cttacaggtt gggaagccgt ggtcggcgct
gcggttgcga cacagaagat 6060cttgtctggt tcagctgata tgaccactct
ggtagatctc ttacctgctc ttttctcccc 6120cggtgccggc atagctggca
tcgtgcttgt cttcatctta agcaattcaa gtgtaaccac 6180atgggctaat
cggctattat ccatgtgtgc caaacaaacc atttgtgaaa actacttctt
6240aagtgaaaga tttggccaac aattaagcaa actttccctg tggcgctctg
tgtaccattg 6300ggcgcaggca cgtgagggat acacacagtg cggcgtgatc
agcgggatct ggagcttcgc 6360cttgtgcatt ctacgcgctg tgtgggattg
ggcggccaag catgtgccac ggttccgtgt 6420gcctatgatt ggctgctcac
ctgcgtggtg cgggcgctgg cttggtaccg gcaccttgtt 6480gaccacctgt
gcgtgtggag aacgtgtgtc ccttcagtgc ctttgctcaa catctgaccc
6540acaactcagt gtgggccgtt ggtgtcggtg tagttggagt gttgggttcc
cattcaaccc 6600gactacgaca ggcactggca ccttacggcc ggacatcagt
gacgccaaca aattgggttt 6660ccggtatggc gttgccgaca tcgtggagct
agagcggcgg ggcgacaaat ggcacgtctg 6720tgcagcatca tgttgcttgg
accgggccag cgttgcatcc gctgtgaagg ccccaccggt 6780cacggctaat
ggtataccta ttaatagctt ttctccacca caaacttatt gcctatctct
6840ctgttccttt gatacagttt gcatgtctac taacttatgt aacccagcta
agaccctgag 6900tgtgtgccaa gaggaggcgg ttgagctgct ggaagagaca
gttgacacag cacaagtagt 6960gatgagccaa aatctggcag cgcgtagacg
cgctgagtat gatgcatggc aggttcgcca 7020agcagttggc gacgagtaca
cgcgtttggc agacgaggat gttgacatga cagcgtcggt 7080gaaaccccca
gtggccaggg ctgctgtggg tagctcaacg ttggatgatg ttagcgtgct
7140gactgtctta cgcgaactcg gcgaccagtg ccaaaatgct atcaaatttg
tagttcaggc 7200ggcttcacgg tttgttccac cagtgcccaa gccacgcacg
cgtgtctcgg gtgtcttgga 7260gcgcgtgcgc atgtgcatgc gcacgcctcc
aatcaagttt gaggccaccg cagtaccaat 7320tcataatata atcccagaag
agtgtcatat tgtgctacgc tgtaccggct gttgtgacca 7380ggccttgacc
gttccgtacg gcacttgctc tctgacttta accaaatatt tgactaacaa
7440acacagtcac tatattccaa aagagaagat agaagaagac acagaaatag
ctgtcatttg 7500cgccgtacca acaaagcgcg caagtaaact tatcactttc
agagcaggtg accgatcagt 7560ctcatgttgt caccccttgc aaactcctat
tagggccctg cttcaaaagt atgggttacc 7620tattgggaag tggtccgact
gcaacgggcc ccttggtgac gacgcccgag tctgtgacgt 7680caatggagtg
acaacttatg aaccatgcat gcaatcctac aattggttcc gatcgattgt
7740ggcaccaaca accccacctt tacctgcaac ccggagcgtg gctggcattt
tgcgcgcaga 7800cacatcgcgc gtctacacca caacagcggt tgatgtctcc
gagcggcagg ctaaggtcac 7860gattgatcaa aagtcagcca aggtggacca
gtgtctccga gacacataca attgctgcct 7920tgccaaggca aagaccttca
gacaatctgg catgtcatat gaggatgctg tgtcaaagat 7980gcgcgcaaac
accacgcgtg atcataacaa cggcatcact tatacagatt tggtctctgg
8040acgcgcaaaa cctgtcgttc agaaaattgt agatcagatg cgcgctggag
tgtacgacgc 8100tccaatgcgc attattccaa aacctgaagt gtttccacga
gacaagtcaa cacggaagcc 8160accacggttc atcgttttcc ctgggtgtgc
cgcacgagtc gcggagaaaa tgatcctggg 8220cgatcctggc gcgataacca
agcacgtgct aggtgatgcc tacgggtttg ccactccgcc 8280gcatgagcgc
gcgcgcctac tggaacaatg gtggaaccgc gcaacggagc cacaagctat
8340cgcggttgat gcagtctgct ttgatagcac catcacggca gaggacatgg
atcgtgaggc 8400caacatcgtg gctgcagcgc atacggaccc ggaaggtgtt
cacggcctat acaattatta 8460caaaagaagc cccatgtgtg atatcacagg
aaaagttgtc ggggtgcgta gctgtcgagc 8520ctcaggtacg cttacaacaa
gcagtggcaa cacgcttact tgctacctca aggttcgcgc 8580agcttgcacg
cgcgccggca ttaaaccaat tggcttacta attcatggag atgacaccct
8640cattatcaca gaacgttgcg ctcaggaaac tctcgatgag ttcagcaacg
cgcttgatga 8700ctatgggttt actcacacca tgcaggtgtc tggggacctc
tcgtctatcg agtgctgcag 8760cgcacgtgtg gacagcgttt gcctccgggg
aggtatgcgt cgcatgctcg tgccacaagc 8820tcgacgtgcg attgcacgcg
ttctcgggga aaagggcgat ccactgggtg ttatcagcag 8880ctatattgtc
atgtatccta ctgcggctgt gactgtctac gttctgatgc ccctgttgtg
8940catgctcatt cgaaatgagc catcgcagac ggggacactt gtaacgttga
cggtccacgg 9000taacagtgtg agcgtgccag tgtggctgct tccaaccatt
attgcaaatt tacatggccg 9060tgacgcacta caggttgtcc gtcacagtgc
agcttccatg gcggaactgt cctcagcgtt 9120ggccttcttt ggcatgagag
ggttgaactg ctggaggcgg agacgccgtg ccatcaggac 9180tgatatgatc
aagttgggcg ggtggaatgc gaatttcgcg cagatgttac tgtggtcacc
9240ggaggtaaga acaccacagc ccgaaccaaa gggcatgtgt ctcttgccac
cggaactatg 9300ggagcgtccg tacgaaaatt tgcacttgag cacgatcgac
cgcaatcgtg gtgctagtcg 9360cttacggttt tggttggttg ctagtgctat
actcgctctg ctttgcttgt aaatcctaaa 9420tcaatgtagt accaggacta
caaggcagga ggtgaagtca gctgtaccca cggctggctg 9480aaaccggggc
ttgacgaccc cccctatccg agttgggcaa ggtaacatca cgggtgtgac
9540gaccccgccc ccccatgtcg cgcgcaagcg cacgggcaag gcagctaggc
tgagagtctg 9600ggcaactctc ccgtacccca cccgaggcta cgcctcgtcc
tggcgaggac cgtaaacata 9660cgtcgtcagc gtggtgacct gacgtatctt
gttaaccact taatggtcgt aactcgaccc 9720ccgtgccggg gatctaagcg
cggcaccgcg aygagagggg tcaacggccc ctttcatt 97782124PRTHuman
Pegivirus 2 2Met Glu Gly Met Pro Thr Gly Thr Leu Ile Val Gln Arg
Asp Gly Ser1 5 10 15Leu His Trp Cys His Ala Arg His His Ser Val Gln
Pro Asp Arg Val 20 25 30Ala Ala Gly Pro Pro Ser Val Thr Ser Val Glu
Arg Asn Met Gly Cys 35 40 45Ser Thr Asp Gln Thr Ile Cys Ser Pro Val
Val Gly Ala Asp Tyr Asn 50 55 60Thr Ser Ser Gly Cys Arg Ala Leu Asn
Gly Ser Tyr His Cys Gly Gly65 70 75 80Gly Ser Cys Arg Ser Pro Ser
Arg Val Gln Val Ala Arg Arg Val Leu 85 90 95Gln Leu Cys Ala Phe Leu
Ala Leu Ile Gly Ser Gly Met Cys Ser Ile 100 105 110Arg Ser Lys Thr
Glu Gly Arg Ile Glu Ser Gly Gln 115 1203191PRTHuman Pegivirus 2
3Ile Leu Gln Ser Gln Arg Ala Cys Trp Thr Gly Glu Gly Phe Ala Phe1 5
10 15Phe Ser Asn Cys Cys Asn Gln Ser Asp Ile Met Trp Cys Leu His
Arg 20 25 30Trp Cys Val Thr Arg Pro Gly Cys Leu Val Cys Thr Gly Asn
Ala Thr 35 40 45His Pro Ile Cys Trp Asp Tyr Leu Gly Ser Gly Val Ser
Arg Arg Pro 50 55 60Ala Arg Arg Met Gly Glu Gly Ala Glu Ala Leu Leu
Arg Leu Ile Gly65 70 75 80Ile Ala Gly Trp Leu Gly Leu Leu Ala Glu
Ser Leu Gly Met Ser Glu 85 90 95Val Tyr Ala Ala Ile Leu Cys Phe Gly
Phe Ile Ala Trp Tyr Gly Trp 100 105 110Gly Ile Pro Lys Thr Leu Val
Cys Thr Val Cys Pro Ala Val Asn Ile 115 120 125Ser Pro Tyr Ser Phe
Leu Ser Pro Asp Thr Ile Ala Phe Gly Thr Trp 130 135 140Ile Leu Gln
Leu Pro Gly Leu Leu Trp Gln Met Phe Val Ser Phe Pro145 150 155
160Ile Leu Tyr Ser Thr Trp Ile Leu Trp Leu Leu Leu Ser Gly Lys Thr
165 170 175Val Ala Val Ile Ala Ile Leu Leu Ala Ser Pro Thr Val Met
Ala 180 185 1904354PRTHuman Pegivirus 2 4Tyr Lys His Gln Ser Glu
Ser Tyr Leu Lys Tyr Cys Thr Ile Thr Asn1 5 10 15Thr Ser Thr Ser Met
Asn Cys Asp Cys Pro Phe Gly Thr Phe Thr Arg 20 25 30Asn Thr Glu Ser
Arg Phe Ser Ile Pro Arg Phe Cys Pro Val Lys Ile 35 40 45Asn Ser Ser
Thr Phe Ile Cys Ser Trp Gly Ser Trp Trp Trp Phe Ala 50 55 60Glu Asn
Ile Thr Arg Pro Tyr Thr Asp Val Gly Met Pro Pro Ala Pro65 70 75
80Ile Ser Ala Leu Cys Tyr Ile Tyr Ser Asn Asn Asp Pro Pro Pro Trp
85 90 95Tyr His Asn Thr Thr Ile Ile Pro Gln Asn Cys Arg Asn Ser Thr
Val 100 105 110Asp Pro Thr Thr Ala Pro Cys Arg Asp Lys Trp Gly Asn
Ala Thr Ala 115 120 125Cys Ile Leu Asp Arg Arg Ser Arg Phe Cys Gly
Asp Cys Tyr Gly Gly 130 135 140Cys Phe Tyr Thr Asn Gly Ser His Asp
Arg Ser Trp Asp Arg Cys Gly145 150 155 160Ile Gly Tyr Arg Asp Gly
Leu Ile Glu Phe Val Gln Leu Gly Gln Ile 165 170 175Arg Pro Asn Ile
Ser Asn Thr Thr Ile Glu Leu Leu Ala Gly Ala Ser 180 185 190Leu Val
Ile Ala Ser Gly Leu Arg Pro Gly Phe Gly Cys Ser Arg Ala 195 200
205His Gly Val Val His Cys Tyr Arg Cys Pro Ser Tyr Arg Asp Leu Glu
210 215 220Gln Phe Gly Pro Gly Leu Gly Lys Trp Val Pro Leu Pro Gly
Glu Pro225 230 235 240Val Pro Glu Leu Cys Ile Asn Pro Gln Trp Ala
Arg Arg Gly Phe Arg 245 250 255Met Ser Asn Asn Pro Leu Ser Leu Leu
Gln Thr Phe Val Glu Asp Ile 260 265 270Phe Leu Ala Pro Phe Cys Asn
Pro Thr Pro Gly Arg Val Arg Val Cys 275 280 285Asn Asn Thr Ala Phe
Tyr Pro Arg Gly Gly Gly Phe Val Gln Leu Ile 290 295 300Gly Asp Val
Gln Val Leu Thr Pro Asn Thr Ala Ser Leu His Ser Leu305 310 315
320Leu Thr Leu Ile Ser Leu Ile Leu Leu Val Cys Val Val Ser Gly Ala
325 330 335Arg Phe Val Pro Leu Ile Ile Ile Phe Phe Trp Ser Ala Arg
His Val 340 345 350Tyr Ala5237PRTHuman Pegivirus 2 5Ser Cys Tyr Leu
Ser Cys Asp Trp Ala Val Cys Asn Asp Ala Phe Cys1 5 10 15Phe Thr Ser
Gly Thr Cys Ala Thr Phe Asn Asp Val Leu Cys Leu Pro 20 25 30Val Ala
Thr Arg Ile Ser Ser Cys Gly His Ala Val Pro Pro Pro Asp 35 40 45Arg
Gly Trp Glu Val Pro Ala Ala Met Ser Trp Val Ile Ser Arg Thr 50 55
60Thr Gly Leu Thr Phe Asp Val Phe Ser Phe Ile Gln Tyr Leu Pro Thr65
70 75 80Val Pro Gly Asn Asn Thr Asn Ile Ile Tyr Cys Gly Glu Pro Thr
Phe 85 90 95Leu Gly Asp Ile Thr Gly Ile Tyr Trp Pro Tyr Phe Leu Pro
Gly Ala 100 105 110Ile Leu Leu Tyr Leu Thr Pro Phe Leu Gly Leu Arg
Leu Met Leu Ala 115 120 125Gly Phe Asn Ile Asp Gly Leu Phe Pro Ile
Arg His Ala Thr Ala Ala 130 135 140Leu Arg Phe Ser Thr Ser Arg Val
Thr Leu Cys Val Val Val Ala Phe145 150 155 160Leu Ile Tyr Ile Leu
Ser His Pro Val Asn Ala Ala Leu Asn Arg Met 165 170 175Phe Leu Ala
Ser Ala Asn Leu Glu Met Ile Leu Ser
Phe Asp Thr Tyr 180 185 190His Glu Thr Val Leu Tyr Ile Leu Cys Leu
Leu Leu Tyr Leu Gln Val 195 200 205Ser Pro Arg Ala Gly Leu Ala Ala
Met Val Ala Ile Lys Leu Ser Arg 210 215 220Gly Leu Leu Phe Ala Val
Val Leu Ala His Gly Val Cys225 230 2356240PRTHuman Pegivirus 2 6Arg
Pro Gly Arg Val Phe Gly Leu Glu Val Cys Ala Asp Ile Ser Trp1 5 10
15Leu Val Glu Phe Thr Gly Asn Cys Thr Trp Tyr Met Ser Cys Val Phe
20 25 30Ser Phe Trp Cys Ala Val Phe Ala Phe Thr Ser Pro Leu Gly Arg
His 35 40 45Tyr Lys Ile Gln Ile Tyr Arg Tyr Trp Ala Gln Val Tyr Ala
Arg Leu 50 55 60Val Leu Ala Val Gly Cys Gly Pro Leu Gly Arg Glu Phe
His Phe Arg65 70 75 80Ala Ser Val Gly Val Leu Trp Cys Gly Ala Cys
Met Leu Trp Pro Arg 85 90 95Glu Cys Ser Glu Ile Ser Leu Val Phe Ile
Leu Cys Ala Leu Thr Val 100 105 110Asp Thr Ile Asp Thr Trp Leu Val
Ala Cys Leu Ser Ala Gly Pro Ser 115 120 125Ala Arg Thr Leu Ala Ile
Leu Ala Asp Asp Met Ala Arg Ile Gly Asp 130 135 140His Arg Ala Leu
Arg Ala Val Leu Arg Cys Phe Gly Ser Arg Gly Thr145 150 155 160Tyr
Ile Tyr Asn His Met Gly Gln Val Ser Glu Arg Val Ala Gln Ala 165 170
175Val Arg Asp Leu Gly Gly Cys Leu Glu Pro Val Val Leu Glu Glu Pro
180 185 190Thr Phe Thr Glu Ile Val Asp Asp Thr Met Ser Leu Val Cys
Gly Gln 195 200 205Leu Leu Gly Gly Lys Pro Val Val Ala Arg Cys Gly
Thr Arg Val Leu 210 215 220Val Gly His Leu Asn Pro Glu Asp Leu Pro
Pro Gly Phe Gln Leu Ser225 230 235 2407628PRTHuman Pegivirus 2 7Ala
Pro Val Val Ile Thr Arg Pro Ser Ile Gly Thr Trp Ser Phe Leu1 5 10
15Lys Ala Thr Leu Thr Gly Arg Ala Glu Thr Pro Gly Ser Gly Gln Ile
20 25 30Val Val Leu Ser Ser Leu Thr Gly Arg Ser Met Gly Thr Ala Val
Asn 35 40 45Gly Thr Leu Tyr Ala Thr Gly His Gly Ala Gly Ala Arg Gly
Leu Ala 50 55 60Thr Cys Ala Gly Leu Arg Thr Pro Leu Tyr Thr Ala Leu
Ser Asp Asp65 70 75 80Val Val Ala Tyr Ser Cys Leu Pro Gly Met Ser
Ser Leu Asp Pro Cys 85 90 95Cys Cys Ser Pro Ser Arg Val Trp Val Met
Asn Asn Asn Gly Gly Leu 100 105 110Val Cys Gly Arg Val Glu Asn Asp
Asp Val Cys Leu Asp Cys Pro Thr 115 120 125His Ile Asp Gln Leu Arg
Gly Ala Ser Gly Ser Pro Val Leu Cys Asp 130 135 140His Gly His Ala
Tyr Ala Leu Met Leu Gly Gly Tyr Ser Thr Ser Gly145 150 155 160Ile
Cys Ala Arg Val Arg Thr Val Arg Pro Trp His Asn Ala Tyr Ser 165 170
175Ser Ser Gly Gly Gln Gly Gly Met Gln Ala Pro Ala Val Thr Pro Thr
180 185 190Tyr Ser Glu Ile Thr Tyr Tyr Ala Pro Thr Gly Ser Gly Lys
Ser Thr 195 200 205Lys Tyr Pro Val Asp Leu Val Lys Gln Gly His Lys
Val Leu Val Leu 210 215 220Leu Pro Ser Val Ala Val Val Lys Ser Met
Ala Pro Tyr Ile Lys Glu225 230 235 240Thr Tyr Lys Ile Arg Pro Glu
Ile Arg Ala Gly Thr Gly Pro Asp Gly 245 250 255Val Thr Val Ile Thr
Gly Glu Asn Leu Ala Tyr Met Thr Tyr Gly Arg 260 265 270Phe Leu Val
Asp Pro Glu Thr Asn Leu Arg Gly Tyr Ala Val Val Ile 275 280 285Cys
Asp Glu Cys His Asp Thr Ser Ser Thr Thr Leu Leu Gly Ile Gly 290 295
300Ala Val Arg Met Tyr Ala Glu Lys Ala Gly Val Lys Thr Val Val
Phe305 310 315 320Ala Thr Ala Thr Pro Ala Gly Ile Gln Val Gln Ser
His Ser Asn Ile 325 330 335Asp Glu Tyr Leu Leu Thr Asp Thr Gly Asp
Val Glu Phe Tyr Gly Ala 340 345 350Lys Ile Lys Met Asp Asn Ile Arg
Thr Gly Arg His Val Ile Phe Cys 355 360 365His Ser Lys Ala Arg Cys
Ala Glu Leu Thr Gln Gln Leu Ser Gly Leu 370 375 380Gly Ile Arg Ala
Val Ser Phe Trp Arg Gly Cys Asp Ile Lys Thr Ile385 390 395 400Pro
Ala Ser Asp Ser Ile Val Val Val Ala Thr Asp Ala Leu Ser Thr 405 410
415Gly Tyr Thr Gly Asn Phe Asp Ser Val Ile Asp Cys Gly Cys Cys Val
420 425 430Glu Gln Thr Val Thr Ile Asp Met Asp Pro Thr Phe Ser Ile
Ser Ala 435 440 445Arg Val Val Pro Cys Thr Ala Ala Leu Arg Met Gln
Arg Arg Gly Arg 450 455 460Thr Gly Arg Gly Arg Arg Gly Ala Tyr Tyr
Thr Thr Ser Pro Gly Ala465 470 475 480Ala Pro Cys Val Ser Val Pro
Asp Ala Asn Val Trp Gln Ala Val Glu 485 490 495Ser Ala Met Val Phe
Tyr Asp Trp Ser Ala Thr Arg Ile Gln Gln Cys 500 505 510Leu Ala Ala
Tyr His Asp Leu Gly Cys Thr Pro Arg Ile Ser Cys Asp 515 520 525Pro
His Thr Pro Val Arg Val Met Asp Thr Leu Arg Ala Tyr Leu Arg 530 535
540Arg Pro Glu Val Thr Thr Ala Ala Leu Ala Gly Glu Gln Trp Pro
Leu545 550 555 560Leu Tyr Gly Ala Gln Leu Cys Ile Cys Lys Glu Thr
Glu Ala His Gly 565 570 575Pro Asp Asp Ser Ile Lys Trp Lys Cys Leu
Leu Asn Asn Ser Asn Lys 580 585 590Thr Pro Leu Leu Tyr Ala Leu Asp
Asn Pro Thr Leu Glu Phe Thr Thr 595 600 605Gln His Asp Leu Thr Arg
Arg Ile Ala Gly Ala Leu Ser Ser Thr Val 610 615 620Phe Val Glu
Thr625841PRTHuman Pegivirus 2 8Gly Tyr Gly Pro Ile Leu Leu Ala Gly
Ala Ala Leu Ala Ala Ser Phe1 5 10 15Ala Phe Ala Gly Ala Thr Gly Ala
Leu Val Pro Ser Ala Val Trp Ser 20 25 30Val Glu Val Arg Pro Ala Gly
Val Thr 35 409262PRTHuman Pegivirus 2 9Arg Pro Asp Ala Thr Asp Glu
Thr Ala Ala Tyr Ala Gln Arg Leu Tyr1 5 10 15Gln Ala Cys Ala Asp Ser
Gly Ile Phe Ala Ser Leu Gln Gly Thr Ala 20 25 30Ser Ala Ala Leu Gly
Lys Leu Ala Asp Ala Ser Arg Gly Ala Ser Gln 35 40 45Tyr Leu Ala Ala
Ala Pro Pro Ser Pro Ala Pro Leu Val Gln Val Leu 50 55 60Gln Phe Leu
Glu Thr Asn Phe Ser Ser Ile Ala Ser Phe Gly Leu Leu65 70 75 80Cys
Ala Gly Cys Gln Ala Gly Glu Cys Phe Thr Ala Leu Ala Gly Leu 85 90
95Val Ser Gly Ala Thr Ala Gly Leu Gly Gly Ala His Lys Trp Leu Leu
100 105 110Ala Ile Ala Gly Thr Trp Leu Val Ser Leu Gln Thr Gly Ser
Arg Gly 115 120 125Gly Met Val Ala Gly Leu Ser Ile Leu Ala Gly Cys
Cys Ile Gly Ser 130 135 140Val Thr Gly Leu Asp Phe Leu Phe Gly Cys
Leu Thr Gly Trp Glu Ala145 150 155 160Val Val Gly Ala Ala Val Ala
Thr Gln Lys Ile Leu Ser Gly Ser Ala 165 170 175Asp Met Thr Thr Leu
Val Asp Leu Leu Pro Ala Leu Phe Ser Pro Gly 180 185 190Ala Gly Ile
Ala Gly Ile Val Leu Val Phe Ile Leu Ser Asn Ser Ser 195 200 205Val
Thr Thr Trp Ala Asn Arg Leu Leu Ser Met Cys Ala Lys Gln Thr 210 215
220Ile Cys Glu Asn Tyr Phe Leu Ser Glu Arg Phe Gly Gln Gln Leu
Ser225 230 235 240Lys Leu Ser Leu Trp Arg Ser Val Tyr His Trp Ala
Gln Ala Arg Glu 245 250 255Gly Tyr Thr Gln Cys Gly 26010458PRTHuman
Pegivirus 2 10Val Ile Ser Gly Ile Trp Ser Phe Ala Leu Cys Ile Leu
Arg Ala Val1 5 10 15Trp Asp Trp Ala Ala Lys His Val Pro Arg Phe Arg
Val Pro Met Ile 20 25 30Gly Cys Ser Pro Ala Trp Cys Gly Arg Trp Leu
Gly Thr Gly Thr Leu 35 40 45Leu Thr Thr Cys Ala Cys Gly Glu Arg Val
Ser Leu Gln Cys Leu Cys 50 55 60Ser Thr Ser Asp Pro Gln Leu Ser Val
Gly Arg Trp Cys Arg Cys Ser65 70 75 80Trp Ser Val Gly Phe Pro Phe
Asn Pro Thr Thr Thr Gly Thr Gly Thr 85 90 95Leu Arg Pro Asp Ile Ser
Asp Ala Asn Lys Leu Gly Phe Arg Tyr Gly 100 105 110Val Ala Asp Ile
Val Glu Leu Glu Arg Arg Gly Asp Lys Trp His Val 115 120 125Cys Ala
Ala Ser Cys Cys Leu Asp Arg Ala Ser Val Ala Ser Ala Val 130 135
140Lys Ala Pro Pro Val Thr Ala Asn Gly Ile Pro Ile Asn Ser Phe
Ser145 150 155 160Pro Pro Gln Thr Tyr Cys Leu Ser Leu Cys Ser Phe
Asp Thr Val Cys 165 170 175Met Ser Thr Asn Leu Cys Asn Pro Ala Lys
Thr Leu Ser Val Cys Gln 180 185 190Glu Glu Ala Val Glu Leu Leu Glu
Glu Thr Val Asp Thr Ala Gln Val 195 200 205Val Met Ser Gln Asn Leu
Ala Ala Arg Arg Arg Ala Glu Tyr Asp Ala 210 215 220Trp Gln Val Arg
Gln Ala Val Gly Asp Glu Tyr Thr Arg Leu Ala Asp225 230 235 240Glu
Asp Val Asp Met Thr Ala Ser Val Lys Pro Pro Val Ala Arg Ala 245 250
255Ala Val Gly Ser Ser Thr Leu Asp Asp Val Ser Val Leu Thr Val Leu
260 265 270Arg Glu Leu Gly Asp Gln Cys Gln Asn Ala Ile Lys Phe Val
Val Gln 275 280 285Ala Ala Ser Arg Phe Val Pro Pro Val Pro Lys Pro
Arg Thr Arg Val 290 295 300Ser Gly Val Leu Glu Arg Val Arg Met Cys
Met Arg Thr Pro Pro Ile305 310 315 320Lys Phe Glu Ala Thr Ala Val
Pro Ile His Asn Ile Ile Pro Glu Glu 325 330 335Cys His Ile Val Leu
Arg Cys Thr Gly Cys Cys Asp Gln Ala Leu Thr 340 345 350Val Pro Tyr
Gly Thr Cys Ser Leu Thr Leu Thr Lys Tyr Leu Thr Asn 355 360 365Lys
His Ser His Tyr Ile Pro Lys Glu Lys Ile Glu Glu Asp Thr Glu 370 375
380Ile Ala Val Ile Cys Ala Val Pro Thr Lys Arg Ala Ser Lys Leu
Ile385 390 395 400Thr Phe Arg Ala Gly Asp Arg Ser Val Ser Cys Cys
His Pro Leu Gln 405 410 415Thr Pro Ile Arg Ala Leu Leu Gln Lys Tyr
Gly Leu Pro Ile Gly Lys 420 425 430Trp Ser Asp Cys Asn Gly Pro Leu
Gly Asp Asp Ala Arg Val Cys Asp 435 440 445Val Asn Gly Val Thr Thr
Tyr Glu Pro Cys 450 45511567PRTHuman Pegivirus 2 11Met Gln Ser Tyr
Asn Trp Phe Arg Ser Ile Val Ala Pro Thr Thr Pro1 5 10 15Pro Leu Pro
Ala Thr Arg Ser Val Ala Gly Ile Leu Arg Ala Asp Thr 20 25 30Ser Arg
Val Tyr Thr Thr Thr Ala Val Asp Val Ser Glu Arg Gln Ala 35 40 45Lys
Val Thr Ile Asp Gln Lys Ser Ala Lys Val Asp Gln Cys Leu Arg 50 55
60Asp Thr Tyr Asn Cys Cys Leu Ala Lys Ala Lys Thr Phe Arg Gln Ser65
70 75 80Gly Met Ser Tyr Glu Asp Ala Val Ser Lys Met Arg Ala Asn Thr
Thr 85 90 95Arg Asp His Asn Asn Gly Ile Thr Tyr Thr Asp Leu Val Ser
Gly Arg 100 105 110Ala Lys Pro Val Val Gln Lys Ile Val Asp Gln Met
Arg Ala Gly Val 115 120 125Tyr Asp Ala Pro Met Arg Ile Ile Pro Lys
Pro Glu Val Phe Pro Arg 130 135 140Asp Lys Ser Thr Arg Lys Pro Pro
Arg Phe Ile Val Phe Pro Gly Cys145 150 155 160Ala Ala Arg Val Ala
Glu Lys Met Ile Leu Gly Asp Pro Gly Ala Ile 165 170 175Thr Lys His
Val Leu Gly Asp Ala Tyr Gly Phe Ala Thr Pro Pro His 180 185 190Glu
Arg Ala Arg Leu Leu Glu Gln Trp Trp Asn Arg Ala Thr Glu Pro 195 200
205Gln Ala Ile Ala Val Asp Ala Val Cys Phe Asp Ser Thr Ile Thr Ala
210 215 220Glu Asp Met Asp Arg Glu Ala Asn Ile Val Ala Ala Ala His
Thr Asp225 230 235 240Pro Glu Gly Val His Gly Leu Tyr Asn Tyr Tyr
Lys Arg Ser Pro Met 245 250 255Cys Asp Ile Thr Gly Lys Val Val Gly
Val Arg Ser Cys Arg Ala Ser 260 265 270Gly Thr Leu Thr Thr Ser Ser
Gly Asn Thr Leu Thr Cys Tyr Leu Lys 275 280 285Val Arg Ala Ala Cys
Thr Arg Ala Gly Ile Lys Pro Ile Gly Leu Leu 290 295 300Ile His Gly
Asp Asp Thr Leu Ile Ile Thr Glu Arg Cys Ala Gln Glu305 310 315
320Thr Leu Asp Glu Phe Ser Asn Ala Leu Asp Asp Tyr Gly Phe Thr His
325 330 335Thr Met Gln Val Ser Gly Asp Leu Ser Ser Ile Glu Cys Cys
Ser Ala 340 345 350Arg Val Asp Ser Val Cys Leu Arg Gly Gly Met Arg
Arg Met Leu Val 355 360 365Pro Gln Ala Arg Arg Ala Ile Ala Arg Val
Leu Gly Glu Lys Gly Asp 370 375 380Pro Leu Gly Val Ile Ser Ser Tyr
Ile Val Met Tyr Pro Thr Ala Ala385 390 395 400Val Thr Val Tyr Val
Leu Met Pro Leu Leu Cys Met Leu Ile Arg Asn 405 410 415Glu Pro Ser
Gln Thr Gly Thr Leu Val Thr Leu Thr Val His Gly Asn 420 425 430Ser
Val Ser Val Pro Val Trp Leu Leu Pro Thr Ile Ile Ala Asn Leu 435 440
445His Gly Arg Asp Ala Leu Gln Val Val Arg His Ser Ala Ala Ser Met
450 455 460Ala Glu Leu Ser Ser Ala Leu Ala Phe Phe Gly Met Arg Gly
Leu Asn465 470 475 480Cys Trp Arg Arg Arg Arg Arg Ala Ile Arg Thr
Asp Met Ile Lys Leu 485 490 495Gly Gly Trp Asn Ala Asn Phe Ala Gln
Met Leu Leu Trp Ser Pro Glu 500 505 510Val Arg Thr Pro Gln Pro Glu
Pro Lys Gly Met Cys Leu Leu Pro Pro 515 520 525Glu Leu Trp Glu Arg
Pro Tyr Glu Asn Leu His Leu Ser Thr Ile Asp 530 535 540Arg Asn Arg
Gly Ala Ser Arg Leu Arg Phe Trp Leu Val Ala Ser Ala545 550 555
560Ile Leu Ala Leu Leu Cys Leu 5651224DNAArtificial
sequenceSynthetic 12tattgctact tcggtacgcc taat 241324DNAArtificial
sequenceSynthetic 13aagggcctag taggacgtgt gaca 241424DNAArtificial
sequenceSynthetic 14cactggggtg agcggaggca gcac 241524DNAArtificial
sequenceSynthetic 15gaggcagcac cgaagtcggg tgaa 241620DNAArtificial
sequenceSynthetic 16tgccacccat cctatctgct 201724DNAArtificial
sequenceSynthetic 17tattgcttgg tatggctggg gtat 241823DNAArtificial
sequenceSynthetic 18atggctgggg tatacctaar aca 231919DNAArtificial
sequenceSynthetic 19tggcgtacaa gcatcaatc 192022DNAArtificial
sequenceSynthetic 20accgatttcc gctttgtgct at 222118DNAArtificial
sequenceSynthetic 21cctgggcttg ggaaatgg 182223DNAArtificial
sequenceSynthetic 22catgggtgat ttcgcggact act 232324DNAArtificial
sequenceSynthetic 23cctcggggac atcacgggca tcta 242424DNAArtificial
sequenceSynthetic 24ctgttaatgc tgcgctcaat agaa 242524DNAArtificial
sequenceSynthetic 25gcgggtattt ggtcttgagg tttg
242624DNAArtificial sequenceSynthetic 26tttggatcac gcggcacata cata
242722DNAArtificial sequenceSynthetic 27acgggtggcg caagcagtca gg
222820DNAArtificial sequenceSynthetic 28gaggagccca cctttactga
202920DNAArtificial sequenceSynthetic 29gaagatctgc cacctggttt
203020DNAArtificial sequenceSynthetic 30tccttcctta aggcgacact
203124DNAArtificial sequenceSynthetic 31gagccgggtt tgggtgatga ataa
243220DNAArtificial sequenceSynthetic 32tgggtgatga ataacaacgg
203320DNAArtificial sequenceSynthetic 33aatgacgacg tctgtttgga
203424DNAArtificial sequenceSynthetic 34acgcgttgat gctcggtggt tact
243520DNAArtificial sequenceSynthetic 35ccagctgtga caccaacata
203620DNAArtificial sequenceSynthetic 36agtcatttgc gacgagtgtc
203720DNAArtificial sequenceSynthetic 37ggcccacggt cctgatgata
203820DNAArtificial sequenceSynthetic 38ggcccayggt ccagacgatr
203920DNAArtificial sequenceSynthetic 39ggcccatggt ccggatgatg
204020DNAArtificial sequenceSynthetic 40ccgtttggag ygttgayaac
204120DNAArtificial sequenceSynthetic 41ctgtttggag cgttgaggtc
204224DNAArtificial sequenceSynthetic 42tacyggcacc ttgttgacca cctg
244321DNAArtificial sequenceSynthetic 43ctagagcggc ggggcgacaa a
214424DNAArtificial sequenceSynthetic 44gaggcggttg agctgctgga agag
244524DNAArtificial sequenceSynthetic 45tagttcaggc ggcttcacgg tttg
244618DNAArtificial sequenceSynthetic 46tgcgccgtac caacaaag
184724DNAArtificial sequenceSynthetic 47tgtcacccct tgcaaactcc tatt
244819DNAArtificial sequenceSynthetic 48agtgtacgac gctccaatg
194922DNAArtificial sequenceSynthetic 49gcacgagtcg cggagaaaat ga
225020DNAArtificial sequenceSynthetic 50acgagtcgcg gagaaaatga
205118DNAArtificial sequenceSynthetic 51cgcgcctact ggaacaat
185224DNAArtificial sequenceSynthetic 52acgcgcttga tgactatggg ttta
245320DNAArtificial sequenceSynthetic 53ttgacggtcc acggtaacag
205420DNAArtificial sequenceSynthetic 54gaggagccca cctttactga
205520DNAArtificial sequenceSynthetic 55tacctccaag caattgtcca
205626DNAArtificial sequenceSynthetic 56caccaaactc attgtgtcat
ccacga 265720DNAArtificial sequenceSynthetic 57gaagatctgc
cacctggttt 205820DNAArtificial sequenceSynthetic 58agtgtcgcct
taaggaagga 205920DNAArtificial sequenceSynthetic 59ccaccggagc
actcagctgg 206020DNAArtificial sequenceSynthetic 60tccttcctta
aggcgacact 206120DNAArtificial sequenceSynthetic 61agggaagaca
acaccacgat 206220DNAArtificial sequenceSynthetic 62aaacaccagg
gtccggccag 206320DNAArtificial sequenceSynthetic 63tgggtgatga
ataacaacgg 206420DNAArtificial sequenceSynthetic 64gatctatgtg
cgtgggacag 206522DNAArtificial sequenceSynthetic 65ccactctgcc
acacaccaac cc 226620DNAArtificial sequenceSynthetic 66aatgacgacg
tctgtttgga 206720DNAArtificial sequenceSynthetic 67catgaccgtg
atcacacaaa 206820DNAArtificial sequenceSynthetic 68ctggtgagcc
cgaagcaccc 206920DNAArtificial sequenceSynthetic 69ccagctgtga
caccaacata 207021DNAArtificial sequenceSynthetic 70tttgactaca
gccacacttg g 217126DNAArtificial sequenceSynthetic 71ccagtggacc
tagtcaaaca gggaca 267220DNAArtificial sequenceSynthetic
72agtcatttgc gacgagtgtc 207320DNAArtificial sequenceSynthetic
73acggtcttca ctccagcttt 207420DNAArtificial sequenceSynthetic
74tcggcataca tgcgcactgc 20759431DNAHuman Pegivirus 2 75gtgtttgacg
tgtgacaatg ccatgaggga tcatgacact ggggtgagcg gaggcagcac 60cgaagtcggg
tgaactcgac tcccagtgcg accacctggc ttggtcgttc atggagggca
120tgcccacggg aacgctgatc gtgcaaaggg atgggtccct gcactggtgc
catgcgcggc 180accactccgt acagcctgat agggtggcgg cgggcccccc
cagtgtgacg tccgtggagc 240gcaacatggg gtgttcaact gatcaaacca
tttgttctcc agtcgtggag gccgactata 300atacctcctc gggctgccgg
gccttaaatg ggagctacca ctgcggtggt ggctcttgcc 360ggtcaccaag
tcgtgtgcag gttgcaggac gagtcctgcg gctgtgcgca ttccttgcgc
420tgatcggatc cggtatgtgt tccatccggt ccaaaaatga agggcgcatt
gagtcagggc 480aaatattgca gtctcagcgc gcatgttgga ctggtgaggg
tttcgctttc ttttctaact 540gttgcaatca atctgacatt atgtggtgtt
tgcaccgttg gtgtgtgaca agacctggct 600gtttggtgtg cacgggcaat
gccactcatc ctgtctgctg ggactatctt gggtccggtg 660tgagtcggcg
gcctgcgcgt cgaatgggtg agggagctga agtgcttctt cgcttgatcg
720gcattgcagg ttggctcggg ctcttagctg aggctcttgg tatgtctgag
atctatgcag 780ctttcctttg ctttggattt attgcttggt atggctgggg
tatacctaag acattggtgt 840gcacagtctg ccctgcagtg aacatttctc
cctatagctt cttatctcca gatactatcg 900catttggtac gtggctacta
caactgcctg gtcttttgtg gcaaatgttt gtcagcttcc 960ctatacttta
cagtacttgg attctttggt tgttgctcag cggcaagact gttgctgtga
1020tagcgatcct tttggctagt cctacggtta tggcatacaa gcatcaagct
gatagctacc 1080tcaaatactg taccataacc aatgcttcaa ctgctatgaa
ctgtgactgc ccctttggaa 1140ctttcactcg caatactgag tctggtttca
ctatacctag attctgtcct gttaaactta 1200atagctctac atttatctgt
tcatgggggt cgtggtggtg gtttgctgag aacatcacac 1260gtccatactc
ggacgttggc atgccgccag caccgatttc cgctttgtgc tatatctatt
1320caaacaatga cccaccttct tggtatcgta acacaactat catacctcag
aactgttaca 1380actctacggc tgatcctacc acagctccat gccgtgacaa
gtggggcaat gcaactgctt 1440gtattcttga ccgccggtcg cggttctgcg
gggactgcta tggcggttgc ttctacacta 1500atggtagtca tgaccgatcc
tgggatcgat gcggaattgg ttaccgtgat ggactcatag 1560agttcgtgca
gctcggtcag attcgaccta acatcgcgaa tacgaccatt gagctcctcg
1620ctggcgcctc gcttgtgatc gcatccggtc ttcgggctgg gtatggttgc
agccgagcgc 1680acggcgtggt gcactgcttt aagtgtcctt cataccgtga
ccttgaacgg ttcgggcccg 1740ggcttgggaa atgggtgcca ttgcctggcg
agcctgtccc ggagttgtgt attaacccgc 1800agtgggcgag gcgcggcttc
cgggtgtcta ataatccctt aagcgtgcta cagaccttcg 1860ttgaggacat
tttcttagcg cctttctgca atccgacgcc tggccgtgta cgtgtgtgta
1920acaatactgc tttctacccg agaggaggcg gctttgtgca gctcatcgga
gacgtccagg 1980tgttaacccc caactctaca tctttgcact ctctgctgac
tttgatatcc cttatcttgt 2040tagtgtgtgt tgtttctggc gcgcgattcg
ttccattggg aatcatattc ttctggagcg 2100tgcgccacgt atatgcttct
tgttacttaa gctgtgattg ggctgtttgc aacgatgcgt 2160tctgtttcac
atctggcact tgtgctacct tcaacgacgt cttgtgtctg ccggttgcgg
2220cgcgcatatc gtcctgtggc catgctgtgc caccgcccga ccgtggttgg
gaggtgcccg 2280cagcgatgtc atgggcgatt tcgcgtacta ccggcttgac
gttcgatgtc ttttccttca 2340tccagtacct tcctactgtg cctggcaaca
attccgatat catttactgt ggtgaaccaa 2400gcttcttcgg ggacatcacg
ggtatctatt ggccttactt tttgcctggc atgttgctct 2460tgtacttgac
tcccctcctg ggtttaaggt taatgcttgc cggctttaat atagatggct
2520tgtttcccat acggcatgcc acggctgcac tgaggttctc gacttcgcgt
gtgaccttga 2580gtgtcgtatt tgctttccta atctatatat tatctcatcc
tgttaatgct gcgctcaata 2640gaatgttcct agcatctgca aatctagaga
tgatcttatc ctttgatacc tatcatgaga 2700ctgttcttta cgtcgtttgt
ctattgctct acctccaggt gtcgccccgt gcgggcttgg 2760ctgctatggt
ggccatcaag ctatctcgag gcctgttatt cgctgtggtg ttggcgcacg
2820gagtgtgccg acctgggcgg gtatttggtc ttgaggtttg cgcggacatc
tcttggttgg 2880tggagtttac tggcaactgc acttggtaca tgtcctgtgt
cttctctttt tggtgcgcag 2940tgtttgcctt caccagtcca cttggacgac
agtataagct tcagatctat cggtactggg 3000cgcaggccta tgccagactc
atcctcgctg tcggttgtgg tcctctcggg agggagttcc 3060atttccgtgc
gagcgtgggc gtgctctggt gtggtgcttg catgctctgg ccccgtgagt
3120gctctgaaat cagcttggtc tttattctgt gtgctctgac tgtggacacc
atagacacat 3180ggttagtagc gtgcttgtcc gcagggccaa gcgcgcgaac
ccttgcaact ctggccgatg 3240acatggcgcg cattggtgac caccgggcgt
tgcgcgccgt gttgcgttgc tttggatcac 3300gtggcacata catatacaac
cacatgggcc aggtctcaga acgggtggcg caagcagtca 3360gggatttcgg
cggttgcttg gaaccagtcg tgttggagga gcccaccttt actgaggtcg
3420tggatgatac aatgaatttg gtgtgtggac aattgcttgg aggtaaaccc
gtggtggccc 3480gctgcggcac gcgtgtctta gtgggacacc tcaaccctga
agacctgcca cctggtttcc 3540agctgagtgc tccggtggtt attaccaaac
caagcattgg tacgtggccc tttcttaagg 3600cgacactcac agggcgtgct
gaaacaccgg gatccggcca gatcgtggtg ttgtcttccc 3660tgacaggtcg
gtcaatgggt actgcagtga atggcacact gtatgcgacc ggccacggtg
3720ctggtgcgcg cggcctagcc acgtgcgctg gtttgaggac gccactttac
acggcattat 3780ctgaagatgt cgtggcctac tcttgccttc cgggcatgag
ctccctagag tcctgcaact 3840gctcgcccag ccgggtttgg gtggtgaaca
acaacggagg gttggtgtgt ggcagagtgg 3900agaaagacga cgtctgtttg
gactgtccca cgcacataga tcaactgcgg ggtgcttcgg 3960ggtcgccggt
tttgtgtgat cacggtcatg catacgcgtt gatgctcggt ggctactcta
4020ccagtggtat ttgtgcgcgt gtccggatag tccggccatg gcagaacgcc
tattcctcct 4080caggggggca aggcggaatg caggcgccag ctgtgacacc
aacatactct gaaatcacct 4140actatgcccc tactggttct ggtaaatcaa
caaaatatcc agtggaccta gtcaagcagg 4200gacacaaagt attagtcctt
ttaccaagtg tggctgtcgt caaaagtatg gctccttaca 4260ttaaggaaaa
atataagatt agacctgaaa ttagagctgg cacagggcct gacggtgtga
4320cggtcatcac tggcgagaac ttggcgtaca tgacctatgg ccgtttcctt
gtagatccgg 4380aaacgaatct gcggggttac gctgtagtca tctgcgacga
gtgccatgac acatcatcca 4440ccacgctact cggcatcggc gcagtgcgca
tgtatgctga gaaagctgga gtgaagaccg 4500ttgtattcgc cacagccact
cctgctggca ttcaagtgca gtcacatccc aacattgatg 4560aatatctatt
gactgataca ggcgacgtgg aattctacgg cgctaaaatt aaattggaca
4620acatcagaac tggtagacat gttatctttt gccactcgaa ggccaggtgt
gcggaactaa 4680cgcagcagct ctccggcctt ggtgttcgtg cagtgagttt
ttggcgcggc tgtgacatca 4740agagcattcc cgcctcagac tctattgttg
tagtggcaac tgatgcattg tccacaggct 4800acacagggaa ctttgattcg
gtcattgact gcgggtgttg cgtagagcaa actgtaacaa 4860ttgacatgga
ccccacgttc tccatctcgg cccgagtggt gccatgcact gctgcattgc
4920gtatgcagcg gcgcggacgc accggtcgtg gcaggagggg agcgtactac
acaaccactc 4980caggagcagc accctgcgtc agcgttcccg atgctaacgt
ctggcaatca gtggagtcag 5040ccatggtctt ttatgattgg agtgctgcca
ggatagagca atgcctggcg gcataccatg 5100atttagggtg cacaccacgc
atcagttgtg acccacacac tccagtgcgg gtgatggaca 5160cactgagggc
gtatctgcgc agacctgagg tgacgaccgc ggctctcgca ggagagcagt
5220ggccgctgct ttacggcgtg cagttgtgca tctgcaaaga gaccgaggcc
cacggtccag 5280acgatggcat caagtggaaa tgcttactca ataacaacaa
caaaacaccc ctgttgtatg 5340ccttagacaa tcctacactg gaattcacta
cccaacatga cttgactcgc cgtatagctg 5400gcgctttatc gagcacagtg
ttcgtggaga caggctacgg ccccatcctc ctcgctggcg 5460ctgctttggc
tgcctccttt gcctttgcgg gcgccactgg agctttagtg ccgtcggccg
5520tttggagcgt tgaaaacggg cttgctggcg tgacccgtcc cgatgcgaca
gacgagaccg 5580cggcctacgc gcagcgcttg taccaagcct gcgcagattc
aggaattctc gccagcttgc 5640agggtacggc gagtgcggca ctgagcagac
tggccgatgc cagtaagggt gctagtcaat 5700atctggcagc cgcgcctcct
tcgcccgccc ccctggtaca ggtgctgcag ttcctcgaga 5760ccaattttag
ctccattgca tctttcggtc tgctctgtgc cggctgtcag gccggcgagt
5820gcttcactgc gcttgccggg ttggtgtccg gtgctacagc tggcttggga
ggtgcccata 5880agtggttgtt agctattgca ggaacttggc tagttagctt
gcagactggg ccccgtggcg 5940gcatggttgc gggtctctca gttctagcag
gctgttgcat cggtagtgtc accgggcttg 6000acttcctgtt tgggtgcctt
acaggttggg aggccgtggt cggtgctgcg gttgcaacgc 6060agaaaatctt
gtctggttcg gctgacatga ccactctggt agatctccta cctgctctct
6120tctcccctgg cgccggcata gctggcgtcg tgcttgtctt tattctaagc
aactcaagtg 6180taaccatgtg ggctaatcgg ctattgtcca tgtgtgcaaa
acaaactatt tgtgaaaatt 6240acttcttaac tgagaaattt ggccaacaat
taagcaaact ttccctgtgg cgctctgtgt 6300accattgggc gcaggcacgt
gaaggataca cacagtgcgg tgtggtcagc gggatctgga 6360gctttgtctt
gtgcattcta cgtgctgtgt gggattgggc ggctaaacat gtgccacggt
6420tccgtgtgcc tatgattggc tgctcacctg cgtggtgcgg gcgctggctt
ggtactggca 6480ccttgttgac cacctgtggg tgtggagaac gtgtatccct
tcagtgcctt tgctcgacat 6540ctgacccaac actcagtgtg ggccgttggt
gttggtgtag ttggcgtgtt gggttcccat 6600tcaacccgac gacgacagcc
accggcactt tacggccgga catcagtgac gccaccaaat 6660tgggcttccg
gtatggtgtc gccgagatcg tggagctaga gcggcggggc aacaaatggc
6720atgtctgtgc agcatcatgt tgcttggacc gggccagcgt tgcatccgcc
gtgagggccc 6780caccggtcac ggccgatggc atacctatca gtaccttttc
tccaccacaa acttacaaac 6840tctctctttg ttcttttgat tcagtttgca
tgactactaa cttatgtaat ccagctaaga 6900ccctgagtgt gtgctcgcag
gaggctgttg agctactgga agaaacagtt gacagagcac 6960aagtagtgat
gtgtcaaaat ctggaggcgc gaagacgcgc tgagtttgat gcatggcaag
7020ttcgcgaagc aattcgcgac gagtacacgc gtttggcaga cgaggatgtt
gacgcgacaa 7080cgtcggtgaa acccccggtg gccaaggctg ctgtgggtag
ctcgacgttg gatgatgtta 7140gcgtgctgac tgtcttgcgc gaactcggtg
accagtgcca aaatgctatc aaatttgtag 7200ttcaggcggc ttcacggttt
gttccaccag tgcccaagcc acgcacgcgt gtctcgggtg 7260tgttggagcg
tgtgcgcatg tgcatgcgca cgccaccaat caagtttgag gctgccgcag
7320taccaattca tgatataatc ccagaagagt gtcacattgt gctacgctgt
accggctgca 7380acgaccaggc cttgactgtt ccgtacggca cttgcactca
gtctttaatc aagcatttga 7440ctagtaaaca cagtcactac attccaaaac
agaagataga agaggacaca gaagtaactg 7500tcatttgcgc cgtaccaaca
acgcgcgcaa gcaaactcat cacattcaga gcaggtgatc 7560gatcagtctc
atgttgtcac cccttgcaaa cccctattag ggccctgctt ctaaagtacg
7620ggttacctat cgggaagtgg tctgactgca acgggcccct tggtgacgat
gctcgagtct 7680gtgacgtcaa tggagtaaca acttatgaac catgcatgca
atcctacagt tggtttcgac 7740cgattgtggc accaacaacc ccacctttgc
ctgcaacccg gaccgtggct ggcattttac 7800gcgcagacac atcgcgcgtt
tacaccacaa cggcggttga cgtctccgag cggcaggcca 7860aggtcacaat
tgatcaaaca tcagccaagg tggatcagtg tttccgagac acatacaatt
7920gctgccttgc taaggcaaag accttcagac aatctggcat gtcatatgag
gatgctgtgt 7980caaagatgcg cgcaaacacc acgcgtgacc ataacaacgg
catcacttat tcagatttgg 8040tctctggacg cgcaaaacct gtcgttcaga
aaattgtaaa tcaaatgcgc gccggagtgt 8100acgacgctcc gatgcgcatt
atcccaaaac ctgaagtgtt ccctcgagac aaaacaacac 8160ggaagccacc
gaggttcatc gttttccctg ggtgcgccgc gcgagtcgcg gagaaaatga
8220tcctgggtga tcctggcgcg ataaccaagc acgtgctagg tgatgcctac
gggtttgcca 8280ctccgccgca tgagcgcgcg cgcctgttgg aacaatggtg
gaaccgcgca acggagccac 8340aagctatcgc ggttgatgcg atctgctttg
atagcaccat cacggcagag gacatggatc 8400gtgaggctaa catcgtggct
gcagcgcata cggaccctga aggtgttcac ggcctatata 8460attattacaa
aagaagcccc atgtgtgaca tcacggggaa ggttgtcgga gtgcgttgct
8520gtcgagcctc gggtacgctt acaacaagca gtggcaacac gcttacttgc
taccttaagg 8580ttcgtgcagc ttgcacgcgc tccggcatta aaccaattgg
cttactaatt catggagatg 8640acaccctcat cgtcacagaa cgttgcgctc
aagagactct cgatgagttc agcaacgcac 8700ttgatgacta tgggttccca
cacaccatcc aggcgtctgg ggacctctcg tctatcgagt 8760gctgtagcgc
acgtgtggac agcgtttgcc tccggggagg tatgcgtcgc atgcttgtgc
8820cacaagctcg acgtgcgatt gcacgcgttc tcggggaaaa gggcgatcca
ctgggtacca 8880tcggtagcta tgttgtcatg tatcccactg cggccgtgac
tgtctacgtg ctattgcccc 8940tgttgtgcat gctcatacga aatgagccat
cacagacggg gacacttgtg acgctgacgg 9000tccacggtaa cagtgtgagt
gtgccagcgt ggctgcttcc aaccatcatt gcaaatttac 9060atggtcgtga
cgcactacag gtagtccgtc acagtgcagc ttccatggcg gaattgtcat
9120cagcgttggc cttctttggc atgagagggt tgaattgctg gaggcggaga
cgccgtgcca 9180ttagggctga tatgatcaag tcgggcgggt ggaatgcgaa
tttcgcgcag atgttactgt 9240ggtcaccgga ggtaagaaca ccacaacccg
aaccaagggg tctgtgtctt ttgccgccgg 9300aactgtggga gcgtccgtac
gaaaatttgc acttgagcac gatcgaccgc aatcgtggtg 9360ctagtcgctt
acggttttgg ttggttgcta gtgctatact cgctctgctt tgcttgtaaa
9420tcttaaatca a 943176124PRTHuman Pegivirus 2 76Met Glu Gly Met
Pro Thr Gly Thr Leu Ile Val Gln Arg Asp Gly Ser1 5 10 15Leu His Trp
Cys His Ala Arg His His Ser Val Gln Pro Asp Arg Val 20 25 30Ala Ala
Gly Pro Pro Ser Val Thr Ser Val Glu Arg Asn Met Gly Cys 35 40 45Ser
Thr Asp Gln Thr Ile Cys Ser Pro Val Val Glu Ala Asp Tyr Asn 50 55
60Thr Ser Ser Gly Cys Arg Ala Leu Asn Gly Ser Tyr His Cys Gly Gly65
70 75 80Gly Ser Cys Arg Ser Pro Ser Arg Val Gln Val Ala Gly Arg Val
Leu 85 90 95Arg Leu Cys Ala Phe Leu Ala Leu Ile Gly Ser Gly Met Cys
Ser Ile 100 105 110Arg Ser Lys Asn Glu Gly Arg Ile Glu Ser Gly Gln
115 12077191PRTHuman Pegivirus 2 77Ile Leu Gln Ser Gln Arg Ala Cys
Trp Thr Gly Glu Gly Phe Ala Phe1 5 10 15Phe Ser Asn Cys Cys Asn Gln
Ser Asp Ile Met Trp Cys Leu His Arg 20 25 30Trp Cys Val Thr Arg Pro
Gly Cys Leu Val Cys Thr Gly Asn Ala Thr 35 40 45His Pro Val Cys Trp
Asp Tyr Leu Gly Ser Gly Val Ser Arg Arg Pro 50
55 60Ala Arg Arg Met Gly Glu Gly Ala Glu Val Leu Leu Arg Leu Ile
Gly65 70 75 80Ile Ala Gly Trp Leu Gly Leu Leu Ala Glu Ala Leu Gly
Met Ser Glu 85 90 95Ile Tyr Ala Ala Phe Leu Cys Phe Gly Phe Ile Ala
Trp Tyr Gly Trp 100 105 110Gly Ile Pro Lys Thr Leu Val Cys Thr Val
Cys Pro Ala Val Asn Ile 115 120 125Ser Pro Tyr Ser Phe Leu Ser Pro
Asp Thr Ile Ala Phe Gly Thr Trp 130 135 140Leu Leu Gln Leu Pro Gly
Leu Leu Trp Gln Met Phe Val Ser Phe Pro145 150 155 160Ile Leu Tyr
Ser Thr Trp Ile Leu Trp Leu Leu Leu Ser Gly Lys Thr 165 170 175Val
Ala Val Ile Ala Ile Leu Leu Ala Ser Pro Thr Val Met Ala 180 185
19078354PRTHuman Pegivirus 2 78Tyr Lys His Gln Ala Asp Ser Tyr Leu
Lys Tyr Cys Thr Ile Thr Asn1 5 10 15Ala Ser Thr Ala Met Asn Cys Asp
Cys Pro Phe Gly Thr Phe Thr Arg 20 25 30Asn Thr Glu Ser Gly Phe Thr
Ile Pro Arg Phe Cys Pro Val Lys Leu 35 40 45Asn Ser Ser Thr Phe Ile
Cys Ser Trp Gly Ser Trp Trp Trp Phe Ala 50 55 60Glu Asn Ile Thr Arg
Pro Tyr Ser Asp Val Gly Met Pro Pro Ala Pro65 70 75 80Ile Ser Ala
Leu Cys Tyr Ile Tyr Ser Asn Asn Asp Pro Pro Ser Trp 85 90 95Tyr Arg
Asn Thr Thr Ile Ile Pro Gln Asn Cys Tyr Asn Ser Thr Ala 100 105
110Asp Pro Thr Thr Ala Pro Cys Arg Asp Lys Trp Gly Asn Ala Thr Ala
115 120 125Cys Ile Leu Asp Arg Arg Ser Arg Phe Cys Gly Asp Cys Tyr
Gly Gly 130 135 140Cys Phe Tyr Thr Asn Gly Ser His Asp Arg Ser Trp
Asp Arg Cys Gly145 150 155 160Ile Gly Tyr Arg Asp Gly Leu Ile Glu
Phe Val Gln Leu Gly Gln Ile 165 170 175Arg Pro Asn Ile Ala Asn Thr
Thr Ile Glu Leu Leu Ala Gly Ala Ser 180 185 190Leu Val Ile Ala Ser
Gly Leu Arg Ala Gly Tyr Gly Cys Ser Arg Ala 195 200 205His Gly Val
Val His Cys Phe Lys Cys Pro Ser Tyr Arg Asp Leu Glu 210 215 220Arg
Phe Gly Pro Gly Leu Gly Lys Trp Val Pro Leu Pro Gly Glu Pro225 230
235 240Val Pro Glu Leu Cys Ile Asn Pro Gln Trp Ala Arg Arg Gly Phe
Arg 245 250 255Val Ser Asn Asn Pro Leu Ser Val Leu Gln Thr Phe Val
Glu Asp Ile 260 265 270Phe Leu Ala Pro Phe Cys Asn Pro Thr Pro Gly
Arg Val Arg Val Cys 275 280 285Asn Asn Thr Ala Phe Tyr Pro Arg Gly
Gly Gly Phe Val Gln Leu Ile 290 295 300Gly Asp Val Gln Val Leu Thr
Pro Asn Ser Thr Ser Leu His Ser Leu305 310 315 320Leu Thr Leu Ile
Ser Leu Ile Leu Leu Val Cys Val Val Ser Gly Ala 325 330 335Arg Phe
Val Pro Leu Gly Ile Ile Phe Phe Trp Ser Val Arg His Val 340 345
350Tyr Ala79237PRTHuman Pegivirus 2 79Ser Cys Tyr Leu Ser Cys Asp
Trp Ala Val Cys Asn Asp Ala Phe Cys1 5 10 15Phe Thr Ser Gly Thr Cys
Ala Thr Phe Asn Asp Val Leu Cys Leu Pro 20 25 30Val Ala Ala Arg Ile
Ser Ser Cys Gly His Ala Val Pro Pro Pro Asp 35 40 45Arg Gly Trp Glu
Val Pro Ala Ala Met Ser Trp Ala Ile Ser Arg Thr 50 55 60Thr Gly Leu
Thr Phe Asp Val Phe Ser Phe Ile Gln Tyr Leu Pro Thr65 70 75 80Val
Pro Gly Asn Asn Ser Asp Ile Ile Tyr Cys Gly Glu Pro Ser Phe 85 90
95Phe Gly Asp Ile Thr Gly Ile Tyr Trp Pro Tyr Phe Leu Pro Gly Met
100 105 110Leu Leu Leu Tyr Leu Thr Pro Leu Leu Gly Leu Arg Leu Met
Leu Ala 115 120 125Gly Phe Asn Ile Asp Gly Leu Phe Pro Ile Arg His
Ala Thr Ala Ala 130 135 140Leu Arg Phe Ser Thr Ser Arg Val Thr Leu
Ser Val Val Phe Ala Phe145 150 155 160Leu Ile Tyr Ile Leu Ser His
Pro Val Asn Ala Ala Leu Asn Arg Met 165 170 175Phe Leu Ala Ser Ala
Asn Leu Glu Met Ile Leu Ser Phe Asp Thr Tyr 180 185 190His Glu Thr
Val Leu Tyr Val Val Cys Leu Leu Leu Tyr Leu Gln Val 195 200 205Ser
Pro Arg Ala Gly Leu Ala Ala Met Val Ala Ile Lys Leu Ser Arg 210 215
220Gly Leu Leu Phe Ala Val Val Leu Ala His Gly Val Cys225 230
23580240PRTHuman Pegivirus 2 80Arg Pro Gly Arg Val Phe Gly Leu Glu
Val Cys Ala Asp Ile Ser Trp1 5 10 15Leu Val Glu Phe Thr Gly Asn Cys
Thr Trp Tyr Met Ser Cys Val Phe 20 25 30Ser Phe Trp Cys Ala Val Phe
Ala Phe Thr Ser Pro Leu Gly Arg Gln 35 40 45Tyr Lys Leu Gln Ile Tyr
Arg Tyr Trp Ala Gln Ala Tyr Ala Arg Leu 50 55 60Ile Leu Ala Val Gly
Cys Gly Pro Leu Gly Arg Glu Phe His Phe Arg65 70 75 80Ala Ser Val
Gly Val Leu Trp Cys Gly Ala Cys Met Leu Trp Pro Arg 85 90 95Glu Cys
Ser Glu Ile Ser Leu Val Phe Ile Leu Cys Ala Leu Thr Val 100 105
110Asp Thr Ile Asp Thr Trp Leu Val Ala Cys Leu Ser Ala Gly Pro Ser
115 120 125Ala Arg Thr Leu Ala Thr Leu Ala Asp Asp Met Ala Arg Ile
Gly Asp 130 135 140His Arg Ala Leu Arg Ala Val Leu Arg Cys Phe Gly
Ser Arg Gly Thr145 150 155 160Tyr Ile Tyr Asn His Met Gly Gln Val
Ser Glu Arg Val Ala Gln Ala 165 170 175Val Arg Asp Phe Gly Gly Cys
Leu Glu Pro Val Val Leu Glu Glu Pro 180 185 190Thr Phe Thr Glu Val
Val Asp Asp Thr Met Asn Leu Val Cys Gly Gln 195 200 205Leu Leu Gly
Gly Lys Pro Val Val Ala Arg Cys Gly Thr Arg Val Leu 210 215 220Val
Gly His Leu Asn Pro Glu Asp Leu Pro Pro Gly Phe Gln Leu Ser225 230
235 24081628PRTHuman Pegivirus 2 81Ala Pro Val Val Ile Thr Lys Pro
Ser Ile Gly Thr Trp Pro Phe Leu1 5 10 15Lys Ala Thr Leu Thr Gly Arg
Ala Glu Thr Pro Gly Ser Gly Gln Ile 20 25 30Val Val Leu Ser Ser Leu
Thr Gly Arg Ser Met Gly Thr Ala Val Asn 35 40 45Gly Thr Leu Tyr Ala
Thr Gly His Gly Ala Gly Ala Arg Gly Leu Ala 50 55 60Thr Cys Ala Gly
Leu Arg Thr Pro Leu Tyr Thr Ala Leu Ser Glu Asp65 70 75 80Val Val
Ala Tyr Ser Cys Leu Pro Gly Met Ser Ser Leu Glu Ser Cys 85 90 95Asn
Cys Ser Pro Ser Arg Val Trp Val Val Asn Asn Asn Gly Gly Leu 100 105
110Val Cys Gly Arg Val Glu Lys Asp Asp Val Cys Leu Asp Cys Pro Thr
115 120 125His Ile Asp Gln Leu Arg Gly Ala Ser Gly Ser Pro Val Leu
Cys Asp 130 135 140His Gly His Ala Tyr Ala Leu Met Leu Gly Gly Tyr
Ser Thr Ser Gly145 150 155 160Ile Cys Ala Arg Val Arg Ile Val Arg
Pro Trp Gln Asn Ala Tyr Ser 165 170 175Ser Ser Gly Gly Gln Gly Gly
Met Gln Ala Pro Ala Val Thr Pro Thr 180 185 190Tyr Ser Glu Ile Thr
Tyr Tyr Ala Pro Thr Gly Ser Gly Lys Ser Thr 195 200 205Lys Tyr Pro
Val Asp Leu Val Lys Gln Gly His Lys Val Leu Val Leu 210 215 220Leu
Pro Ser Val Ala Val Val Lys Ser Met Ala Pro Tyr Ile Lys Glu225 230
235 240Lys Tyr Lys Ile Arg Pro Glu Ile Arg Ala Gly Thr Gly Pro Asp
Gly 245 250 255Val Thr Val Ile Thr Gly Glu Asn Leu Ala Tyr Met Thr
Tyr Gly Arg 260 265 270Phe Leu Val Asp Pro Glu Thr Asn Leu Arg Gly
Tyr Ala Val Val Ile 275 280 285Cys Asp Glu Cys His Asp Thr Ser Ser
Thr Thr Leu Leu Gly Ile Gly 290 295 300Ala Val Arg Met Tyr Ala Glu
Lys Ala Gly Val Lys Thr Val Val Phe305 310 315 320Ala Thr Ala Thr
Pro Ala Gly Ile Gln Val Gln Ser His Pro Asn Ile 325 330 335Asp Glu
Tyr Leu Leu Thr Asp Thr Gly Asp Val Glu Phe Tyr Gly Ala 340 345
350Lys Ile Lys Leu Asp Asn Ile Arg Thr Gly Arg His Val Ile Phe Cys
355 360 365His Ser Lys Ala Arg Cys Ala Glu Leu Thr Gln Gln Leu Ser
Gly Leu 370 375 380Gly Val Arg Ala Val Ser Phe Trp Arg Gly Cys Asp
Ile Lys Ser Ile385 390 395 400Pro Ala Ser Asp Ser Ile Val Val Val
Ala Thr Asp Ala Leu Ser Thr 405 410 415Gly Tyr Thr Gly Asn Phe Asp
Ser Val Ile Asp Cys Gly Cys Cys Val 420 425 430Glu Gln Thr Val Thr
Ile Asp Met Asp Pro Thr Phe Ser Ile Ser Ala 435 440 445Arg Val Val
Pro Cys Thr Ala Ala Leu Arg Met Gln Arg Arg Gly Arg 450 455 460Thr
Gly Arg Gly Arg Arg Gly Ala Tyr Tyr Thr Thr Thr Pro Gly Ala465 470
475 480Ala Pro Cys Val Ser Val Pro Asp Ala Asn Val Trp Gln Ser Val
Glu 485 490 495Ser Ala Met Val Phe Tyr Asp Trp Ser Ala Ala Arg Ile
Glu Gln Cys 500 505 510Leu Ala Ala Tyr His Asp Leu Gly Cys Thr Pro
Arg Ile Ser Cys Asp 515 520 525Pro His Thr Pro Val Arg Val Met Asp
Thr Leu Arg Ala Tyr Leu Arg 530 535 540Arg Pro Glu Val Thr Thr Ala
Ala Leu Ala Gly Glu Gln Trp Pro Leu545 550 555 560Leu Tyr Gly Val
Gln Leu Cys Ile Cys Lys Glu Thr Glu Ala His Gly 565 570 575Pro Asp
Asp Gly Ile Lys Trp Lys Cys Leu Leu Asn Asn Asn Asn Lys 580 585
590Thr Pro Leu Leu Tyr Ala Leu Asp Asn Pro Thr Leu Glu Phe Thr Thr
595 600 605Gln His Asp Leu Thr Arg Arg Ile Ala Gly Ala Leu Ser Ser
Thr Val 610 615 620Phe Val Glu Thr6258241PRTHuman Pegivirus 2 82Gly
Tyr Gly Pro Ile Leu Leu Ala Gly Ala Ala Leu Ala Ala Ser Phe1 5 10
15Ala Phe Ala Gly Ala Thr Gly Ala Leu Val Pro Ser Ala Val Trp Ser
20 25 30Val Glu Asn Gly Leu Ala Gly Val Thr 35 4083262PRTHuman
Pegivirus 2 83Arg Pro Asp Ala Thr Asp Glu Thr Ala Ala Tyr Ala Gln
Arg Leu Tyr1 5 10 15Gln Ala Cys Ala Asp Ser Gly Ile Leu Ala Ser Leu
Gln Gly Thr Ala 20 25 30Ser Ala Ala Leu Ser Arg Leu Ala Asp Ala Ser
Lys Gly Ala Ser Gln 35 40 45Tyr Leu Ala Ala Ala Pro Pro Ser Pro Ala
Pro Leu Val Gln Val Leu 50 55 60Gln Phe Leu Glu Thr Asn Phe Ser Ser
Ile Ala Ser Phe Gly Leu Leu65 70 75 80Cys Ala Gly Cys Gln Ala Gly
Glu Cys Phe Thr Ala Leu Ala Gly Leu 85 90 95Val Ser Gly Ala Thr Ala
Gly Leu Gly Gly Ala His Lys Trp Leu Leu 100 105 110Ala Ile Ala Gly
Thr Trp Leu Val Ser Leu Gln Thr Gly Pro Arg Gly 115 120 125Gly Met
Val Ala Gly Leu Ser Val Leu Ala Gly Cys Cys Ile Gly Ser 130 135
140Val Thr Gly Leu Asp Phe Leu Phe Gly Cys Leu Thr Gly Trp Glu
Ala145 150 155 160Val Val Gly Ala Ala Val Ala Thr Gln Lys Ile Leu
Ser Gly Ser Ala 165 170 175Asp Met Thr Thr Leu Val Asp Leu Leu Pro
Ala Leu Phe Ser Pro Gly 180 185 190Ala Gly Ile Ala Gly Val Val Leu
Val Phe Ile Leu Ser Asn Ser Ser 195 200 205Val Thr Met Trp Ala Asn
Arg Leu Leu Ser Met Cys Ala Lys Gln Thr 210 215 220Ile Cys Glu Asn
Tyr Phe Leu Thr Glu Lys Phe Gly Gln Gln Leu Ser225 230 235 240Lys
Leu Ser Leu Trp Arg Ser Val Tyr His Trp Ala Gln Ala Arg Glu 245 250
255Gly Tyr Thr Gln Cys Gly 26084458PRTHuman Pegivirus 2 84Val Val
Ser Gly Ile Trp Ser Phe Val Leu Cys Ile Leu Arg Ala Val1 5 10 15Trp
Asp Trp Ala Ala Lys His Val Pro Arg Phe Arg Val Pro Met Ile 20 25
30Gly Cys Ser Pro Ala Trp Cys Gly Arg Trp Leu Gly Thr Gly Thr Leu
35 40 45Leu Thr Thr Cys Gly Cys Gly Glu Arg Val Ser Leu Gln Cys Leu
Cys 50 55 60Ser Thr Ser Asp Pro Thr Leu Ser Val Gly Arg Trp Cys Trp
Cys Ser65 70 75 80Trp Arg Val Gly Phe Pro Phe Asn Pro Thr Thr Thr
Ala Thr Gly Thr 85 90 95Leu Arg Pro Asp Ile Ser Asp Ala Thr Lys Leu
Gly Phe Arg Tyr Gly 100 105 110Val Ala Glu Ile Val Glu Leu Glu Arg
Arg Gly Asn Lys Trp His Val 115 120 125Cys Ala Ala Ser Cys Cys Leu
Asp Arg Ala Ser Val Ala Ser Ala Val 130 135 140Arg Ala Pro Pro Val
Thr Ala Asp Gly Ile Pro Ile Ser Thr Phe Ser145 150 155 160Pro Pro
Gln Thr Tyr Lys Leu Ser Leu Cys Ser Phe Asp Ser Val Cys 165 170
175Met Thr Thr Asn Leu Cys Asn Pro Ala Lys Thr Leu Ser Val Cys Ser
180 185 190Gln Glu Ala Val Glu Leu Leu Glu Glu Thr Val Asp Arg Ala
Gln Val 195 200 205Val Met Cys Gln Asn Leu Glu Ala Arg Arg Arg Ala
Glu Phe Asp Ala 210 215 220Trp Gln Val Arg Glu Ala Ile Arg Asp Glu
Tyr Thr Arg Leu Ala Asp225 230 235 240Glu Asp Val Asp Ala Thr Thr
Ser Val Lys Pro Pro Val Ala Lys Ala 245 250 255Ala Val Gly Ser Ser
Thr Leu Asp Asp Val Ser Val Leu Thr Val Leu 260 265 270Arg Glu Leu
Gly Asp Gln Cys Gln Asn Ala Ile Lys Phe Val Val Gln 275 280 285Ala
Ala Ser Arg Phe Val Pro Pro Val Pro Lys Pro Arg Thr Arg Val 290 295
300Ser Gly Val Leu Glu Arg Val Arg Met Cys Met Arg Thr Pro Pro
Ile305 310 315 320Lys Phe Glu Ala Ala Ala Val Pro Ile His Asp Ile
Ile Pro Glu Glu 325 330 335Cys His Ile Val Leu Arg Cys Thr Gly Cys
Asn Asp Gln Ala Leu Thr 340 345 350Val Pro Tyr Gly Thr Cys Thr Gln
Ser Leu Ile Lys His Leu Thr Ser 355 360 365Lys His Ser His Tyr Ile
Pro Lys Gln Lys Ile Glu Glu Asp Thr Glu 370 375 380Val Thr Val Ile
Cys Ala Val Pro Thr Thr Arg Ala Ser Lys Leu Ile385 390 395 400Thr
Phe Arg Ala Gly Asp Arg Ser Val Ser Cys Cys His Pro Leu Gln 405 410
415Thr Pro Ile Arg Ala Leu Leu Leu Lys Tyr Gly Leu Pro Ile Gly Lys
420 425 430Trp Ser Asp Cys Asn Gly Pro Leu Gly Asp Asp Ala Arg Val
Cys Asp 435 440 445Val Asn Gly Val Thr Thr Tyr Glu Pro Cys 450
45585567PRTHuman Pegivirus 2 85Met Gln Ser Tyr Ser Trp Phe Arg Pro
Ile Val Ala Pro Thr Thr Pro1 5 10 15Pro Leu Pro Ala Thr Arg Thr Val
Ala Gly Ile Leu Arg Ala Asp Thr 20 25 30Ser Arg Val Tyr Thr Thr Thr
Ala Val Asp Val Ser Glu Arg Gln Ala 35 40 45Lys Val Thr Ile Asp Gln
Thr Ser Ala Lys Val Asp Gln Cys Phe Arg 50 55 60Asp Thr Tyr Asn Cys
Cys Leu Ala Lys Ala Lys Thr Phe Arg Gln Ser65 70 75 80Gly Met Ser
Tyr Glu Asp Ala Val Ser Lys Met Arg Ala Asn Thr Thr 85 90 95Arg
Asp
His Asn Asn Gly Ile Thr Tyr Ser Asp Leu Val Ser Gly Arg 100 105
110Ala Lys Pro Val Val Gln Lys Ile Val Asn Gln Met Arg Ala Gly Val
115 120 125Tyr Asp Ala Pro Met Arg Ile Ile Pro Lys Pro Glu Val Phe
Pro Arg 130 135 140Asp Lys Thr Thr Arg Lys Pro Pro Arg Phe Ile Val
Phe Pro Gly Cys145 150 155 160Ala Ala Arg Val Ala Glu Lys Met Ile
Leu Gly Asp Pro Gly Ala Ile 165 170 175Thr Lys His Val Leu Gly Asp
Ala Tyr Gly Phe Ala Thr Pro Pro His 180 185 190Glu Arg Ala Arg Leu
Leu Glu Gln Trp Trp Asn Arg Ala Thr Glu Pro 195 200 205Gln Ala Ile
Ala Val Asp Ala Ile Cys Phe Asp Ser Thr Ile Thr Ala 210 215 220Glu
Asp Met Asp Arg Glu Ala Asn Ile Val Ala Ala Ala His Thr Asp225 230
235 240Pro Glu Gly Val His Gly Leu Tyr Asn Tyr Tyr Lys Arg Ser Pro
Met 245 250 255Cys Asp Ile Thr Gly Lys Val Val Gly Val Arg Cys Cys
Arg Ala Ser 260 265 270Gly Thr Leu Thr Thr Ser Ser Gly Asn Thr Leu
Thr Cys Tyr Leu Lys 275 280 285Val Arg Ala Ala Cys Thr Arg Ser Gly
Ile Lys Pro Ile Gly Leu Leu 290 295 300Ile His Gly Asp Asp Thr Leu
Ile Val Thr Glu Arg Cys Ala Gln Glu305 310 315 320Thr Leu Asp Glu
Phe Ser Asn Ala Leu Asp Asp Tyr Gly Phe Pro His 325 330 335Thr Ile
Gln Ala Ser Gly Asp Leu Ser Ser Ile Glu Cys Cys Ser Ala 340 345
350Arg Val Asp Ser Val Cys Leu Arg Gly Gly Met Arg Arg Met Leu Val
355 360 365Pro Gln Ala Arg Arg Ala Ile Ala Arg Val Leu Gly Glu Lys
Gly Asp 370 375 380Pro Leu Gly Thr Ile Gly Ser Tyr Val Val Met Tyr
Pro Thr Ala Ala385 390 395 400Val Thr Val Tyr Val Leu Leu Pro Leu
Leu Cys Met Leu Ile Arg Asn 405 410 415Glu Pro Ser Gln Thr Gly Thr
Leu Val Thr Leu Thr Val His Gly Asn 420 425 430Ser Val Ser Val Pro
Ala Trp Leu Leu Pro Thr Ile Ile Ala Asn Leu 435 440 445His Gly Arg
Asp Ala Leu Gln Val Val Arg His Ser Ala Ala Ser Met 450 455 460Ala
Glu Leu Ser Ser Ala Leu Ala Phe Phe Gly Met Arg Gly Leu Asn465 470
475 480Cys Trp Arg Arg Arg Arg Arg Ala Ile Arg Ala Asp Met Ile Lys
Ser 485 490 495Gly Gly Trp Asn Ala Asn Phe Ala Gln Met Leu Leu Trp
Ser Pro Glu 500 505 510Val Arg Thr Pro Gln Pro Glu Pro Arg Gly Leu
Cys Leu Leu Pro Pro 515 520 525Glu Leu Trp Glu Arg Pro Tyr Glu Asn
Leu His Leu Ser Thr Ile Asp 530 535 540Arg Asn Arg Gly Ala Ser Arg
Leu Arg Phe Trp Leu Val Ala Ser Ala545 550 555 560Ile Leu Ala Leu
Leu Cys Leu 5658645PRTHuman Pegivirus 2 86Gly Gly Ser Cys Arg Ser
Pro Ser Arg Val Gln Val Ala Arg Arg Val1 5 10 15Leu Gln Leu Ser Ala
Phe Leu Ala Leu Ile Gly Ser Gly Met Ser Ser 20 25 30Ile Arg Ser Lys
Thr Glu Gly Arg Ile Glu Ser Gly Gln 35 40 458741PRTHuman Pegivirus
2 87Arg Asp Gly Ser Leu His Trp Ser His Ala Arg His His Ser Val
Gln1 5 10 15Pro Asp Arg Val Ala Ala Gly Pro Pro Ser Val Thr Ser Val
Glu Arg 20 25 30Asn Met Gly Ser Ser Thr Asp Gln Thr 35
408831PRTHuman Pegivirus 2 88Ser Met Asn Ser Asp Ser Pro Phe Gly
Thr Phe Thr Arg Asn Thr Glu1 5 10 15Ser Arg Phe Ser Ile Pro Arg Phe
Ser Pro Val Lys Ile Asn Ser 20 25 308940PRTHuman Pegivirus 2 89Gln
Ala Pro Ala Val Thr Pro Thr Tyr Ser Glu Ile Thr Tyr Tyr Ala1 5 10
15Pro Thr Gly Ser Gly Lys Ser Thr Lys Tyr Pro Val Asp Leu Val Lys
20 25 30Gln Gly His Lys Val Leu Val Leu 35 409032PRTHuman Pegivirus
2 90Val Lys Ser Met Ala Pro Tyr Ile Lys Glu Thr Tyr Lys Ile Arg
Pro1 5 10 15Glu Ile Arg Ala Gly Thr Gly Pro Asp Gly Val Thr Val Ile
Thr Gly 20 25 309121PRTHuman Pegivirus 2 91Pro Glu Thr Asn Leu Arg
Gly Tyr Ala Val Val Ile Ser Asp Glu Ser1 5 10 15His Asp Thr Ser Ser
209234PRTHuman Pegivirus 2 92Pro Cys Thr Ala Ala Leu Arg Met Gln
Arg Arg Gly Arg Thr Gly Arg1 5 10 15Gly Arg Arg Gly Ala Tyr Tyr Thr
Thr Ser Pro Gly Ala Ala Pro Cys 20 25 30Val Ser9332PRTHuman
Pegivirus 2 93Leu Ser Glu Arg Phe Gly Gln Gln Leu Ser Lys Leu Ser
Leu Trp Arg1 5 10 15Ser Val Tyr His Trp Ala Gln Ala Arg Glu Gly Tyr
Thr Gln Cys Gly 20 25 309440PRTHuman Pegivirus 2 94Asn Pro Thr Thr
Thr Gly Thr Gly Thr Leu Arg Pro Asp Ile Ser Asp1 5 10 15Ala Asn Lys
Leu Gly Phe Arg Tyr Gly Val Ala Asp Ile Val Glu Leu 20 25 30Glu Arg
Arg Gly Asp Lys Trp His 35 409533PRTHuman Pegivirus 2 95Gln Asn Leu
Ala Ala Arg Arg Arg Ala Glu Tyr Asp Ala Trp Gln Val1 5 10 15Arg Gln
Ala Val Gly Asp Glu Tyr Thr Arg Leu Ala Asp Glu Asp Val 20 25
30Asp9631PRTHuman Pegivirus 2 96Arg Phe Val Pro Pro Val Pro Lys Pro
Arg Thr Arg Val Ser Gly Val1 5 10 15Leu Glu Arg Val Arg Met Cys Met
Arg Thr Pro Pro Ile Lys Phe 20 25 309722PRTHuman Pegivirus 2 97Asn
Thr Thr Arg Asp His Asn Asn Gly Ile Thr Tyr Thr Asp Leu Val1 5 10
15Ser Gly Arg Ala Lys Pro 209835PRTHuman Pegivirus 2 98Asp Ala Pro
Met Arg Ile Ile Pro Lys Pro Glu Val Phe Pro Arg Asp1 5 10 15Lys Ser
Thr Arg Lys Pro Pro Arg Phe Ile Val Phe Pro Gly Cys Ala 20 25 30Ala
Arg Val 359920PRTHuman Pegivirus 2 99Met Pro Leu Leu Cys Met Leu
Ile Arg Asn Glu Pro Ser Gln Thr Gly1 5 10 15Thr Leu Val Thr
2010045PRTHuman Pegivirus 2 100Gly Gly Ser Cys Arg Ser Pro Ser Arg
Val Gln Val Ala Arg Arg Val1 5 10 15Leu Gln Leu Ser Ala Phe Leu Ala
Leu Ile Gly Ser Gly Met Ser Ser 20 25 30Ile Arg Ser Lys Thr Glu Gly
Arg Ile Glu Ser Gly Gln 35 40 4510123PRTHuman Pegivirus 2 101Gly
Gly Ser Cys Arg Ser Pro Ser Arg Val Gln Val Ala Arg Arg Val1 5 10
15Leu Gln Leu Ser Ala Phe Leu 2010226PRTHuman Pegivirus 2 102Ser
Ala Phe Leu Ala Leu Ile Gly Ser Gly Met Ser Ser Ile Arg Ser1 5 10
15Lys Thr Glu Gly Arg Ile Glu Ser Gly Gln 20 2510320PRTHuman
Pegivirus 2 103Ala Arg Arg Val Leu Gln Leu Ser Ala Phe Leu Ala Leu
Ile Gly Ser1 5 10 15Gly Met Ser Ser 2010445PRTHuman Pegivirus 2
104Gly Gly Ser Cys Arg Ser Pro Ser Arg Val Gln Val Ala Arg Arg Val1
5 10 15Leu Gln Leu Cys Ala Phe Leu Ala Leu Ile Gly Ser Gly Met Cys
Ser 20 25 30Ile Arg Ser Lys Thr Glu Gly Arg Ile Glu Ser Gly Gln 35
40 4510523PRTHuman Pegivirus 2 105Gly Gly Ser Cys Arg Ser Pro Ser
Arg Val Gln Val Ala Arg Arg Val1 5 10 15Leu Gln Leu Cys Ala Phe Leu
2010626PRTHuman Pegivirus 2 106Cys Ala Phe Leu Ala Leu Ile Gly Ser
Gly Met Cys Ser Ile Arg Ser1 5 10 15Lys Thr Glu Gly Arg Ile Glu Ser
Gly Gln 20 2510720PRTHuman Pegivirus 2 107Ala Arg Arg Val Leu Gln
Leu Cys Ala Phe Leu Ala Leu Ile Gly Ser1 5 10 15Gly Met Cys Ser
2010845PRTHuman Pegivirus 2 108Gly Gly Ser Cys Arg Ser Pro Ser Arg
Val Gln Val Ala Gly Arg Val1 5 10 15Leu Arg Leu Cys Ala Phe Leu Ala
Leu Ile Gly Ser Gly Met Cys Ser 20 25 30Ile Arg Ser Lys Asn Glu Gly
Arg Ile Glu Ser Gly Gln 35 40 4510923PRTHuman Pegivirus 2 109Gly
Gly Ser Cys Arg Ser Pro Ser Arg Val Gln Val Ala Gly Arg Val1 5 10
15Leu Arg Leu Cys Ala Phe Leu 2011016PRTHuman Pegivirus 2 110Met
Cys Ser Ile Arg Ser Lys Asn Glu Gly Arg Ile Glu Ser Gly Gln1 5 10
1511120PRTHuman Pegivirus 2 111Val Ala Gly Arg Val Leu Arg Leu Cys
Ala Phe Leu Ala Leu Ile Gly1 5 10 15Ser Gly Met Cys 2011241PRTHuman
Pegivirus 2 112Arg Asp Gly Ser Leu His Trp Ser His Ala Arg His His
Ser Val Gln1 5 10 15Pro Asp Arg Val Ala Ala Gly Pro Pro Ser Val Thr
Ser Val Glu Arg 20 25 30Asn Met Gly Ser Ser Thr Asp Gln Thr 35
4011323PRTHuman Pegivirus 2 113Arg Asp Gly Ser Leu His Trp Ser His
Ala Arg His His Ser Val Gln1 5 10 15Pro Asp Arg Val Ala Ala Gly
2011441PRTHuman Pegivirus 2 114Arg Asp Gly Ser Leu His Trp Cys His
Ala Arg His His Ser Val Gln1 5 10 15Pro Asp Arg Val Ala Ala Gly Pro
Pro Ser Val Thr Ser Val Glu Arg 20 25 30Asn Met Gly Ser Ser Thr Asp
Gln Thr 35 4011523PRTHuman Pegivirus 2 115Arg Asp Gly Ser Leu His
Trp Cys His Ala Arg His His Ser Val Gln1 5 10 15Pro Asp Arg Val Ala
Ala Gly 2011622PRTHuman Pegivirus 2 116Val Ala Ala Gly Pro Pro Ser
Val Thr Ser Val Glu Arg Asn Met Gly1 5 10 15Ser Ser Thr Asp Gln Thr
2011721PRTHuman Pegivirus 2 117Arg His His Ser Val Gln Pro Asp Arg
Val Ala Ala Gly Pro Pro Ser1 5 10 15Val Thr Ser Val Glu
2011831PRTHuman Pegivirus 2 118Ser Met Asn Ser Asp Ser Pro Phe Gly
Thr Phe Thr Arg Asn Thr Glu1 5 10 15Ser Arg Phe Ser Ile Pro Arg Phe
Ser Pro Val Lys Ile Asn Ser 20 25 3011931PRTHuman Pegivirus 2
119Ser Met Asn Cys Asp Cys Pro Phe Gly Thr Phe Thr Arg Asn Thr Glu1
5 10 15Ser Arg Phe Ser Ile Pro Arg Phe Cys Pro Val Lys Ile Asn Ser
20 25 3012019PRTHuman Pegivirus 2 120Ser Met Asn Ser Asp Ser Pro
Phe Gly Thr Phe Thr Arg Asn Thr Glu1 5 10 15Ser Arg
Phe12119PRTHuman Pegivirus 2 121Ser Met Asn Cys Asp Cys Pro Phe Gly
Thr Phe Thr Arg Asn Thr Glu1 5 10 15Ser Arg Phe12215PRTHuman
Pegivirus 2 122Ser Arg Phe Ser Ile Pro Arg Phe Ser Pro Val Lys Ile
Asn Ser1 5 10 1512316PRTHuman Pegivirus 2 123Phe Gly Thr Phe Thr
Arg Asn Thr Glu Ser Arg Phe Ser Ile Pro Arg1 5 10 1512431PRTHuman
Pegivirus 2 124Ala Met Asn Cys Asp Cys Pro Phe Gly Thr Phe Thr Arg
Asn Thr Glu1 5 10 15Ser Gly Phe Thr Ile Pro Arg Phe Cys Pro Val Lys
Leu Asn Ser 20 25 3012519PRTHuman Pegivirus 2 125Ala Met Asn Cys
Asp Cys Pro Phe Gly Thr Phe Thr Arg Asn Thr Glu1 5 10 15Ser Gly
Phe12615PRTHuman Pegivirus 2 126Ser Gly Phe Thr Ile Pro Arg Phe Cys
Pro Val Lys Leu Asn Ser1 5 10 1512716PRTHuman Pegivirus 2 127Phe
Gly Thr Phe Thr Arg Asn Thr Glu Ser Gly Phe Thr Ile Pro Arg1 5 10
1512831PRTHuman Pegivirus 2 128Ala Met Asn Cys Asp Cys Pro Phe Gly
Thr Phe Thr Arg Asn Thr Glu1 5 10 15Ser Gly Phe Ser Ile Ser Ile Asp
Ser Val Leu Leu Lys Ser Ile 20 25 3012940PRTHuman Pegivirus 2
129Gln Ala Pro Ala Val Thr Pro Thr Tyr Ser Glu Ile Thr Tyr Tyr Ala1
5 10 15Pro Thr Gly Ser Gly Lys Ser Thr Lys Tyr Pro Val Asp Leu Val
Lys 20 25 30Gln Gly His Lys Val Leu Val Leu 35 4013024PRTHuman
Pegivirus 2 130Gln Ala Pro Ala Val Thr Pro Thr Tyr Ser Glu Ile Thr
Tyr Tyr Ala1 5 10 15Pro Thr Gly Ser Gly Lys Ser Thr 2013120PRTHuman
Pegivirus 2 131Gly Lys Ser Thr Lys Tyr Pro Val Asp Leu Val Lys Gln
Gly His Lys1 5 10 15Val Leu Val Leu 2013223PRTHuman Pegivirus 2
132Ile Thr Tyr Tyr Ala Pro Thr Gly Ser Gly Lys Ser Thr Lys Tyr Pro1
5 10 15Val Asp Leu Val Lys Gln Gly 2013332PRTHuman Pegivirus 2
133Val Lys Ser Met Ala Pro Tyr Ile Lys Glu Thr Tyr Lys Ile Arg Pro1
5 10 15Glu Ile Arg Ala Gly Thr Gly Pro Asp Gly Val Thr Val Ile Thr
Gly 20 25 3013418PRTHuman Pegivirus 2 134Val Lys Ser Met Ala Pro
Tyr Ile Lys Glu Thr Tyr Lys Ile Arg Pro1 5 10 15Glu
Ile13517PRTHuman Pegivirus 2 135Pro Glu Ile Arg Ala Gly Thr Gly Pro
Asp Gly Val Thr Val Ile Thr1 5 10 15Gly13619PRTHuman Pegivirus 2
136Ile Lys Glu Thr Tyr Lys Ile Arg Pro Glu Ile Arg Ala Gly Thr Gly1
5 10 15Pro Asp Gly13732PRTHuman Pegivirus 2 137Val Lys Ser Met Ala
Pro Tyr Ile Lys Glu Lys Tyr Lys Ile Arg Pro1 5 10 15Glu Ile Arg Ala
Gly Thr Gly Pro Asp Gly Val Thr Val Ile Thr Gly 20 25
3013818PRTHuman Pegivirus 2 138Val Lys Ser Met Ala Pro Tyr Ile Lys
Glu Lys Tyr Lys Ile Arg Pro1 5 10 15Glu Ile13919PRTHuman Pegivirus
2 139Ile Lys Glu Lys Tyr Lys Ile Arg Pro Glu Ile Arg Ala Gly Thr
Gly1 5 10 15Pro Asp Gly14040PRTHuman Pegivirus 2 140Leu Val Asp Pro
Glu Thr Asn Leu Arg Gly Tyr Ala Val Val Ile Cys1 5 10 15Asp Glu Cys
His Asp Thr Ser Ser Thr Thr Leu Leu Gly Ile Gly Ala 20 25 30Val Arg
Met Tyr Ala Glu Lys Ala 35 4014124PRTHuman Pegivirus 2 141Leu Val
Asp Pro Glu Thr Asn Leu Arg Gly Tyr Ala Val Val Ile Cys1 5 10 15Asp
Glu Cys His Asp Thr Ser Ser 2014225PRTHuman Pegivirus 2 142Thr Asn
Leu Arg Gly Tyr Ala Val Val Ile Cys Asp Glu Cys His Asp1 5 10 15Thr
Ser Ser Thr Thr Leu Leu Gly Ile 20 2514320PRTHuman Pegivirus 2
143Asp Thr Ser Ser Thr Thr Leu Leu Gly Ile Gly Ala Val Arg Met Tyr1
5 10 15Ala Glu Lys Ala 2014421PRTHuman Pegivirus 2 144Pro Glu Thr
Asn Leu Arg Gly Tyr Ala Val Val Ile Ser Asp Glu Ser1 5 10 15His Asp
Thr Ser Ser 2014514PRTHuman Pegivirus 2 145Pro Glu Thr Asn Leu Arg
Gly Tyr Ala Val Val Ile Ser Asp1 5 1014611PRTHuman Pegivirus 2
146Val Ile Ser Asp Glu Ser His Asp Thr Ser Ser1 5 1014721PRTHuman
Pegivirus 2 147Pro Glu Thr Asn Leu Arg Gly Tyr Ala Val Val Ile Cys
Asp Glu Cys1 5 10 15His Asp Thr Ser Ser 2014814PRTHuman Pegivirus 2
148Pro Glu Thr Asn Leu Arg Gly Tyr Ala Val Val Ile Cys Asp1 5
1014917PRTHuman Pegivirus 2 149Arg Arg Gly Phe Ala Val Val Ile Cys
Val Glu Cys His Glu His Ile1 5 10 15Thr15034PRTHuman Pegivirus 2
150Pro Cys Thr Ala Ala Leu Arg Met Gln Arg Arg Gly Arg Thr Gly Arg1
5 10 15Gly Arg Arg Gly Ala Tyr Tyr Thr Thr Ser Pro Gly Ala Ala Pro
Cys 20 25 30Val Ser15120PRTHuman Pegivirus 2 151Pro Cys Thr Ala Ala
Leu Arg Met Gln Arg Arg Gly Arg Thr Gly Arg1 5 10 15Gly Arg Arg Gly
2015218PRTHuman Pegivirus 2 152Gly Arg Arg Gly Ala Tyr Tyr Thr Thr
Ser Pro Gly Ala Ala Pro Cys1 5 10 15Val
Ser15319PRTHuman Pegivirus 2 153Arg Arg Gly Arg Thr Gly Arg Gly Arg
Arg Gly Ala Tyr Tyr Thr Thr1 5 10 15Ser Pro Gly15433PRTHuman
Pegivirus 2 154Pro Cys Thr Ala Ala Leu Arg Met Gln Arg Arg Gly Arg
Thr Gly Arg1 5 10 15Gly Arg Arg Gly Ala Tyr Tyr Thr Thr Thr Pro Gly
Ala Ala Pro Cys 20 25 30Val15517PRTHuman Pegivirus 2 155Gly Arg Arg
Gly Ala Tyr Tyr Thr Thr Thr Pro Gly Ala Ala Pro Cys1 5 10
15Val15619PRTHuman Pegivirus 2 156Arg Arg Gly Arg Thr Gly Arg Gly
Arg Arg Gly Ala Tyr Tyr Thr Thr1 5 10 15Thr Pro Gly15732PRTHuman
Pegivirus 2 157Leu Ser Glu Arg Phe Gly Gln Gln Leu Ser Lys Leu Ser
Leu Trp Arg1 5 10 15Ser Val Tyr His Trp Ala Gln Ala Arg Glu Gly Tyr
Thr Gln Cys Gly 20 25 3015818PRTHuman Pegivirus 2 158Leu Ser Glu
Arg Phe Gly Gln Gln Leu Ser Lys Leu Ser Leu Trp Arg1 5 10 15Ser
Val15917PRTHuman Pegivirus 2 159Arg Ser Val Tyr His Trp Ala Gln Ala
Arg Glu Gly Tyr Thr Gln Cys1 5 10 15Gly16019PRTHuman Pegivirus 2
160Leu Ser Lys Leu Ser Leu Trp Arg Ser Val Tyr His Trp Ala Gln Ala1
5 10 15Arg Glu Gly16132PRTHuman Pegivirus 2 161Leu Thr Glu Lys Phe
Gly Gln Gln Leu Ser Lys Leu Ser Leu Trp Arg1 5 10 15Ser Val Tyr His
Trp Ala Gln Ala Arg Glu Gly Tyr Thr Gln Cys Gly 20 25
3016218PRTHuman Pegivirus 2 162Leu Thr Glu Lys Phe Gly Gln Gln Leu
Ser Lys Leu Ser Leu Trp Arg1 5 10 15Ser Val16340PRTHuman Pegivirus
2 163Asn Pro Thr Thr Thr Gly Thr Gly Thr Leu Arg Pro Asp Ile Ser
Asp1 5 10 15Ala Asn Lys Leu Gly Phe Arg Tyr Gly Val Ala Asp Ile Val
Glu Leu 20 25 30Glu Arg Arg Gly Asp Lys Trp His 35 4016424PRTHuman
Pegivirus 2 164Phe Asn Pro Thr Thr Thr Gly Thr Gly Thr Leu Arg Pro
Asp Ile Ser1 5 10 15Asp Ala Asn Lys Leu Gly Phe Arg 2016520PRTHuman
Pegivirus 2 165Gly Phe Arg Tyr Gly Val Ala Asp Ile Val Glu Leu Glu
Arg Arg Gly1 5 10 15Asp Lys Trp His 2016619PRTHuman Pegivirus 2
166Arg Pro Asp Ile Ser Asp Ala Asn Lys Leu Gly Phe Arg Tyr Gly Val1
5 10 15Ala Asp Ile16740PRTHuman Pegivirus 2 167Asn Pro Thr Thr Thr
Ala Thr Gly Thr Leu Arg Pro Asp Ile Ser Asp1 5 10 15Ala Thr Lys Leu
Gly Phe Arg Tyr Gly Val Ala Glu Ile Val Glu Leu 20 25 30Glu Arg Arg
Gly Asn Lys Trp His 35 4016823PRTHuman Pegivirus 2 168Asn Pro Thr
Thr Thr Ala Thr Gly Thr Leu Arg Pro Asp Ile Ser Asp1 5 10 15Ala Thr
Lys Leu Gly Phe Arg 2016920PRTHuman Pegivirus 2 169Gly Phe Arg Tyr
Gly Val Ala Glu Ile Val Glu Leu Glu Arg Arg Gly1 5 10 15Asn Lys Trp
His 2017019PRTHuman Pegivirus 2 170Arg Pro Asp Ile Ser Asp Ala Thr
Lys Leu Gly Phe Arg Tyr Gly Val1 5 10 15Ala Glu Ile17133PRTHuman
Pegivirus 2 171Gln Asn Leu Ala Ala Arg Arg Arg Ala Glu Tyr Asp Ala
Trp Gln Val1 5 10 15Arg Gln Ala Val Gly Asp Glu Tyr Thr Arg Leu Ala
Asp Glu Asp Val 20 25 30Asp17220PRTHuman Pegivirus 2 172Gln Asn Leu
Ala Ala Arg Arg Arg Ala Glu Tyr Asp Ala Trp Gln Val1 5 10 15Arg Gln
Ala Val 2017317PRTHuman Pegivirus 2 173Arg Gln Ala Val Gly Asp Glu
Tyr Thr Arg Leu Ala Asp Glu Asp Val1 5 10 15Asp17419PRTHuman
Pegivirus 2 174Arg Ala Glu Tyr Asp Ala Trp Gln Val Arg Gln Ala Val
Gly Asp Glu1 5 10 15Tyr Thr Arg17533PRTHuman Pegivirus 2 175Gln Asn
Leu Glu Ala Arg Arg Arg Ala Glu Phe Asp Ala Trp Gln Val1 5 10 15Arg
Glu Ala Ile Arg Asp Glu Tyr Thr Arg Leu Ala Asp Glu Asp Val 20 25
30Asp17620PRTHuman Pegivirus 2 176Gln Asn Leu Glu Ala Arg Arg Arg
Ala Glu Phe Asp Ala Trp Gln Val1 5 10 15Arg Glu Ala Ile
2017717PRTHuman Pegivirus 2 177Arg Glu Ala Ile Arg Asp Glu Tyr Thr
Arg Leu Ala Asp Glu Asp Val1 5 10 15Asp17819PRTHuman Pegivirus 2
178Arg Ala Glu Phe Asp Ala Trp Gln Val Arg Glu Ala Ile Arg Asp Glu1
5 10 15Tyr Thr Arg17923PRTHuman Pegivirus 2 179Phe Glu Ala Trp Gln
Val Arg Glu Ala Ile Arg Asp Glu Tyr Thr Arg1 5 10 15Leu Ala Asp Glu
Asp Val Asp 2018031PRTHuman Pegivirus 2 180Arg Phe Val Pro Pro Val
Pro Lys Pro Arg Thr Arg Val Ser Gly Val1 5 10 15Leu Glu Arg Val Arg
Met Cys Met Arg Thr Pro Pro Ile Lys Phe 20 25 3018116PRTHuman
Pegivirus 2 181Arg Phe Val Pro Pro Val Pro Lys Pro Arg Thr Arg Val
Ser Gly Val1 5 10 1518218PRTHuman Pegivirus 2 182Ser Gly Val Leu
Glu Arg Val Arg Met Cys Met Arg Thr Pro Pro Ile1 5 10 15Lys
Phe18315PRTHuman Pegivirus 2 183Lys Pro Arg Thr Arg Val Ser Gly Val
Leu Glu Arg Val Arg Met1 5 10 1518417PRTHuman Pegivirus 2 184Arg
Thr Arg Val Ser Gly Val Leu Glu Arg Val Arg Met Cys Met Thr1 5 10
15Thr18522PRTHuman Pegivirus 2 185Asn Thr Thr Arg Asp His Asn Asn
Gly Ile Thr Tyr Thr Asp Leu Val1 5 10 15Ser Gly Arg Ala Lys Pro
2018614PRTHuman Pegivirus 2 186Asn Thr Thr Arg Asp His Asn Asn Gly
Ile Thr Tyr Thr Asp1 5 1018711PRTHuman Pegivirus 2 187Tyr Thr Asp
Leu Val Ser Gly Arg Ala Lys Pro1 5 1018822PRTHuman Pegivirus 2
188Asn Thr Thr Arg Asp His Asn Asn Gly Ile Thr Tyr Ser Asp Leu Val1
5 10 15Ser Gly Arg Ala Lys Pro 2018914PRTHuman Pegivirus 2 189Asn
Thr Thr Arg Asp His Asn Asn Gly Ile Thr Tyr Ser Asp1 5
1019011PRTHuman Pegivirus 2 190Tyr Ser Asp Leu Val Ser Gly Arg Ala
Lys Pro1 5 1019135PRTHuman Pegivirus 2 191Asp Ala Pro Met Arg Ile
Ile Pro Lys Pro Glu Val Phe Pro Arg Asp1 5 10 15Lys Ser Thr Arg Lys
Pro Pro Arg Phe Ile Val Phe Pro Gly Cys Ala 20 25 30Ala Arg Val
3519224PRTHuman Pegivirus 2 192Asp Ala Pro Met Arg Ile Ile Pro Lys
Pro Glu Val Phe Pro Arg Asp1 5 10 15Lys Ser Thr Arg Lys Pro Pro Arg
2019320PRTHuman Pegivirus 2 193Asp Lys Ser Thr Arg Lys Pro Pro Arg
Phe Ile Val Phe Pro Gly Cys1 5 10 15Ala Ala Arg Val 2019420PRTHuman
Pegivirus 2 194Ile Pro Lys Pro Glu Val Phe Pro Arg Asp Lys Ser Thr
Arg Lys Pro1 5 10 15Pro Arg Phe Ile 2019535PRTHuman Pegivirus 2
195Asp Ala Pro Met Arg Ile Ile Pro Lys Pro Glu Val Phe Pro Arg Asp1
5 10 15Lys Thr Thr Arg Lys Pro Pro Arg Phe Ile Val Phe Pro Gly Cys
Ala 20 25 30Ala Arg Val 3519624PRTHuman Pegivirus 2 196Asp Ala Pro
Met Arg Ile Ile Pro Lys Pro Glu Val Phe Pro Arg Asp1 5 10 15Lys Thr
Thr Arg Lys Pro Pro Arg 2019720PRTHuman Pegivirus 2 197Asp Lys Thr
Thr Arg Lys Pro Pro Arg Phe Ile Val Phe Pro Gly Cys1 5 10 15Ala Ala
Arg Val 2019820PRTHuman Pegivirus 2 198Ile Pro Lys Pro Glu Val Phe
Pro Arg Asp Lys Thr Thr Arg Lys Pro1 5 10 15Pro Arg Phe Ile
2019920PRTHuman Pegivirus 2 199Met Pro Leu Leu Cys Met Leu Ile Arg
Asn Glu Pro Ser Gln Thr Gly1 5 10 15Thr Leu Val Thr 2020015PRTHuman
Pegivirus 2 200Met Pro Leu Leu Cys Met Leu Ile Arg Asn Glu Pro Ser
Gln Thr1 5 10 1520115PRTHuman Pegivirus 2 201Met Leu Ile Arg Asn
Glu Pro Ser Gln Thr Gly Thr Leu Val Thr1 5 10 1520220PRTHuman
Pegivirus 2 202Leu Pro Leu Leu Cys Met Leu Ile Arg Asn Glu Pro Ser
Gln Thr Gly1 5 10 15Thr Leu Val Thr 2020315PRTHuman Pegivirus 2
203Leu Pro Leu Leu Cys Met Leu Ile Arg Asn Glu Pro Ser Gln Thr1 5
10 1520420PRTHuman Pegivirus 2 204Leu Pro Leu Leu Cys Met Leu Ile
Arg Asn Glu Pro Ser Gln Thr Gly1 5 10 15Thr Leu Val Thr
2020539PRTHuman Pegivirus 2 205Ala Glu Ala Ala Pro Lys Ser Gly Glu
Leu Asp Ser Gln Cys Asp His1 5 10 15Leu Ala Trp Ser Phe Met Glu Gly
Met Pro Thr Gly Thr Leu Ile Val 20 25 30Gln Arg Asp Gly Ser Leu His
3520623PRTHuman Pegivirus 2 206Ala Glu Ala Ala Pro Lys Ser Gly Glu
Leu Asp Ser Gln Cys Asp His1 5 10 15Leu Ala Trp Ser Phe Met Glu
2020719PRTHuman Pegivirus 2 207Phe Met Glu Gly Met Pro Thr Gly Thr
Leu Ile Val Gln Arg Asp Gly1 5 10 15Ser Leu His20817PRTHuman
Pegivirus 2 208Gln Cys Asp His Leu Ala Trp Ser Phe Met Glu Gly Met
Pro Thr Gly1 5 10 15Thr20950PRTHuman Pegivirus 2 209Ser Val Glu Val
Arg Pro Ala Gly Val Thr Arg Pro Asp Ala Thr Asp1 5 10 15Glu Thr Ala
Ala Tyr Ala Gln Arg Leu Tyr Gln Ala Cys Ala Asp Ser 20 25 30Gly Ile
Phe Ala Ser Leu Gln Gly Thr Ala Ser Ala Ala Leu Gly Lys 35 40 45Leu
Ala 5021031PRTHuman Pegivirus 2 210Ser Val Glu Val Arg Pro Ala Gly
Val Thr Arg Pro Asp Ala Thr Asp1 5 10 15Glu Thr Ala Ala Tyr Ala Gln
Arg Leu Tyr Gln Ala Cys Ala Asp 20 25 3021123PRTHuman Pegivirus 2
211Ala Cys Ala Asp Ser Gly Ile Phe Ala Ser Leu Gln Gly Thr Ala Ser1
5 10 15Ala Ala Leu Gly Lys Leu Ala 2021232PRTHuman Pegivirus 2
212Val Thr Arg Pro Asp Ala Thr Asp Glu Thr Ala Ala Tyr Ala Gln Arg1
5 10 15Leu Tyr Gln Ala Cys Ala Asp Ser Gly Ile Phe Ala Ser Leu Gln
Gly 20 25 3021350PRTHuman Pegivirus 2 213Ser Val Glu Asn Gly Leu
Ala Gly Val Thr Arg Pro Asp Ala Thr Asp1 5 10 15Glu Thr Ala Ala Tyr
Ala Gln Arg Leu Tyr Gln Ala Cys Ala Asp Ser 20 25 30Gly Ile Leu Ala
Ser Leu Gln Gly Thr Ala Ser Ala Ala Leu Ser Arg 35 40 45Leu Ala
5021431PRTHuman Pegivirus 2 214Ser Val Glu Asn Gly Leu Ala Gly Val
Thr Arg Pro Asp Ala Thr Asp1 5 10 15Glu Thr Ala Ala Tyr Ala Gln Arg
Leu Tyr Gln Ala Cys Ala Asp 20 25 3021523PRTHuman Pegivirus 2
215Ala Cys Ala Asp Ser Gly Ile Leu Ala Ser Leu Gln Gly Thr Ala Ser1
5 10 15Ala Ala Leu Ser Arg Leu Ala 2021632PRTHuman Pegivirus 2
216Val Thr Arg Pro Asp Ala Thr Asp Glu Thr Ala Ala Tyr Ala Gln Arg1
5 10 15Leu Tyr Gln Ala Cys Ala Asp Ser Gly Ile Leu Ala Ser Leu Gln
Gly 20 25 3021739PRTHuman Pegivirus 2 217Ala Glu Ala Ala Pro Lys
Ser Gly Glu Leu Asp Ser Gln Cys Asp His1 5 10 15Leu Ala Trp Ser Phe
Met Glu Gly Met Pro Thr Gly Thr Leu Ile Val 20 25 30Gln Arg Asp Gly
Ser Leu His 3521850PRTHuman Pegivirus 2 218Ser Val Glu Val Arg Pro
Ala Gly Val Thr Arg Pro Asp Ala Thr Asp1 5 10 15Glu Thr Ala Ala Tyr
Ala Gln Arg Leu Tyr Gln Ala Cys Ala Asp Ser 20 25 30Gly Ile Phe Ala
Ser Leu Gln Gly Thr Ala Ser Ala Ala Leu Gly Lys 35 40 45Leu Ala
5021918DNAArtificial sequenceSynthetic 219gagtcacgcg gggtgctt
1822026DNAArtificial sequenceSynthetic 220cttaatataa ggggccatac
ttttga 2622120DNAArtificial sequenceSynthetic 221tggaaccagt
cgtgttggag 2022220DNAArtificial sequenceSynthetic 222gaacagcagc
aggggtctag 2022320DNAArtificial sequenceSynthetic 223ctagacccct
gctgctgttc 2022420DNAArtificial sequenceSynthetic 224tgactacagc
cacacttggt 2022522DNAArtificial sequenceSynthetic 225atatgggagc
taccactgcg gt 2222626DNAArtificial sequenceSynthetic 226taacaggaca
gaatctaggt atggag 2622724DNAArtificial sequenceSynthetic
227tgtctattgc tctacctcca ggtg 2422823DNAArtificial
sequenceSynthetic 228ttccaaagca acgtaacacg gcg 2322923DNAArtificial
sequenceSynthetic 229cgccgtgtta cgttgctttg gaa 2323024DNAArtificial
sequenceSynthetic 230ttaccagaac cagtaggggc atag
2423128DNAArtificial sequenceSynthetic 231acagtcacat tccaacattg
atgaatac 2823221DNAArtificial sequenceSynthetic 232tgctcccctt
ctaccacgac c 2123322DNAArtificial sequenceSynthetic 233taggcgtgtg
gttttccggt ct 2223422DNAArtificial sequenceSynthetic 234ccaatcccac
acagcgcgta ga 2223522DNAArtificial sequenceSynthetic 235tctacgcgct
gtgtgggatt gg 2223623DNAArtificial sequenceSynthetic 236gggtctcaaa
cttgattgga ggc 2323724DNAArtificial sequenceSynthetic 237ctatgcccct
actggttctg gtaa 2423828DNAArtificial sequenceSynthetic
238gtattcatca atgttggaat gtgactgt 2823924DNAArtificial
sequenceSynthetic 239acttattgac tgacacaggc gacg
2424024DNAArtificial sequenceSynthetic 240tgcatgcgca atgcagcagt
acat 2424125DNAArtificial sequenceSynthetic 241gtgcccataa
gtggctatta gctat 2524227DNAArtificial sequenceSynthetic
242tgcttaattg ttggccaaat ctttcac 2724324DNAArtificial
sequenceSynthetic 243acgattccgt gtgcctatga ttgg
2424427DNAArtificial sequenceSynthetic 244ctgggttaca taagttagta
gacatgc 2724521DNAArtificial sequenceSynthetic 245tctccgcctc
cagcagttca a 2124625DNAArtificial sequenceSynthetic 246agccgcagta
ggatacatga caata 2524726DNAArtificial sequenceSynthetic
247tgtgatatca caggaaaagt tgtcgg 2624823DNAArtificial
sequenceSynthetic 248acagtcacag ccgcagtagg ata 2324925DNAArtificial
sequenceSynthetic 249gagaaaatga tcctgggcga tcctg
2525023DNAArtificial sequenceSynthetic 250gccgtgatgg tgctatcaaa gca
2325120DNAArtificial sequenceSynthetic 251gggacacctc aaccctgaag
2025220DNAArtificial sequenceSynthetic 252tcactgcggt acccattgac
2025322DNAArtificial sequenceSynthetic 253atatgggagc taccactgcg gt
2225426DNAArtificial sequenceSynthetic 254ggtacagtat ttgaggtagc
tttcag 2625524DNAArtificial sequenceSynthetic 255ctttttggtg
cgcagtgttt gcct 2425624DNAArtificial sequenceSynthetic
256tgtcagggaa gacaacacca cgat 2425722DNAArtificial
sequenceSynthetic 257acactcacag
ggcgtgctga aa 2225823DNAArtificial sequenceSynthetic 258acgccaagtt
ctcaccagtg atg 2325925DNAArtificial sequenceSynthetic 259gtgtggctgt
agtcaaaagt atggc 2526022DNAArtificial sequenceSynthetic
260cagcagtaca tggcaccact cg 2226122DNAArtificial sequenceSynthetic
261ttcggtcatc gactgcgggt gt 2226225DNAArtificial sequenceSynthetic
262ccagccaagt tcctgcaata gctaa 2526322DNAArtificial
sequenceSynthetic 263ttcactgcgc ttgctggctt gg 2226421DNAArtificial
sequenceSynthetic 264ccgtaaggtg ccagtgcctg t 2126527DNAArtificial
sequenceSynthetic 265aagcatcaat ctgaaagcta cctcaaa
2726625DNAArtificial sequenceSynthetic 266tgaatcttat agtgtcgtcc
aagtg 2526720DNAArtificial sequenceSynthetic 267ctgtgactgc
ccctttggaa 2026820DNAArtificial sequenceSynthetic 268catgccaacg
tccgtgtatg 2026924DNAArtificial sequenceSynthetic 269aacccttgca
attctggccg atga 2427022DNAArtificial sequenceSynthetic
270agatccctga ctgcttgcgc ca 2227122DNAArtificial sequenceSynthetic
271cccacggtcc tgatgatagc at 2227223DNAArtificial sequenceSynthetic
272gctaaccagc caagttcctg caa 2327330DNAArtificial sequenceSynthetic
273aagtgaaaga tttggccaac aattaagcaa 3027420DNAArtificial
sequenceSynthetic 274ccacgcaggt gagcagccaa 2027525DNAArtificial
sequenceSynthetic 275acaattatta caaaagaagc cccat
2527619DNAArtificial sequenceSynthetic 276agtcacagcc gcagtagga
1927720DNAArtificial sequenceSynthetic 277ttgacatgac agcgtcggtg
2027820DNAArtificial sequenceSynthetic 278agcgcgcatc tgatctacaa
2027919DNAArtificial sequenceSynthetic 279aacagcggtt gatgtctcc
1928019DNAArtificial sequenceSynthetic 280aaagatgcgc gcaaacacc
1928120DNAArtificial sequenceSynthetic 281agcgcgcatc tgatctacaa
2028224DNAArtificial sequenceSynthetic 282gctagtgcta tactcgctct
gctt 2428323DNAArtificial sequenceSynthetic 283atgatgcatg
gcaggttcgc caa 2328423DNAArtificial sequenceSynthetic 284gatcaatcgt
gaccttagcc tgc 2328523DNAArtificial sequenceSynthetic 285aactcggcga
ccagtgccaa aat 2328622DNAArtificial sequenceSynthetic 286tgtctgcgcg
caaaatgcca gc 2228724DNAArtificial sequenceSynthetic 287ggcaaagacc
ttcagacaat ctgg 2428823DNAArtificial sequenceSynthetic
288caccccgaca acttttcctg tga 2328923DNAArtificial sequenceSynthetic
289ttgatcgtgc aaagggatgg gtc 2329023DNAArtificial sequenceSynthetic
290ctaacagtcc aagccaacct gca 2329122DNAArtificial sequenceSynthetic
291gccatgaggg atcatgacac tg 2229222DNAArtificial sequenceSynthetic
292tttgcacgat cagcgttccc gt 2229326DNAArtificial sequenceSynthetic
293ctgtcctgtt actccatacc tagatt 2629421DNAArtificial
sequenceSynthetic 294cgacacctgg aggtagagca a 2129522DNAArtificial
sequenceSynthetic 295cctccaatca agtttgagac cc 2229620DNAArtificial
sequenceSynthetic 296gtacactcca gcgcgcatct 2029720DNAArtificial
sequenceSynthetic 297accaagtgtg gctgtagtca 2029820DNAArtificial
sequenceSynthetic 298cccctgttgt atgcctagcc 202999867DNAHuman
Pegivirus 2misc_feature(1284)..(1284)N = A, C, T, or
Gmisc_feature(1572)..(1572)N = A, C, T, or
Gmisc_feature(1745)..(1745)N = A, C, T, or
Gmisc_feature(1815)..(1815)N = A, C, T, or
Gmisc_feature(2052)..(2052)N = A, C, T, or
Gmisc_feature(2424)..(2424)N = A, C, T, or
Gmisc_feature(2562)..(2562)N = A, C, T, or
Gmisc_feature(2793)..(2793)N = A, C, T, or
Gmisc_feature(3291)..(3291)N = A, C, T, or
Gmisc_feature(3312)..(3312)N = A, C, T, or
Gmisc_feature(3336)..(3336)N = A, C, T, or
Gmisc_feature(3499)..(3499)N = A, C, T, or
Gmisc_feature(3711)..(3711)N = A, C, T, or
Gmisc_feature(3919)..(3919)N = A, C, T, or
Gmisc_feature(3987)..(3987)N = A, C, T, or
Gmisc_feature(4389)..(4389)N = A, C, T, or
Gmisc_feature(4890)..(4890)N = A, C, T, or
Gmisc_feature(5160)..(5160)N = A, C, T, or
Gmisc_feature(5368)..(5368)N = A, C, T, or
Gmisc_feature(5707)..(5707)N = A, C, T, or
Gmisc_feature(5710)..(5710)N = A, C, T, or
Gmisc_feature(5727)..(5727)N = A, C, T, or
Gmisc_feature(5746)..(5746)N = A, C, T, or
Gmisc_feature(5838)..(5838)N = A, C, T, or
Gmisc_feature(6036)..(6036)N = A, C, T, or
Gmisc_feature(6213)..(6213)N = A, C, T, or
Gmisc_feature(6309)..(6309)N = A, C, T, or
Gmisc_feature(6423)..(6423)N = A, C, T, or
Gmisc_feature(6486)..(6486)N = A, C, T, or
Gmisc_feature(6489)..(6489)N = A, C, T, or
Gmisc_feature(6999)..(6999)N = A, C, T, or
Gmisc_feature(7083)..(7083)N = A, C, T, or
Gmisc_feature(8100)..(8100)N = A, C, T, or
Gmisc_feature(8253)..(8253)N = A, C, T, or
Gmisc_feature(8497)..(8497)N = A, C, T, or
Gmisc_feature(8512)..(8512)N = A, C, T, or
Gmisc_feature(8997)..(8997)N = A, C, T, or
Gmisc_feature(9072)..(9072)N = A, C, T, or
Gmisc_feature(9134)..(9134)N = A, C, T, or
Gmisc_feature(9147)..(9147)N = A, C, T, or
Gmisc_feature(9336)..(9336)N = A, C, T, or
Gmisc_feature(9339)..(9339)N = A, C, T, or
Gmisc_feature(9348)..(9348)N = A, C, T, or
Gmisc_feature(9359)..(9359)N = A, C, T, or
Gmisc_feature(9363)..(9364)N = A, C, T, or
Gmisc_feature(9375)..(9375)N = A, C, T, or G 299aactgttgtt
gtagcaatgc gcatattgct acttcggtac gcctaattgg taggcgcccg 60gccgaccggc
cccgcaaggg cctagtagga cgtgtgacaa tgccatgagg gatcatgaca
120ctggggtgag cggaggcagc accgaagtcg ggtgaactcg actcccagtg
cgaccacctg 180gcttggtcgt tcatggaggg catgcccacg ggaacgctga
tcgtgcaaag ggatgggtcc 240ctgcactggt gccatgcgcg gcaccactcc
gtacagcctg atagggtggc ggcgggcccc 300cccagtgtga cgtccgtgga
gcgcaacatg gggtgttcaa ctgatcaaac catttgttct 360ccagtcgtgg
gggccgacta taatacctcc tcgggctgcc gggccttaaa tgggagctac
420cactgcggtg gtggctcttg ccggtcacca agtcgtgtgc aggttgcgag
acgagtcttg 480cagctgtgcg cattccttgc gctgatcgga tccggtatgt
gttcgatccg gtccaaaact 540gaagggcgca ttgagtcagg gcaaatattg
cagtctcagc gcgcatgttg gactggtgag 600ggttttgctt tcttttctaa
ctgttgcaat caatctgata ttatgtggtg tttgcaccgt 660tggtgtgtga
caagacctgg ctgtttagtg tgcacgggca atgccactca tcctgtctgc
720tgggactatc ttgggtccgg tgtaagtcgg cggcctgcgc gtcgaatggg
tgagggagct 780gaagtgcttc ttcgcttgat cggcattgca ggttggctcg
ggctgttagc tgaggctctt 840ggtatgtccg aaatctatgc agctattctt
tgctttggat ttattgcttg gtatggctgg 900ggtataccta aaacattggt
gtgcacagtc tgccctgcag tgaacatttc tccctatagc 960ttcttatctc
cagatactat cgcatttggt acgtggatac tacaactacc tggtcttttg
1020tggcaaatgt ttgtcagctt ccctatactt tacagcactt ggattctttg
gttgttgctc 1080agcggcaaga ctgttgctgt gatagcgatc cttttggcta
gtcctacggt tatggcgtac 1140aagcatcaat ctgaaagcta cctcaaatac
tgtaccataa ccaatgcttc aactgctatg 1200aactgtgact gcccctttgg
aacctttact cgcaatactg agtctcgttt ctctatacct 1260agattctgtc
ctgttaaaat taanagctct acatttatct gttcatgggg gtcgtggtgg
1320tggtttgctg agaacatcac acgtccatac tcggacgttg gcatgccacc
agcaccgatt 1380tccgctttgt gctatatcta ttcaaacaat gacccacctc
cttggtatca taacacaact 1440atcatacctc agaactgtcg caactctacg
gttgatccta ccacagctcc atgccgtgac 1500aagtggggca atgcaactgc
ttgtattctt gaccgccggt cgcggttctg cggggactgc 1560tatggcggtt
gnttctatac taatggtagt catgatcgat cctgggatcg atgcgggatt
1620ggttaccgtg atggactcat agagttcgtg cagctcggtc agattcgacc
taacatctcg 1680aatacgacca ttgagctcct cgctggcgcc tcgcttgtga
tcgcatccgg tcttcggcct 1740gggtntggtt gcagccgagc gcatggcgtg
gtgcactgct ataggtgtcc ttcataccgt 1800gaccttgaac agttnggtcc
tgggcttggg aaatgggtgc cattgcccgg cgagcctgtc 1860ccggagttgt
gtatcaaccc tcagtgggcg aggcgcggct tccggatgtc taataatcct
1920ctgagcttgc tacagacctt cgttgaggac attttcctag cgcctttttg
taatccgacg 1980cctggccgtg tacgtgtgtg taacaatacc gctttctatc
caagaggagg cggctttgtg 2040cagctcatcg gngacgtcca ggtgctaacc
cctaacactg catctttaca ctctctgctg 2100actttgatat ctcttatctt
gttggtgtgt gttgtttctg gtgcgcgatt cgttccacta 2160ataatcatat
ttttctggag cgcgcgccat gtatatgctt cttgttactt aagctgtgat
2220tgggctgttt gcaacgatgc gttctgtttc acatctggca cttgtgctac
cttcaatgac 2280gtcttgtgtc tgccggttgc gacgcgcata tcgtcctgtg
gccatgctgt gccaccgccc 2340gaccgtggtt gggaggtgcc tgcggcgatg
tcatgggtga tttcgcggac tactggcttg 2400acgttcgatg tcttttcctt
catncagtat cttcctactg tgcctggcaa caacaccgat 2460atcatttact
gtggtgaacc aaccttcttc ggggacatca cgggcatcta ttggccttac
2520tttttgcctg gcgtgttgct cttgtacttg actcccttcc tnggtttaag
gttaatgctt 2580gccggcttta atatagatgg cttgtttccc atacggcatg
ccacggctgc actgaggttc 2640tcgacttcgc gtgtgacctt gagtgtcgta
tctgcttttc taatctatat attatctcac 2700cctgttaatg ctgcgctcaa
tagaatgttc ttagcatctg caaatttaga gatgatctta 2760tcctttgata
cctatcatga gactgttctt tanatcgttt gtctattgct ctacctccag
2820gtgtcgcccc gtgcgggctt ggccgctatg gtggccatca agctatctcg
aggcctgtta 2880ttcgctgtgg tgttggcgca cggagtgtgc cgacctgggc
gggtatttgg tcttgaggtt 2940tgcgcggaca tctcttggtt ggtggagttt
actggcaact gcacttggta catgtcctgt 3000gtcttctctt tttggtgcgc
agtgtttgcc ttcaccagtc cacttggacg acagtataag 3060cttcagatct
atcggtactg ggcgcaggtc tatgccagac tcatcctcgc tgtcggttgt
3120ggtcctctcg ggcgagagtt ccatttccgc gcaagcgtgg gcgtgctttg
gtgtggtgct 3180tgcatgctct ggccccgtga gtgctctgaa atcagcttgg
tcttcattct gtgtgctctg 3240acagtggaca ccatagacac atggttagta
gcgtgcttgt ccgcagggcc nagtgcgcga 3300acccttgcaa cnctggccga
tgacatggcg cgcatnggtg accaccgggc gttgcgcgcc 3360gtgttgcgtt
gctttggatc acgtggcaca tacatataca accacatggg ccaggtctca
3420gaacgggtgg cgcaagcagt cagggatctc ggcggttgct tggaaccagt
cgtgttggag 3480gagcccacct ttactgagnt cgtggatgat acaatgagtt
tggtgtgtgg acaattgctt 3540ggaggtaaac ccgtggtggc ccgctgcggc
acgcgtgtct tggtgggaca cctcaaccct 3600gaagatctgc cacctggttt
ccagctgagt gctccggtgg ttattaccaa accaagcatt 3660ggtacgtggt
ccttccttaa ggcgacactc acagggcgtg ctgaaacacc nggatccggc
3720cagatcgtgg tgttgtcttc cctgacaggt cggtcaatgg gtactgcagt
gaatggcaca 3780ctgtatgcga ccggccatgg tgccggtgcg cgcggcctag
ccacgtgcgc tggtttgagg 3840acgccacttt acacggcatt atctgatgat
gtcgtggcct actcttgcct tccgggcatg 3900agttccctag agccctgcng
ctgttcgccg agccgggttt gggtgatgaa caacaacgga 3960gggttggtgt
gtggcagagt ggagaangac gacgtctgtt tggactgtcc cacgcacata
4020gatcaactgc ggggtgcttc ggggtcaccg gttttgtgtg atcacggtca
tgcatacgcg 4080ttgatgctcg gtggttactc taccagtggt atttgtgcgc
gtgtccggat agtccggcca 4140tggcagaacg cctattcctc ctcagggggg
caaggcggaa tgcaggcgcc agctgtgaca 4200ccaacatact ctgaaatcac
ctactatgcc cctactggtt ctggtaagtc aacaaaatat 4260ccagtggacc
tagtcaaaca gggacacaaa gtattggtcc ttataccaag tgtggctgtc
4320gtcaaaagta tggcccctta cattaaggag acatataaga ttagacctga
aattagagct 4380ggcacaggnc ctgacggtgt gacggtcatc actggtgaga
acttggcgta catgacctat 4440ggccgcttcc ttgtggatcc ggagacgaat
ctgcggggtt atgccgtagt catttgcgac 4500gagtgccacg acacatcatc
caccacgcta ctcggcattg gcgcagtgcg catgtatgcc 4560gagaaagctg
gagtgaagac cgttgtattc gccacagcca ctcctgctgg cattcaagta
4620cagccacatc ccaacattga tgaatattta ttgactgaca caggcgacgt
ggaattctac 4680ggcgccaaaa tcaaattgga caacatcaga actggtagac
atgttatctt ttgccactcg 4740aaggccaggt gtgcggaact aacgcagcag
ctctccggcc ttggtgttcg tgcagtgagt 4800ttttggcgcg gctgtgacat
caaaaccatt cccgcctcag actctattgt tgtagtggca 4860actgatgcat
tgtccacagg ctacacaggn aactttgatt cggtcatcga ctgcgggtgt
4920tgcgtagagc aaactgtgac aattgacatg gaccctacgt tctccatctc
ggcccgagtg 4980gtgccatgta ctgctgcatt gcgtatgcag cggcgcggac
gtaccggtcg tggcagaagg 5040ggagcgtact acacaaccac tccaggagca
gcaccctgcg tcagcgttcc cgatgctaac 5100gtctggcaag cagtggagag
cgccatggtc ttttatgatt ggagtgctgc caggatacan 5160cagtgcctgg
cggcatacca tgatttaggg tgcacaccac gcatcagttg tgacccacac
5220actccagtgc gggtgatgga cacactgagg gcgtacctgc gcagacctga
ggtgacgact 5280gcggctctcg caggagagca gtggccgctg ctttacggtg
tgcagttgtg catctgcaaa 5340gagaccgagg cccacggtcc agacgatngc
atcaagtgga agtgcttact caacaacagt 5400aacaaaacac ccctgttgta
tgccttagac aatcctacac tggaattcac tacccaacat 5460gacttgactc
gccgtatagc cggcgcttta tcgagcacag tgttcgtgga gacaggctac
5520ggccccatcc tccttgctgg cgccgctttg gctgcctcct tcgcctttgc
gggcgccact 5580ggagctttag tgccgtcggc cgtttggagc gttgacaacg
ggcttgctgg cgtgacccgt 5640cccgacgcga cagacgagac cgcggcctac
gcgcagcgct tgtaccaagc ctgcgcagat 5700tcagganttn tcgccagctt
gcagggnacg gcgagtgcgg cgctgngcaa actggccgat 5760gccagtaggg
gtgctagtca atatctggca gccgcgcctc cttcgcccgc ccccctggta
5820caggtgctgc agttcctnga gaccaacttt agctccattg catctttcgg
tctgctctgt 5880gctggctgtc aggctggcga gtgcttcact gcgcttgccg
ggttggtgtc cggtgctaca 5940gctggcttgg gaggtgccca taagtggttg
ttagctattg caggaacttg gctagttagc 6000ttgcagactg ggccccgtgg
cggcatggtt gcgggnctct cggttctagc aggctgttgc 6060atcggtagtg
tcaccgggct tgacttcctg tttgggtgcc ttacaggttg ggaggccgtg
6120gtcggtgctg cggttgcgac acagaagatc ttgtctggtt cggctgatat
gaccactctg 6180gtagatctct tacctgctct cttctcccct ggngccggca
tagctggcat cgtgcttgtc 6240tttattctaa gcaactcaag tgtaaccacg
tgggctaatc ggctattgtc catgtgtgca 6300aaacaaacna tttgtgaaaa
ctacttctta actgagaaat ttggccaaca attaagcaaa 6360ctttccctgt
ggcgctctgt gtaccattgg gcgcaggcac gtgagggata cacacagtgc
6420ggngtggtca gcgggatctg gagctttgtc ttgtgcattc tacgcgctgt
gtgggattgg 6480gcggcnaanc atgtgccacg gttccgtgtg cctatgattg
gctgctcacc tgcgtggtgc 6540gggcgctggc ttggtaccgg caccttgttg
accacctgtg ggtgtggaga acgtgtgtcc 6600cttcagtgcc tttgctcaac
atctgaccca acactcagtg tgggccgttg gtgtcggtgt 6660agttggagtg
ttgggttccc attcaacccg actacgacag ccaccggcac tttacggccg
6720gacatcagtg acgccactaa attgggtttc cggtatggtg ttgccgagat
cgtggagcta 6780gagcggcggg gcgacaaatg gcatgtctgt gcagcatcat
gttgcttgga ccgagccagc 6840gttgcatccg ccgtgaaggc cccaccggtc
acggccaatg gtatacctat cagtaccttt 6900tctccaccac aaacttacag
cctctctctc tgttcttttg attcagtttg catgtctact 6960aacttatgta
acccagctaa gaccctgagt gtgtgctcnc aggaggctgt tgagctactg
7020gaagaaacag ttgacacagc acaagtaatg atgtgtcaaa atctggaggc
gcgaagacgc 7080gcngagtatg atgcatggca agttcgccaa gcagttggcg
acgagtacac gcgtttggca 7140gacgaggatg ttgacacgac aacgtcggtg
aaacccccgg tggccagggc tgctgtgggt 7200agctcaacgt tggatgatgt
tagcgtgctg actgtcttgc gcgaactcgg cgaccaatgc 7260caaaatgcta
tcaaatttgt agttcaggcg gcttcacggt ttgttccacc agtgcccaag
7320ccacgcacgc gtgtctcggg tgtgttggag cgtgtgcgca tgtgcatgcg
cacgccacca 7380atcaagtttg aggccaccgc agtaccaatt cataacataa
tcccagaaga gtgtcacatt 7440gtgctacgct gtaccggctg taacgaccag
gccttgactg ttccgtacgg cacttgcact 7500cagactttaa tcaaacattt
gactaacaaa cacagccact acattccaaa acagaagata 7560gaagaagaca
cagaagtaac tgtcatttgc gccgtaccaa caaagcgcgc aagtaaactc
7620atcactttca gagcaggtga tcgatcagtc tcatgttgtc accccttgca
aactcctatt 7680agggccctgc ttctaaagta cgggttacct atcgggaagt
ggtccgactg caacgggccc 7740cttggtgacg acgcccgagt ctgtgacgtc
aatggagtaa caacttatga accatgcatg 7800caatcctaca gttggttccg
accgattgtg gcaccaacaa ccccaccttt acctgcaacc 7860cggagcgtgg
ctggcatttt acgcgcagac acatcgcgcg tttacaccac aacggcggtt
7920gacgtctccg agcggcaggc taaggtcaca attgatcaaa catcagccaa
ggtggatcag 7980tgtttccgag acacatacaa ttgctgcctt gctaaggcaa
agaccttcag acaatctggc 8040atgtcatatg aggatgctgt gtcaaagatg
cgcgcaaaca ccacgcgtga ccataacaan 8100ggcatcactt attcagattt
ggtctctgga cgcgcaaaac ctgtcgttca gaaaattgta 8160gatcaaatgc
gcgctggagt gtacgacgct ccaatgcgca ttatcccaaa acctgaagtg
8220ttccctcgag acaagtcaac acggaagcca ccncggttca tcgttttccc
tgggtgcgcc 8280gcgcgagtcg cggagaaaat gatcctgggc gatcctggcg
cgataaccaa gcacgtgcta 8340ggtgatgcct acgggtttgc cactccgccg
catgagcgcg cgcgcctatt ggaacaatgg 8400tggaaccgcg caacggagcc
acaagctatc gcggttgatg cgatctgctt tgatagcacc 8460atcacggcag
aggacatgga tcgtgaggcc aacatcntgg ctgcagcgca tncggaccct
8520gaaggtgttc acggcctata caattattac aaaagaagcc ccatgtgtga
catcacagga 8580aaagttgtcg gggtgcgttg ctgtcgagcc tcaggtacgc
ttacaacaag cagtggcaac 8640acgcttactt gctacctcaa ggttcgtgca
gcttgcacgc gcgccggcat taaaccaatt 8700ggcttactaa ttcatggaga
tgacaccctc attatcacag aacgttgcgc tcaagaaact 8760ctcgatgagt
tcagcaacgc acttgatgac tatgggttcc ctcacaccat ccaggtgtct
8820ggggacctct cgtctgtcga gtgctgtagc gcacgtgtgg acagcgtttg
cctccgggga 8880ggtatgcgtc gcatgctcgt gccacaagct cgacgtgcga
ttgcacgcgt tctcggggaa 8940aagggcgatc cactgggtgt catcagcagc
tatattgtca tgtatcctac tgcggcngtg 9000actgtctacg tgctattgcc
cctgttgtgc atgctcattc gaaatgagcc
atcgcagacg 9060gggacacttg tnacgctgac ggtccacggt aacagtgtga
gcgtgccagt gtggctgctt 9120ccaaccatca ttgnaaattt acatggncgt
gacgcactac aggtagtccg tcacagtgca 9180gcttccatgg cggaactgtc
atcagcgttg gccttctttg gcatgagagg gttgaactgc 9240tggaggcgga
gacgccgtgc catcaggact gatatgatca agttgggcgg gtggaatgcg
9300aatttcgcgc agatgttact gtggtcaccg gaggtnagna caccacancc
cgaaccaang 9360ggnntgtgtc tcttnccacc ggaactatgg gagcgtccgt
acgaaaattt gcacttgagc 9420acgatcgacc gcaatcgtgg tgctagtcgc
ttacggtttt ggttggttgc tagtgctata 9480ctcgctctgc tttgcttgta
aatcctaaat caatgtagta ccaggactac aaggcaggag 9540gtgaagtcag
ctgtacccac ggctggctga aaccggggct tgacgacccc ccctatccga
9600gttgggcaag gtaacatcac gggtgtgacg accccgcccc cccatgtcgc
gcgtaagcgc 9660acgggcaagg cagctaggct gagagtctgg gcaactctcc
cgtaccccac ccgaggctac 9720gcctcgtcct ggcgaggacc gtaaacatac
gtcgtcagcg tggtgacctg acgtatcttg 9780ttaaccactt aatggtcgta
actcgacccc cgtgccgggg atctaagcgc ggcaccgcga 9840tgaggggggt
caacggcccc tttcatt 98673009867DNAHuman Pegivirus 2 300aactgttgtt
gtagcaatgc gcatattgct acttcggtac gcctaattgg taggcgcccg 60gccgaccggc
cccgcaaggg cctagtagga cgtgtgacaa tgccatgagg gatcatgaca
120ctggggtgag cggaggcagc accgaagtcg ggtgaactcg actcccagtg
cgaccacctg 180gcttggtcgt tcatggaggg catgcccacg ggaacgctga
tcgtgcaaag ggatgggtcc 240ctgcactggt gccatgcgcg gcaccactcc
gtacagcctg atagggtggc ggcgggcccc 300cccagtgtga cgtccgtgga
gcgcaacatg gggtgttcaa ctgatcaaac catttgttct 360ccagtcgtgg
aggccgacta taatacctcc tcgggctgcc gggccttaaa tgggagctac
420cactgcggtg gtggctcttg ccggtcacca agtcgtgtgc aggttgcagg
acgagtcctg 480cggctgtgcg cattccttgc gctgatcgga tccggtatgt
gttccatccg gtccaaaaat 540gaagggcgca ttgagtcagg gcaaatattg
cagtctcagc gcgcatgttg gactggtgag 600ggtttcgctt tcttttctaa
ctgttgcaat caatctgaca ttatgtggtg tttgcaccgt 660tggtgtgtga
caagacctgg ctgtttggtg tgcacgggca atgccactca tcctgtctgc
720tgggactatc ttgggtccgg tgtgagtcgg cggcctgcgc gtcgaatggg
tgagggagct 780gaagtgcttc ttcgcttgat cggcattgca ggttggctcg
ggctcttagc tgaggctctt 840ggtatgtctg agatctatgc agctttcctt
tgctttggat ttattgcttg gtatggctgg 900ggtataccta agacattggt
gtgcacagtc tgccctgcag tgaacatttc tccctatagc 960ttcttatctc
cagatactat cgcatttggt acgtggctac tacaactgcc tggtcttttg
1020tggcaaatgt ttgtcagctt ccctatactt tacagtactt ggattctttg
gttgttgctc 1080agcggcaaga ctgttgctgt gatagcgatc cttttggcta
gtcctacggt tatggcatac 1140aagcatcaag ctgatagcta cctcaaatac
tgtaccataa ccaatgcttc aactgctatg 1200aactgtgact gcccctttgg
aactttcact cgcaatactg agtctggttt cactatacct 1260agattctgtc
ctgttaaact taatagctct acatttatct gttcatgggg gtcgtggtgg
1320tggtttgctg agaacatcac acgtccatac tcggacgttg gcatgccgcc
agcaccgatt 1380tccgctttgt gctatatcta ttcaaacaat gacccacctt
cttggtatcg taacacaact 1440atcatacctc agaactgtta caactctacg
gctgatccta ccacagctcc atgccgtgac 1500aagtggggca atgcaactgc
ttgtattctt gaccgccggt cgcggttctg cggggactgc 1560tatggcggtt
gcttctacac taatggtagt catgaccgat cctgggatcg atgcggaatt
1620ggttaccgtg atggactcat agagttcgtg cagctcggtc agattcgacc
taacatcgcg 1680aatacgacca ttgagctcct cgctggcgcc tcgcttgtga
tcgcatccgg tcttcgggct 1740gggtatggtt gcagccgagc gcacggcgtg
gtgcactgct ttaagtgtcc ttcataccgt 1800gaccttgaac ggttcgggcc
cgggcttggg aaatgggtgc cattgcctgg cgagcctgtc 1860ccggagttgt
gtattaaccc gcagtgggcg aggcgcggct tccgggtgtc taataatccc
1920ttaagcgtgc tacagacctt cgttgaggac attttcttag cgcctttctg
caatccgacg 1980cctggccgtg tacgtgtgtg taacaatact gctttctacc
cgagaggagg cggctttgtg 2040cagctcatcg gagacgtcca ggtgttaacc
cccaactcta catctttgca ctctctgctg 2100actttgatat cccttatctt
gttagtgtgt gttgtttctg gcgcgcgatt cgttccattg 2160ggaatcatat
tcttctggag cgtgcgccac gtatatgctt cttgttactt aagctgtgat
2220tgggctgttt gcaacgatgc gttctgtttc acatctggca cttgtgctac
cttcaacgac 2280gtcttgtgtc tgccggttgc ggcgcgcata tcgtcctgtg
gccatgctgt gccaccgccc 2340gaccgtggtt gggaggtgcc cgcagcgatg
tcatgggcga tttcgcgtac taccggcttg 2400acgttcgatg tcttttcctt
catccagtac cttcctactg tgcctggcaa caattccgat 2460atcatttact
gtggtgaacc aagcttcttc ggggacatca cgggtatcta ttggccttac
2520tttttgcctg gcatgttgct cttgtacttg actcccctcc tgggtttaag
gttaatgctt 2580gccggcttta atatagatgg cttgtttccc atacggcatg
ccacggctgc actgaggttc 2640tcgacttcgc gtgtgacctt gagtgtcgta
tttgctttcc taatctatat attatctcat 2700cctgttaatg ctgcgctcaa
tagaatgttc ctagcatctg caaatctaga gatgatctta 2760tcctttgata
cctatcatga gactgttctt tacgtcgttt gtctattgct ctacctccag
2820gtgtcgcccc gtgcgggctt ggctgctatg gtggccatca agctatctcg
aggcctgtta 2880ttcgctgtgg tgttggcgca cggagtgtgc cgacctgggc
gggtatttgg tcttgaggtt 2940tgcgcggaca tctcttggtt ggtggagttt
actggcaact gcacttggta catgtcctgt 3000gtcttctctt tttggtgcgc
agtgtttgcc ttcaccagtc cacttggacg acagtataag 3060cttcagatct
atcggtactg ggcgcaggcc tatgccagac tcatcctcgc tgtcggttgt
3120ggtcctctcg ggagggagtt ccatttccgt gcgagcgtgg gcgtgctctg
gtgtggtgct 3180tgcatgctct ggccccgtga gtgctctgaa atcagcttgg
tctttattct gtgtgctctg 3240actgtggaca ccatagacac atggttagta
gcgtgcttgt ccgcagggcc aagcgcgcga 3300acccttgcaa ctctggccga
tgacatggcg cgcattggtg accaccgggc gttgcgcgcc 3360gtgttgcgtt
gctttggatc acgtggcaca tacatataca accacatggg ccaggtctca
3420gaacgggtgg cgcaagcagt cagggatttc ggcggttgct tggaaccagt
cgtgttggag 3480gagcccacct ttactgaggt cgtggatgat acaatgaatt
tggtgtgtgg acaattgctt 3540ggaggtaaac ccgtggtggc ccgctgcggc
acgcgtgtct tagtgggaca cctcaaccct 3600gaagacctgc cacctggttt
ccagctgagt gctccggtgg ttattaccaa accaagcatt 3660ggtacgtggc
cctttcttaa ggcgacactc acagggcgtg ctgaaacacc gggatccggc
3720cagatcgtgg tgttgtcttc cctgacaggt cggtcaatgg gtactgcagt
gaatggcaca 3780ctgtatgcga ccggccacgg tgctggtgcg cgcggcctag
ccacgtgcgc tggtttgagg 3840acgccacttt acacggcatt atctgaagat
gtcgtggcct actcttgcct tccgggcatg 3900agctccctag agtcctgcaa
ctgctcgccc agccgggttt gggtggtgaa caacaacgga 3960gggttggtgt
gtggcagagt ggagaaagac gacgtctgtt tggactgtcc cacgcacata
4020gatcaactgc ggggtgcttc ggggtcgccg gttttgtgtg atcacggtca
tgcatacgcg 4080ttgatgctcg gtggctactc taccagtggt atttgtgcgc
gtgtccggat agtccggcca 4140tggcagaacg cctattcctc ctcagggggg
caaggcggaa tgcaggcgcc agctgtgaca 4200ccaacatact ctgaaatcac
ctactatgcc cctactggtt ctggtaaatc aacaaaatat 4260ccagtggacc
tagtcaagca gggacacaaa gtattagtcc ttttaccaag tgtggctgtc
4320gtcaaaagta tggctcctta cattaaggaa aaatataaga ttagacctga
aattagagct 4380ggcacagggc ctgacggtgt gacggtcatc actggcgaga
acttggcgta catgacctat 4440ggccgtttcc ttgtagatcc ggaaacgaat
ctgcggggtt acgctgtagt catctgcgac 4500gagtgccatg acacatcatc
caccacgcta ctcggcatcg gcgcagtgcg catgtatgct 4560gagaaagctg
gagtgaagac cgttgtattc gccacagcca ctcctgctgg cattcaagtg
4620cagtcacatc ccaacattga tgaatatcta ttgactgata caggcgacgt
ggaattctac 4680ggcgctaaaa ttaaattgga caacatcaga actggtagac
atgttatctt ttgccactcg 4740aaggccaggt gtgcggaact aacgcagcag
ctctccggcc ttggtgttcg tgcagtgagt 4800ttttggcgcg gctgtgacat
caagagcatt cccgcctcag actctattgt tgtagtggca 4860actgatgcat
tgtccacagg ctacacaggg aactttgatt cggtcattga ctgcgggtgt
4920tgcgtagagc aaactgtaac aattgacatg gaccccacgt tctccatctc
ggcccgagtg 4980gtgccatgca ctgctgcatt gcgtatgcag cggcgcggac
gcaccggtcg tggcaggagg 5040ggagcgtact acacaaccac tccaggagca
gcaccctgcg tcagcgttcc cgatgctaac 5100gtctggcaat cagtggagtc
agccatggtc ttttatgatt ggagtgctgc caggatagag 5160caatgcctgg
cggcatacca tgatttaggg tgcacaccac gcatcagttg tgacccacac
5220actccagtgc gggtgatgga cacactgagg gcgtatctgc gcagacctga
ggtgacgacc 5280gcggctctcg caggagagca gtggccgctg ctttacggcg
tgcagttgtg catctgcaaa 5340gagaccgagg cccacggtcc agacgatggc
atcaagtgga aatgcttact caataacaac 5400aacaaaacac ccctgttgta
tgccttagac aatcctacac tggaattcac tacccaacat 5460gacttgactc
gccgtatagc tggcgcttta tcgagcacag tgttcgtgga gacaggctac
5520ggccccatcc tcctcgctgg cgctgctttg gctgcctcct ttgcctttgc
gggcgccact 5580ggagctttag tgccgtcggc cgtttggagc gttgaaaacg
ggcttgctgg cgtgacccgt 5640cccgatgcga cagacgagac cgcggcctac
gcgcagcgct tgtaccaagc ctgcgcagat 5700tcaggaattc tcgccagctt
gcagggtacg gcgagtgcgg cactgagcag actggccgat 5760gccagtaagg
gtgctagtca atatctggca gccgcgcctc cttcgcccgc ccccctggta
5820caggtgctgc agttcctcga gaccaatttt agctccattg catctttcgg
tctgctctgt 5880gccggctgtc aggccggcga gtgcttcact gcgcttgccg
ggttggtgtc cggtgctaca 5940gctggcttgg gaggtgccca taagtggttg
ttagctattg caggaacttg gctagttagc 6000ttgcagactg ggccccgtgg
cggcatggtt gcgggtctct cagttctagc aggctgttgc 6060atcggtagtg
tcaccgggct tgacttcctg tttgggtgcc ttacaggttg ggaggccgtg
6120gtcggtgctg cggttgcaac gcagaaaatc ttgtctggtt cggctgacat
gaccactctg 6180gtagatctcc tacctgctct cttctcccct ggcgccggca
tagctggcgt cgtgcttgtc 6240tttattctaa gcaactcaag tgtaaccatg
tgggctaatc ggctattgtc catgtgtgca 6300aaacaaacta tttgtgaaaa
ttacttctta actgagaaat ttggccaaca attaagcaaa 6360ctttccctgt
ggcgctctgt gtaccattgg gcgcaggcac gtgaaggata cacacagtgc
6420ggtgtggtca gcgggatctg gagctttgtc ttgtgcattc tacgtgctgt
gtgggattgg 6480gcggctaaac atgtgccacg gttccgtgtg cctatgattg
gctgctcacc tgcgtggtgc 6540gggcgctggc ttggtactgg caccttgttg
accacctgtg ggtgtggaga acgtgtatcc 6600cttcagtgcc tttgctcgac
atctgaccca acactcagtg tgggccgttg gtgttggtgt 6660agttggcgtg
ttgggttccc attcaacccg acgacgacag ccaccggcac tttacggccg
6720gacatcagtg acgccaccaa attgggcttc cggtatggtg tcgccgagat
cgtggagcta 6780gagcggcggg gcaacaaatg gcatgtctgt gcagcatcat
gttgcttgga ccgggccagc 6840gttgcatccg ccgtgagggc cccaccggtc
acggccgatg gcatacctat cagtaccttt 6900tctccaccac aaacttacaa
actctctctt tgttcttttg attcagtttg catgactact 6960aacttatgta
atccagctaa gaccctgagt gtgtgctcgc aggaggctgt tgagctactg
7020gaagaaacag ttgacagagc acaagtagtg atgtgtcaaa atctggaggc
gcgaagacgc 7080gctgagtttg atgcatggca agttcgcgaa gcaattcgcg
acgagtacac gcgtttggca 7140gacgaggatg ttgacgcgac aacgtcggtg
aaacccccgg tggccaaggc tgctgtgggt 7200agctcgacgt tggatgatgt
tagcgtgctg actgtcttgc gcgaactcgg tgaccagtgc 7260caaaatgcta
tcaaatttgt agttcaggcg gcttcacggt ttgttccacc agtgcccaag
7320ccacgcacgc gtgtctcggg tgtgttggag cgtgtgcgca tgtgcatgcg
cacgccacca 7380atcaagtttg aggctgccgc agtaccaatt catgatataa
tcccagaaga gtgtcacatt 7440gtgctacgct gtaccggctg caacgaccag
gccttgactg ttccgtacgg cacttgcact 7500cagtctttaa tcaagcattt
gactagtaaa cacagtcact acattccaaa acagaagata 7560gaagaggaca
cagaagtaac tgtcatttgc gccgtaccaa caacgcgcgc aagcaaactc
7620atcacattca gagcaggtga tcgatcagtc tcatgttgtc accccttgca
aacccctatt 7680agggccctgc ttctaaagta cgggttacct atcgggaagt
ggtctgactg caacgggccc 7740cttggtgacg atgctcgagt ctgtgacgtc
aatggagtaa caacttatga accatgcatg 7800caatcctaca gttggtttcg
accgattgtg gcaccaacaa ccccaccttt gcctgcaacc 7860cggaccgtgg
ctggcatttt acgcgcagac acatcgcgcg tttacaccac aacggcggtt
7920gacgtctccg agcggcaggc caaggtcaca attgatcaaa catcagccaa
ggtggatcag 7980tgtttccgag acacatacaa ttgctgcctt gctaaggcaa
agaccttcag acaatctggc 8040atgtcatatg aggatgctgt gtcaaagatg
cgcgcaaaca ccacgcgtga ccataacaac 8100ggcatcactt attcagattt
ggtctctgga cgcgcaaaac ctgtcgttca gaaaattgta 8160aatcaaatgc
gcgccggagt gtacgacgct ccgatgcgca ttatcccaaa acctgaagtg
8220ttccctcgag acaaaacaac acggaagcca ccgaggttca tcgttttccc
tgggtgcgcc 8280gcgcgagtcg cggagaaaat gatcctgggt gatcctggcg
cgataaccaa gcacgtgcta 8340ggtgatgcct acgggtttgc cactccgccg
catgagcgcg cgcgcctgtt ggaacaatgg 8400tggaaccgcg caacggagcc
acaagctatc gcggttgatg cgatctgctt tgatagcacc 8460atcacggcag
aggacatgga tcgtgaggct aacatcgtgg ctgcagcgca tacggaccct
8520gaaggtgttc acggcctata taattattac aaaagaagcc ccatgtgtga
catcacgggg 8580aaggttgtcg gagtgcgttg ctgtcgagcc tcgggtacgc
ttacaacaag cagtggcaac 8640acgcttactt gctaccttaa ggttcgtgca
gcttgcacgc gctccggcat taaaccaatt 8700ggcttactaa ttcatggaga
tgacaccctc atcgtcacag aacgttgcgc tcaagagact 8760ctcgatgagt
tcagcaacgc acttgatgac tatgggttcc cacacaccat ccaggcgtct
8820ggggacctct cgtctatcga gtgctgtagc gcacgtgtgg acagcgtttg
cctccgggga 8880ggtatgcgtc gcatgcttgt gccacaagct cgacgtgcga
ttgcacgcgt tctcggggaa 8940aagggcgatc cactgggtac catcggtagc
tatgttgtca tgtatcccac tgcggccgtg 9000actgtctacg tgctattgcc
cctgttgtgc atgctcatac gaaatgagcc atcacagacg 9060gggacacttg
tgacgctgac ggtccacggt aacagtgtga gtgtgccagc gtggctgctt
9120ccaaccatca ttgcaaattt acatggtcgt gacgcactac aggtagtccg
tcacagtgca 9180gcttccatgg cggaattgtc atcagcgttg gccttctttg
gcatgagagg gttgaattgc 9240tggaggcgga gacgccgtgc cattagggct
gatatgatca agtcgggcgg gtggaatgcg 9300aatttcgcgc agatgttact
gtggtcaccg gaggtaagaa caccacaacc cgaaccaagg 9360ggtctgtgtc
ttttgccgcc ggaactgtgg gagcgtccgt acgaaaattt gcacttgagc
9420acgatcgacc gcaatcgtgg tgctagtcgc ttacggtttt ggttggttgc
tagtgctata 9480ctcgctctgc tttgcttgta aatcctaaat caatgtagta
ccaggactac aaggcaggag 9540gtgaagtcag ctgtacccac ggctggctga
aaccggggct tgacgacccc ccctatccga 9600gttgggcaag gtaacatcac
gggtgtgacg accccgcccc cccatgtcgc gcgtaagcgc 9660acgggcaagg
cagctaggct gagagtctgg gcaactctcc cgtaccccac ccgaggctac
9720gcctcgtcct ggcgaggacc gtaaacatac gtcgtcagcg tggtgacctg
acgtatcttg 9780ttaaccactt aatggtcgta actcgacccc cgtgccgggg
atctaagcgc ggcaccgcga 9840tgaggggggt caacggcccc tttcatt
98673019867DNAHuman Pegivirus 2 301aactgttgtt gtagcaatgc gcatattgct
acttcggtac gcctaattgg taggcgcccg 60gccgaccggc cccgcaaggg cctagtagga
cgtgtgacaa tgccatgagg gatcatgaca 120ctggggtgag cggaggcagc
accgaagtcg ggtgaactcg actcccagtg cgaccacctg 180gcttggtcgt
tcatggaggg catgcccacg ggaacgctga tcgtgcaaag ggatgggtcc
240ctgcactggt gccatgcgcg gcaccactcc gtacagcctg atagggtggc
ggcgggcccc 300cccagtgtga cgtccgtgga gcgcaacatg gggtgttcaa
ctgatcaaac catttgttct 360ccagtcgtgg gggccgacta taatacctcc
tcgggctgcc gggccttaaa tgggagttac 420cactgcggtg gtggctcttg
ccggtcacca agctgtgtgc aggttgcgag acgagtcttg 480cagctgtgcg
cactccttgc gctgatcgga tccggtatgt gttcgatccg gtccaaaact
540gaagggcgca ttgagtcagg gcaaatattg cagtctcagc gcgcatgttg
gactggtgag 600ggtttcgctt tcttttctaa ctgttgcaat caatctgata
tcatgtggtg tttgcaccgt 660tggtgtgtga caagacctgg ctgtttggtg
tgcacgggca atgccactca tcctgtctgc 720tgggactatc tcgggtccgg
cgtaagtcgg cggcctgctc gtcgaatggg tgagggagct 780gaagtgcttc
ttcgcttgat cggcgctgca ggctggcttg ggctgttagc tgaggctctt
840ggtatgtccg aaatctatgc agctattctt tgctttgggt ttattgcttg
gtatggctgg 900ggtataccta agacattggt gtgcacagtc tgccctgcag
taaacatttc tccctatagc 960ttcttatctc cagatactat cgcatttggt
acgtggatac tacaactacc tggtcttttg 1020tggcagatgt ttgtcaactt
ccctatactt tacagcactt ggattctttg gttgttgctc 1080agcggcaaga
ctgttgctgt gatagcgatc cttctggcta gtcctacggt tatggcgtac
1140aagcatcaat ctgacagcta cctcaaatac tgtaccataa ccaatgcttc
aactgctatg 1200aactgtgact gcccctttgg aacctttact cgcaatactg
agtctcgttt ctctatacct 1260agattctgtc ctgttaaaat taaaagctct
acatttatct gctcatgggg gtcgtggtgg 1320tggtttgcgg agaacatcac
gcgtccatac wcrgacgttg gcrtgccacc agcaccgatt 1380tcygctttgt
gctatatcta ttcwaacaat gacccacctc cttggtatca taacacaact
1440atcatacctc agaaytgtcg caactctacg gtkgatccta ccacarctcc
atgccgygac 1500aagtggggya aygcaactgc ttgtattctt gaccgccgkt
cgcggttctg cggggactgc 1560tatggcggtt gtttctatac taatggtact
catgatcgat cctgggatcg atgtgggatt 1620ggttaccgtg atggactcat
agagttsgtg cagctcggtc agattsgacc taacatctss 1680aatacgacca
ttgagctcct cgcwggcgcc tcgcttgtga tcgcatccgg tcttcggcct
1740gggtttggtt gcagccgagc gcatggcgtg gtgcactgct ataggtgtcc
ttcataccgt 1800gaccttgaac agtttggtcc tgggcttggg aaatgggtgc
cattgcccgg cgagcctgtc 1860ccggagctgt gtatcaaccc tcagtgggcg
aggcgcggct tccggatgtc taataatcct 1920ctgagcttgc tacagacctt
cgttgaggac attttcctag cgcctttttg taatccgacg 1980cctggccgtg
tacgtgtgtg taacaatacc gctttctatc caagaggagg cggctttgtg
2040cagctcatcg gggacgtcca ggtgctaacc cctaacactg catctttaca
ctctctgctg 2100actttgatat ctcttatctt gttggtgtgt gttgtttctg
gtgcgcgatt cggtccacta 2160ataatcatat ttttctggag cgcgcgccay
gtatatgctt cttgttactt aagctgkgat 2220tgggctgttt gcaacgatgc
gttctgtttc acatctggya cttgtgctac tttcaatgac 2280gtcttgtgtc
tgccggttgc aacgcacata tcgtcctgtg gccacgctgt accgcccccc
2340gaccgyggtt gggaggtgcc tgcggcgatg tcatgggtga tttcgcggac
tactggcttg 2400acgttcgatg tcttctcttt catccagtat ttccctactg
tgcctggcaa taacactgat 2460attatctact gtggtgaacc waccttcttt
ggggacatca cgggcattta ttggccttac 2520tttttgcctg gcgtgttgct
cttgtacttg actcccttcc tgggttttag gttaatgctt 2580gctggcttta
atatagatgg cttgtttccc atacggcatg ccacggctgc actgaggttc
2640tcgacttcgc gtgcgacctt gagtgtcgta tctgcttttc taatctatat
attatctcac 2700cctgttaayg ctgcgctcaa tagaatgttc ytagcatctg
caaacttaga gatgatctta 2760tcctttgata cctatcacga gactgttctt
tacatctttt gtctattcct ctacctccag 2820gtgtcgcccc gtgcgggctt
ggccgctatg gtggccatca agctatctcg gggcctgcta 2880ttcgctctgg
tgttggcgca aggcgtgtgc cgacctgggc gggtgtttgg tcttgaggtt
2940tgcgcggacg tctcttggtt ggtggagttt actggcaact gcacttggta
catgtcctgc 3000atcttctcct tttggtgcgc agtgtttgcc ttcaccagtc
cacttggacg acagtataag 3060cttcagatct accggtactg ggcacaggtc
tatgccagac tcatcctcgc tgtcrgttgt 3120ggtcctctcg gacgagagtt
ccatttccgc gcaagcgtgg gcgtgctttg gtgtggtgct 3180tgcatgctct
ggccccgtga gtgctctgaa atcagcttgg ctctcattct gtgtgctctg
3240acagtggaca ccatagacac atggctagta gcgtgcttgt ccgcagggcc
gagtgcgcga 3300gcccttgcaa cgctggccga cgacatggtg cgcatgggtg
accaccgggc gttgcgcgcc 3360gtgttgcgtt gctttggatc acgtggcaca
tacatataca accacatggg ccaggtctca 3420gaacgggtgg cgcaagcagt
cagggatctc ggcggttgct tggaaccagt cgtgttggag 3480gagcccacct
ttactgaggt cgtggatgat acaatgagta agatatgtgg acaattgctt
3540ggtggtaaac ccgtggtggc ccgctgcggc acgcgtgtct tggtgggaca
cctcaaccct 3600gaagatctgc cacctggttt ccagctaagt gctccggtgg
ttattaccaa accaagcatt 3660ggtacgtggt ccttccttaa ggcgacactc
acagggcgcg ctgaaacacc cggatccggc 3720cagatcgtgg tgttgtcttc
cctgacaggt cggtcgatgg gtactgcagt gartggcaca 3780ctgtatgcga
ccggccatgg tgctggtgca cgcggcctag ccacgtgtgc tggtttgagg
3840acgcctctct acacggcatt atctgatgat gtcgtggcct actcttgcct
tccgggcatg 3900agttccctag agccctgccg ctgttcgccg agccgggttt
gggtgatgaa caacaatgga 3960gggttggtgt gtggcagagt ggagaatgaa
gacgtctgtt tggactgtcc cacgcacata 4020gatcaactgc ggggtgcttc
gggttcaccg gttttgtgtg atcacggtca tgcatacgcg 4080ttgatgctcg
gtggttactc taccagtggt atttgtgcgc gtgtccggat agtccggcca
4140tggcagaacg cctattcctc ctcagggggg cagggcggga tgcaggcgcc
agctgtgaca 4200ccaacatact ctgaaatcac ctactacgcc cctacyggtt
ctggtaagtc
aacaaaatat 4260ccagtggacc tagtcaaaca gggacacaag gtattggtca
tcataccaag tgtgtctgtc 4320gtcaagagta tggcccctta cattaaggag
acatataaga ttagacctga aattagagct 4380ggcacagggc ctgacggtgt
gacggttatc actggtgaga acttggcgta catgacctac 4440ggccgcttcc
tcgtggatcc ggagacgaat ctgcggggtt atgccgttgt catttgcgac
4500gagtgccatg acacatcatc taccacgcta ctcggtattg gcgcagtgcg
catgtatgcc 4560gagaaagctg gagtgaagac cgttgtattc gccacagcca
ctcctgctgg cattcaagta 4620cagccacatc ccaacattga tgaatattta
ttgactgaca caggcgacgt ggatttctac 4680ggcgccaaaa tcaaattgga
taacatcaga actggtagac atgttatctt ttgccactcg 4740aaggccaggt
gtgcggaact aacgcagcag ctctccggcc ttggtgttcg tgcagtgagt
4800ttttggcgag gctgtgacat caaaaccatt cccgcctcag actctattgt
cgtagtggca 4860actgatgcat tgtccacagg ctacacaggg aactttgatt
cggtcatcga ctgcgggtgt 4920tgcgtagagc aaactgtgac aattgacatg
gatcccacgt tctccatctc agcccgagtg 4980gtgccatgta ctgctgcatt
gcgtatgcag cggcgcggac gtactggtcg cggcagaagg 5040ggggcgtact
acacaaccac tccaggagca gcaccctgtg tcagcgttcc cgacgctaac
5100gtctggcaag cagtggagag cgccatggtc ttttatgatt ggaacgccgc
caggatacag 5160cagtgcctgg cagcatacca tgatttaggg tgtacaccac
gcctcagttg tgatccatgc 5220actccagtgc gggtgatgga cacactgagr
gcgtacctgc gcagacctga ggtgacgact 5280gcggctctcg caggagagca
gtggccgctg ctttacggtg tgcagttgtg catctgcaag 5340gagactgagg
cccacggtcc agacgatrgc atcaagtgga agtgcttact caacaacagt
5400aayaaaacac ccctgttgta tgccttagac aatcctacac tggaattcac
tacccaacat 5460gacttgactc gccgtatagc cggcgcttta tcgagcacag
tgttcgtgga gacaggctac 5520ggccccatcc tccttgctgg cgctgcattg
gctgcctcct tcgcctttgc gggcgccact 5580ggagccttag tgccgtcggc
cgtttggagc gttgataacg gggttgctgg cgtgacccgt 5640cccgacgcga
cagacgagac cgcggcctac gcgcagcgct tgtaccaagc ctgcgcagat
5700tcaggacttc tcgccagctt gcagggcacg gcgagtgcgg cgctgagcaa
actggccgat 5760gccagtaggg gtgctagtca atatctggca agcgcgcctc
cctcgcccgc ccccctggta 5820caggtgctgc agttccttga gaccaacttt
agctccatcg catccttcgg tctgctctgc 5880gctggttgtc aggctggcga
gtgcttcaca gcgcttgccg ggttggtgtc cggtgctacg 5940gctggcttgg
gaggagccca taagtggttg ttagccattg caggaacttg gctagttagt
6000ctgcagacag gggcccgtgg cggcatggtt gcgggcctct cggtcctggc
aggctgttgc 6060atcggtagtg tcactgggct tgacttcctg tttgggtgcc
ttacaggttg ggaggccgtg 6120gttggtgctg cggttgcgac acagaaaatc
ttgtctggtt cggctgatat gaccactctg 6180ttagatctct tacctgcttt
tttctcccct ggtgccggcg tagctggcat cgtgcttgtc 6240tttattctaa
gcaactcaag tgtaaccacg tgggctaatc ggctattgtc catgtgtgca
6300aaacaaacca tttgtgacaa ctacttctta tctgacaaat ttggccaaca
attaagtaaa 6360ctttccttgt ggcgcactct gtatcgttgg gcggaggcac
gtgagggata cacacagtgt 6420ggtgtggtcg gcgggatctg gagctttgtc
ttgtgcattc tacgcgctgt gtgggattgg 6480gcggctaaac atgtgccacg
gttccgtgtg cccatgattg gctgctcacc tgcgtggtgc 6540gggcgctggc
ttggtactgg caccttgttg accacctgcg ggtgtggaga acgtgtgtct
6600cttcagtgtc tttgctcaac gtctgatcca ttgctcagag tgggtcgttg
gtgtcggtgt 6660agttggagtg ttgggttccc attcaacccg actacgacag
ccaccggcac tttacggccg 6720gacattagcg acgccactag attgggtttc
cggtatggca ttgccgagat cgtggagctg 6780gagctgcggg agcacaaatg
gcacgtctgt gcagcatcat gttgcttgga ccgagctagt 6840gttgcatccg
ctgtgaaggc cccaccggtc acagccaatg gtatacctat cagtaccttt
6900tctccaccac aaacttacag cctctctctc tgttcttttg attcagtttg
catgtctact 6960aatctatgta acccagccaa gaccctgagt gtgtgctcac
aggaggccgt tgagctgctg 7020gaagaaacag ttgacacggc acaagtgatg
atgtgtcaaa atctggaggc gcgaagacgc 7080gccgaatatg atgcatggca
agttcgccac gcagttggcg acgagtacac gcgtttggca 7140gacgaggatg
ttgacacgac aacgtcggtg aaacccccgg cggccagggc tgctgtggat
7200agctcaacgt tggaagatgt tagcgtgctg actgttttgc gcgagctcgg
cgaccaatgc 7260caaaatgcta tcaaatttgt agttcaggcg gcttcacggt
ttgttccacc tgtgcccaga 7320ccgcgcacgc gcgtctcggg cgtgttggag
cgtgtgcgca tgtgcatgcg cacgccacca 7380atcaagtttg aggccaccgc
agtaccaatt cataacataa tcccagaaga gtgtcacatc 7440gtgctacgct
gtactggctg taacgaccag gccttgactg ttccgtacgg cacttgcact
7500cagtctttaa ttagacattt gaccaacaaa cacaaccact atattccaaa
acagaagata 7560gaagaagaca cagaagtaac tgtcatttgc gccgtaccaa
caaagcgcgc aagtaaactc 7620atcactttca gagcaggtga tcgatcagtc
tcatgttgcc accccttaca aactcctatt 7680agggccctgc ttctaaagta
cgggttacct atcgggacgt ggtccgactg caacggaccc 7740cttggtgacg
acgcccgagt ctgtgacgtc aatggagtga caacttatga accatgcatg
7800caatcctaca gttggttccg accaattgtg gcacctacaa ccccaccttt
acctgtaacc 7860cggagcgtgg ctgggatttt acgcgcagac acatcgcgcg
tttacaccac aacggcggtc 7920gacgtctccg agcggcagtc taaggtcaca
attgatcaaa catcagccaa ggtggaccag 7980tggttccgtg acacatacaa
ctgttgcctt gctaaggcaa aaaccttcag acaatctggc 8040atgtcatatg
aggatgctgt gtcaaaaatg cgcgcaaaca ccacgcgtga ccataacact
8100ggcatcactt attcagattt ggtctctgga cgcgcaaaac ctgccgttca
aaaaattgta 8160gatcaaatgc gtgctggagt gtacgacgct ccaatgcgca
ttatcccaaa acctgaagtg 8220ttcccccgag acaagtcaac acggaagcca
ccgcggttca tcgtttttcc tgggtgcgcc 8280gcgcgagtcg cggagaaaat
gatcctgggc gatcctggcg cgataaccaa gcacgtgcta 8340ggtgatgcct
acgggtttgc cactccgccg catgaacgcg cgcgcctatt ggagcaatgg
8400tggaaccgtg caacggagcc acaagctatc gcggttgatg cgatctgctt
tgatagcacc 8460atcacagcag aggacatgga tcgcgaggcc aacatcctgg
ctgcggcgca ttcggaccct 8520gaaggtgttc acggcctata caattattac
aaaagaagcc ccatgtgtga tatcacagga 8580aatgttgtcg gagtgcgttg
ctgtagagcc tcaggtacgc ttacaacaag cagtggcaac 8640acgcttactt
gctacctcaa ggttcgtgca gcttgcacgc gcgccggcat taaaccaatt
8700ggcttgctaa ttcatgggga tgataccctc atcatcacag aacgttgcgc
tcaagagact 8760ctcgatgagt tcagcaatgc acttaatgac tacgggttcc
ctcacacctt ccaggcgtct 8820ggggacctct cgtcagttga gtgctgtagc
gcacgtgtgg acagcgtttg cctccgggga 8880ggtatgcgtc gcatgctcgt
gccacaagct cgacgtgcga ttgcacgcgt tctcggggaa 8940aagggcgatc
cactgggtgt catcagcagc tatattgtca tgtatcctac tgcggccgtg
9000actgtctacg tgctattgcc cctgttgtgc atgctcattc gaaatgagcc
atcgcagacg 9060gggacacttg taacgctgac ggtccacggt aacagtgtga
gcgtgccagt gtggctgctt 9120ccaaccatca ttgtaaattt acatggccgt
gacgcactgc aggtagttcg tcacactgca 9180gcttccatgg cggagctgtc
atcagcgttg gccttctttg gcatgagagg gttgaactgc 9240tggaggcgga
gacgccgtgc catcaggact gacatgatca agttgggcgg gtggaatgcg
9300aatttcgcgc agatgttact gtggtcaccg gaggtgagga caccacaacc
cgaaccaagg 9360ggtgtgtgtc tcttaccacc ggaactatgg gagcgtccgt
acgaaaattt gcacttgagc 9420acgatcgacc gcaatcgtgg tgctagtcgc
ctacggtttt ggttggttgc cagtgctata 9480ctcgctctgc tttgcttgta
aatcctaaat caatgtagta ccaggactac aaggcaggag 9540gtgaagtcag
ctgtacccac ggctggctga aaccggggct tgacgacccc ccctatccga
9600gttgggcaag gtaacatcac gggtgtgacg accccgcccc cccatgtcgc
gcgtaagcgc 9660acgggcaagg cagctaggct gagagtctgg gcaactctcc
cgtaccccac ccgaggctac 9720gcctcgtcct ggcgaggacc gtaaacatac
gtcgtcagcg tggtgacctg acgtatcttg 9780ttaaccactt aatggtcgta
actcgacccc cgtgccgggg atctaagcgc ggcaccgcga 9840tgaggggggt
caacggcccc tttcatt 98673029867DNAHuman Pegivirus
2misc_feature(1)..(11)N = A, C, T, or Gmisc_feature(6209)..(6214)N
= A, C, T, or G 302nnnnnnnnnn ntagcaatgc gcatattgct acttcggtac
gcctgattgg taggcgcccg 60gccgaccggc cccgcaaggg cctagtagga cgtgtgacaa
tgccatgagg gatcatgaca 120ctggggtgag cggaggcagc accgaagtcg
ggtgaactcg actcccagtg cgaccacctg 180gcttggtcgt tcatggaggg
catgcccacg ggaacgctga tcgtgcaaag ggatgggtcc 240ctgcactggt
gccatgcgcg gcaccactcc gtacagcctg atagggtggc ggcgggcccc
300cccagtgtga cgtccgtgga gcgcaacatg gggtgttcaa ctgatcaaac
catttgttct 360ccagtcgtgg gggccgacta taatacctcc tcgggctgcc
gggccttaaa tgggagcgac 420cactgcggtg gaagctcttg ccggtcacca
agtcgtgtgc aggctgctag acgagtcttg 480cagctgtgcg cattccttgc
gttgatcgga tccggtatgt gttcgatccg gtccaaaact 540gaagggcgca
ttgagtcggg gcaaatattg cagtctcagc gcgcatgttg gactggtgag
600ggttttgctt tcttttctaa ctgttgcaat caatctgata ttatgtggtg
tttgcaccgt 660tggtgtgtga caagacctgg ctgtttagtg tgcacgggca
atgccaccca tcctgtctgc 720tgggactatc ttgggtccgg tgtaagtcgg
cggcctgcgc gccgattggg cgagggtgct 780gaaatgcttc ttcgcttgat
cggcattgca ggttggctcg gcttgttagc tgaggctctt 840ggtatgtccg
aaatgtatgc agctatcctt tgctttgggt ttattgcttg gtatggctgg
900ggtataccta aaacattggt gtgcacagtc tgccctgcgg taaacatttc
tccctatagc 960tttttatctc cagacactat cgcatttggt acrtggatac
tacaactacc tggtcttttg 1020tggcaaatgt ttgtcaactt tcctatactt
tacagcactt ggattctttg gttgttgctt 1080agtggcaaga ctgttgctgt
gatagcaatc cttttggcta gtcctacggt tatggcgtac 1140aagcatccat
ctgaaagcta cctcaaatac tgtaccataa ccaatgcttc agctgctatg
1200aactgtgact gcccctttgg aacctttact cgcaatactg agtctcgttt
ctctatacct 1260agattctgtc ctgttaaaat tgagagttct acatttatct
gctcatgggg gtcgtggtgg 1320tggtttgctg agaacatcac acgtccatac
tcggacgtcg gcatgccgcc agcaccgatt 1380tccgctttgt gctatatcta
ttcaaacaat gacccacctc cttggtatta taatacaact 1440attatacctc
agaaytgtcg caactctwcg gttgatccya ccacagctcc atgccgtgac
1500aagtggggca atgcaactgc ttgtattctt gaccgccggt cgcggttctg
cggggactgc 1560tatggcggct gcttctatac taatggtagt catgatcgat
cctgggatcg atgcgggatt 1620ggttaccgtg atggactcat agagtttgtg
cagcttggtc agattcgacc taacatctcg 1680aacacgacca ttgagctcct
cgctggtgcc tcgcttgtga ttgcatccgg tcttcggcct 1740gggtacggtt
gcagtcgagc gcatggcgtg gtgcactgct ataggtgtcc ttcataccgt
1800gaccttgaac agttcggtcc tgggctcggg aaatgggtgc cgttgcctgg
tgagcctgtc 1860ccggagttgt gtatcaaccc tcaatgggcg aggcgcggct
tccgggtgtc taacaatcct 1920ttaagcttga tacagacctt tgttgaggac
atcttcctag cgcctttttg yamtccgacg 1980cctggtcgtg tacgtgtgtg
taacaatact gctttctatc caagaggagg cggctttgtg 2040cagctcatcg
gagacgtcca ggtgctgacc cctaacactg catctttaca ctctctgctg
2100accttgatat cccttatctt gctagtgtgt gttgtctctg gcgcgcgatt
cgtcccatta 2160atcatcatat ttttctggag cgtgcgccat gtatatgctt
cctgttactt gagctgtgat 2220tgggctgttt gcaatgatgc attctgtttc
acttctggta cttgtgctac cttcaatgac 2280gtcttgtgtc tgccggttgc
gacgcgtata tcgtcctgtg ggcatgctgt gccaccgccc 2340gaccgtggtt
gggaggtgcc tgcggcgctg tcaygggkga tttcgcggac tacyggcttg
2400acgttcgatg tcttttcttt cattcagtat cttcctactg tgcctggcaa
caattccgat 2460atcatttact gtggtgaacc gacctttttc ggggatctca
cgggcatcta ttggccttac 2520tttttgcctg gtgtgttgct tttgtacttg
actcccttcc ttggtttaag gttaatgctt 2580gccggcttta atatagatgg
cttatttccc atacggcatg ccacggctgc actgaggttc 2640tcgacttcgc
gtgtgaccct gagtgtcgtg gctgcctttc taatctatat attatcccac
2700cctgttaatg ctgcgctcaa tagaatgttc ttagcatctg caaatttaga
gatgattcta 2760tcctttgata cttatcatga gactatcctt tatatcgtct
gcctaatgct ctacctccag 2820gtgtcacccc gtgcgggctt ggccgccatg
gtggccatca agctatctcg aggtctgtta 2880ttcgctgtgg tgttggcgca
cggagtgtgc cgacccgggc gggtatttgg tcttgaggtt 2940tgcgcggaca
tctcctggtt ggtggagttt actggcaact gcacttggta tatgtcctgt
3000gtcttctctt tttggtgcgc agtgtttgcc ttcaccagtc cacttggacg
acagtataag 3060cttcagatct atcggtactg ggcgcaggtc tatgccagac
tcatcctcgc tgtcggttgt 3120ggtcctctcg gccgagagtt ccatttccgc
gcaagcgtgg gcgtactttg gtgtggcgct 3180tgcatgctct ggccccgtga
gtgctctgaa atcagcttgg tcttcattct gtgtgctctg 3240acagtggaca
ccatagacac atggttagta gcgtgcttgt ccgcagggcc aagtgcgcga
3300acccttgcaa cgctggccga tgacatggcg cgcatgggtg acaaccgggc
gttgcgcgcc 3360gtgttgtgtt gctttggatc acgtggcaca tacatataca
accacatggg ccaggtctca 3420gaacgggtgg cgcgagcagt cagggatctc
ggcggttgct tggaaccagt cgtgttggag 3480gagcccacct ttactgagrt
cgtggatgat acamtgartt tagtgtgtgg acaattgctt 3540ggaggtaaac
ctgtggtggc ccgctgcggc acgcgtgtct tggtgggaca cctcaaccct
3600gaagatctgc cacctggttt ccagctgagt gctccggtgg ttatyaccaa
accaagcatt 3660ggtacgtggt ccttccttaa ggcgacactc acagggcgtk
stgaracacc gggatccggc 3720cagatcgtgg tgctgtcttc cctgacaggt
cggtcaatgg gtactgcagt gaatgrcaca 3780ctgtatgcga ccggccatgg
tgccggtgcg cgtggcctag ccacgtgcgc tggtttgagg 3840acgccacttt
acacggcatt atctgatgat gtcgtggcct attcttgcct tccgggcatg
3900agttccctgg agccctgccg ctgtacgccg agccgggttt gggtgatgaa
caacaatgga 3960gggttggtgt gtggcagagt agagaaggac gacgtctgtt
tggactgtcc cacgcacata 4020gatcaactgc ggggtgcttc ggggtcaccg
gttttgtgtg atcacggtca tgcatacgcg 4080ttgatgctcg gtggttactc
taccagtggt atttgtgcgc gtgtccggat agttcagcca 4140tggcagaacg
cctattcctc ctccgggggg caaggcggaa tgcaggcgcc agctgtgaca
4200ccaacatact ctgaaatcac ctactatgcc cctaccggtt ctggtaagtc
aacaaaatat 4260ccagtggacc tggtcaaaca gggacacaag gtattggtca
tcataccaag tgtgactgtc 4320gttaaaagta tggccaatta tattaaggag
acatacaaga tcagacctga aattagagct 4380ggcactggcc ctgacggtgt
gacggtcatc actggtgaaa gcttggcgta catgacctat 4440ggccgcttcc
ttgtggatcc ggagacgaat ctgcgaggct acgccgtagt catttgcgac
4500gagtgccacg acacatcatc caccacgcta ctcggcatag gcgcggtgcg
catgtttgct 4560gagaaagctg gagtgaggac cgttgtattc gccacagcca
cccctgctgg cattcaagta 4620cagccacatc ctaacattga tgaatattta
ttgactgaca caggcgacgt ggacttctac 4680ggcgccaaaa tcaaattgga
caacatcaga actggtagac atgttatctt ttgtcactcg 4740aaggccaggt
gtgcggaact aacgcagcag ctctccggcc ttggtgttcg tgcagtgagt
4800ttttggcgcg gctgtgacat caaaaccatt cccgcctcag actctattgt
tgtagtggca 4860actgatgcat tgtccacagg ctacacagga aattttgatt
cggtcatcga ctgcgggtgt 4920tgcgtagaac aaactgtgac aattgacatg
gaccctacgt tttccatctc ggcgcgagtg 4980gtgccatgca ctgctgcatt
gcgtatgcag cggcgcggac gtaccggtcg tggcagaagg 5040ggagcgtatt
acacaaccac tccaggagca gcaccttgcg tcagcgttcc cgatgctaac
5100gtctggcaag cagtggaaag cgccatggtt ttttatgact ggggtgctgc
caggatacaa 5160caatgcctgg cggcatacca tgatctaggg tgcacaccac
gcatcagttg ygatccacac 5220actccagtgc gggtgatgga cacactgcgg
gcgtacctgc gcagacctga ggtgacgact 5280gcagctctck caggagagca
gtggccgctg ctttatggtg tgcagttgtg catctgcaaa 5340gaaaccgagg
cccatggtcc agacgatagc atcaagtgga agtgcttact caacaacagt
5400aataaaacac ccctgctgta tgccttagac aatcctacac tggatttcac
tacccaacat 5460gacttgactc gccgtatagc cggcgctcta tcgagcacag
tgttcgtgga gacaggctac 5520ggccccatcc tcctcgctgg cgccgctttg
gctgcctcct tcgcctttgc gggtgctaca 5580ggagctttag tgccgtcggc
cgtttggagt gttgacaacg ggttagctgg cgtgacccgt 5640cccgacgcga
cagacgaaac cgcggcctac gcgcagcgct tgtaccaagc ctgcgcagat
5700tcaggacttt tcgccagctt gcagggcaca gcgagtgcgg cgctgggcaa
actggctgat 5760gccagtaggg gtgctagtca atatctggca gccgcgcctc
cttcgcccgc ccccctggtg 5820caggtgctgc atttccttga gaccaacttt
agttccattg catctttcgg tctgctctgt 5880gctggttgtc aggctggtga
gtgcttcact gcgcttgccg ggctggtgtc cggtgctaca 5940gctggcttgg
gaggtgccca taagtggttg ttagctattg caggaacctg gctagttagc
6000ttgcagactg ggccccgtgg cggcatggtt gcgggtctct cggttctagc
aggctgttgc 6060atcggtagtg tcaccgggct cgacttcctg tttgggtgcc
ttacaggttg ggaggccgtg 6120gttggcgctg cggttgcgac gcagaagatc
ttgtctggtt cagctgatat gaccactctg 6180gtagatctct tacctgctat
cttctcccnn nnnnccggca tagctggcat cgtgcttgtc 6240tttattctaa
gtaactcaag tgtaaccacg tgggctaatc ggctactgtc catgtgtgca
6300aaacaaacta tttgtgataa ctacttctta actgagaaat ttggccatca
attaagcaaa 6360ctttccctgt ggcgcgctgt gtaccattgg gcgcaggcac
gtgagggata cacacagtgc 6420ggcgtggtca gcgggatctg gagctttgtc
ttgtgcatcc tgcgggctgt gtgggattgg 6480gcggccaagc atgtgccacg
gttccgtgtg cctatgatcg gctgctcacc tgcatggtgc 6540gggcgctggc
ttggtaccgg caccctgttg accacctgtg ggtgtggaga acgtgtgtcc
6600cttcagtgcc tctgctcaac gtctgaccca acactcagtg tgggccgttg
gtgtcggtgt 6660agttggagtg tcgggttccc attcaacccg actacgacag
ccaccggcaa tttacggccg 6720gacattagtg acgccactaa attgggtttc
cgatatggta tagccgagat cgtggaactg 6780gagcggcggg gtgacaaatg
gcatgtctgt gcagcatcat gttgcttgga ccgagccagc 6840gttgcatccg
ccgtgaaggc cccaccggtc acggccaatg gtatacctat cggtactttt
6900tctccaccac aaacttacag cctctctctc tgttcttttg attcagtttg
catgtctagt 6960aacttatgta acccagctaa gaccctgagt gtgtgctccc
aggaggctgt cgagctactg 7020gaagaaacag ttgataaagc acaagtaatg
atgtgtcaaa atctggaggc gcgaagacgc 7080gcagagtatg atgcatggca
ggttcgccaa gcagttggcg acgagtacac gcgtttggca 7140gacgaggatg
ttgacgcgac aacgtcggtg aaacccccgg tggccagggc tgctgtgggt
7200agctcaacgt tggatgacgt tagcgtgctg actgtcttgc gcgaactcgg
cgaccaatgc 7260caaaatgcta tcaaatttgt agttcaggcg gcttcacggt
ttgttccacc agtgcccaag 7320ccgcgcacgc gcgtctcggg tgtgctggag
cgtgtgcgca tgtgcatgcg cacgccacca 7380atcaaatttg aggccaccgc
agtaccaatt cataacataa tcccagaagt gtgtcacatt 7440gtgctacgct
gtaccggctg taacgaccag gccttgactg ttccgtacgg cacttgcact
7500cagactttaa tcaaacattt gactaacaaa cacagccact acattccaaa
acagaagata 7560gaagaagaca cagaagtaac tgtcatttgc gccgtaccaa
caacgcgcgc aagtaagctc 7620atcactttca gagcaggtga tcgatcagtc
tcatgttgtc accccttgca aactcctgtt 7680aggaccctgc ttctaaagta
tgggttgcct atcgggaagt ggtccgactg caacggcccc 7740cttggtgacg
atgcccgagt ctgtgacatc aatggagtaa caacttatga accatgcatg
7800caatcctata gttggttccg accaattgta gcgccaacaa ccccaccttt
acctgcaacc 7860cggagcgtgg ctggcatttt acgcgcggac acatcgcgcg
tttacaccac aacggcggtt 7920gacgtctccg agcggcaggc taaggtcaca
attgatcaaa catcagccaa ggtggatcag 7980tgtttccgag acacatacaa
ttgctgcctt gctaaggcaa agaccttcaa acaatctggc 8040atgtcatatg
aggatgctgt gtyaaagatg cgcgcaaaca ccacgcgtga ccataacaat
8100ggcaccactt attcagattt ggtctctgga cgcgcaaaac ctgtcgttca
gaaaattgta 8160gatcaaatgc gcgctggagt gtacgacgct ccaatgcgca
ttatcccaaa acctgaagtg 8220ttccctcggg acaagtcaac acggaagcca
ccacggttca tcgttttccc tgggtgcgcc 8280gcgcgagtcg cggagaaaat
gatcctgggc gatcctggcg cgataaccaa gcacgtgctt 8340ggtgatgcct
atgggtttgc cactccgccg catgagcgcg cgcgtctatt ggaacaatgg
8400tggaaccgtg caacggagcc acaagctatc gcggttgatg cgatctgctt
tgatagcact 8460atcacggcag aggacatgga tcgcgaggcc aacatcatgg
ctgcggcgca ttcggaccct 8520gaaggtgttc atggcctgta caagtattac
aaaagaagcc ccatgtgtga catcacggga 8580aaagttgtcg gggtgcgttg
ctgtcgagcc tcaggtacgc ttacaacaag cagtggcaac 8640acgcttactt
gctatctcaa ggttcgcgca gcttgcacgc gcgccggcat taaaccaatt
8700ggcttactaa ttcatggaga tgataccctc attgtcacag aacgttgcgc
tcaagaaact 8760ctcgatgagt tcagcagcgc actcgatgac tatgggttcc
ctcacacctt gcaggtgtct 8820ggggacctct cgtctgttga gtgctgtagc
gcacgtgtgg acagcgtttg cctccgggga 8880ggtatgcgtc gcatgctcgt
gccacaagct cgacgtgcga ttgcacgcgt tctcggggag 8940aagggtgatc
cactgggtgt catcagtagc tatatcgtca tgtatccyac tgcggctgtg
9000actgtctacg tgctattgcc cctgttatgc atgctcattc ggaatgagcc
atcgcagacg 9060gggacgattg tgacgctgac ggtccacggy aacagtgtga
gcgtgccggt gtggctgctt 9120ccaaccatca ttgtaaattt acatggtcgt
gacgcactac aggtagtccg tcacagtgca 9180gcttccatgg cggaactgtc
gtcagcgttg gccttctttg gcatgagagg gttgaactgc 9240tggaggcgga
gacgccgcgc catcaggact gatatgatca rgttgggcgg gtggattgcg
9300aatttcgcgc
agatgttact gtggtcaccg gaggtgagga caccacagcc cgaaccgaag
9360ggcttgtgtc tcttaccacc ggaactatgg gagcgtccgt acgaaaattt
gcamttgagc 9420acggtcgacc gtaatcgtgg tgctagtcgc ttacggtttt
ggctggttgc aagtgctata 9480ctcgctctgc tttgcttgta aatcctaaat
caatgtagta ccaggactac aaggcaggag 9540gtgaagtcag ctgtacccac
ggctggctga aaccggggct tgacgacccc ccctatccga 9600gttgggcaag
gtaacatcac gggtgtgacg accccgcccc cccatgtcgc gcgtaagcgc
9660acgggcaagg cagctaggct gagagtctgg gcaactctcc cgtaccccac
ccgaggctac 9720gcctcgtcct ggcgaggacc gtaaacatac gtcgtcagcg
tggtgacctg acgtatcttg 9780ttaaccactt aatggtcgta actcgacccc
cgtgccgggg atctaagcgc ggcaccgcga 9840tgaggggggt caacggcccc tttcatt
98673039867DNAHuman Pegivirus 2misc_feature(1)..(23)N=A, C, T or G
303nnnnnnnnnn nnnnnnnnnn nnntattgct acttcggtac gcctaattgg
taggcgcccg 60gccgaccggc cccgcaaggg cctagtagga cgtgtgacaa tgccatgagg
gatcatgaca 120ctggggtgag cggaggcagc accgaagtcg ggtgaactcg
actcccagtg cgaccacctg 180gcttggtcgt tcatggaggg catgcccacg
ggaacgctga tcgtgcaaag ggatgggtcc 240ctgcactggt gccatgcgcg
gcaccactcc gtacagcctg atagggtggc ggcgggcccc 300cccagtgtga
cgtccgtgga gcgcaacatg gggtgttcaa ctgatcaaac catttgttct
360ccagtcgtgg gggccgacta taatacctcc tcgggctgcc gggccttaaa
tgggagctac 420cactgcggtg gtggctcttg ccggtcacca agtcgtgtgc
aggttgcgag acgagtcttg 480cagctgtgcg cattccttgc gctgatcgga
tccggtatgt gttcgatccg gtccaaaact 540gaagggcgca ttgagtcagg
gcaaatattg cagtctcagc gcgcatgttg gactggtgag 600ggttttgctt
tcttttctaa ctgttgcaat caatctgata ttatgtggtg tttgcaccgt
660tggtgtgtga caagacctgg ctgtttagtg tgcacgggca atgccaccca
tcctatctgc 720tgggactatc ttggatccgg tgtaagtcgg cggcctgcac
gtcgaatggg tgagggagct 780gaagcgcttc ttcgcttgat cggcattgca
ggttggcttg gactgttagc tgagtccctt 840ggtatgtccg aagtctatgc
agctattctt tgctttggat ttattgcttg gtatggctgg 900ggtataccta
aaacactggt gtgcaccgtc tgccctgcag tgaacatttc tccctatagc
960ttcttatctc cagatactat cgcatttggt acgtggatac tacaactacc
tggtcttttg 1020tggcaaatgt ttgttagctt ccctatactc tacagcactt
ggattctttg gttgttgctc 1080agcggcaaga ctgttgctgt gatagcaatc
cttctggcta gtcctacggt tatggcgtac 1140aagcatcaat ctgaaagcta
cctcaaatac tgtaccataa ccaatacttc aacttctatg 1200aactgtgact
gcccctttgg aacctttact cgcaatactg agtctcgttt ctccatacct
1260agattctgtc ctgttaaaat caatagctct acatttattt gttcatgggg
gtcgtggtgg 1320tggtttgctg aaaacatcac gcgtccatac acggacgttg
gcatgccacc agcaccgatt 1380tccgctttgt gctatatcta ttctaacaat
gacccacctc cttggtatca taacacaact 1440atcatacctc agaactgtcg
caactctacg gtggatccta ccacagctcc atgccgtgac 1500aagtggggca
acgcaactgc ttgtattctt gaccgccggt cgcggttctg cggggactgc
1560tatggcggtt gtttctatac taatggtagt catgatcgat cctgggatcg
atgcgggatt 1620ggttaccgtg atggactcat agagttcgtg cagctcggtc
agattcgacc taacatctcg 1680aatacgacca ttgagctcct cgctggcgcc
tcgcttgtga tcgcatccgg tcttcggcct 1740gggtttggtt gcagccgagc
gcatggcgtg gtgcactgct ataggtgtcc ttcataccgt 1800gaccttgaac
agtttggtcc tgggcttggg aaatgggtgc cattgcccgg cgagcctgtc
1860ccggagttgt gtatcaaccc tcagtgggcg aggcgcggct tccggatgtc
taataatcct 1920ctgagcttgc tacagacctt cgttgaggac attttcctag
cgcctttttg taatccgacg 1980cctggccgtg tacgtgtgtg taacaatacc
gctttctatc caagaggagg cggctttgtg 2040cagctcatcg gggacgtcca
ggtgctaacc cctaacactg catctttaca ctctctgctg 2100actttgatat
ctcttatctt gttggtgtgt gttgtttctg gtgcgcgatt cgttccacta
2160ataatcatat ttttctggag cgcgcgccat gtatatgctt cttgttactt
aagctgtgat 2220tgggctgttt gcaacgatgc gttctgtttc acatctggca
cttgtgccac cttcaatgac 2280gtcttgtgtc tgccggttgc gacgcgcata
tcgtcctgtg gtcatgctgt gccacctccc 2340gaccgtggtt gggaggtgcc
tgcggcgatg tcatgggtga tttcgcggac tactggcctg 2400acgttcgatg
tcttttcctt cattcagtac cttcctactg tgcctggcaa caacaccaat
2460atcatttact gtggtgaacc aaccttcctc ggggacatca cgggcatcta
ttggccttac 2520tttttgcctg gcgcaatcct cttgtacttg actcccttcc
taggtttaag gttaatgctt 2580gccggcttca atatagatgg cttgtttccc
atacggcatg ccacggctgc actgaggttt 2640tcgacttctc gtgtgacctt
gtgtgtcgta gttgctttcc taatctatat attatctcac 2700cctgttaatg
ctgcgctcaa tagaatgttc ttagcatctg caaatttaga gatgatctta
2760tcttttgata cctatcatga gactgttctt tatatccttt gtctattgct
ctacctccag 2820gtgtcgcccc gtgcgggctt ggccgctatg gtggccatca
agctatctcg aggcctgtta 2880ttcgctgtgg tgttggcgca cggtgtgtgc
cgacctgggc gggtatttgg tcttgaggtt 2940tgcgcggaca tctcttggtt
ggtggagttt actggcaatt gcacttggta catgtcctgt 3000gtcttctctt
tttggtgcgc agtgtttgcc ttcaccagtc cacttggacg acactataag
3060attcagatct atcggtactg ggcgcaggtc tatgccagac tcgtcctcgc
tgtcggttgt 3120ggtcctctcg gtcgagagtt ccatttccgt gcaagtgtgg
gcgtgctgtg gtgtggagct 3180tgcatgctct ggccccgtga gtgctctgaa
atcagcctgg tcttcattct gtgtgctctg 3240acagtggaca ccatagacac
atggttagta gcgtgcttgt ccgcagggcc gagtgcgcga 3300acccttgcaa
ttctggccga tgacatggcg cgcattggtg accaccgggc gttgcgcgcc
3360gtgttacgtt gctttggatc acgcggcaca tacatataca accacatggg
ccaagtctcg 3420gaacgggtgg cgcaagcagt cagggatctc ggcggttgct
tggaaccagt cgtgttggag 3480gagcccacct ttactgagat cgtggatgac
acaatgagtt tggtgtgtgg acaattgctt 3540ggaggtaaac ctgtggtggc
ccgctgcggc acgcgtgtct tggtgggaca cctcaaccct 3600gaagatctgc
cacctggttt ccagctgagt gctccggtgg ttattaccag gccaagcatt
3660ggtacgtggt ccttccttaa ggcgacactc acagggcgtg ctgaaacacc
agggtccggc 3720cagatcgtgg tgttgtcttc cctgacaggt cggtcaatgg
gtaccgcagt gaatggcaca 3780ctgtatgcga ccggccatgg tgccggcgcg
cgcggcctag ccacgtgcgc tggtttgagg 3840acgccacttt acacggcatt
atctgatgat gtcgtggcct attcttgcct tccgggcatg 3900agttccctag
acccctgctg ctgttcgccg agccgggttt gggtgatgaa taacaacgga
3960gggttggtgt gtggcagagt ggagaatgac gacgtctgtt tggactgtcc
cacgcacata 4020gatcaactgc ggggtgcttc gggctcacca gttttgtgtg
atcacggtca tgcatacgcg 4080ttgatgctcg gtggttactc taccagtggt
atttgtgcac gcgtccggac ggtccggcca 4140tggcataacg cctattcctc
ctcggggggg caaggcggaa tgcaggcgcc agctgtgaca 4200ccaacatact
ctgaaatcac ctactatgcc cctactggtt ctggtaagtc aacaaaatat
4260ccagtggacc tagtcaaaca gggacacaaa gtattggtcc ttttaccaag
tgtggctgta 4320gtcaaaagta tggcccctta tattaaggag acatataaga
tcagacccga aattagagct 4380ggcacaggtc ctgacggtgt gacggtcatc
actggtgaga acttggcgta catgacctat 4440ggccgcttcc ttgtggatcc
ggagacgaat ctgcggggct atgctgtagt catttgcgac 4500gagtgtcacg
acacatcatc caccacgcta ctcggcattg gcgcagtgcg catgtatgcc
4560gagaaagctg gagtgaagac cgttgtattc gccacagcca cccctgctgg
cattcaagta 4620cagtcacatt ccaacattga tgaatactta ttgactgaca
caggcgacgt ggaattttac 4680ggcgccaaaa tcaaaatgga caacatcaga
actggtagac atgttatctt ttgccactcg 4740aaggccaggt gtgcggaact
aacgcagcag ctctccggcc ttggcattcg tgcagtgagt 4800ttttggcgcg
gctgtgacat caaaaccatt cccgcctcag actccattgt tgtggtggca
4860actgatgcat tgtccacggg ctacacagga aactttgatt cggtcatcga
ctgcgggtgt 4920tgcgtggagc aaactgtgac aattgacatg gaccctacgt
tctccatctc ggcccgagtg 4980gtgccatgta ctgctgcatt gcgcatgcag
cggcgcggac gtaccggtcg tggtagaagg 5040ggagcgtact acacaacttc
tccaggagca gcaccctgcg tcagcgttcc cgatgctaac 5100gtctggcaag
cagtggagag cgccatggtc ttttatgatt ggagtgctac caggatacaa
5160cagtgcctgg cggcatacca tgatttgggg tgcacaccac gcatcagctg
tgacccacac 5220actccagtgc gggtgatgga cacactgagg gcgtacctgc
gcagacctga ggtgacgact 5280gcagctctcg caggagagca gtggccgctg
ctttatggtg cgcagttgtg catctgcaaa 5340gagaccgagg cccacggtcc
tgatgatagc atcaagtgga agtgcttact caacaacagt 5400aacaaaacac
ccctgttgta tgccttagac aatcctacac tggaattcac aacccaacat
5460gacttgactc gccgtatagc cggcgctcta tcgagcacag tgttcgtgga
gacaggctac 5520ggccccatcc tccttgctgg cgccgctttg gctgcctcct
tcgcctttgc gggcgccact 5580ggagctttag tgccgtcggc tgtttggagc
gttgaggtca ggcctgctgg cgtgacccgt 5640cccgacgcga cagacgagac
cgcggcctac gcacagcgct tgtaccaagc ctgtgcagat 5700tcaggaattt
tcgccagctt gcagggtacg gcgagtgcgg cgctgggcaa actggccgac
5760gccagtaggg gtgctagtca atatctggca gccgcgcctc cttcacccgc
ccccctggta 5820caggtgttgc agttcctcga gaccaacttt agctccattg
catctttcgg cctgctctgt 5880gctggctgcc aggctggcga gtgcttcact
gcgcttgctg gcttggtgtc cggtgctaca 5940gctggcttgg ggggtgccca
taagtggcta ttagctattg caggaacttg gctggttagc 6000ttgcagaccg
ggtcccgtgg cggcatggtt gcgggcctct cgattctagc gggctgttgc
6060atcggtagtg tcaccgggct tgacttcctg tttgggtgcc ttacaggttg
ggaagccgtg 6120gtcggcgctg cggttgcgac acagaagatc ttgtctggtt
cagctgatat gaccactctg 6180gtagatctct tacctgctct tttctccccc
ggtgccggca tagctggcat cgtgcttgtc 6240ttcatcttaa gcaattcaag
tgtaaccaca tgggctaatc ggctattatc catgtgtgcc 6300aaacaaacca
tttgtgaaaa ctacttctta agtgaaagat ttggccaaca attaagcaaa
6360ctttccctgt ggcgctctgt gtaccattgg gcgcaggcac gtgagggata
cacacagtgc 6420ggcgtgatca gcgggatctg gagcttcgcc ttgtgcattc
tacgcgctgt gtgggattgg 6480gcggccaagc atgtgccacg gttccgtgtg
cctatgattg gctgctcacc tgcgtggtgc 6540gggcgctggc ttggtaccgg
caccttgttg accacctgtg cgtgtggaga acgtgtgtcc 6600cttcagtgcc
tttgctcaac atctgaccca caactcagtg tgggccgttg gtgtcggtgt
6660agttggagtg ttgggttccc attcaacccg actacgacag gcactggcac
cttacggccg 6720gacatcagtg acgccaacaa attgggtttc cggtatggcg
ttgccgacat cgtggagcta 6780gagcggcggg gcgacaaatg gcacgtctgt
gcagcatcat gttgcttgga ccgggccagc 6840gttgcatccg ctgtgaaggc
cccaccggtc acggctaatg gtatacctat taatagcttt 6900tctccaccac
aaacttattg cctatctctc tgttcctttg atacagtttg catgtctact
6960aacttatgta acccagctaa gaccctgagt gtgtgccaag aggaggcggt
tgagctgctg 7020gaagagacag ttgacacagc acaagtagtg atgagccaaa
atctggcagc gcgtagacgc 7080gctgagtatg atgcatggca ggttcgccaa
gcagttggcg acgagtacac gcgtttggca 7140gacgaggatg ttgacatgac
agcgtcggtg aaacccccag tggccagggc tgctgtgggt 7200agctcaacgt
tggatgatgt tagcgtgctg actgtcttac gcgaactcgg cgaccagtgc
7260caaaatgcta tcaaatttgt agttcaggcg gcttcacggt ttgttccacc
agtgcccaag 7320ccacgcacgc gtgtctcggg tgtcttggag cgcgtgcgca
tgtgcatgcg cacgcctcca 7380atcaagtttg aggccaccgc agtaccaatt
cataatataa tcccagaaga gtgtcatatt 7440gtgctacgct gtaccggctg
ttgtgaccag gccttgaccg ttccgtacgg cacttgctct 7500ctgactttaa
ccaaatattt gactaacaaa cacagtcact atattccaaa agagaagata
7560gaagaagaca cagaaatagc tgtcatttgc gccgtaccaa caaagcgcgc
aagtaaactt 7620atcactttca gagcaggtga ccgatcagtc tcatgttgtc
accccttgca aactcctatt 7680agggccctgc ttcaaaagta tgggttacct
attgggaagt ggtccgactg caacgggccc 7740cttggtgacg acgcccgagt
ctgtgacgtc aatggagtga caacttatga accatgcatg 7800caatcctaca
attggttccg atcgattgtg gcaccaacaa ccccaccttt acctgcaacc
7860cggagcgtgg ctggcatttt gcgcgcagac acatcgcgcg tctacaccac
aacagcggtt 7920gatgtctccg agcggcaggc taaggtcacg attgatcaaa
agtcagccaa ggtggaccag 7980tgtctccgag acacatacaa ttgctgcctt
gccaaggcaa agaccttcag acaatctggc 8040atgtcatatg aggatgctgt
gtcaaagatg cgcgcaaaca ccacgcgtga tcataacaac 8100ggcatcactt
atacagattt ggtctctgga cgcgcaaaac ctgtcgttca gaaaattgta
8160gatcagatgc gcgctggagt gtacgacgct ccaatgcgca ttattccaaa
acctgaagtg 8220tttccacgag acaagtcaac acggaagcca ccacggttca
tcgttttccc tgggtgtgcc 8280gcacgagtcg cggagaaaat gatcctgggc
gatcctggcg cgataaccaa gcacgtgcta 8340ggtgatgcct acgggtttgc
cactccgccg catgagcgcg cgcgcctact ggaacaatgg 8400tggaaccgcg
caacggagcc acaagctatc gcggttgatg cagtctgctt tgatagcacc
8460atcacggcag aggacatgga tcgtgaggcc aacatcgtgg ctgcagcgca
tacggacccg 8520gaaggtgttc acggcctata caattattac aaaagaagcc
ccatgtgtga tatcacagga 8580aaagttgtcg gggtgcgtag ctgtcgagcc
tcaggtacgc ttacaacaag cagtggcaac 8640acgcttactt gctacctcaa
ggttcgcgca gcttgcacgc gcgccggcat taaaccaatt 8700ggcttactaa
ttcatggaga tgacaccctc attatcacag aacgttgcgc tcaggaaact
8760ctcgatgagt tcagcaacgc gcttgatgac tatgggttta ctcacaccat
gcaggtgtct 8820ggggacctct cgtctatcga gtgctgcagc gcacgtgtgg
acagcgtttg cctccgggga 8880ggtatgcgtc gcatgctcgt gccacaagct
cgacgtgcga ttgcacgcgt tctcggggaa 8940aagggcgatc cactgggtgt
tatcagcagc tatattgtca tgtatcctac tgcggctgtg 9000actgtctacg
ttctgatgcc cctgttgtgc atgctcattc gaaatgagcc atcgcagacg
9060gggacacttg taacgttgac ggtccacggt aacagtgtga gcgtgccagt
gtggctgctt 9120ccaaccatta ttgcaaattt acatggccgt gacgcactac
aggttgtccg tcacagtgca 9180gcttccatgg cggaactgtc ctcagcgttg
gccttctttg gcatgagagg gttgaactgc 9240tggaggcgga gacgccgtgc
catcaggact gatatgatca agttgggcgg gtggaatgcg 9300aatttcgcgc
agatgttact gtggtcaccg gaggtaagaa caccacagcc cgaaccaaag
9360ggcatgtgtc tcttgccacc ggaactatgg gagcgtccgt acgaaaattt
gcacttgagc 9420acgatcgacc gcaatcgtgg tgctagtcgc ttacggtttt
ggttggttgc tagtgctata 9480ctcgctctgc tttgcttgta aatcctaaat
caatgtagta ccaggactac aaggcaggag 9540gtgaagtcag ctgtacccac
ggctggctga aaccggggct tgacgacccc ccctatccga 9600gttgggcaag
gtaacatcac gggtgtgacg accccgcccc cccatgtcgc gcgcaagcgc
9660acgggcaagg cagctaggct gagagtctgg gcaactctcc cgtaccccac
ccgaggctac 9720gcctcgtcct ggcgaggacc gtaaacatac gtcgtcagcg
tggtgacctg acgtatcttg 9780ttaaccactt aatggtcgta actcgacccc
cgtgccgggg atctaagcgc ggcaccgcga 9840tgagaggggt caacggcccc tttcatt
986730479PRTHuman Pegivirus 2 304Met Gly Cys Ser Thr Asp Gln Thr
Ile Cys Ser Pro Val Val Gly Ala1 5 10 15Asp Tyr Asn Thr Ser Ser Gly
Cys Arg Ala Leu Asn Gly Ser Tyr His 20 25 30Cys Gly Gly Gly Ser Cys
Arg Ser Pro Ser Arg Val Gln Val Ala Arg 35 40 45Arg Val Leu Gln Leu
Cys Ala Phe Leu Ala Leu Ile Gly Ser Gly Met 50 55 60Cys Ser Ile Arg
Ser Lys Thr Glu Gly Arg Ile Glu Ser Gly Gln65 70 75305191PRTHuman
Pegivirus 2 305Ile Leu Gln Ser Gln Arg Ala Cys Trp Thr Gly Glu Gly
Phe Ala Phe1 5 10 15Phe Ser Asn Cys Cys Asn Gln Ser Asp Ile Met Trp
Cys Leu His Arg 20 25 30Trp Cys Val Thr Arg Pro Gly Cys Leu Val Cys
Thr Gly Asn Ala Thr 35 40 45His Pro Val Cys Trp Asp Tyr Leu Gly Ser
Gly Val Ser Arg Arg Pro 50 55 60Ala Arg Arg Met Gly Glu Gly Ala Glu
Val Leu Leu Arg Leu Ile Gly65 70 75 80Ile Ala Gly Trp Leu Gly Leu
Leu Ala Glu Ala Leu Gly Met Ser Glu 85 90 95Ile Tyr Ala Ala Ile Leu
Cys Phe Gly Phe Ile Ala Trp Tyr Gly Trp 100 105 110Gly Ile Pro Lys
Thr Leu Val Cys Thr Val Cys Pro Ala Val Asn Ile 115 120 125Ser Pro
Tyr Ser Phe Leu Ser Pro Asp Thr Ile Ala Phe Gly Thr Trp 130 135
140Ile Leu Gln Leu Pro Gly Leu Leu Trp Gln Met Phe Val Ser Phe
Pro145 150 155 160Ile Leu Tyr Ser Thr Trp Ile Leu Trp Leu Leu Leu
Ser Gly Lys Thr 165 170 175Val Ala Val Ile Ala Ile Leu Leu Ala Ser
Pro Thr Val Met Ala 180 185 190306354PRTHuman Pegivirus
2MISC_FEATURE(49)..(49)Xaa = any amino
acidMISC_FEATURE(145)..(145)Xaa = any amino
acidMISC_FEATURE(203)..(203)Xaa = any amino
acidMISC_FEATURE(226)..(226)Xaa = any amino acid 306Tyr Lys His Gln
Ser Glu Ser Tyr Leu Lys Tyr Cys Thr Ile Thr Asn1 5 10 15Ala Ser Thr
Ala Met Asn Cys Asp Cys Pro Phe Gly Thr Phe Thr Arg 20 25 30Asn Thr
Glu Ser Arg Phe Ser Ile Pro Arg Phe Cys Pro Val Lys Ile 35 40 45Xaa
Ser Ser Thr Phe Ile Cys Ser Trp Gly Ser Trp Trp Trp Phe Ala 50 55
60Glu Asn Ile Thr Arg Pro Tyr Ser Asp Val Gly Met Pro Pro Ala Pro65
70 75 80Ile Ser Ala Leu Cys Tyr Ile Tyr Ser Asn Asn Asp Pro Pro Pro
Trp 85 90 95Tyr His Asn Thr Thr Ile Ile Pro Gln Asn Cys Arg Asn Ser
Thr Val 100 105 110Asp Pro Thr Thr Ala Pro Cys Arg Asp Lys Trp Gly
Asn Ala Thr Ala 115 120 125Cys Ile Leu Asp Arg Arg Ser Arg Phe Cys
Gly Asp Cys Tyr Gly Gly 130 135 140Xaa Phe Tyr Thr Asn Gly Ser His
Asp Arg Ser Trp Asp Arg Cys Gly145 150 155 160Ile Gly Tyr Arg Asp
Gly Leu Ile Glu Phe Val Gln Leu Gly Gln Ile 165 170 175Arg Pro Asn
Ile Ser Asn Thr Thr Ile Glu Leu Leu Ala Gly Ala Ser 180 185 190Leu
Val Ile Ala Ser Gly Leu Arg Pro Gly Xaa Gly Cys Ser Arg Ala 195 200
205His Gly Val Val His Cys Tyr Arg Cys Pro Ser Tyr Arg Asp Leu Glu
210 215 220Gln Xaa Gly Pro Gly Leu Gly Lys Trp Val Pro Leu Pro Gly
Glu Pro225 230 235 240Val Pro Glu Leu Cys Ile Asn Pro Gln Trp Ala
Arg Arg Gly Phe Arg 245 250 255Met Ser Asn Asn Pro Leu Ser Leu Leu
Gln Thr Phe Val Glu Asp Ile 260 265 270Phe Leu Ala Pro Phe Cys Asn
Pro Thr Pro Gly Arg Val Arg Val Cys 275 280 285Asn Asn Thr Ala Phe
Tyr Pro Arg Gly Gly Gly Phe Val Gln Leu Ile 290 295 300Gly Asp Val
Gln Val Leu Thr Pro Asn Thr Ala Ser Leu His Ser Leu305 310 315
320Leu Thr Leu Ile Ser Leu Ile Leu Leu Val Cys Val Val Ser Gly Ala
325 330 335Arg Phe Val Pro Leu Ile Ile Ile Phe Phe Trp Ser Ala Arg
His Val 340 345 350Tyr Ala307237PRTHuman Pegivirus
2MISC_FEATURE(75)..(75)Xaa = any amino
acidMISC_FEATURE(198)..(198)Xaa = any amino acid 307Ser Cys Tyr Leu
Ser Cys Asp Trp Ala Val Cys Asn Asp Ala Phe Cys1 5 10 15Phe Thr Ser
Gly Thr Cys Ala Thr Phe Asn Asp Val Leu Cys Leu Pro 20 25 30Val Ala
Thr Arg Ile Ser Ser Cys Gly His Ala Val Pro Pro Pro Asp 35 40 45Arg
Gly Trp Glu Val Pro Ala Ala Met Ser Trp Val Ile Ser Arg Thr 50
55 60Thr Gly Leu Thr Phe Asp Val Phe Ser Phe Xaa Gln Tyr Leu Pro
Thr65 70 75 80Val Pro Gly Asn Asn Thr Asp Ile Ile Tyr Cys Gly Glu
Pro Thr Phe 85 90 95Phe Gly Asp Ile Thr Gly Ile Tyr Trp Pro Tyr Phe
Leu Pro Gly Val 100 105 110Leu Leu Leu Tyr Leu Thr Pro Phe Leu Gly
Leu Arg Leu Met Leu Ala 115 120 125Gly Phe Asn Ile Asp Gly Leu Phe
Pro Ile Arg His Ala Thr Ala Ala 130 135 140Leu Arg Phe Ser Thr Ser
Arg Val Thr Leu Ser Val Val Ser Ala Phe145 150 155 160Leu Ile Tyr
Ile Leu Ser His Pro Val Asn Ala Ala Leu Asn Arg Met 165 170 175Phe
Leu Ala Ser Ala Asn Leu Glu Met Ile Leu Ser Phe Asp Thr Tyr 180 185
190His Glu Thr Val Leu Xaa Ile Val Cys Leu Leu Leu Tyr Leu Gln Val
195 200 205Ser Pro Arg Ala Gly Leu Ala Ala Met Val Ala Ile Lys Leu
Ser Arg 210 215 220Gly Leu Leu Phe Ala Val Val Leu Ala His Gly Val
Cys225 230 235308240PRTHuman Pegivirus 2MISC_FEATURE(142)..(142)Xaa
= any amino acidMISC_FEATURE(197)..(197)Xaa = any amino acid 308Arg
Pro Gly Arg Val Phe Gly Leu Glu Val Cys Ala Asp Ile Ser Trp1 5 10
15Leu Val Glu Phe Thr Gly Asn Cys Thr Trp Tyr Met Ser Cys Val Phe
20 25 30Ser Phe Trp Cys Ala Val Phe Ala Phe Thr Ser Pro Leu Gly Arg
Gln 35 40 45Tyr Lys Leu Gln Ile Tyr Arg Tyr Trp Ala Gln Val Tyr Ala
Arg Leu 50 55 60Ile Leu Ala Val Gly Cys Gly Pro Leu Gly Arg Glu Phe
His Phe Arg65 70 75 80Ala Ser Val Gly Val Leu Trp Cys Gly Ala Cys
Met Leu Trp Pro Arg 85 90 95Glu Cys Ser Glu Ile Ser Leu Val Phe Ile
Leu Cys Ala Leu Thr Val 100 105 110Asp Thr Ile Asp Thr Trp Leu Val
Ala Cys Leu Ser Ala Gly Pro Ser 115 120 125Ala Arg Thr Leu Ala Thr
Leu Ala Asp Asp Met Ala Arg Xaa Gly Asp 130 135 140His Arg Ala Leu
Arg Ala Val Leu Arg Cys Phe Gly Ser Arg Gly Thr145 150 155 160Tyr
Ile Tyr Asn His Met Gly Gln Val Ser Glu Arg Val Ala Gln Ala 165 170
175Val Arg Asp Leu Gly Gly Cys Leu Glu Pro Val Val Leu Glu Glu Pro
180 185 190Thr Phe Thr Glu Xaa Val Asp Asp Thr Met Ser Leu Val Cys
Gly Gln 195 200 205Leu Leu Gly Gly Lys Pro Val Val Ala Arg Cys Gly
Thr Arg Val Leu 210 215 220Val Gly His Leu Asn Pro Glu Asp Leu Pro
Pro Gly Phe Gln Leu Ser225 230 235 240309628PRTHuman Pegivirus
2MISC_FEATURE(97)..(97)Xaa = any amino
acidMISC_FEATURE(119)..(119)Xaa = any amino
acidMISC_FEATURE(510)..(510)Xaa = any amino
acidMISC_FEATURE(580)..(580)Xaa = any amino acid 309Ala Pro Val Val
Ile Thr Lys Pro Ser Ile Gly Thr Trp Ser Phe Leu1 5 10 15Lys Ala Thr
Leu Thr Gly Arg Ala Glu Thr Pro Gly Ser Gly Gln Ile 20 25 30Val Val
Leu Ser Ser Leu Thr Gly Arg Ser Met Gly Thr Ala Val Asn 35 40 45Gly
Thr Leu Tyr Ala Thr Gly His Gly Ala Gly Ala Arg Gly Leu Ala 50 55
60Thr Cys Ala Gly Leu Arg Thr Pro Leu Tyr Thr Ala Leu Ser Asp Asp65
70 75 80Val Val Ala Tyr Ser Cys Leu Pro Gly Met Ser Ser Leu Glu Pro
Cys 85 90 95Xaa Cys Ser Pro Ser Arg Val Trp Val Met Asn Asn Asn Gly
Gly Leu 100 105 110Val Cys Gly Arg Val Glu Xaa Asp Asp Val Cys Leu
Asp Cys Pro Thr 115 120 125His Ile Asp Gln Leu Arg Gly Ala Ser Gly
Ser Pro Val Leu Cys Asp 130 135 140His Gly His Ala Tyr Ala Leu Met
Leu Gly Gly Tyr Ser Thr Ser Gly145 150 155 160Ile Cys Ala Arg Val
Arg Ile Val Arg Pro Trp Gln Asn Ala Tyr Ser 165 170 175Ser Ser Gly
Gly Gln Gly Gly Met Gln Ala Pro Ala Val Thr Pro Thr 180 185 190Tyr
Ser Glu Ile Thr Tyr Tyr Ala Pro Thr Gly Ser Gly Lys Ser Thr 195 200
205Lys Tyr Pro Val Asp Leu Val Lys Gln Gly His Lys Val Leu Val Leu
210 215 220Ile Pro Ser Val Ala Val Val Lys Ser Met Ala Pro Tyr Ile
Lys Glu225 230 235 240Thr Tyr Lys Ile Arg Pro Glu Ile Arg Ala Gly
Thr Gly Pro Asp Gly 245 250 255Val Thr Val Ile Thr Gly Glu Asn Leu
Ala Tyr Met Thr Tyr Gly Arg 260 265 270Phe Leu Val Asp Pro Glu Thr
Asn Leu Arg Gly Tyr Ala Val Val Ile 275 280 285Cys Asp Glu Cys His
Asp Thr Ser Ser Thr Thr Leu Leu Gly Ile Gly 290 295 300Ala Val Arg
Met Tyr Ala Glu Lys Ala Gly Val Lys Thr Val Val Phe305 310 315
320Ala Thr Ala Thr Pro Ala Gly Ile Gln Val Gln Pro His Pro Asn Ile
325 330 335Asp Glu Tyr Leu Leu Thr Asp Thr Gly Asp Val Glu Phe Tyr
Gly Ala 340 345 350Lys Ile Lys Leu Asp Asn Ile Arg Thr Gly Arg His
Val Ile Phe Cys 355 360 365His Ser Lys Ala Arg Cys Ala Glu Leu Thr
Gln Gln Leu Ser Gly Leu 370 375 380Gly Val Arg Ala Val Ser Phe Trp
Arg Gly Cys Asp Ile Lys Thr Ile385 390 395 400Pro Ala Ser Asp Ser
Ile Val Val Val Ala Thr Asp Ala Leu Ser Thr 405 410 415Gly Tyr Thr
Gly Asn Phe Asp Ser Val Ile Asp Cys Gly Cys Cys Val 420 425 430Glu
Gln Thr Val Thr Ile Asp Met Asp Pro Thr Phe Ser Ile Ser Ala 435 440
445Arg Val Val Pro Cys Thr Ala Ala Leu Arg Met Gln Arg Arg Gly Arg
450 455 460Thr Gly Arg Gly Arg Arg Gly Ala Tyr Tyr Thr Thr Thr Pro
Gly Ala465 470 475 480Ala Pro Cys Val Ser Val Pro Asp Ala Asn Val
Trp Gln Ala Val Glu 485 490 495Ser Ala Met Val Phe Tyr Asp Trp Ser
Ala Ala Arg Ile Xaa Gln Cys 500 505 510Leu Ala Ala Tyr His Asp Leu
Gly Cys Thr Pro Arg Ile Ser Cys Asp 515 520 525Pro His Thr Pro Val
Arg Val Met Asp Thr Leu Arg Ala Tyr Leu Arg 530 535 540Arg Pro Glu
Val Thr Thr Ala Ala Leu Ala Gly Glu Gln Trp Pro Leu545 550 555
560Leu Tyr Gly Val Gln Leu Cys Ile Cys Lys Glu Thr Glu Ala His Gly
565 570 575Pro Asp Asp Xaa Ile Lys Trp Lys Cys Leu Leu Asn Asn Ser
Asn Lys 580 585 590Thr Pro Leu Leu Tyr Ala Leu Asp Asn Pro Thr Leu
Glu Phe Thr Thr 595 600 605Gln His Asp Leu Thr Arg Arg Ile Ala Gly
Ala Leu Ser Ser Thr Val 610 615 620Phe Val Glu Thr62531041PRTHuman
Pegivirus 2 310Gly Tyr Gly Pro Ile Leu Leu Ala Gly Ala Ala Leu Ala
Ala Ser Phe1 5 10 15Ala Phe Ala Gly Ala Thr Gly Ala Leu Val Pro Ser
Ala Val Trp Ser 20 25 30Val Asp Asn Gly Leu Ala Gly Val Thr 35
40311262PRTHuman Pegivirus 2MISC_FEATURE(24)..(25)Xaa = any amino
acidMISC_FEATURE(37)..(37)Xaa = any amino acid 311Arg Pro Asp Ala
Thr Asp Glu Thr Ala Ala Tyr Ala Gln Arg Leu Tyr1 5 10 15Gln Ala Cys
Ala Asp Ser Gly Xaa Xaa Ala Ser Leu Gln Gly Thr Ala 20 25 30Ser Ala
Ala Leu Xaa Lys Leu Ala Asp Ala Ser Arg Gly Ala Ser Gln 35 40 45Tyr
Leu Ala Ala Ala Pro Pro Ser Pro Ala Pro Leu Val Gln Val Leu 50 55
60Gln Phe Leu Glu Thr Asn Phe Ser Ser Ile Ala Ser Phe Gly Leu Leu65
70 75 80Cys Ala Gly Cys Gln Ala Gly Glu Cys Phe Thr Ala Leu Ala Gly
Leu 85 90 95Val Ser Gly Ala Thr Ala Gly Leu Gly Gly Ala His Lys Trp
Leu Leu 100 105 110Ala Ile Ala Gly Thr Trp Leu Val Ser Leu Gln Thr
Gly Pro Arg Gly 115 120 125Gly Met Val Ala Gly Leu Ser Val Leu Ala
Gly Cys Cys Ile Gly Ser 130 135 140Val Thr Gly Leu Asp Phe Leu Phe
Gly Cys Leu Thr Gly Trp Glu Ala145 150 155 160Val Val Gly Ala Ala
Val Ala Thr Gln Lys Ile Leu Ser Gly Ser Ala 165 170 175Asp Met Thr
Thr Leu Val Asp Leu Leu Pro Ala Leu Phe Ser Pro Gly 180 185 190Ala
Gly Ile Ala Gly Ile Val Leu Val Phe Ile Leu Ser Asn Ser Ser 195 200
205Val Thr Thr Trp Ala Asn Arg Leu Leu Ser Met Cys Ala Lys Gln Thr
210 215 220Ile Cys Glu Asn Tyr Phe Leu Thr Glu Lys Phe Gly Gln Gln
Leu Ser225 230 235 240Lys Leu Ser Leu Trp Arg Ser Val Tyr His Trp
Ala Gln Ala Arg Glu 245 250 255Gly Tyr Thr Gln Cys Gly
260312458PRTHuman Pegivirus 2MISC_FEATURE(22)..(22)Xaa = any amino
acid 312Val Val Ser Gly Ile Trp Ser Phe Val Leu Cys Ile Leu Arg Ala
Val1 5 10 15Trp Asp Trp Ala Ala Xaa His Val Pro Arg Phe Arg Val Pro
Met Ile 20 25 30Gly Cys Ser Pro Ala Trp Cys Gly Arg Trp Leu Gly Thr
Gly Thr Leu 35 40 45Leu Thr Thr Cys Gly Cys Gly Glu Arg Val Ser Leu
Gln Cys Leu Cys 50 55 60Ser Thr Ser Asp Pro Thr Leu Ser Val Gly Arg
Trp Cys Arg Cys Ser65 70 75 80Trp Ser Val Gly Phe Pro Phe Asn Pro
Thr Thr Thr Ala Thr Gly Thr 85 90 95Leu Arg Pro Asp Ile Ser Asp Ala
Thr Lys Leu Gly Phe Arg Tyr Gly 100 105 110Val Ala Glu Ile Val Glu
Leu Glu Arg Arg Gly Asp Lys Trp His Val 115 120 125Cys Ala Ala Ser
Cys Cys Leu Asp Arg Ala Ser Val Ala Ser Ala Val 130 135 140Lys Ala
Pro Pro Val Thr Ala Asn Gly Ile Pro Ile Ser Thr Phe Ser145 150 155
160Pro Pro Gln Thr Tyr Ser Leu Ser Leu Cys Ser Phe Asp Ser Val Cys
165 170 175Met Ser Thr Asn Leu Cys Asn Pro Ala Lys Thr Leu Ser Val
Cys Ser 180 185 190Gln Glu Ala Val Glu Leu Leu Glu Glu Thr Val Asp
Thr Ala Gln Val 195 200 205Met Met Cys Gln Asn Leu Glu Ala Arg Arg
Arg Ala Glu Tyr Asp Ala 210 215 220Trp Gln Val Arg Gln Ala Val Gly
Asp Glu Tyr Thr Arg Leu Ala Asp225 230 235 240Glu Asp Val Asp Thr
Thr Thr Ser Val Lys Pro Pro Val Ala Arg Ala 245 250 255Ala Val Gly
Ser Ser Thr Leu Asp Asp Val Ser Val Leu Thr Val Leu 260 265 270Arg
Glu Leu Gly Asp Gln Cys Gln Asn Ala Ile Lys Phe Val Val Gln 275 280
285Ala Ala Ser Arg Phe Val Pro Pro Val Pro Lys Pro Arg Thr Arg Val
290 295 300Ser Gly Val Leu Glu Arg Val Arg Met Cys Met Arg Thr Pro
Pro Ile305 310 315 320Lys Phe Glu Ala Thr Ala Val Pro Ile His Asn
Ile Ile Pro Glu Glu 325 330 335Cys His Ile Val Leu Arg Cys Thr Gly
Cys Asn Asp Gln Ala Leu Thr 340 345 350Val Pro Tyr Gly Thr Cys Thr
Gln Thr Leu Ile Lys His Leu Thr Asn 355 360 365Lys His Ser His Tyr
Ile Pro Lys Gln Lys Ile Glu Glu Asp Thr Glu 370 375 380Val Thr Val
Ile Cys Ala Val Pro Thr Lys Arg Ala Ser Lys Leu Ile385 390 395
400Thr Phe Arg Ala Gly Asp Arg Ser Val Ser Cys Cys His Pro Leu Gln
405 410 415Thr Pro Ile Arg Ala Leu Leu Leu Lys Tyr Gly Leu Pro Ile
Gly Lys 420 425 430Trp Ser Asp Cys Asn Gly Pro Leu Gly Asp Asp Ala
Arg Val Cys Asp 435 440 445Val Asn Gly Val Thr Thr Tyr Glu Pro Cys
450 455313567PRTHuman Pegivirus 2MISC_FEATURE(101)..(101)Xaa = any
amino acidMISC_FEATURE(234)..(234)Xaa = any amino
acidMISC_FEATURE(239)..(239)Xaa = any amino
acidMISC_FEATURE(446)..(446)Xaa = any amino
acidMISC_FEATURE(514)..(514)Xaa = any amino
acidMISC_FEATURE(517)..(517)Xaa = any amino
acidMISC_FEATURE(521)..(521)Xaa = any amino
acidMISC_FEATURE(523)..(523)Xaa = any amino
acidMISC_FEATURE(526)..(526)Xaa = any amino acid 313Met Gln Ser Tyr
Ser Trp Phe Arg Pro Ile Val Ala Pro Thr Thr Pro1 5 10 15Pro Leu Pro
Ala Thr Arg Ser Val Ala Gly Ile Leu Arg Ala Asp Thr 20 25 30Ser Arg
Val Tyr Thr Thr Thr Ala Val Asp Val Ser Glu Arg Gln Ala 35 40 45Lys
Val Thr Ile Asp Gln Thr Ser Ala Lys Val Asp Gln Cys Phe Arg 50 55
60Asp Thr Tyr Asn Cys Cys Leu Ala Lys Ala Lys Thr Phe Arg Gln Ser65
70 75 80Gly Met Ser Tyr Glu Asp Ala Val Ser Lys Met Arg Ala Asn Thr
Thr 85 90 95Arg Asp His Asn Xaa Gly Ile Thr Tyr Ser Asp Leu Val Ser
Gly Arg 100 105 110Ala Lys Pro Val Val Gln Lys Ile Val Asp Gln Met
Arg Ala Gly Val 115 120 125Tyr Asp Ala Pro Met Arg Ile Ile Pro Lys
Pro Glu Val Phe Pro Arg 130 135 140Asp Lys Ser Thr Arg Lys Pro Pro
Arg Phe Ile Val Phe Pro Gly Cys145 150 155 160Ala Ala Arg Val Ala
Glu Lys Met Ile Leu Gly Asp Pro Gly Ala Ile 165 170 175Thr Lys His
Val Leu Gly Asp Ala Tyr Gly Phe Ala Thr Pro Pro His 180 185 190Glu
Arg Ala Arg Leu Leu Glu Gln Trp Trp Asn Arg Ala Thr Glu Pro 195 200
205Gln Ala Ile Ala Val Asp Ala Ile Cys Phe Asp Ser Thr Ile Thr Ala
210 215 220Glu Asp Met Asp Arg Glu Ala Asn Ile Xaa Ala Ala Ala His
Xaa Asp225 230 235 240Pro Glu Gly Val His Gly Leu Tyr Asn Tyr Tyr
Lys Arg Ser Pro Met 245 250 255Cys Asp Ile Thr Gly Lys Val Val Gly
Val Arg Cys Cys Arg Ala Ser 260 265 270Gly Thr Leu Thr Thr Ser Ser
Gly Asn Thr Leu Thr Cys Tyr Leu Lys 275 280 285Val Arg Ala Ala Cys
Thr Arg Ala Gly Ile Lys Pro Ile Gly Leu Leu 290 295 300Ile His Gly
Asp Asp Thr Leu Ile Ile Thr Glu Arg Cys Ala Gln Glu305 310 315
320Thr Leu Asp Glu Phe Ser Asn Ala Leu Asp Asp Tyr Gly Phe Pro His
325 330 335Thr Ile Gln Val Ser Gly Asp Leu Ser Ser Val Glu Cys Cys
Ser Ala 340 345 350Arg Val Asp Ser Val Cys Leu Arg Gly Gly Met Arg
Arg Met Leu Val 355 360 365Pro Gln Ala Arg Arg Ala Ile Ala Arg Val
Leu Gly Glu Lys Gly Asp 370 375 380Pro Leu Gly Val Ile Ser Ser Tyr
Ile Val Met Tyr Pro Thr Ala Ala385 390 395 400Val Thr Val Tyr Val
Leu Leu Pro Leu Leu Cys Met Leu Ile Arg Asn 405 410 415Glu Pro Ser
Gln Thr Gly Thr Leu Val Thr Leu Thr Val His Gly Asn 420 425 430Ser
Val Ser Val Pro Val Trp Leu Leu Pro Thr Ile Ile Xaa Asn Leu 435 440
445His Gly Arg Asp Ala Leu Gln Val Val Arg His Ser Ala Ala Ser Met
450 455 460Ala Glu Leu Ser Ser Ala Leu Ala Phe Phe Gly Met Arg Gly
Leu Asn465 470 475 480Cys Trp Arg Arg Arg Arg Arg Ala Ile Arg Thr
Asp Met Ile Lys Leu 485 490 495Gly Gly Trp Asn Ala Asn Phe Ala Gln
Met Leu Leu Trp Ser Pro Glu
500 505 510Val Xaa Thr Pro Xaa Pro Glu Pro Xaa Gly Xaa Cys Leu Xaa
Pro Pro 515 520 525Glu Leu Trp Glu Arg Pro Tyr Glu Asn Leu His Leu
Ser Thr Ile Asp 530 535 540Arg Asn Arg Gly Ala Ser Arg Leu Arg Phe
Trp Leu Val Ala Ser Ala545 550 555 560Ile Leu Ala Leu Leu Cys Leu
56531479PRTHuman Pegivirus 2 314Met Gly Cys Ser Thr Asp Gln Thr Ile
Cys Ser Pro Val Val Glu Ala1 5 10 15Asp Tyr Asn Thr Ser Ser Gly Cys
Arg Ala Leu Asn Gly Ser Tyr His 20 25 30Cys Gly Gly Gly Ser Cys Arg
Ser Pro Ser Arg Val Gln Val Ala Gly 35 40 45Arg Val Leu Arg Leu Cys
Ala Phe Leu Ala Leu Ile Gly Ser Gly Met 50 55 60Cys Ser Ile Arg Ser
Lys Asn Glu Gly Arg Ile Glu Ser Gly Gln65 70 75315191PRTHuman
Pegivirus 2 315Ile Leu Gln Ser Gln Arg Ala Cys Trp Thr Gly Glu Gly
Phe Ala Phe1 5 10 15Phe Ser Asn Cys Cys Asn Gln Ser Asp Ile Met Trp
Cys Leu His Arg 20 25 30Trp Cys Val Thr Arg Pro Gly Cys Leu Val Cys
Thr Gly Asn Ala Thr 35 40 45His Pro Val Cys Trp Asp Tyr Leu Gly Ser
Gly Val Ser Arg Arg Pro 50 55 60Ala Arg Arg Met Gly Glu Gly Ala Glu
Val Leu Leu Arg Leu Ile Gly65 70 75 80Ile Ala Gly Trp Leu Gly Leu
Leu Ala Glu Ala Leu Gly Met Ser Glu 85 90 95Ile Tyr Ala Ala Phe Leu
Cys Phe Gly Phe Ile Ala Trp Tyr Gly Trp 100 105 110Gly Ile Pro Lys
Thr Leu Val Cys Thr Val Cys Pro Ala Val Asn Ile 115 120 125Ser Pro
Tyr Ser Phe Leu Ser Pro Asp Thr Ile Ala Phe Gly Thr Trp 130 135
140Leu Leu Gln Leu Pro Gly Leu Leu Trp Gln Met Phe Val Ser Phe
Pro145 150 155 160Ile Leu Tyr Ser Thr Trp Ile Leu Trp Leu Leu Leu
Ser Gly Lys Thr 165 170 175Val Ala Val Ile Ala Ile Leu Leu Ala Ser
Pro Thr Val Met Ala 180 185 190316354PRTHuman Pegivirus 2 316Tyr
Lys His Gln Ala Asp Ser Tyr Leu Lys Tyr Cys Thr Ile Thr Asn1 5 10
15Ala Ser Thr Ala Met Asn Cys Asp Cys Pro Phe Gly Thr Phe Thr Arg
20 25 30Asn Thr Glu Ser Gly Phe Thr Ile Pro Arg Phe Cys Pro Val Lys
Leu 35 40 45Asn Ser Ser Thr Phe Ile Cys Ser Trp Gly Ser Trp Trp Trp
Phe Ala 50 55 60Glu Asn Ile Thr Arg Pro Tyr Ser Asp Val Gly Met Pro
Pro Ala Pro65 70 75 80Ile Ser Ala Leu Cys Tyr Ile Tyr Ser Asn Asn
Asp Pro Pro Ser Trp 85 90 95Tyr Arg Asn Thr Thr Ile Ile Pro Gln Asn
Cys Tyr Asn Ser Thr Ala 100 105 110Asp Pro Thr Thr Ala Pro Cys Arg
Asp Lys Trp Gly Asn Ala Thr Ala 115 120 125Cys Ile Leu Asp Arg Arg
Ser Arg Phe Cys Gly Asp Cys Tyr Gly Gly 130 135 140Cys Phe Tyr Thr
Asn Gly Ser His Asp Arg Ser Trp Asp Arg Cys Gly145 150 155 160Ile
Gly Tyr Arg Asp Gly Leu Ile Glu Phe Val Gln Leu Gly Gln Ile 165 170
175Arg Pro Asn Ile Ala Asn Thr Thr Ile Glu Leu Leu Ala Gly Ala Ser
180 185 190Leu Val Ile Ala Ser Gly Leu Arg Ala Gly Tyr Gly Cys Ser
Arg Ala 195 200 205His Gly Val Val His Cys Phe Lys Cys Pro Ser Tyr
Arg Asp Leu Glu 210 215 220Arg Phe Gly Pro Gly Leu Gly Lys Trp Val
Pro Leu Pro Gly Glu Pro225 230 235 240Val Pro Glu Leu Cys Ile Asn
Pro Gln Trp Ala Arg Arg Gly Phe Arg 245 250 255Val Ser Asn Asn Pro
Leu Ser Val Leu Gln Thr Phe Val Glu Asp Ile 260 265 270Phe Leu Ala
Pro Phe Cys Asn Pro Thr Pro Gly Arg Val Arg Val Cys 275 280 285Asn
Asn Thr Ala Phe Tyr Pro Arg Gly Gly Gly Phe Val Gln Leu Ile 290 295
300Gly Asp Val Gln Val Leu Thr Pro Asn Ser Thr Ser Leu His Ser
Leu305 310 315 320Leu Thr Leu Ile Ser Leu Ile Leu Leu Val Cys Val
Val Ser Gly Ala 325 330 335Arg Phe Val Pro Leu Gly Ile Ile Phe Phe
Trp Ser Val Arg His Val 340 345 350Tyr Ala317237PRTHuman Pegivirus
2 317Ser Cys Tyr Leu Ser Cys Asp Trp Ala Val Cys Asn Asp Ala Phe
Cys1 5 10 15Phe Thr Ser Gly Thr Cys Ala Thr Phe Asn Asp Val Leu Cys
Leu Pro 20 25 30Val Ala Ala Arg Ile Ser Ser Cys Gly His Ala Val Pro
Pro Pro Asp 35 40 45Arg Gly Trp Glu Val Pro Ala Ala Met Ser Trp Ala
Ile Ser Arg Thr 50 55 60Thr Gly Leu Thr Phe Asp Val Phe Ser Phe Ile
Gln Tyr Leu Pro Thr65 70 75 80Val Pro Gly Asn Asn Ser Asp Ile Ile
Tyr Cys Gly Glu Pro Ser Phe 85 90 95Phe Gly Asp Ile Thr Gly Ile Tyr
Trp Pro Tyr Phe Leu Pro Gly Met 100 105 110Leu Leu Leu Tyr Leu Thr
Pro Leu Leu Gly Leu Arg Leu Met Leu Ala 115 120 125Gly Phe Asn Ile
Asp Gly Leu Phe Pro Ile Arg His Ala Thr Ala Ala 130 135 140Leu Arg
Phe Ser Thr Ser Arg Val Thr Leu Ser Val Val Phe Ala Phe145 150 155
160Leu Ile Tyr Ile Leu Ser His Pro Val Asn Ala Ala Leu Asn Arg Met
165 170 175Phe Leu Ala Ser Ala Asn Leu Glu Met Ile Leu Ser Phe Asp
Thr Tyr 180 185 190His Glu Thr Val Leu Tyr Val Val Cys Leu Leu Leu
Tyr Leu Gln Val 195 200 205Ser Pro Arg Ala Gly Leu Ala Ala Met Val
Ala Ile Lys Leu Ser Arg 210 215 220Gly Leu Leu Phe Ala Val Val Leu
Ala His Gly Val Cys225 230 235318240PRTHuman Pegivirus 2 318Arg Pro
Gly Arg Val Phe Gly Leu Glu Val Cys Ala Asp Ile Ser Trp1 5 10 15Leu
Val Glu Phe Thr Gly Asn Cys Thr Trp Tyr Met Ser Cys Val Phe 20 25
30Ser Phe Trp Cys Ala Val Phe Ala Phe Thr Ser Pro Leu Gly Arg Gln
35 40 45Tyr Lys Leu Gln Ile Tyr Arg Tyr Trp Ala Gln Ala Tyr Ala Arg
Leu 50 55 60Ile Leu Ala Val Gly Cys Gly Pro Leu Gly Arg Glu Phe His
Phe Arg65 70 75 80Ala Ser Val Gly Val Leu Trp Cys Gly Ala Cys Met
Leu Trp Pro Arg 85 90 95Glu Cys Ser Glu Ile Ser Leu Val Phe Ile Leu
Cys Ala Leu Thr Val 100 105 110Asp Thr Ile Asp Thr Trp Leu Val Ala
Cys Leu Ser Ala Gly Pro Ser 115 120 125Ala Arg Thr Leu Ala Thr Leu
Ala Asp Asp Met Ala Arg Ile Gly Asp 130 135 140His Arg Ala Leu Arg
Ala Val Leu Arg Cys Phe Gly Ser Arg Gly Thr145 150 155 160Tyr Ile
Tyr Asn His Met Gly Gln Val Ser Glu Arg Val Ala Gln Ala 165 170
175Val Arg Asp Phe Gly Gly Cys Leu Glu Pro Val Val Leu Glu Glu Pro
180 185 190Thr Phe Thr Glu Val Val Asp Asp Thr Met Asn Leu Val Cys
Gly Gln 195 200 205Leu Leu Gly Gly Lys Pro Val Val Ala Arg Cys Gly
Thr Arg Val Leu 210 215 220Val Gly His Leu Asn Pro Glu Asp Leu Pro
Pro Gly Phe Gln Leu Ser225 230 235 240319628PRTHuman Pegivirus 2
319Ala Pro Val Val Ile Thr Lys Pro Ser Ile Gly Thr Trp Pro Phe Leu1
5 10 15Lys Ala Thr Leu Thr Gly Arg Ala Glu Thr Pro Gly Ser Gly Gln
Ile 20 25 30Val Val Leu Ser Ser Leu Thr Gly Arg Ser Met Gly Thr Ala
Val Asn 35 40 45Gly Thr Leu Tyr Ala Thr Gly His Gly Ala Gly Ala Arg
Gly Leu Ala 50 55 60Thr Cys Ala Gly Leu Arg Thr Pro Leu Tyr Thr Ala
Leu Ser Glu Asp65 70 75 80Val Val Ala Tyr Ser Cys Leu Pro Gly Met
Ser Ser Leu Glu Ser Cys 85 90 95Asn Cys Ser Pro Ser Arg Val Trp Val
Val Asn Asn Asn Gly Gly Leu 100 105 110Val Cys Gly Arg Val Glu Lys
Asp Asp Val Cys Leu Asp Cys Pro Thr 115 120 125His Ile Asp Gln Leu
Arg Gly Ala Ser Gly Ser Pro Val Leu Cys Asp 130 135 140His Gly His
Ala Tyr Ala Leu Met Leu Gly Gly Tyr Ser Thr Ser Gly145 150 155
160Ile Cys Ala Arg Val Arg Ile Val Arg Pro Trp Gln Asn Ala Tyr Ser
165 170 175Ser Ser Gly Gly Gln Gly Gly Met Gln Ala Pro Ala Val Thr
Pro Thr 180 185 190Tyr Ser Glu Ile Thr Tyr Tyr Ala Pro Thr Gly Ser
Gly Lys Ser Thr 195 200 205Lys Tyr Pro Val Asp Leu Val Lys Gln Gly
His Lys Val Leu Val Leu 210 215 220Leu Pro Ser Val Ala Val Val Lys
Ser Met Ala Pro Tyr Ile Lys Glu225 230 235 240Lys Tyr Lys Ile Arg
Pro Glu Ile Arg Ala Gly Thr Gly Pro Asp Gly 245 250 255Val Thr Val
Ile Thr Gly Glu Asn Leu Ala Tyr Met Thr Tyr Gly Arg 260 265 270Phe
Leu Val Asp Pro Glu Thr Asn Leu Arg Gly Tyr Ala Val Val Ile 275 280
285Cys Asp Glu Cys His Asp Thr Ser Ser Thr Thr Leu Leu Gly Ile Gly
290 295 300Ala Val Arg Met Tyr Ala Glu Lys Ala Gly Val Lys Thr Val
Val Phe305 310 315 320Ala Thr Ala Thr Pro Ala Gly Ile Gln Val Gln
Ser His Pro Asn Ile 325 330 335Asp Glu Tyr Leu Leu Thr Asp Thr Gly
Asp Val Glu Phe Tyr Gly Ala 340 345 350Lys Ile Lys Leu Asp Asn Ile
Arg Thr Gly Arg His Val Ile Phe Cys 355 360 365His Ser Lys Ala Arg
Cys Ala Glu Leu Thr Gln Gln Leu Ser Gly Leu 370 375 380Gly Val Arg
Ala Val Ser Phe Trp Arg Gly Cys Asp Ile Lys Ser Ile385 390 395
400Pro Ala Ser Asp Ser Ile Val Val Val Ala Thr Asp Ala Leu Ser Thr
405 410 415Gly Tyr Thr Gly Asn Phe Asp Ser Val Ile Asp Cys Gly Cys
Cys Val 420 425 430Glu Gln Thr Val Thr Ile Asp Met Asp Pro Thr Phe
Ser Ile Ser Ala 435 440 445Arg Val Val Pro Cys Thr Ala Ala Leu Arg
Met Gln Arg Arg Gly Arg 450 455 460Thr Gly Arg Gly Arg Arg Gly Ala
Tyr Tyr Thr Thr Thr Pro Gly Ala465 470 475 480Ala Pro Cys Val Ser
Val Pro Asp Ala Asn Val Trp Gln Ser Val Glu 485 490 495Ser Ala Met
Val Phe Tyr Asp Trp Ser Ala Ala Arg Ile Glu Gln Cys 500 505 510Leu
Ala Ala Tyr His Asp Leu Gly Cys Thr Pro Arg Ile Ser Cys Asp 515 520
525Pro His Thr Pro Val Arg Val Met Asp Thr Leu Arg Ala Tyr Leu Arg
530 535 540Arg Pro Glu Val Thr Thr Ala Ala Leu Ala Gly Glu Gln Trp
Pro Leu545 550 555 560Leu Tyr Gly Val Gln Leu Cys Ile Cys Lys Glu
Thr Glu Ala His Gly 565 570 575Pro Asp Asp Gly Ile Lys Trp Lys Cys
Leu Leu Asn Asn Asn Asn Lys 580 585 590Thr Pro Leu Leu Tyr Ala Leu
Asp Asn Pro Thr Leu Glu Phe Thr Thr 595 600 605Gln His Asp Leu Thr
Arg Arg Ile Ala Gly Ala Leu Ser Ser Thr Val 610 615 620Phe Val Glu
Thr62532041PRTHuman Pegivirus 2 320Gly Tyr Gly Pro Ile Leu Leu Ala
Gly Ala Ala Leu Ala Ala Ser Phe1 5 10 15Ala Phe Ala Gly Ala Thr Gly
Ala Leu Val Pro Ser Ala Val Trp Ser 20 25 30Val Glu Asn Gly Leu Ala
Gly Val Thr 35 40321262PRTHuman Pegivirus 2 321Arg Pro Asp Ala Thr
Asp Glu Thr Ala Ala Tyr Ala Gln Arg Leu Tyr1 5 10 15Gln Ala Cys Ala
Asp Ser Gly Ile Leu Ala Ser Leu Gln Gly Thr Ala 20 25 30Ser Ala Ala
Leu Ser Arg Leu Ala Asp Ala Ser Lys Gly Ala Ser Gln 35 40 45Tyr Leu
Ala Ala Ala Pro Pro Ser Pro Ala Pro Leu Val Gln Val Leu 50 55 60Gln
Phe Leu Glu Thr Asn Phe Ser Ser Ile Ala Ser Phe Gly Leu Leu65 70 75
80Cys Ala Gly Cys Gln Ala Gly Glu Cys Phe Thr Ala Leu Ala Gly Leu
85 90 95Val Ser Gly Ala Thr Ala Gly Leu Gly Gly Ala His Lys Trp Leu
Leu 100 105 110Ala Ile Ala Gly Thr Trp Leu Val Ser Leu Gln Thr Gly
Pro Arg Gly 115 120 125Gly Met Val Ala Gly Leu Ser Val Leu Ala Gly
Cys Cys Ile Gly Ser 130 135 140Val Thr Gly Leu Asp Phe Leu Phe Gly
Cys Leu Thr Gly Trp Glu Ala145 150 155 160Val Val Gly Ala Ala Val
Ala Thr Gln Lys Ile Leu Ser Gly Ser Ala 165 170 175Asp Met Thr Thr
Leu Val Asp Leu Leu Pro Ala Leu Phe Ser Pro Gly 180 185 190Ala Gly
Ile Ala Gly Val Val Leu Val Phe Ile Leu Ser Asn Ser Ser 195 200
205Val Thr Met Trp Ala Asn Arg Leu Leu Ser Met Cys Ala Lys Gln Thr
210 215 220Ile Cys Glu Asn Tyr Phe Leu Thr Glu Lys Phe Gly Gln Gln
Leu Ser225 230 235 240Lys Leu Ser Leu Trp Arg Ser Val Tyr His Trp
Ala Gln Ala Arg Glu 245 250 255Gly Tyr Thr Gln Cys Gly
260322458PRTHuman Pegivirus 2 322Val Val Ser Gly Ile Trp Ser Phe
Val Leu Cys Ile Leu Arg Ala Val1 5 10 15Trp Asp Trp Ala Ala Lys His
Val Pro Arg Phe Arg Val Pro Met Ile 20 25 30Gly Cys Ser Pro Ala Trp
Cys Gly Arg Trp Leu Gly Thr Gly Thr Leu 35 40 45Leu Thr Thr Cys Gly
Cys Gly Glu Arg Val Ser Leu Gln Cys Leu Cys 50 55 60Ser Thr Ser Asp
Pro Thr Leu Ser Val Gly Arg Trp Cys Trp Cys Ser65 70 75 80Trp Arg
Val Gly Phe Pro Phe Asn Pro Thr Thr Thr Ala Thr Gly Thr 85 90 95Leu
Arg Pro Asp Ile Ser Asp Ala Thr Lys Leu Gly Phe Arg Tyr Gly 100 105
110Val Ala Glu Ile Val Glu Leu Glu Arg Arg Gly Asn Lys Trp His Val
115 120 125Cys Ala Ala Ser Cys Cys Leu Asp Arg Ala Ser Val Ala Ser
Ala Val 130 135 140Arg Ala Pro Pro Val Thr Ala Asp Gly Ile Pro Ile
Ser Thr Phe Ser145 150 155 160Pro Pro Gln Thr Tyr Lys Leu Ser Leu
Cys Ser Phe Asp Ser Val Cys 165 170 175Met Thr Thr Asn Leu Cys Asn
Pro Ala Lys Thr Leu Ser Val Cys Ser 180 185 190Gln Glu Ala Val Glu
Leu Leu Glu Glu Thr Val Asp Arg Ala Gln Val 195 200 205Val Met Cys
Gln Asn Leu Glu Ala Arg Arg Arg Ala Glu Phe Asp Ala 210 215 220Trp
Gln Val Arg Glu Ala Ile Arg Asp Glu Tyr Thr Arg Leu Ala Asp225 230
235 240Glu Asp Val Asp Ala Thr Thr Ser Val Lys Pro Pro Val Ala Lys
Ala 245 250 255Ala Val Gly Ser Ser Thr Leu Asp Asp Val Ser Val Leu
Thr Val Leu 260 265 270Arg Glu Leu Gly Asp Gln Cys Gln Asn Ala Ile
Lys Phe Val Val Gln 275 280 285Ala Ala Ser Arg Phe Val Pro Pro Val
Pro Lys Pro Arg Thr Arg Val 290 295 300Ser Gly Val Leu Glu Arg Val
Arg Met Cys Met Arg Thr Pro Pro Ile305 310 315 320Lys Phe Glu Ala
Ala Ala Val Pro Ile His Asp Ile Ile Pro Glu Glu 325 330 335Cys His
Ile Val Leu Arg Cys Thr Gly Cys Asn Asp Gln Ala Leu Thr 340 345
350Val Pro Tyr Gly Thr Cys Thr Gln Ser Leu Ile
Lys His Leu Thr Ser 355 360 365Lys His Ser His Tyr Ile Pro Lys Gln
Lys Ile Glu Glu Asp Thr Glu 370 375 380Val Thr Val Ile Cys Ala Val
Pro Thr Thr Arg Ala Ser Lys Leu Ile385 390 395 400Thr Phe Arg Ala
Gly Asp Arg Ser Val Ser Cys Cys His Pro Leu Gln 405 410 415Thr Pro
Ile Arg Ala Leu Leu Leu Lys Tyr Gly Leu Pro Ile Gly Lys 420 425
430Trp Ser Asp Cys Asn Gly Pro Leu Gly Asp Asp Ala Arg Val Cys Asp
435 440 445Val Asn Gly Val Thr Thr Tyr Glu Pro Cys 450
455323567PRTHuman Pegivirus 2 323Met Gln Ser Tyr Ser Trp Phe Arg
Pro Ile Val Ala Pro Thr Thr Pro1 5 10 15Pro Leu Pro Ala Thr Arg Thr
Val Ala Gly Ile Leu Arg Ala Asp Thr 20 25 30Ser Arg Val Tyr Thr Thr
Thr Ala Val Asp Val Ser Glu Arg Gln Ala 35 40 45Lys Val Thr Ile Asp
Gln Thr Ser Ala Lys Val Asp Gln Cys Phe Arg 50 55 60Asp Thr Tyr Asn
Cys Cys Leu Ala Lys Ala Lys Thr Phe Arg Gln Ser65 70 75 80Gly Met
Ser Tyr Glu Asp Ala Val Ser Lys Met Arg Ala Asn Thr Thr 85 90 95Arg
Asp His Asn Asn Gly Ile Thr Tyr Ser Asp Leu Val Ser Gly Arg 100 105
110Ala Lys Pro Val Val Gln Lys Ile Val Asn Gln Met Arg Ala Gly Val
115 120 125Tyr Asp Ala Pro Met Arg Ile Ile Pro Lys Pro Glu Val Phe
Pro Arg 130 135 140Asp Lys Thr Thr Arg Lys Pro Pro Arg Phe Ile Val
Phe Pro Gly Cys145 150 155 160Ala Ala Arg Val Ala Glu Lys Met Ile
Leu Gly Asp Pro Gly Ala Ile 165 170 175Thr Lys His Val Leu Gly Asp
Ala Tyr Gly Phe Ala Thr Pro Pro His 180 185 190Glu Arg Ala Arg Leu
Leu Glu Gln Trp Trp Asn Arg Ala Thr Glu Pro 195 200 205Gln Ala Ile
Ala Val Asp Ala Ile Cys Phe Asp Ser Thr Ile Thr Ala 210 215 220Glu
Asp Met Asp Arg Glu Ala Asn Ile Val Ala Ala Ala His Thr Asp225 230
235 240Pro Glu Gly Val His Gly Leu Tyr Asn Tyr Tyr Lys Arg Ser Pro
Met 245 250 255Cys Asp Ile Thr Gly Lys Val Val Gly Val Arg Cys Cys
Arg Ala Ser 260 265 270Gly Thr Leu Thr Thr Ser Ser Gly Asn Thr Leu
Thr Cys Tyr Leu Lys 275 280 285Val Arg Ala Ala Cys Thr Arg Ser Gly
Ile Lys Pro Ile Gly Leu Leu 290 295 300Ile His Gly Asp Asp Thr Leu
Ile Val Thr Glu Arg Cys Ala Gln Glu305 310 315 320Thr Leu Asp Glu
Phe Ser Asn Ala Leu Asp Asp Tyr Gly Phe Pro His 325 330 335Thr Ile
Gln Ala Ser Gly Asp Leu Ser Ser Ile Glu Cys Cys Ser Ala 340 345
350Arg Val Asp Ser Val Cys Leu Arg Gly Gly Met Arg Arg Met Leu Val
355 360 365Pro Gln Ala Arg Arg Ala Ile Ala Arg Val Leu Gly Glu Lys
Gly Asp 370 375 380Pro Leu Gly Thr Ile Gly Ser Tyr Val Val Met Tyr
Pro Thr Ala Ala385 390 395 400Val Thr Val Tyr Val Leu Leu Pro Leu
Leu Cys Met Leu Ile Arg Asn 405 410 415Glu Pro Ser Gln Thr Gly Thr
Leu Val Thr Leu Thr Val His Gly Asn 420 425 430Ser Val Ser Val Pro
Ala Trp Leu Leu Pro Thr Ile Ile Ala Asn Leu 435 440 445His Gly Arg
Asp Ala Leu Gln Val Val Arg His Ser Ala Ala Ser Met 450 455 460Ala
Glu Leu Ser Ser Ala Leu Ala Phe Phe Gly Met Arg Gly Leu Asn465 470
475 480Cys Trp Arg Arg Arg Arg Arg Ala Ile Arg Ala Asp Met Ile Lys
Ser 485 490 495Gly Gly Trp Asn Ala Asn Phe Ala Gln Met Leu Leu Trp
Ser Pro Glu 500 505 510Val Arg Thr Pro Gln Pro Glu Pro Arg Gly Leu
Cys Leu Leu Pro Pro 515 520 525Glu Leu Trp Glu Arg Pro Tyr Glu Asn
Leu His Leu Ser Thr Ile Asp 530 535 540Arg Asn Arg Gly Ala Ser Arg
Leu Arg Phe Trp Leu Val Ala Ser Ala545 550 555 560Ile Leu Ala Leu
Leu Cys Leu 56532479PRTHuman Pegivirus 2 324Met Gly Cys Ser Thr Asp
Gln Thr Ile Cys Ser Pro Val Val Gly Ala1 5 10 15Asp Tyr Asn Thr Ser
Ser Gly Cys Arg Ala Leu Asn Gly Ser Tyr His 20 25 30Cys Gly Gly Gly
Ser Cys Arg Ser Pro Ser Cys Val Gln Val Ala Arg 35 40 45Arg Val Leu
Gln Leu Cys Ala Leu Leu Ala Leu Ile Gly Ser Gly Met 50 55 60Cys Ser
Ile Arg Ser Lys Thr Glu Gly Arg Ile Glu Ser Gly Gln65 70
75325191PRTHuman Pegivirus 2 325Ile Leu Gln Ser Gln Arg Ala Cys Trp
Thr Gly Glu Gly Phe Ala Phe1 5 10 15Phe Ser Asn Cys Cys Asn Gln Ser
Asp Ile Met Trp Cys Leu His Arg 20 25 30Trp Cys Val Thr Arg Pro Gly
Cys Leu Val Cys Thr Gly Asn Ala Thr 35 40 45His Pro Val Cys Trp Asp
Tyr Leu Gly Ser Gly Val Ser Arg Arg Pro 50 55 60Ala Arg Arg Met Gly
Glu Gly Ala Glu Val Leu Leu Arg Leu Ile Gly65 70 75 80Ala Ala Gly
Trp Leu Gly Leu Leu Ala Glu Ala Leu Gly Met Ser Glu 85 90 95Ile Tyr
Ala Ala Ile Leu Cys Phe Gly Phe Ile Ala Trp Tyr Gly Trp 100 105
110Gly Ile Pro Lys Thr Leu Val Cys Thr Val Cys Pro Ala Val Asn Ile
115 120 125Ser Pro Tyr Ser Phe Leu Ser Pro Asp Thr Ile Ala Phe Gly
Thr Trp 130 135 140Ile Leu Gln Leu Pro Gly Leu Leu Trp Gln Met Phe
Val Asn Phe Pro145 150 155 160Ile Leu Tyr Ser Thr Trp Ile Leu Trp
Leu Leu Leu Ser Gly Lys Thr 165 170 175Val Ala Val Ile Ala Ile Leu
Leu Ala Ser Pro Thr Val Met Ala 180 185 190326354PRTHuman Pegivirus
2MISC_FEATURE(72)..(72)Xaa = any amino
acidMISC_FEATURE(76)..(76)Xaa = any amino
acidMISC_FEATURE(117)..(117)Xaa = any amino
acidMISC_FEATURE(170)..(170)Xaa = any amino
acidMISC_FEATURE(177)..(177)Xaa = any amino
acidMISC_FEATURE(181)..(181)Xaa = any amino acid 326Tyr Lys His Gln
Ser Asp Ser Tyr Leu Lys Tyr Cys Thr Ile Thr Asn1 5 10 15Ala Ser Thr
Ala Met Asn Cys Asp Cys Pro Phe Gly Thr Phe Thr Arg 20 25 30Asn Thr
Glu Ser Arg Phe Ser Ile Pro Arg Phe Cys Pro Val Lys Ile 35 40 45Lys
Ser Ser Thr Phe Ile Cys Ser Trp Gly Ser Trp Trp Trp Phe Ala 50 55
60Glu Asn Ile Thr Arg Pro Tyr Xaa Asp Val Gly Xaa Pro Pro Ala Pro65
70 75 80Ile Ser Ala Leu Cys Tyr Ile Tyr Ser Asn Asn Asp Pro Pro Pro
Trp 85 90 95Tyr His Asn Thr Thr Ile Ile Pro Gln Asn Cys Arg Asn Ser
Thr Val 100 105 110Asp Pro Thr Thr Xaa Pro Cys Arg Asp Lys Trp Gly
Asn Ala Thr Ala 115 120 125Cys Ile Leu Asp Arg Arg Ser Arg Phe Cys
Gly Asp Cys Tyr Gly Gly 130 135 140Cys Phe Tyr Thr Asn Gly Thr His
Asp Arg Ser Trp Asp Arg Cys Gly145 150 155 160Ile Gly Tyr Arg Asp
Gly Leu Ile Glu Xaa Val Gln Leu Gly Gln Ile 165 170 175Xaa Pro Asn
Ile Xaa Asn Thr Thr Ile Glu Leu Leu Ala Gly Ala Ser 180 185 190Leu
Val Ile Ala Ser Gly Leu Arg Pro Gly Phe Gly Cys Ser Arg Ala 195 200
205His Gly Val Val His Cys Tyr Arg Cys Pro Ser Tyr Arg Asp Leu Glu
210 215 220Gln Phe Gly Pro Gly Leu Gly Lys Trp Val Pro Leu Pro Gly
Glu Pro225 230 235 240Val Pro Glu Leu Cys Ile Asn Pro Gln Trp Ala
Arg Arg Gly Phe Arg 245 250 255Met Ser Asn Asn Pro Leu Ser Leu Leu
Gln Thr Phe Val Glu Asp Ile 260 265 270Phe Leu Ala Pro Phe Cys Asn
Pro Thr Pro Gly Arg Val Arg Val Cys 275 280 285Asn Asn Thr Ala Phe
Tyr Pro Arg Gly Gly Gly Phe Val Gln Leu Ile 290 295 300Gly Asp Val
Gln Val Leu Thr Pro Asn Thr Ala Ser Leu His Ser Leu305 310 315
320Leu Thr Leu Ile Ser Leu Ile Leu Leu Val Cys Val Val Ser Gly Ala
325 330 335Arg Phe Gly Pro Leu Ile Ile Ile Phe Phe Trp Ser Ala Arg
His Val 340 345 350Tyr Ala327237PRTHuman Pegivirus
2MISC_FEATURE(6)..(6)Xaa = any amino acid 327Ser Cys Tyr Leu Ser
Xaa Asp Trp Ala Val Cys Asn Asp Ala Phe Cys1 5 10 15Phe Thr Ser Gly
Thr Cys Ala Thr Phe Asn Asp Val Leu Cys Leu Pro 20 25 30Val Ala Thr
His Ile Ser Ser Cys Gly His Ala Val Pro Pro Pro Asp 35 40 45Arg Gly
Trp Glu Val Pro Ala Ala Met Ser Trp Val Ile Ser Arg Thr 50 55 60Thr
Gly Leu Thr Phe Asp Val Phe Ser Phe Ile Gln Tyr Phe Pro Thr65 70 75
80Val Pro Gly Asn Asn Thr Asp Ile Ile Tyr Cys Gly Glu Pro Thr Phe
85 90 95Phe Gly Asp Ile Thr Gly Ile Tyr Trp Pro Tyr Phe Leu Pro Gly
Val 100 105 110Leu Leu Leu Tyr Leu Thr Pro Phe Leu Gly Phe Arg Leu
Met Leu Ala 115 120 125Gly Phe Asn Ile Asp Gly Leu Phe Pro Ile Arg
His Ala Thr Ala Ala 130 135 140Leu Arg Phe Ser Thr Ser Arg Ala Thr
Leu Ser Val Val Ser Ala Phe145 150 155 160Leu Ile Tyr Ile Leu Ser
His Pro Val Asn Ala Ala Leu Asn Arg Met 165 170 175Phe Leu Ala Ser
Ala Asn Leu Glu Met Ile Leu Ser Phe Asp Thr Tyr 180 185 190His Glu
Thr Val Leu Tyr Ile Phe Cys Leu Phe Leu Tyr Leu Gln Val 195 200
205Ser Pro Arg Ala Gly Leu Ala Ala Met Val Ala Ile Lys Leu Ser Arg
210 215 220Gly Leu Leu Phe Ala Leu Val Leu Ala Gln Gly Val Cys225
230 235328240PRTHuman Pegivirus 2MISC_FEATURE(69)..(69)Xaa = any
amino acid 328Arg Pro Gly Arg Val Phe Gly Leu Glu Val Cys Ala Asp
Val Ser Trp1 5 10 15Leu Val Glu Phe Thr Gly Asn Cys Thr Trp Tyr Met
Ser Cys Ile Phe 20 25 30Ser Phe Trp Cys Ala Val Phe Ala Phe Thr Ser
Pro Leu Gly Arg Gln 35 40 45Tyr Lys Leu Gln Ile Tyr Arg Tyr Trp Ala
Gln Val Tyr Ala Arg Leu 50 55 60Ile Leu Ala Val Xaa Cys Gly Pro Leu
Gly Arg Glu Phe His Phe Arg65 70 75 80Ala Ser Val Gly Val Leu Trp
Cys Gly Ala Cys Met Leu Trp Pro Arg 85 90 95Glu Cys Ser Glu Ile Ser
Leu Ala Leu Ile Leu Cys Ala Leu Thr Val 100 105 110Asp Thr Ile Asp
Thr Trp Leu Val Ala Cys Leu Ser Ala Gly Pro Ser 115 120 125Ala Arg
Ala Leu Ala Thr Leu Ala Asp Asp Met Val Arg Met Gly Asp 130 135
140His Arg Ala Leu Arg Ala Val Leu Arg Cys Phe Gly Ser Arg Gly
Thr145 150 155 160Tyr Ile Tyr Asn His Met Gly Gln Val Ser Glu Arg
Val Ala Gln Ala 165 170 175Val Arg Asp Leu Gly Gly Cys Leu Glu Pro
Val Val Leu Glu Glu Pro 180 185 190Thr Phe Thr Glu Val Val Asp Asp
Thr Met Ser Lys Ile Cys Gly Gln 195 200 205Leu Leu Gly Gly Lys Pro
Val Val Ala Arg Cys Gly Thr Arg Val Leu 210 215 220Val Gly His Leu
Asn Pro Glu Asp Leu Pro Pro Gly Phe Gln Leu Ser225 230 235
240329628PRTHuman Pegivirus 2MISC_FEATURE(48)..(48)Xaa = any amino
acidMISC_FEATURE(580)..(580)Xaa = any amino acid 329Ala Pro Val Val
Ile Thr Lys Pro Ser Ile Gly Thr Trp Ser Phe Leu1 5 10 15Lys Ala Thr
Leu Thr Gly Arg Ala Glu Thr Pro Gly Ser Gly Gln Ile 20 25 30Val Val
Leu Ser Ser Leu Thr Gly Arg Ser Met Gly Thr Ala Val Xaa 35 40 45Gly
Thr Leu Tyr Ala Thr Gly His Gly Ala Gly Ala Arg Gly Leu Ala 50 55
60Thr Cys Ala Gly Leu Arg Thr Pro Leu Tyr Thr Ala Leu Ser Asp Asp65
70 75 80Val Val Ala Tyr Ser Cys Leu Pro Gly Met Ser Ser Leu Glu Pro
Cys 85 90 95Arg Cys Ser Pro Ser Arg Val Trp Val Met Asn Asn Asn Gly
Gly Leu 100 105 110Val Cys Gly Arg Val Glu Asn Glu Asp Val Cys Leu
Asp Cys Pro Thr 115 120 125His Ile Asp Gln Leu Arg Gly Ala Ser Gly
Ser Pro Val Leu Cys Asp 130 135 140His Gly His Ala Tyr Ala Leu Met
Leu Gly Gly Tyr Ser Thr Ser Gly145 150 155 160Ile Cys Ala Arg Val
Arg Ile Val Arg Pro Trp Gln Asn Ala Tyr Ser 165 170 175Ser Ser Gly
Gly Gln Gly Gly Met Gln Ala Pro Ala Val Thr Pro Thr 180 185 190Tyr
Ser Glu Ile Thr Tyr Tyr Ala Pro Thr Gly Ser Gly Lys Ser Thr 195 200
205Lys Tyr Pro Val Asp Leu Val Lys Gln Gly His Lys Val Leu Val Ile
210 215 220Ile Pro Ser Val Ser Val Val Lys Ser Met Ala Pro Tyr Ile
Lys Glu225 230 235 240Thr Tyr Lys Ile Arg Pro Glu Ile Arg Ala Gly
Thr Gly Pro Asp Gly 245 250 255Val Thr Val Ile Thr Gly Glu Asn Leu
Ala Tyr Met Thr Tyr Gly Arg 260 265 270Phe Leu Val Asp Pro Glu Thr
Asn Leu Arg Gly Tyr Ala Val Val Ile 275 280 285Cys Asp Glu Cys His
Asp Thr Ser Ser Thr Thr Leu Leu Gly Ile Gly 290 295 300Ala Val Arg
Met Tyr Ala Glu Lys Ala Gly Val Lys Thr Val Val Phe305 310 315
320Ala Thr Ala Thr Pro Ala Gly Ile Gln Val Gln Pro His Pro Asn Ile
325 330 335Asp Glu Tyr Leu Leu Thr Asp Thr Gly Asp Val Asp Phe Tyr
Gly Ala 340 345 350Lys Ile Lys Leu Asp Asn Ile Arg Thr Gly Arg His
Val Ile Phe Cys 355 360 365His Ser Lys Ala Arg Cys Ala Glu Leu Thr
Gln Gln Leu Ser Gly Leu 370 375 380Gly Val Arg Ala Val Ser Phe Trp
Arg Gly Cys Asp Ile Lys Thr Ile385 390 395 400Pro Ala Ser Asp Ser
Ile Val Val Val Ala Thr Asp Ala Leu Ser Thr 405 410 415Gly Tyr Thr
Gly Asn Phe Asp Ser Val Ile Asp Cys Gly Cys Cys Val 420 425 430Glu
Gln Thr Val Thr Ile Asp Met Asp Pro Thr Phe Ser Ile Ser Ala 435 440
445Arg Val Val Pro Cys Thr Ala Ala Leu Arg Met Gln Arg Arg Gly Arg
450 455 460Thr Gly Arg Gly Arg Arg Gly Ala Tyr Tyr Thr Thr Thr Pro
Gly Ala465 470 475 480Ala Pro Cys Val Ser Val Pro Asp Ala Asn Val
Trp Gln Ala Val Glu 485 490 495Ser Ala Met Val Phe Tyr Asp Trp Asn
Ala Ala Arg Ile Gln Gln Cys 500 505 510Leu Ala Ala Tyr His Asp Leu
Gly Cys Thr Pro Arg Leu Ser Cys Asp 515 520 525Pro Cys Thr Pro Val
Arg Val Met Asp Thr Leu Arg Ala Tyr Leu Arg 530 535 540Arg Pro Glu
Val Thr Thr Ala Ala Leu Ala Gly Glu Gln Trp Pro Leu545 550 555
560Leu Tyr Gly Val Gln Leu Cys Ile Cys Lys Glu Thr Glu Ala His Gly
565 570 575Pro Asp Asp Xaa Ile Lys Trp Lys Cys Leu Leu Asn Asn Ser
Asn Lys 580 585 590Thr Pro Leu Leu Tyr Ala Leu Asp Asn Pro Thr Leu
Glu Phe Thr Thr 595 600 605Gln His Asp Leu Thr Arg Arg Ile Ala Gly
Ala Leu
Ser Ser Thr Val 610 615 620Phe Val Glu Thr62533041PRTHuman
Pegivirus 2 330Gly Tyr Gly Pro Ile Leu Leu Ala Gly Ala Ala Leu Ala
Ala Ser Phe1 5 10 15Ala Phe Ala Gly Ala Thr Gly Ala Leu Val Pro Ser
Ala Val Trp Ser 20 25 30Val Asp Asn Gly Val Ala Gly Val Thr 35
40331262PRTHuman Pegivirus 2 331Arg Pro Asp Ala Thr Asp Glu Thr Ala
Ala Tyr Ala Gln Arg Leu Tyr1 5 10 15Gln Ala Cys Ala Asp Ser Gly Leu
Leu Ala Ser Leu Gln Gly Thr Ala 20 25 30Ser Ala Ala Leu Ser Lys Leu
Ala Asp Ala Ser Arg Gly Ala Ser Gln 35 40 45Tyr Leu Ala Ser Ala Pro
Pro Ser Pro Ala Pro Leu Val Gln Val Leu 50 55 60Gln Phe Leu Glu Thr
Asn Phe Ser Ser Ile Ala Ser Phe Gly Leu Leu65 70 75 80Cys Ala Gly
Cys Gln Ala Gly Glu Cys Phe Thr Ala Leu Ala Gly Leu 85 90 95Val Ser
Gly Ala Thr Ala Gly Leu Gly Gly Ala His Lys Trp Leu Leu 100 105
110Ala Ile Ala Gly Thr Trp Leu Val Ser Leu Gln Thr Gly Ala Arg Gly
115 120 125Gly Met Val Ala Gly Leu Ser Val Leu Ala Gly Cys Cys Ile
Gly Ser 130 135 140Val Thr Gly Leu Asp Phe Leu Phe Gly Cys Leu Thr
Gly Trp Glu Ala145 150 155 160Val Val Gly Ala Ala Val Ala Thr Gln
Lys Ile Leu Ser Gly Ser Ala 165 170 175Asp Met Thr Thr Leu Leu Asp
Leu Leu Pro Ala Phe Phe Ser Pro Gly 180 185 190Ala Gly Val Ala Gly
Ile Val Leu Val Phe Ile Leu Ser Asn Ser Ser 195 200 205Val Thr Thr
Trp Ala Asn Arg Leu Leu Ser Met Cys Ala Lys Gln Thr 210 215 220Ile
Cys Asp Asn Tyr Phe Leu Ser Asp Lys Phe Gly Gln Gln Leu Ser225 230
235 240Lys Leu Ser Leu Trp Arg Thr Leu Tyr Arg Trp Ala Glu Ala Arg
Glu 245 250 255Gly Tyr Thr Gln Cys Gly 260332458PRTHuman Pegivirus
2 332Val Val Gly Gly Ile Trp Ser Phe Val Leu Cys Ile Leu Arg Ala
Val1 5 10 15Trp Asp Trp Ala Ala Lys His Val Pro Arg Phe Arg Val Pro
Met Ile 20 25 30Gly Cys Ser Pro Ala Trp Cys Gly Arg Trp Leu Gly Thr
Gly Thr Leu 35 40 45Leu Thr Thr Cys Gly Cys Gly Glu Arg Val Ser Leu
Gln Cys Leu Cys 50 55 60Ser Thr Ser Asp Pro Leu Leu Arg Val Gly Arg
Trp Cys Arg Cys Ser65 70 75 80Trp Ser Val Gly Phe Pro Phe Asn Pro
Thr Thr Thr Ala Thr Gly Thr 85 90 95Leu Arg Pro Asp Ile Ser Asp Ala
Thr Arg Leu Gly Phe Arg Tyr Gly 100 105 110Ile Ala Glu Ile Val Glu
Leu Glu Leu Arg Glu His Lys Trp His Val 115 120 125Cys Ala Ala Ser
Cys Cys Leu Asp Arg Ala Ser Val Ala Ser Ala Val 130 135 140Lys Ala
Pro Pro Val Thr Ala Asn Gly Ile Pro Ile Ser Thr Phe Ser145 150 155
160Pro Pro Gln Thr Tyr Ser Leu Ser Leu Cys Ser Phe Asp Ser Val Cys
165 170 175Met Ser Thr Asn Leu Cys Asn Pro Ala Lys Thr Leu Ser Val
Cys Ser 180 185 190Gln Glu Ala Val Glu Leu Leu Glu Glu Thr Val Asp
Thr Ala Gln Val 195 200 205Met Met Cys Gln Asn Leu Glu Ala Arg Arg
Arg Ala Glu Tyr Asp Ala 210 215 220Trp Gln Val Arg His Ala Val Gly
Asp Glu Tyr Thr Arg Leu Ala Asp225 230 235 240Glu Asp Val Asp Thr
Thr Thr Ser Val Lys Pro Pro Ala Ala Arg Ala 245 250 255Ala Val Asp
Ser Ser Thr Leu Glu Asp Val Ser Val Leu Thr Val Leu 260 265 270Arg
Glu Leu Gly Asp Gln Cys Gln Asn Ala Ile Lys Phe Val Val Gln 275 280
285Ala Ala Ser Arg Phe Val Pro Pro Val Pro Arg Pro Arg Thr Arg Val
290 295 300Ser Gly Val Leu Glu Arg Val Arg Met Cys Met Arg Thr Pro
Pro Ile305 310 315 320Lys Phe Glu Ala Thr Ala Val Pro Ile His Asn
Ile Ile Pro Glu Glu 325 330 335Cys His Ile Val Leu Arg Cys Thr Gly
Cys Asn Asp Gln Ala Leu Thr 340 345 350Val Pro Tyr Gly Thr Cys Thr
Gln Ser Leu Ile Arg His Leu Thr Asn 355 360 365Lys His Asn His Tyr
Ile Pro Lys Gln Lys Ile Glu Glu Asp Thr Glu 370 375 380Val Thr Val
Ile Cys Ala Val Pro Thr Lys Arg Ala Ser Lys Leu Ile385 390 395
400Thr Phe Arg Ala Gly Asp Arg Ser Val Ser Cys Cys His Pro Leu Gln
405 410 415Thr Pro Ile Arg Ala Leu Leu Leu Lys Tyr Gly Leu Pro Ile
Gly Thr 420 425 430Trp Ser Asp Cys Asn Gly Pro Leu Gly Asp Asp Ala
Arg Val Cys Asp 435 440 445Val Asn Gly Val Thr Thr Tyr Glu Pro Cys
450 455333567PRTHuman Pegivirus 2 333Met Gln Ser Tyr Ser Trp Phe
Arg Pro Ile Val Ala Pro Thr Thr Pro1 5 10 15Pro Leu Pro Val Thr Arg
Ser Val Ala Gly Ile Leu Arg Ala Asp Thr 20 25 30Ser Arg Val Tyr Thr
Thr Thr Ala Val Asp Val Ser Glu Arg Gln Ser 35 40 45Lys Val Thr Ile
Asp Gln Thr Ser Ala Lys Val Asp Gln Trp Phe Arg 50 55 60Asp Thr Tyr
Asn Cys Cys Leu Ala Lys Ala Lys Thr Phe Arg Gln Ser65 70 75 80Gly
Met Ser Tyr Glu Asp Ala Val Ser Lys Met Arg Ala Asn Thr Thr 85 90
95Arg Asp His Asn Thr Gly Ile Thr Tyr Ser Asp Leu Val Ser Gly Arg
100 105 110Ala Lys Pro Ala Val Gln Lys Ile Val Asp Gln Met Arg Ala
Gly Val 115 120 125Tyr Asp Ala Pro Met Arg Ile Ile Pro Lys Pro Glu
Val Phe Pro Arg 130 135 140Asp Lys Ser Thr Arg Lys Pro Pro Arg Phe
Ile Val Phe Pro Gly Cys145 150 155 160Ala Ala Arg Val Ala Glu Lys
Met Ile Leu Gly Asp Pro Gly Ala Ile 165 170 175Thr Lys His Val Leu
Gly Asp Ala Tyr Gly Phe Ala Thr Pro Pro His 180 185 190Glu Arg Ala
Arg Leu Leu Glu Gln Trp Trp Asn Arg Ala Thr Glu Pro 195 200 205Gln
Ala Ile Ala Val Asp Ala Ile Cys Phe Asp Ser Thr Ile Thr Ala 210 215
220Glu Asp Met Asp Arg Glu Ala Asn Ile Leu Ala Ala Ala His Ser
Asp225 230 235 240Pro Glu Gly Val His Gly Leu Tyr Asn Tyr Tyr Lys
Arg Ser Pro Met 245 250 255Cys Asp Ile Thr Gly Asn Val Val Gly Val
Arg Cys Cys Arg Ala Ser 260 265 270Gly Thr Leu Thr Thr Ser Ser Gly
Asn Thr Leu Thr Cys Tyr Leu Lys 275 280 285Val Arg Ala Ala Cys Thr
Arg Ala Gly Ile Lys Pro Ile Gly Leu Leu 290 295 300Ile His Gly Asp
Asp Thr Leu Ile Ile Thr Glu Arg Cys Ala Gln Glu305 310 315 320Thr
Leu Asp Glu Phe Ser Asn Ala Leu Asn Asp Tyr Gly Phe Pro His 325 330
335Thr Phe Gln Ala Ser Gly Asp Leu Ser Ser Val Glu Cys Cys Ser Ala
340 345 350Arg Val Asp Ser Val Cys Leu Arg Gly Gly Met Arg Arg Met
Leu Val 355 360 365Pro Gln Ala Arg Arg Ala Ile Ala Arg Val Leu Gly
Glu Lys Gly Asp 370 375 380Pro Leu Gly Val Ile Ser Ser Tyr Ile Val
Met Tyr Pro Thr Ala Ala385 390 395 400Val Thr Val Tyr Val Leu Leu
Pro Leu Leu Cys Met Leu Ile Arg Asn 405 410 415Glu Pro Ser Gln Thr
Gly Thr Leu Val Thr Leu Thr Val His Gly Asn 420 425 430Ser Val Ser
Val Pro Val Trp Leu Leu Pro Thr Ile Ile Val Asn Leu 435 440 445His
Gly Arg Asp Ala Leu Gln Val Val Arg His Thr Ala Ala Ser Met 450 455
460Ala Glu Leu Ser Ser Ala Leu Ala Phe Phe Gly Met Arg Gly Leu
Asn465 470 475 480Cys Trp Arg Arg Arg Arg Arg Ala Ile Arg Thr Asp
Met Ile Lys Leu 485 490 495Gly Gly Trp Asn Ala Asn Phe Ala Gln Met
Leu Leu Trp Ser Pro Glu 500 505 510Val Arg Thr Pro Gln Pro Glu Pro
Arg Gly Val Cys Leu Leu Pro Pro 515 520 525Glu Leu Trp Glu Arg Pro
Tyr Glu Asn Leu His Leu Ser Thr Ile Asp 530 535 540Arg Asn Arg Gly
Ala Ser Arg Leu Arg Phe Trp Leu Val Ala Ser Ala545 550 555 560Ile
Leu Ala Leu Leu Cys Leu 56533479PRTHuman Pegivirus 2 334Met Gly Cys
Ser Thr Asp Gln Thr Ile Cys Ser Pro Val Val Gly Ala1 5 10 15Asp Tyr
Asn Thr Ser Ser Gly Cys Arg Ala Leu Asn Gly Ser Asp His 20 25 30Cys
Gly Gly Ser Ser Cys Arg Ser Pro Ser Arg Val Gln Ala Ala Arg 35 40
45Arg Val Leu Gln Leu Cys Ala Phe Leu Ala Leu Ile Gly Ser Gly Met
50 55 60Cys Ser Ile Arg Ser Lys Thr Glu Gly Arg Ile Glu Ser Gly
Gln65 70 75335191PRTHuman Pegivirus 2 335Ile Leu Gln Ser Gln Arg
Ala Cys Trp Thr Gly Glu Gly Phe Ala Phe1 5 10 15Phe Ser Asn Cys Cys
Asn Gln Ser Asp Ile Met Trp Cys Leu His Arg 20 25 30Trp Cys Val Thr
Arg Pro Gly Cys Leu Val Cys Thr Gly Asn Ala Thr 35 40 45His Pro Val
Cys Trp Asp Tyr Leu Gly Ser Gly Val Ser Arg Arg Pro 50 55 60Ala Arg
Arg Leu Gly Glu Gly Ala Glu Met Leu Leu Arg Leu Ile Gly65 70 75
80Ile Ala Gly Trp Leu Gly Leu Leu Ala Glu Ala Leu Gly Met Ser Glu
85 90 95Met Tyr Ala Ala Ile Leu Cys Phe Gly Phe Ile Ala Trp Tyr Gly
Trp 100 105 110Gly Ile Pro Lys Thr Leu Val Cys Thr Val Cys Pro Ala
Val Asn Ile 115 120 125Ser Pro Tyr Ser Phe Leu Ser Pro Asp Thr Ile
Ala Phe Gly Thr Trp 130 135 140Ile Leu Gln Leu Pro Gly Leu Leu Trp
Gln Met Phe Val Asn Phe Pro145 150 155 160Ile Leu Tyr Ser Thr Trp
Ile Leu Trp Leu Leu Leu Ser Gly Lys Thr 165 170 175Val Ala Val Ile
Ala Ile Leu Leu Ala Ser Pro Thr Val Met Ala 180 185
190336354PRTHuman Pegivirus 2MISC_FEATURE(111)..(111)Xaa = any
amino acidMISC_FEATURE(279)..(279)Xaa = any amino acid 336Tyr Lys
His Pro Ser Glu Ser Tyr Leu Lys Tyr Cys Thr Ile Thr Asn1 5 10 15Ala
Ser Ala Ala Met Asn Cys Asp Cys Pro Phe Gly Thr Phe Thr Arg 20 25
30Asn Thr Glu Ser Arg Phe Ser Ile Pro Arg Phe Cys Pro Val Lys Ile
35 40 45Glu Ser Ser Thr Phe Ile Cys Ser Trp Gly Ser Trp Trp Trp Phe
Ala 50 55 60Glu Asn Ile Thr Arg Pro Tyr Ser Asp Val Gly Met Pro Pro
Ala Pro65 70 75 80Ile Ser Ala Leu Cys Tyr Ile Tyr Ser Asn Asn Asp
Pro Pro Pro Trp 85 90 95Tyr Tyr Asn Thr Thr Ile Ile Pro Gln Asn Cys
Arg Asn Ser Xaa Val 100 105 110Asp Pro Thr Thr Ala Pro Cys Arg Asp
Lys Trp Gly Asn Ala Thr Ala 115 120 125Cys Ile Leu Asp Arg Arg Ser
Arg Phe Cys Gly Asp Cys Tyr Gly Gly 130 135 140Cys Phe Tyr Thr Asn
Gly Ser His Asp Arg Ser Trp Asp Arg Cys Gly145 150 155 160Ile Gly
Tyr Arg Asp Gly Leu Ile Glu Phe Val Gln Leu Gly Gln Ile 165 170
175Arg Pro Asn Ile Ser Asn Thr Thr Ile Glu Leu Leu Ala Gly Ala Ser
180 185 190Leu Val Ile Ala Ser Gly Leu Arg Pro Gly Tyr Gly Cys Ser
Arg Ala 195 200 205His Gly Val Val His Cys Tyr Arg Cys Pro Ser Tyr
Arg Asp Leu Glu 210 215 220Gln Phe Gly Pro Gly Leu Gly Lys Trp Val
Pro Leu Pro Gly Glu Pro225 230 235 240Val Pro Glu Leu Cys Ile Asn
Pro Gln Trp Ala Arg Arg Gly Phe Arg 245 250 255Val Ser Asn Asn Pro
Leu Ser Leu Ile Gln Thr Phe Val Glu Asp Ile 260 265 270Phe Leu Ala
Pro Phe Cys Xaa Pro Thr Pro Gly Arg Val Arg Val Cys 275 280 285Asn
Asn Thr Ala Phe Tyr Pro Arg Gly Gly Gly Phe Val Gln Leu Ile 290 295
300Gly Asp Val Gln Val Leu Thr Pro Asn Thr Ala Ser Leu His Ser
Leu305 310 315 320Leu Thr Leu Ile Ser Leu Ile Leu Leu Val Cys Val
Val Ser Gly Ala 325 330 335Arg Phe Val Pro Leu Ile Ile Ile Phe Phe
Trp Ser Val Arg His Val 340 345 350Tyr Ala337237PRTHuman Pegivirus
2MISC_FEATURE(59)..(60)Xaa = any amino acid 337Ser Cys Tyr Leu Ser
Cys Asp Trp Ala Val Cys Asn Asp Ala Phe Cys1 5 10 15Phe Thr Ser Gly
Thr Cys Ala Thr Phe Asn Asp Val Leu Cys Leu Pro 20 25 30Val Ala Thr
Arg Ile Ser Ser Cys Gly His Ala Val Pro Pro Pro Asp 35 40 45Arg Gly
Trp Glu Val Pro Ala Ala Leu Ser Xaa Xaa Ile Ser Arg Thr 50 55 60Thr
Gly Leu Thr Phe Asp Val Phe Ser Phe Ile Gln Tyr Leu Pro Thr65 70 75
80Val Pro Gly Asn Asn Ser Asp Ile Ile Tyr Cys Gly Glu Pro Thr Phe
85 90 95Phe Gly Asp Leu Thr Gly Ile Tyr Trp Pro Tyr Phe Leu Pro Gly
Val 100 105 110Leu Leu Leu Tyr Leu Thr Pro Phe Leu Gly Leu Arg Leu
Met Leu Ala 115 120 125Gly Phe Asn Ile Asp Gly Leu Phe Pro Ile Arg
His Ala Thr Ala Ala 130 135 140Leu Arg Phe Ser Thr Ser Arg Val Thr
Leu Ser Val Val Ala Ala Phe145 150 155 160Leu Ile Tyr Ile Leu Ser
His Pro Val Asn Ala Ala Leu Asn Arg Met 165 170 175Phe Leu Ala Ser
Ala Asn Leu Glu Met Ile Leu Ser Phe Asp Thr Tyr 180 185 190His Glu
Thr Ile Leu Tyr Ile Val Cys Leu Met Leu Tyr Leu Gln Val 195 200
205Ser Pro Arg Ala Gly Leu Ala Ala Met Val Ala Ile Lys Leu Ser Arg
210 215 220Gly Leu Leu Phe Ala Val Val Leu Ala His Gly Val Cys225
230 235338240PRTHuman Pegivirus 2MISC_FEATURE(197)..(197)Xaa = any
amino acidMISC_FEATURE(202)..(203)Xaa = any amino acid 338Arg Pro
Gly Arg Val Phe Gly Leu Glu Val Cys Ala Asp Ile Ser Trp1 5 10 15Leu
Val Glu Phe Thr Gly Asn Cys Thr Trp Tyr Met Ser Cys Val Phe 20 25
30Ser Phe Trp Cys Ala Val Phe Ala Phe Thr Ser Pro Leu Gly Arg Gln
35 40 45Tyr Lys Leu Gln Ile Tyr Arg Tyr Trp Ala Gln Val Tyr Ala Arg
Leu 50 55 60Ile Leu Ala Val Gly Cys Gly Pro Leu Gly Arg Glu Phe His
Phe Arg65 70 75 80Ala Ser Val Gly Val Leu Trp Cys Gly Ala Cys Met
Leu Trp Pro Arg 85 90 95Glu Cys Ser Glu Ile Ser Leu Val Phe Ile Leu
Cys Ala Leu Thr Val 100 105 110Asp Thr Ile Asp Thr Trp Leu Val Ala
Cys Leu Ser Ala Gly Pro Ser 115 120 125Ala Arg Thr Leu Ala Thr Leu
Ala Asp Asp Met Ala Arg Met Gly Asp 130 135 140Asn Arg Ala Leu Arg
Ala Val Leu Cys Cys Phe Gly Ser Arg Gly Thr145 150 155 160Tyr Ile
Tyr Asn His Met Gly Gln Val Ser Glu Arg Val Ala Arg Ala 165 170
175Val Arg Asp Leu Gly Gly Cys Leu Glu Pro Val Val Leu Glu Glu Pro
180 185 190Thr Phe Thr Glu Xaa Val Asp Asp Thr Xaa Xaa Leu Val Cys
Gly Gln 195 200 205Leu Leu Gly Gly Lys Pro Val Val Ala Arg Cys Gly
Thr Arg
Val Leu 210 215 220Val Gly His Leu Asn Pro Glu Asp Leu Pro Pro Gly
Phe Gln Leu Ser225 230 235 240339628PRTHuman Pegivirus
2MISC_FEATURE(24)..(24)Xaa = any amino
acidMISC_FEATURE(49)..(49)Xaa = any amino
acidMISC_FEATURE(554)..(554)Xaa = any amino acid 339Ala Pro Val Val
Ile Thr Lys Pro Ser Ile Gly Thr Trp Ser Phe Leu1 5 10 15Lys Ala Thr
Leu Thr Gly Arg Xaa Glu Thr Pro Gly Ser Gly Gln Ile 20 25 30Val Val
Leu Ser Ser Leu Thr Gly Arg Ser Met Gly Thr Ala Val Asn 35 40 45Xaa
Thr Leu Tyr Ala Thr Gly His Gly Ala Gly Ala Arg Gly Leu Ala 50 55
60Thr Cys Ala Gly Leu Arg Thr Pro Leu Tyr Thr Ala Leu Ser Asp Asp65
70 75 80Val Val Ala Tyr Ser Cys Leu Pro Gly Met Ser Ser Leu Glu Pro
Cys 85 90 95Arg Cys Thr Pro Ser Arg Val Trp Val Met Asn Asn Asn Gly
Gly Leu 100 105 110Val Cys Gly Arg Val Glu Lys Asp Asp Val Cys Leu
Asp Cys Pro Thr 115 120 125His Ile Asp Gln Leu Arg Gly Ala Ser Gly
Ser Pro Val Leu Cys Asp 130 135 140His Gly His Ala Tyr Ala Leu Met
Leu Gly Gly Tyr Ser Thr Ser Gly145 150 155 160Ile Cys Ala Arg Val
Arg Ile Val Gln Pro Trp Gln Asn Ala Tyr Ser 165 170 175Ser Ser Gly
Gly Gln Gly Gly Met Gln Ala Pro Ala Val Thr Pro Thr 180 185 190Tyr
Ser Glu Ile Thr Tyr Tyr Ala Pro Thr Gly Ser Gly Lys Ser Thr 195 200
205Lys Tyr Pro Val Asp Leu Val Lys Gln Gly His Lys Val Leu Val Ile
210 215 220Ile Pro Ser Val Thr Val Val Lys Ser Met Ala Asn Tyr Ile
Lys Glu225 230 235 240Thr Tyr Lys Ile Arg Pro Glu Ile Arg Ala Gly
Thr Gly Pro Asp Gly 245 250 255Val Thr Val Ile Thr Gly Glu Ser Leu
Ala Tyr Met Thr Tyr Gly Arg 260 265 270Phe Leu Val Asp Pro Glu Thr
Asn Leu Arg Gly Tyr Ala Val Val Ile 275 280 285Cys Asp Glu Cys His
Asp Thr Ser Ser Thr Thr Leu Leu Gly Ile Gly 290 295 300Ala Val Arg
Met Phe Ala Glu Lys Ala Gly Val Arg Thr Val Val Phe305 310 315
320Ala Thr Ala Thr Pro Ala Gly Ile Gln Val Gln Pro His Pro Asn Ile
325 330 335Asp Glu Tyr Leu Leu Thr Asp Thr Gly Asp Val Asp Phe Tyr
Gly Ala 340 345 350Lys Ile Lys Leu Asp Asn Ile Arg Thr Gly Arg His
Val Ile Phe Cys 355 360 365His Ser Lys Ala Arg Cys Ala Glu Leu Thr
Gln Gln Leu Ser Gly Leu 370 375 380Gly Val Arg Ala Val Ser Phe Trp
Arg Gly Cys Asp Ile Lys Thr Ile385 390 395 400Pro Ala Ser Asp Ser
Ile Val Val Val Ala Thr Asp Ala Leu Ser Thr 405 410 415Gly Tyr Thr
Gly Asn Phe Asp Ser Val Ile Asp Cys Gly Cys Cys Val 420 425 430Glu
Gln Thr Val Thr Ile Asp Met Asp Pro Thr Phe Ser Ile Ser Ala 435 440
445Arg Val Val Pro Cys Thr Ala Ala Leu Arg Met Gln Arg Arg Gly Arg
450 455 460Thr Gly Arg Gly Arg Arg Gly Ala Tyr Tyr Thr Thr Thr Pro
Gly Ala465 470 475 480Ala Pro Cys Val Ser Val Pro Asp Ala Asn Val
Trp Gln Ala Val Glu 485 490 495Ser Ala Met Val Phe Tyr Asp Trp Gly
Ala Ala Arg Ile Gln Gln Cys 500 505 510Leu Ala Ala Tyr His Asp Leu
Gly Cys Thr Pro Arg Ile Ser Cys Asp 515 520 525Pro His Thr Pro Val
Arg Val Met Asp Thr Leu Arg Ala Tyr Leu Arg 530 535 540Arg Pro Glu
Val Thr Thr Ala Ala Leu Xaa Gly Glu Gln Trp Pro Leu545 550 555
560Leu Tyr Gly Val Gln Leu Cys Ile Cys Lys Glu Thr Glu Ala His Gly
565 570 575Pro Asp Asp Ser Ile Lys Trp Lys Cys Leu Leu Asn Asn Ser
Asn Lys 580 585 590Thr Pro Leu Leu Tyr Ala Leu Asp Asn Pro Thr Leu
Asp Phe Thr Thr 595 600 605Gln His Asp Leu Thr Arg Arg Ile Ala Gly
Ala Leu Ser Ser Thr Val 610 615 620Phe Val Glu Thr62534041PRTHuman
Pegivirus 2 340Gly Tyr Gly Pro Ile Leu Leu Ala Gly Ala Ala Leu Ala
Ala Ser Phe1 5 10 15Ala Phe Ala Gly Ala Thr Gly Ala Leu Val Pro Ser
Ala Val Trp Ser 20 25 30Val Asp Asn Gly Leu Ala Gly Val Thr 35
40341262PRTHuman Pegivirus 2MISC_FEATURE(191)..(193)Xaa = any amino
acid 341Arg Pro Asp Ala Thr Asp Glu Thr Ala Ala Tyr Ala Gln Arg Leu
Tyr1 5 10 15Gln Ala Cys Ala Asp Ser Gly Leu Phe Ala Ser Leu Gln Gly
Thr Ala 20 25 30Ser Ala Ala Leu Gly Lys Leu Ala Asp Ala Ser Arg Gly
Ala Ser Gln 35 40 45Tyr Leu Ala Ala Ala Pro Pro Ser Pro Ala Pro Leu
Val Gln Val Leu 50 55 60His Phe Leu Glu Thr Asn Phe Ser Ser Ile Ala
Ser Phe Gly Leu Leu65 70 75 80Cys Ala Gly Cys Gln Ala Gly Glu Cys
Phe Thr Ala Leu Ala Gly Leu 85 90 95Val Ser Gly Ala Thr Ala Gly Leu
Gly Gly Ala His Lys Trp Leu Leu 100 105 110Ala Ile Ala Gly Thr Trp
Leu Val Ser Leu Gln Thr Gly Pro Arg Gly 115 120 125Gly Met Val Ala
Gly Leu Ser Val Leu Ala Gly Cys Cys Ile Gly Ser 130 135 140Val Thr
Gly Leu Asp Phe Leu Phe Gly Cys Leu Thr Gly Trp Glu Ala145 150 155
160Val Val Gly Ala Ala Val Ala Thr Gln Lys Ile Leu Ser Gly Ser Ala
165 170 175Asp Met Thr Thr Leu Val Asp Leu Leu Pro Ala Ile Phe Ser
Xaa Xaa 180 185 190Xaa Gly Ile Ala Gly Ile Val Leu Val Phe Ile Leu
Ser Asn Ser Ser 195 200 205Val Thr Thr Trp Ala Asn Arg Leu Leu Ser
Met Cys Ala Lys Gln Thr 210 215 220Ile Cys Asp Asn Tyr Phe Leu Thr
Glu Lys Phe Gly His Gln Leu Ser225 230 235 240Lys Leu Ser Leu Trp
Arg Ala Val Tyr His Trp Ala Gln Ala Arg Glu 245 250 255Gly Tyr Thr
Gln Cys Gly 260342458PRTHuman Pegivirus 2 342Val Val Ser Gly Ile
Trp Ser Phe Val Leu Cys Ile Leu Arg Ala Val1 5 10 15Trp Asp Trp Ala
Ala Lys His Val Pro Arg Phe Arg Val Pro Met Ile 20 25 30Gly Cys Ser
Pro Ala Trp Cys Gly Arg Trp Leu Gly Thr Gly Thr Leu 35 40 45Leu Thr
Thr Cys Gly Cys Gly Glu Arg Val Ser Leu Gln Cys Leu Cys 50 55 60Ser
Thr Ser Asp Pro Thr Leu Ser Val Gly Arg Trp Cys Arg Cys Ser65 70 75
80Trp Ser Val Gly Phe Pro Phe Asn Pro Thr Thr Thr Ala Thr Gly Asn
85 90 95Leu Arg Pro Asp Ile Ser Asp Ala Thr Lys Leu Gly Phe Arg Tyr
Gly 100 105 110Ile Ala Glu Ile Val Glu Leu Glu Arg Arg Gly Asp Lys
Trp His Val 115 120 125Cys Ala Ala Ser Cys Cys Leu Asp Arg Ala Ser
Val Ala Ser Ala Val 130 135 140Lys Ala Pro Pro Val Thr Ala Asn Gly
Ile Pro Ile Gly Thr Phe Ser145 150 155 160Pro Pro Gln Thr Tyr Ser
Leu Ser Leu Cys Ser Phe Asp Ser Val Cys 165 170 175Met Ser Ser Asn
Leu Cys Asn Pro Ala Lys Thr Leu Ser Val Cys Ser 180 185 190Gln Glu
Ala Val Glu Leu Leu Glu Glu Thr Val Asp Lys Ala Gln Val 195 200
205Met Met Cys Gln Asn Leu Glu Ala Arg Arg Arg Ala Glu Tyr Asp Ala
210 215 220Trp Gln Val Arg Gln Ala Val Gly Asp Glu Tyr Thr Arg Leu
Ala Asp225 230 235 240Glu Asp Val Asp Ala Thr Thr Ser Val Lys Pro
Pro Val Ala Arg Ala 245 250 255Ala Val Gly Ser Ser Thr Leu Asp Asp
Val Ser Val Leu Thr Val Leu 260 265 270Arg Glu Leu Gly Asp Gln Cys
Gln Asn Ala Ile Lys Phe Val Val Gln 275 280 285Ala Ala Ser Arg Phe
Val Pro Pro Val Pro Lys Pro Arg Thr Arg Val 290 295 300Ser Gly Val
Leu Glu Arg Val Arg Met Cys Met Arg Thr Pro Pro Ile305 310 315
320Lys Phe Glu Ala Thr Ala Val Pro Ile His Asn Ile Ile Pro Glu Val
325 330 335Cys His Ile Val Leu Arg Cys Thr Gly Cys Asn Asp Gln Ala
Leu Thr 340 345 350Val Pro Tyr Gly Thr Cys Thr Gln Thr Leu Ile Lys
His Leu Thr Asn 355 360 365Lys His Ser His Tyr Ile Pro Lys Gln Lys
Ile Glu Glu Asp Thr Glu 370 375 380Val Thr Val Ile Cys Ala Val Pro
Thr Thr Arg Ala Ser Lys Leu Ile385 390 395 400Thr Phe Arg Ala Gly
Asp Arg Ser Val Ser Cys Cys His Pro Leu Gln 405 410 415Thr Pro Val
Arg Thr Leu Leu Leu Lys Tyr Gly Leu Pro Ile Gly Lys 420 425 430Trp
Ser Asp Cys Asn Gly Pro Leu Gly Asp Asp Ala Arg Val Cys Asp 435 440
445Ile Asn Gly Val Thr Thr Tyr Glu Pro Cys 450 455343567PRTHuman
Pegivirus 2MISC_FEATURE(89)..(89)Xaa = any amino
acidMISC_FEATURE(495)..(495)Xaa = any amino
acidMISC_FEATURE(539)..(539)Xaa = any amino acid 343Met Gln Ser Tyr
Ser Trp Phe Arg Pro Ile Val Ala Pro Thr Thr Pro1 5 10 15Pro Leu Pro
Ala Thr Arg Ser Val Ala Gly Ile Leu Arg Ala Asp Thr 20 25 30Ser Arg
Val Tyr Thr Thr Thr Ala Val Asp Val Ser Glu Arg Gln Ala 35 40 45Lys
Val Thr Ile Asp Gln Thr Ser Ala Lys Val Asp Gln Cys Phe Arg 50 55
60Asp Thr Tyr Asn Cys Cys Leu Ala Lys Ala Lys Thr Phe Lys Gln Ser65
70 75 80Gly Met Ser Tyr Glu Asp Ala Val Xaa Lys Met Arg Ala Asn Thr
Thr 85 90 95Arg Asp His Asn Asn Gly Thr Thr Tyr Ser Asp Leu Val Ser
Gly Arg 100 105 110Ala Lys Pro Val Val Gln Lys Ile Val Asp Gln Met
Arg Ala Gly Val 115 120 125Tyr Asp Ala Pro Met Arg Ile Ile Pro Lys
Pro Glu Val Phe Pro Arg 130 135 140Asp Lys Ser Thr Arg Lys Pro Pro
Arg Phe Ile Val Phe Pro Gly Cys145 150 155 160Ala Ala Arg Val Ala
Glu Lys Met Ile Leu Gly Asp Pro Gly Ala Ile 165 170 175Thr Lys His
Val Leu Gly Asp Ala Tyr Gly Phe Ala Thr Pro Pro His 180 185 190Glu
Arg Ala Arg Leu Leu Glu Gln Trp Trp Asn Arg Ala Thr Glu Pro 195 200
205Gln Ala Ile Ala Val Asp Ala Ile Cys Phe Asp Ser Thr Ile Thr Ala
210 215 220Glu Asp Met Asp Arg Glu Ala Asn Ile Met Ala Ala Ala His
Ser Asp225 230 235 240Pro Glu Gly Val His Gly Leu Tyr Lys Tyr Tyr
Lys Arg Ser Pro Met 245 250 255Cys Asp Ile Thr Gly Lys Val Val Gly
Val Arg Cys Cys Arg Ala Ser 260 265 270Gly Thr Leu Thr Thr Ser Ser
Gly Asn Thr Leu Thr Cys Tyr Leu Lys 275 280 285Val Arg Ala Ala Cys
Thr Arg Ala Gly Ile Lys Pro Ile Gly Leu Leu 290 295 300Ile His Gly
Asp Asp Thr Leu Ile Val Thr Glu Arg Cys Ala Gln Glu305 310 315
320Thr Leu Asp Glu Phe Ser Ser Ala Leu Asp Asp Tyr Gly Phe Pro His
325 330 335Thr Leu Gln Val Ser Gly Asp Leu Ser Ser Val Glu Cys Cys
Ser Ala 340 345 350Arg Val Asp Ser Val Cys Leu Arg Gly Gly Met Arg
Arg Met Leu Val 355 360 365Pro Gln Ala Arg Arg Ala Ile Ala Arg Val
Leu Gly Glu Lys Gly Asp 370 375 380Pro Leu Gly Val Ile Ser Ser Tyr
Ile Val Met Tyr Pro Thr Ala Ala385 390 395 400Val Thr Val Tyr Val
Leu Leu Pro Leu Leu Cys Met Leu Ile Arg Asn 405 410 415Glu Pro Ser
Gln Thr Gly Thr Ile Val Thr Leu Thr Val His Gly Asn 420 425 430Ser
Val Ser Val Pro Val Trp Leu Leu Pro Thr Ile Ile Val Asn Leu 435 440
445His Gly Arg Asp Ala Leu Gln Val Val Arg His Ser Ala Ala Ser Met
450 455 460Ala Glu Leu Ser Ser Ala Leu Ala Phe Phe Gly Met Arg Gly
Leu Asn465 470 475 480Cys Trp Arg Arg Arg Arg Arg Ala Ile Arg Thr
Asp Met Ile Xaa Leu 485 490 495Gly Gly Trp Ile Ala Asn Phe Ala Gln
Met Leu Leu Trp Ser Pro Glu 500 505 510Val Arg Thr Pro Gln Pro Glu
Pro Lys Gly Leu Cys Leu Leu Pro Pro 515 520 525Glu Leu Trp Glu Arg
Pro Tyr Glu Asn Leu Xaa Leu Ser Thr Val Asp 530 535 540Arg Asn Arg
Gly Ala Ser Arg Leu Arg Phe Trp Leu Val Ala Ser Ala545 550 555
560Ile Leu Ala Leu Leu Cys Leu 56534479PRTHuman Pegivirus 2 344Met
Gly Cys Ser Thr Asp Gln Thr Ile Cys Ser Pro Val Val Gly Ala1 5 10
15Asp Tyr Asn Thr Ser Ser Gly Cys Arg Ala Leu Asn Gly Ser Tyr His
20 25 30Cys Gly Gly Gly Ser Cys Arg Ser Pro Ser Arg Val Gln Val Ala
Arg 35 40 45Arg Val Leu Gln Leu Cys Ala Phe Leu Ala Leu Ile Gly Ser
Gly Met 50 55 60Cys Ser Ile Arg Ser Lys Thr Glu Gly Arg Ile Glu Ser
Gly Gln65 70 75345191PRTHuman Pegivirus 2 345Ile Leu Gln Ser Gln
Arg Ala Cys Trp Thr Gly Glu Gly Phe Ala Phe1 5 10 15Phe Ser Asn Cys
Cys Asn Gln Ser Asp Ile Met Trp Cys Leu His Arg 20 25 30Trp Cys Val
Thr Arg Pro Gly Cys Leu Val Cys Thr Gly Asn Ala Thr 35 40 45His Pro
Ile Cys Trp Asp Tyr Leu Gly Ser Gly Val Ser Arg Arg Pro 50 55 60Ala
Arg Arg Met Gly Glu Gly Ala Glu Ala Leu Leu Arg Leu Ile Gly65 70 75
80Ile Ala Gly Trp Leu Gly Leu Leu Ala Glu Ser Leu Gly Met Ser Glu
85 90 95Val Tyr Ala Ala Ile Leu Cys Phe Gly Phe Ile Ala Trp Tyr Gly
Trp 100 105 110Gly Ile Pro Lys Thr Leu Val Cys Thr Val Cys Pro Ala
Val Asn Ile 115 120 125Ser Pro Tyr Ser Phe Leu Ser Pro Asp Thr Ile
Ala Phe Gly Thr Trp 130 135 140Ile Leu Gln Leu Pro Gly Leu Leu Trp
Gln Met Phe Val Ser Phe Pro145 150 155 160Ile Leu Tyr Ser Thr Trp
Ile Leu Trp Leu Leu Leu Ser Gly Lys Thr 165 170 175Val Ala Val Ile
Ala Ile Leu Leu Ala Ser Pro Thr Val Met Ala 180 185
190346354PRTHuman Pegivirus 2 346Tyr Lys His Gln Ser Glu Ser Tyr
Leu Lys Tyr Cys Thr Ile Thr Asn1 5 10 15Thr Ser Thr Ser Met Asn Cys
Asp Cys Pro Phe Gly Thr Phe Thr Arg 20 25 30Asn Thr Glu Ser Arg Phe
Ser Ile Pro Arg Phe Cys Pro Val Lys Ile 35 40 45Asn Ser Ser Thr Phe
Ile Cys Ser Trp Gly Ser Trp Trp Trp Phe Ala 50 55 60Glu Asn Ile Thr
Arg Pro Tyr Thr Asp Val Gly Met Pro Pro Ala Pro65 70 75 80Ile Ser
Ala Leu Cys Tyr Ile Tyr Ser Asn Asn Asp Pro Pro Pro Trp 85 90 95Tyr
His Asn Thr Thr Ile Ile Pro Gln Asn Cys Arg Asn Ser Thr Val 100 105
110Asp Pro Thr Thr Ala Pro Cys Arg Asp Lys Trp Gly Asn Ala Thr Ala
115 120 125Cys Ile Leu Asp Arg Arg Ser Arg Phe Cys Gly Asp Cys Tyr
Gly Gly 130 135 140Cys Phe Tyr Thr Asn Gly Ser His Asp Arg Ser Trp
Asp Arg Cys Gly145
150 155 160Ile Gly Tyr Arg Asp Gly Leu Ile Glu Phe Val Gln Leu Gly
Gln Ile 165 170 175Arg Pro Asn Ile Ser Asn Thr Thr Ile Glu Leu Leu
Ala Gly Ala Ser 180 185 190Leu Val Ile Ala Ser Gly Leu Arg Pro Gly
Phe Gly Cys Ser Arg Ala 195 200 205His Gly Val Val His Cys Tyr Arg
Cys Pro Ser Tyr Arg Asp Leu Glu 210 215 220Gln Phe Gly Pro Gly Leu
Gly Lys Trp Val Pro Leu Pro Gly Glu Pro225 230 235 240Val Pro Glu
Leu Cys Ile Asn Pro Gln Trp Ala Arg Arg Gly Phe Arg 245 250 255Met
Ser Asn Asn Pro Leu Ser Leu Leu Gln Thr Phe Val Glu Asp Ile 260 265
270Phe Leu Ala Pro Phe Cys Asn Pro Thr Pro Gly Arg Val Arg Val Cys
275 280 285Asn Asn Thr Ala Phe Tyr Pro Arg Gly Gly Gly Phe Val Gln
Leu Ile 290 295 300Gly Asp Val Gln Val Leu Thr Pro Asn Thr Ala Ser
Leu His Ser Leu305 310 315 320Leu Thr Leu Ile Ser Leu Ile Leu Leu
Val Cys Val Val Ser Gly Ala 325 330 335Arg Phe Val Pro Leu Ile Ile
Ile Phe Phe Trp Ser Ala Arg His Val 340 345 350Tyr
Ala347237PRTHuman Pegivirus 2 347Ser Cys Tyr Leu Ser Cys Asp Trp
Ala Val Cys Asn Asp Ala Phe Cys1 5 10 15Phe Thr Ser Gly Thr Cys Ala
Thr Phe Asn Asp Val Leu Cys Leu Pro 20 25 30Val Ala Thr Arg Ile Ser
Ser Cys Gly His Ala Val Pro Pro Pro Asp 35 40 45Arg Gly Trp Glu Val
Pro Ala Ala Met Ser Trp Val Ile Ser Arg Thr 50 55 60Thr Gly Leu Thr
Phe Asp Val Phe Ser Phe Ile Gln Tyr Leu Pro Thr65 70 75 80Val Pro
Gly Asn Asn Thr Asn Ile Ile Tyr Cys Gly Glu Pro Thr Phe 85 90 95Leu
Gly Asp Ile Thr Gly Ile Tyr Trp Pro Tyr Phe Leu Pro Gly Ala 100 105
110Ile Leu Leu Tyr Leu Thr Pro Phe Leu Gly Leu Arg Leu Met Leu Ala
115 120 125Gly Phe Asn Ile Asp Gly Leu Phe Pro Ile Arg His Ala Thr
Ala Ala 130 135 140Leu Arg Phe Ser Thr Ser Arg Val Thr Leu Cys Val
Val Val Ala Phe145 150 155 160Leu Ile Tyr Ile Leu Ser His Pro Val
Asn Ala Ala Leu Asn Arg Met 165 170 175Phe Leu Ala Ser Ala Asn Leu
Glu Met Ile Leu Ser Phe Asp Thr Tyr 180 185 190His Glu Thr Val Leu
Tyr Ile Leu Cys Leu Leu Leu Tyr Leu Gln Val 195 200 205Ser Pro Arg
Ala Gly Leu Ala Ala Met Val Ala Ile Lys Leu Ser Arg 210 215 220Gly
Leu Leu Phe Ala Val Val Leu Ala His Gly Val Cys225 230
235348240PRTHuman Pegivirus 2 348Arg Pro Gly Arg Val Phe Gly Leu
Glu Val Cys Ala Asp Ile Ser Trp1 5 10 15Leu Val Glu Phe Thr Gly Asn
Cys Thr Trp Tyr Met Ser Cys Val Phe 20 25 30Ser Phe Trp Cys Ala Val
Phe Ala Phe Thr Ser Pro Leu Gly Arg His 35 40 45Tyr Lys Ile Gln Ile
Tyr Arg Tyr Trp Ala Gln Val Tyr Ala Arg Leu 50 55 60Val Leu Ala Val
Gly Cys Gly Pro Leu Gly Arg Glu Phe His Phe Arg65 70 75 80Ala Ser
Val Gly Val Leu Trp Cys Gly Ala Cys Met Leu Trp Pro Arg 85 90 95Glu
Cys Ser Glu Ile Ser Leu Val Phe Ile Leu Cys Ala Leu Thr Val 100 105
110Asp Thr Ile Asp Thr Trp Leu Val Ala Cys Leu Ser Ala Gly Pro Ser
115 120 125Ala Arg Thr Leu Ala Ile Leu Ala Asp Asp Met Ala Arg Ile
Gly Asp 130 135 140His Arg Ala Leu Arg Ala Val Leu Arg Cys Phe Gly
Ser Arg Gly Thr145 150 155 160Tyr Ile Tyr Asn His Met Gly Gln Val
Ser Glu Arg Val Ala Gln Ala 165 170 175Val Arg Asp Leu Gly Gly Cys
Leu Glu Pro Val Val Leu Glu Glu Pro 180 185 190Thr Phe Thr Glu Ile
Val Asp Asp Thr Met Ser Leu Val Cys Gly Gln 195 200 205Leu Leu Gly
Gly Lys Pro Val Val Ala Arg Cys Gly Thr Arg Val Leu 210 215 220Val
Gly His Leu Asn Pro Glu Asp Leu Pro Pro Gly Phe Gln Leu Ser225 230
235 240349628PRTHuman Pegivirus 2 349Ala Pro Val Val Ile Thr Arg
Pro Ser Ile Gly Thr Trp Ser Phe Leu1 5 10 15Lys Ala Thr Leu Thr Gly
Arg Ala Glu Thr Pro Gly Ser Gly Gln Ile 20 25 30Val Val Leu Ser Ser
Leu Thr Gly Arg Ser Met Gly Thr Ala Val Asn 35 40 45Gly Thr Leu Tyr
Ala Thr Gly His Gly Ala Gly Ala Arg Gly Leu Ala 50 55 60Thr Cys Ala
Gly Leu Arg Thr Pro Leu Tyr Thr Ala Leu Ser Asp Asp65 70 75 80Val
Val Ala Tyr Ser Cys Leu Pro Gly Met Ser Ser Leu Asp Pro Cys 85 90
95Cys Cys Ser Pro Ser Arg Val Trp Val Met Asn Asn Asn Gly Gly Leu
100 105 110Val Cys Gly Arg Val Glu Asn Asp Asp Val Cys Leu Asp Cys
Pro Thr 115 120 125His Ile Asp Gln Leu Arg Gly Ala Ser Gly Ser Pro
Val Leu Cys Asp 130 135 140His Gly His Ala Tyr Ala Leu Met Leu Gly
Gly Tyr Ser Thr Ser Gly145 150 155 160Ile Cys Ala Arg Val Arg Thr
Val Arg Pro Trp His Asn Ala Tyr Ser 165 170 175Ser Ser Gly Gly Gln
Gly Gly Met Gln Ala Pro Ala Val Thr Pro Thr 180 185 190Tyr Ser Glu
Ile Thr Tyr Tyr Ala Pro Thr Gly Ser Gly Lys Ser Thr 195 200 205Lys
Tyr Pro Val Asp Leu Val Lys Gln Gly His Lys Val Leu Val Leu 210 215
220Leu Pro Ser Val Ala Val Val Lys Ser Met Ala Pro Tyr Ile Lys
Glu225 230 235 240Thr Tyr Lys Ile Arg Pro Glu Ile Arg Ala Gly Thr
Gly Pro Asp Gly 245 250 255Val Thr Val Ile Thr Gly Glu Asn Leu Ala
Tyr Met Thr Tyr Gly Arg 260 265 270Phe Leu Val Asp Pro Glu Thr Asn
Leu Arg Gly Tyr Ala Val Val Ile 275 280 285Cys Asp Glu Cys His Asp
Thr Ser Ser Thr Thr Leu Leu Gly Ile Gly 290 295 300Ala Val Arg Met
Tyr Ala Glu Lys Ala Gly Val Lys Thr Val Val Phe305 310 315 320Ala
Thr Ala Thr Pro Ala Gly Ile Gln Val Gln Ser His Ser Asn Ile 325 330
335Asp Glu Tyr Leu Leu Thr Asp Thr Gly Asp Val Glu Phe Tyr Gly Ala
340 345 350Lys Ile Lys Met Asp Asn Ile Arg Thr Gly Arg His Val Ile
Phe Cys 355 360 365His Ser Lys Ala Arg Cys Ala Glu Leu Thr Gln Gln
Leu Ser Gly Leu 370 375 380Gly Ile Arg Ala Val Ser Phe Trp Arg Gly
Cys Asp Ile Lys Thr Ile385 390 395 400Pro Ala Ser Asp Ser Ile Val
Val Val Ala Thr Asp Ala Leu Ser Thr 405 410 415Gly Tyr Thr Gly Asn
Phe Asp Ser Val Ile Asp Cys Gly Cys Cys Val 420 425 430Glu Gln Thr
Val Thr Ile Asp Met Asp Pro Thr Phe Ser Ile Ser Ala 435 440 445Arg
Val Val Pro Cys Thr Ala Ala Leu Arg Met Gln Arg Arg Gly Arg 450 455
460Thr Gly Arg Gly Arg Arg Gly Ala Tyr Tyr Thr Thr Ser Pro Gly
Ala465 470 475 480Ala Pro Cys Val Ser Val Pro Asp Ala Asn Val Trp
Gln Ala Val Glu 485 490 495Ser Ala Met Val Phe Tyr Asp Trp Ser Ala
Thr Arg Ile Gln Gln Cys 500 505 510Leu Ala Ala Tyr His Asp Leu Gly
Cys Thr Pro Arg Ile Ser Cys Asp 515 520 525Pro His Thr Pro Val Arg
Val Met Asp Thr Leu Arg Ala Tyr Leu Arg 530 535 540Arg Pro Glu Val
Thr Thr Ala Ala Leu Ala Gly Glu Gln Trp Pro Leu545 550 555 560Leu
Tyr Gly Ala Gln Leu Cys Ile Cys Lys Glu Thr Glu Ala His Gly 565 570
575Pro Asp Asp Ser Ile Lys Trp Lys Cys Leu Leu Asn Asn Ser Asn Lys
580 585 590Thr Pro Leu Leu Tyr Ala Leu Asp Asn Pro Thr Leu Glu Phe
Thr Thr 595 600 605Gln His Asp Leu Thr Arg Arg Ile Ala Gly Ala Leu
Ser Ser Thr Val 610 615 620Phe Val Glu Thr62535041PRTHuman
Pegivirus 2 350Gly Tyr Gly Pro Ile Leu Leu Ala Gly Ala Ala Leu Ala
Ala Ser Phe1 5 10 15Ala Phe Ala Gly Ala Thr Gly Ala Leu Val Pro Ser
Ala Val Trp Ser 20 25 30Val Glu Val Arg Pro Ala Gly Val Thr 35
40351262PRTHuman Pegivirus 2 351Arg Pro Asp Ala Thr Asp Glu Thr Ala
Ala Tyr Ala Gln Arg Leu Tyr1 5 10 15Gln Ala Cys Ala Asp Ser Gly Ile
Phe Ala Ser Leu Gln Gly Thr Ala 20 25 30Ser Ala Ala Leu Gly Lys Leu
Ala Asp Ala Ser Arg Gly Ala Ser Gln 35 40 45Tyr Leu Ala Ala Ala Pro
Pro Ser Pro Ala Pro Leu Val Gln Val Leu 50 55 60Gln Phe Leu Glu Thr
Asn Phe Ser Ser Ile Ala Ser Phe Gly Leu Leu65 70 75 80Cys Ala Gly
Cys Gln Ala Gly Glu Cys Phe Thr Ala Leu Ala Gly Leu 85 90 95Val Ser
Gly Ala Thr Ala Gly Leu Gly Gly Ala His Lys Trp Leu Leu 100 105
110Ala Ile Ala Gly Thr Trp Leu Val Ser Leu Gln Thr Gly Ser Arg Gly
115 120 125Gly Met Val Ala Gly Leu Ser Ile Leu Ala Gly Cys Cys Ile
Gly Ser 130 135 140Val Thr Gly Leu Asp Phe Leu Phe Gly Cys Leu Thr
Gly Trp Glu Ala145 150 155 160Val Val Gly Ala Ala Val Ala Thr Gln
Lys Ile Leu Ser Gly Ser Ala 165 170 175Asp Met Thr Thr Leu Val Asp
Leu Leu Pro Ala Leu Phe Ser Pro Gly 180 185 190Ala Gly Ile Ala Gly
Ile Val Leu Val Phe Ile Leu Ser Asn Ser Ser 195 200 205Val Thr Thr
Trp Ala Asn Arg Leu Leu Ser Met Cys Ala Lys Gln Thr 210 215 220Ile
Cys Glu Asn Tyr Phe Leu Ser Glu Arg Phe Gly Gln Gln Leu Ser225 230
235 240Lys Leu Ser Leu Trp Arg Ser Val Tyr His Trp Ala Gln Ala Arg
Glu 245 250 255Gly Tyr Thr Gln Cys Gly 260352458PRTHuman Pegivirus
2 352Val Ile Ser Gly Ile Trp Ser Phe Ala Leu Cys Ile Leu Arg Ala
Val1 5 10 15Trp Asp Trp Ala Ala Lys His Val Pro Arg Phe Arg Val Pro
Met Ile 20 25 30Gly Cys Ser Pro Ala Trp Cys Gly Arg Trp Leu Gly Thr
Gly Thr Leu 35 40 45Leu Thr Thr Cys Ala Cys Gly Glu Arg Val Ser Leu
Gln Cys Leu Cys 50 55 60Ser Thr Ser Asp Pro Gln Leu Ser Val Gly Arg
Trp Cys Arg Cys Ser65 70 75 80Trp Ser Val Gly Phe Pro Phe Asn Pro
Thr Thr Thr Gly Thr Gly Thr 85 90 95Leu Arg Pro Asp Ile Ser Asp Ala
Asn Lys Leu Gly Phe Arg Tyr Gly 100 105 110Val Ala Asp Ile Val Glu
Leu Glu Arg Arg Gly Asp Lys Trp His Val 115 120 125Cys Ala Ala Ser
Cys Cys Leu Asp Arg Ala Ser Val Ala Ser Ala Val 130 135 140Lys Ala
Pro Pro Val Thr Ala Asn Gly Ile Pro Ile Asn Ser Phe Ser145 150 155
160Pro Pro Gln Thr Tyr Cys Leu Ser Leu Cys Ser Phe Asp Thr Val Cys
165 170 175Met Ser Thr Asn Leu Cys Asn Pro Ala Lys Thr Leu Ser Val
Cys Gln 180 185 190Glu Glu Ala Val Glu Leu Leu Glu Glu Thr Val Asp
Thr Ala Gln Val 195 200 205Val Met Ser Gln Asn Leu Ala Ala Arg Arg
Arg Ala Glu Tyr Asp Ala 210 215 220Trp Gln Val Arg Gln Ala Val Gly
Asp Glu Tyr Thr Arg Leu Ala Asp225 230 235 240Glu Asp Val Asp Met
Thr Ala Ser Val Lys Pro Pro Val Ala Arg Ala 245 250 255Ala Val Gly
Ser Ser Thr Leu Asp Asp Val Ser Val Leu Thr Val Leu 260 265 270Arg
Glu Leu Gly Asp Gln Cys Gln Asn Ala Ile Lys Phe Val Val Gln 275 280
285Ala Ala Ser Arg Phe Val Pro Pro Val Pro Lys Pro Arg Thr Arg Val
290 295 300Ser Gly Val Leu Glu Arg Val Arg Met Cys Met Arg Thr Pro
Pro Ile305 310 315 320Lys Phe Glu Ala Thr Ala Val Pro Ile His Asn
Ile Ile Pro Glu Glu 325 330 335Cys His Ile Val Leu Arg Cys Thr Gly
Cys Cys Asp Gln Ala Leu Thr 340 345 350Val Pro Tyr Gly Thr Cys Ser
Leu Thr Leu Thr Lys Tyr Leu Thr Asn 355 360 365Lys His Ser His Tyr
Ile Pro Lys Glu Lys Ile Glu Glu Asp Thr Glu 370 375 380Ile Ala Val
Ile Cys Ala Val Pro Thr Lys Arg Ala Ser Lys Leu Ile385 390 395
400Thr Phe Arg Ala Gly Asp Arg Ser Val Ser Cys Cys His Pro Leu Gln
405 410 415Thr Pro Ile Arg Ala Leu Leu Gln Lys Tyr Gly Leu Pro Ile
Gly Lys 420 425 430Trp Ser Asp Cys Asn Gly Pro Leu Gly Asp Asp Ala
Arg Val Cys Asp 435 440 445Val Asn Gly Val Thr Thr Tyr Glu Pro Cys
450 455353567PRTHuman Pegivirus 2 353Met Gln Ser Tyr Asn Trp Phe
Arg Ser Ile Val Ala Pro Thr Thr Pro1 5 10 15Pro Leu Pro Ala Thr Arg
Ser Val Ala Gly Ile Leu Arg Ala Asp Thr 20 25 30Ser Arg Val Tyr Thr
Thr Thr Ala Val Asp Val Ser Glu Arg Gln Ala 35 40 45Lys Val Thr Ile
Asp Gln Lys Ser Ala Lys Val Asp Gln Cys Leu Arg 50 55 60Asp Thr Tyr
Asn Cys Cys Leu Ala Lys Ala Lys Thr Phe Arg Gln Ser65 70 75 80Gly
Met Ser Tyr Glu Asp Ala Val Ser Lys Met Arg Ala Asn Thr Thr 85 90
95Arg Asp His Asn Asn Gly Ile Thr Tyr Thr Asp Leu Val Ser Gly Arg
100 105 110Ala Lys Pro Val Val Gln Lys Ile Val Asp Gln Met Arg Ala
Gly Val 115 120 125Tyr Asp Ala Pro Met Arg Ile Ile Pro Lys Pro Glu
Val Phe Pro Arg 130 135 140Asp Lys Ser Thr Arg Lys Pro Pro Arg Phe
Ile Val Phe Pro Gly Cys145 150 155 160Ala Ala Arg Val Ala Glu Lys
Met Ile Leu Gly Asp Pro Gly Ala Ile 165 170 175Thr Lys His Val Leu
Gly Asp Ala Tyr Gly Phe Ala Thr Pro Pro His 180 185 190Glu Arg Ala
Arg Leu Leu Glu Gln Trp Trp Asn Arg Ala Thr Glu Pro 195 200 205Gln
Ala Ile Ala Val Asp Ala Val Cys Phe Asp Ser Thr Ile Thr Ala 210 215
220Glu Asp Met Asp Arg Glu Ala Asn Ile Val Ala Ala Ala His Thr
Asp225 230 235 240Pro Glu Gly Val His Gly Leu Tyr Asn Tyr Tyr Lys
Arg Ser Pro Met 245 250 255Cys Asp Ile Thr Gly Lys Val Val Gly Val
Arg Ser Cys Arg Ala Ser 260 265 270Gly Thr Leu Thr Thr Ser Ser Gly
Asn Thr Leu Thr Cys Tyr Leu Lys 275 280 285Val Arg Ala Ala Cys Thr
Arg Ala Gly Ile Lys Pro Ile Gly Leu Leu 290 295 300Ile His Gly Asp
Asp Thr Leu Ile Ile Thr Glu Arg Cys Ala Gln Glu305 310 315 320Thr
Leu Asp Glu Phe Ser Asn Ala Leu Asp Asp Tyr Gly Phe Thr His 325 330
335Thr Met Gln Val Ser Gly Asp Leu Ser Ser Ile Glu Cys Cys Ser Ala
340 345 350Arg Val Asp Ser Val Cys Leu Arg Gly Gly Met Arg Arg Met
Leu Val 355 360 365Pro Gln Ala Arg Arg Ala Ile Ala Arg Val Leu Gly
Glu Lys Gly Asp 370 375 380Pro Leu Gly Val Ile Ser Ser Tyr Ile Val
Met Tyr
Pro Thr Ala Ala385 390 395 400Val Thr Val Tyr Val Leu Met Pro Leu
Leu Cys Met Leu Ile Arg Asn 405 410 415Glu Pro Ser Gln Thr Gly Thr
Leu Val Thr Leu Thr Val His Gly Asn 420 425 430Ser Val Ser Val Pro
Val Trp Leu Leu Pro Thr Ile Ile Ala Asn Leu 435 440 445His Gly Arg
Asp Ala Leu Gln Val Val Arg His Ser Ala Ala Ser Met 450 455 460Ala
Glu Leu Ser Ser Ala Leu Ala Phe Phe Gly Met Arg Gly Leu Asn465 470
475 480Cys Trp Arg Arg Arg Arg Arg Ala Ile Arg Thr Asp Met Ile Lys
Leu 485 490 495Gly Gly Trp Asn Ala Asn Phe Ala Gln Met Leu Leu Trp
Ser Pro Glu 500 505 510Val Arg Thr Pro Gln Pro Glu Pro Lys Gly Met
Cys Leu Leu Pro Pro 515 520 525Glu Leu Trp Glu Arg Pro Tyr Glu Asn
Leu His Leu Ser Thr Ile Asp 530 535 540Arg Asn Arg Gly Ala Ser Arg
Leu Arg Phe Trp Leu Val Ala Ser Ala545 550 555 560Ile Leu Ala Leu
Leu Cys Leu 5653549718DNAHuman Pegivirus 2misc_feature(16)..(19)N =
A, C, T or Gmisc_feature(35)..(135)N = A, C, T or
Gmisc_feature(157)..(264)N = A, C, T or Gmisc_feature(280)..(319)N
= A, C, T or Gmisc_feature(335)..(340)N = A, C, T or
Gmisc_feature(359)..(460)N = A, C, T or Gmisc_feature(483)..(516)N
= A, C, T or Gmisc_feature(559)..(625)N = A, C, T or
Gmisc_feature(646)..(671)N = A, C, T or
Gmisc_feature(1336)..(1577)N = A, C, T or
Gmisc_feature(1605)..(1629)N = A, C, T or
Gmisc_feature(1790)..(1815)N = A, C, T or
Gmisc_feature(1996)..(2001)N = A, C, T or
Gmisc_feature(2067)..(2103)N = A, C, T or
Gmisc_feature(2633)..(2641)N = A, C, T or
Gmisc_feature(2669)..(2670)N = A, C, T or
Gmisc_feature(2706)..(2716)N = A, C, T or
Gmisc_feature(2733)..(2907)N = A, C, T or
Gmisc_feature(2927)..(2965)N = A, C, T or
Gmisc_feature(2981)..(3059)N = A, C, T or
Gmisc_feature(3171)..(3386)N = A, C, T or
Gmisc_feature(3402)..(3425)N = A, C, T or
Gmisc_feature(3450)..(3524)N = A, C, T or
Gmisc_feature(3450)..(3580)N = A, C, T or
Gmisc_feature(3608)..(3679)N = A, C, T or
Gmisc_feature(3699)..(3713)N = A, C, T or
Gmisc_feature(3732)..(3813)N = A, C, T or
Gmisc_feature(3834)..(3862)N = A, C, T or
Gmisc_feature(3900)..(3967)N = A, C, T or
Gmisc_feature(3992)..(4178)N = A, C, T or
Gmisc_feature(4206)..(4240)N = A, C, T or
Gmisc_feature(4257)..(4359)N = A, C, T or
Gmisc_feature(4378)..(4386)N = A, C, T or
Gmisc_feature(4595)..(4748)N = A, C, T or
Gmisc_feature(4812)..(4890)N = A, C, T or
Gmisc_feature(4916)..(4974)N = A, C, T or
Gmisc_feature(5393)..(5411)N = A, C, T or
Gmisc_feature(5458)..(5463)N = A, C, T or
Gmisc_feature(5490)..(5535)N = A, C, T or
Gmisc_feature(5551)..(5614)N = A, C, T or
Gmisc_feature(5630)..(5786)N = A, C, T or
Gmisc_feature(5852)..(5855)N = A, C, T or
Gmisc_feature(5883)..(5884)N = A, C, T or
Gmisc_feature(6034)..(6086)N = A, C, T or
Gmisc_feature(6121)..(6121)N = A, C, T or
Gmisc_feature(6137)..(6184)N = A, C, T or
Gmisc_feature(6137)..(6327)N = A, C, T or
Gmisc_feature(6343)..(6386)N = A, C, T or
Gmisc_feature(6627)..(6637)N = A, C, T or
Gmisc_feature(6659)..(6662)N = A, C, T or
Gmisc_feature(6869)..(6903)N = A, C, T or
Gmisc_feature(6939)..(6952)N = A, C, T or
Gmisc_feature(6971)..(7009)N = A, C, T or
Gmisc_feature(7150)..(7246)N = A, C, T or
Gmisc_feature(7462)..(7470)N = A, C, T or
Gmisc_feature(7911)..(7968)N = A, C, T or
Gmisc_feature(7987)..(8114)N = A, C, T or
Gmisc_feature(8139)..(8175)N = A, C, T or
Gmisc_feature(8198)..(8386)N = A, C, T or
Gmisc_feature(8413)..(8442)N = A, C, T or
Gmisc_feature(8483)..(8487)N = A, C, T or
Gmisc_feature(8506)..(8513)N = A, C, T or
Gmisc_feature(8529)..(8732)N = A, C, T or
Gmisc_feature(8748)..(8799)N = A, C, T or
Gmisc_feature(8977)..(9017)N = A, C, T or
Gmisc_feature(9052)..(9383)N = A, C, T or
Gmisc_feature(9402)..(9476)N = A, C, T or
Gmisc_feature(9530)..(9718)N = A, C, T or G 354gtgtttgaca
atgccnnnng ggatcatgtc actgnnnnnn nnnnnnnnnn nnnnnnnnnn 60nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
120nnnnnnnnnn nnnnngcaga cagggatggg tccctgnnnn nnnnnnnnnn
nnnnnnnnnn 180nnnnnnnnnn nnnnnnnggt ggcggcgggc ccnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 240nnnnnnnnnn nnnnnnnnnn nnnnatttgt
tctccagtcn nnnnnnnnnn nnnnnnnnnn 300nnnnnnnnnn nnnnnnnnnt
aaatgggagc taccnnnnnn gtggtggctc ttgcctgtnn 360nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
420nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn gcattgagtc
agggcaaact 480atnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnntttt
ctccttcatt tcaactttgg 540tgaatctgat attatgtgnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 600nnnnnnnnnn nnnnnnnnnn
nnnnnatctg ctgggactat cttggnnnnn nnnnnnnnnn 660nnnnnnnnnn
ngttgaatgg gtgagggagg aagcaatgtc ttcgcttgat cggcattgca
720ggttggcttg gactgtgtga gtctcttggt atgtctgaga tctacgcagc
tattctttgc 780tttggattta ttgcttggta tggctggggt atacctaaaa
cattggtgtg cacagtctgc 840cctgcagtga acatttcccc ctatagcttc
ttatctccag atactatcgc atttggtacg 900tggatactac aactacctga
tcttttgtgg caaatgattg tcagattccc tatactttac 960agtacttgga
ttctttggtt gttgctcagc ggcaagactg ttgctgtgat agcgatcctt
1020ttggctagtc ctacggttat ggcatacaac tcaatctgaa agctaacctc
aaatatgaac 1080cataaccaat actatcaact gctatgaact gtgactgccc
ctttggaact ttcactcgca 1140atactgagtc tggtttctca atatccatag
attctgtcct gttaaaatca atagctctca 1200tttatttgtt catgggggtc
gtggtggtgg tttgctgcac agatcacccg tccatactac 1260gggacgttct
caccccgcca gcaccgattt ccgctttgtg ctatatctat tcaagcaatg
1320acccaccttc ttggtnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 1380nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 1440nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nttctatact aatggtagtn 1500nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1560nnnnnnnnnn
nnnnnnncct aacatctcga atactacctt tcgannnnnn nnnnnnnnnn
1620nnnnnnnnna tcgcatccgg tcttcgggct gggtatggtt gcagccgagc
gcacggcgtg 1680gtgcactgct ttaagtgtcc ttcataccgt gacctgtgaa
tccatttggt cctgggcttg 1740ggaaatgggt gccattgcct ggcgagcctg
tcccggagtt gtgtattaan nnnnnnnnnn 1800nnnnnnnnnn nnnnnggtgt
ctaacaatgc tttcagcttg ttgcagacct tcgttgagga 1860cattttccta
gcaccctttt gcaacccgac gcctgggcgt gtacgtgtgt gtaacaacac
1920tgctttctat ccgagaggag gcggttttgt gcagctcatc ggagacgtcc
aggtgctaac 1980ccctaacact gcatcnnnnn nctctgtgct gactttgata
tcgtctaact cttgtgtggt 2040gtgtgttgtt tgtgtgtgcg cattgtnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2100nnngtatatt cttttttttt
attaatttgt tattgggctg tttgcaacga tgcgttctgt 2160ttcacatctg
gcacttgtgc tacctcaatg acgtcttgtg tctgccggtt gcgacgcgta
2220tatcgtcctg tggccatgct gtgccacctc ccgaccgtgg ttgggaggtg
cctgcggcaa 2280tgtcatgggc gatttcgcgg actactggtt tgacgttcga
tgtcttttcc tttgtccagt 2340atcttcctac tgtgcctggc aacaataccg
atatcattta ctgtggtgaa ccaaccttct 2400ttggggacat cacgggcatc
tattggcctt acttcttgcc tggcgtgcta ctgttgtact 2460tgactccttt
cttgggctta aggttaatgc ttgctggctt caatatagat ggcttgtttc
2520ccatacggca tgccacggct gcactgaggt tctcgacttc acgtgtgacc
ttgtgtgtcg 2580tactcgcctt cctaatctat atattatctc atcctgtcaa
tgctgcactc aannnnnnnn 2640ncttgagcat ctgcaaattt agagatgann
ttatcttttg ataccttata tgagactgtt 2700cttatnnnnn nnnnnnattg
ctctacctcc agnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2760nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
2820nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 2880nnnnnnnnnn nnnnnnnnnn nnnnnnncca tgtgtcttct
ctttttnnnn nnnnnnnnnn 2940nnnnnnnnnn nnnnnnnnnn nnnnnactat
aagattcaga nnnnnnnnnn nnnnnnnnnn 3000nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnc 3060cgctgaagtg
tgggcgtgct tggagagtag cttgcatgct ctggcacgtg agtgctctga
3120aatcagcctg tgtcttcatt ctgtgtgctc tgacatagac accatagaca
catnnnnnnn 3180nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 3240nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn caccaggcac 3300atacatatac acaccacatg
ggccaagnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3360nnnnnnnnnn
nnnnnnnnnn nnnnnnttgg aggagcccac cnnnnnnnnn nnnnnnnnnn
3420nnnnntgtgt ttgagtgtgt ggacaattgn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 3480nnnnnnnnnn nnnnnnnnnn nnnntcaacc ctgaagatcn
nnnncctggt ttccagctgn 3540nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn gtccttcctt aaggctgaca 3600tctaacannn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3660nnnnnnnnnn
nnnnnnnnng cagtgaatgg cacacatgnn nnnnnnnnnn nnnggcgccg
3720gcgcgcgcgg cnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 3780nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnntgagttc
cctagacccc tgcnnnnnnn 3840nnnnnnnnnn nnnnnnnnnn nngaataaga
cagtgagaat tggtgtgtgg cagagggagn 3900nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3960nnnnnnnttt
gtgtgatcac ggctcactgc annnnnnnnn nnnnnnnnnn nnnnnnnnnn
4020nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 4080nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 4140nnnnnnnnnn nctggttctg gtaagtcaac
aaannnnnca gtggacctag tcaaacaggg 4200acacannnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn aaaagtatgg ccccttnnnn 4260nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnngctggc acaggtcctn nnnnnnnnnn
4320nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnc cgcttccttg
tggagccnnn 4380nnnnnntcgg cgggggttcg ctgtagtcat ttgcgtcgag
tgccatgaac acatcactcc 4440accacgcgac tcggcggcgg ctcagtgcgc
atgtatggag aaagctggag tgaagtccgt 4500tgtatttgcg acagacactt
ctgctggcat tcaagtacag tcacattctc aacattgatg 4560aatatctatt
gactgataca ggcgacgtgg aattnnnnnn nnnnnnnnnn nnnnnnnnnn
4620nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 4680nnnagcagct ctccggcctt ggacagtcgt gcagtgagtt
ttnnnnnnnn nnnnnnnnnn 4740nnnnnnnntc ccgcctcaga cacacatgtt
gtggtggcaa ctgcatgaat ttcaccgcta 4800cacaggaaac tnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4860nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn ccaagatggt gccatgtact gcagcnnnnn
4920nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnctactc 4980caggagcagc accctgcgtc ggcgttcccg acgccaacgt
ctggcaagca gtggagtcag 5040ccatggtctt ttatgattgg agtgctgcca
ggatacagca gtgtctggcg gcataccatg 5100atttagggtg tacaccacgc
atcagctgtg acccacacac tccagtgcgg gtgatggaca 5160cactgagggc
gtatctgcgc aggcccgagg tgacgactgc agctctcgca ggagagcagt
5220ggccgctgct ttacggtgtg cagctgtgca tctgcaaaga gaccgaggcc
cacggtccag 5280acgatggtat caagtggaag tgcttactca acaacagtaa
taaaacaccc ctgttgtacg 5340ccttagacaa tcctcacact ggaattcaca
ctcaacatga cttgactcga ccnnnnnnnn 5400nnnnnnnnnn ngagagcagt
gttcgtggag agaggcaacg gccccatcct ccttgctnnn 5460nnngctttgg
ctgcctccat cgcctttgcn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
5520nnnnnnnnnn nnnnngtcag gcctgctggc nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 5580nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnncagatt
caggaatttn nnnnnnnnnn 5640nnnnnnnnnn nnnnnnnggt gctgggcaaa
ctggcnnnnn nnnnnnnnnn nnnnnnnnnn 5700nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5760nnnnnnnnnn
nnnnnnnnnn nnnnnncgga cctgctctgt caccctggct gccaggctgg
5820cgggggcatt ctggcttttc tgtagctggt gnnnngtgac tccagccctg
gcttgggggg 5880tgnncataag tggttgttag ctattgcagg aacttggcta
gttagcttgc agactgggcc 5940ccgtggcggc atggttgcgg gcctctcagt
tctagcaggc tgttgcatcg gtagtgtcac 6000cgggcttgac ttcctgtttg
ggtgccttac aggnnnnnnn nnnnnnnnnn nnnnnnnnnn 6060nnnnnnnnnn
nnnnnnnnnn nnnnnncagt agatatctac tcactctggt agatctctac
6120ntgctctttt ctccccnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 6180nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 6240nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6300nnnnnnnnnn nnnnnnnnnn
nnnnnnngtg agggatacac acnnnnnnnn nnnnnnnnnn 6360nnnnnnnnnn
nnnnnnnnnn nnnnnnctac gtgctgtgtg ggattgggcg gctcaaacat
6420gtgccacggt tccgtgtgcc tatgattggc tgctcacctg cgtggtgcgg
gcgctggctt 6480ggtactggca ccttgttgac cacctgtggg tgtggagaac
gtgtatccct tcagtgcctt 6540tgctcaacat ctgacccaca ctcagtgtgg
gccgttggtg ttggtgtagt tggagtgttg 6600ggttccattc aacacgacga
cgacagnnnn nnnnnnntta cggccggaca tcgtgacgnn 6660nncaaattgg
gtttccggtt tggtttcgcc gaattcgtgg atttagagcg ccggggaaac
6720aaatggcatg tctgtgcagc atcatgttgc ttggaccggg ccagcgttgc
atccgctgtg 6780aaggccccac cggtcacggc cgatggcata cctatcagta
ccttttctcc accacaaact 6840tattcccaat ctctctgttc ctttgaacnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6900nnnagaccct gagtgtgtgc
caagagatgc tgtctgagnn nnnnnnnnnn nnagttgaca 6960cagcacatgt
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnng tttgaagcat
7020ggcaagttcg cgaagcaatt cgcgacgagt acacgcgttt ggcagacgag
gatgttgacg 7080cgacaacgtc ggtgaaaccc ccggtggcca gggctgctgt
gggtagctcg acgttggatg 7140atgttagcgn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 7200nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnacgc acgcgtgtct 7260cgggtgtgtt
ggagcgcgtg cgcatgtgca tgcgcaccca cactcaagtt tgaggccatc
7320ccagtaccaa ttcatagtat aatcccaaaa gagtgtcaca ttgtgctacg
ctgtaccggc 7380tgtaatgacc aggccttgac tgttccgtac ggcacctgca
ctcagacttt agccaaacat 7440ctgactaaca aacacaacac tnnnnnnnnn
aaagaagata gaagaagaaa acaaataagg 7500ctgtcatttg cgctgtacca
acaaagcgcg caaccaaact catcactttc agagcaggtg 7560accgatcagt
ctcatgttgt caccccttgc aaactcctgt tagggccctg cttgaaaagt
7620acgggttacc tatcgggaag tggtccgact gcaacggccc gcttggtgac
gatgcccgag 7680tctgtgacgt caatggagta acaacttatg aaccatgcat
ggagtcctac agttggttcc 7740gaccaattgt ggcaccaaca accccacctt
tacctgcaac ccggagtgtg gctggcattt 7800tacgcgcaga cacatcgcgc
gtttacatca caacggcggt tgacgtcccc gagcggcagg 7860ctaaggtcac
aatcgatcaa acatcagcca aggtggatca gtgtttccga nnnnnnnnnn
7920nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnat
gggatgctgt 7980gtcaaannnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 8040nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 8100nnnnnnnnnn nnnntgccag
tactccaaaa cctgaagtnn nnnnnnnnnn nnnnnnnnnn 8160nnnnnnnnnn
nnnnnttctc gttttccctg ggtgtgcnnn nnnnnnnnnn nnnnnnnnnn
8220nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 8280nnnnnnnnnn nnnnnnnnnn nnnnnactgg ctaacaatgg
tggaaccnnn nnnnnnnnnn 8340nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnttac aggaaaggaa 8400gccccatgtg tgnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nngctgtcga gcctcagcgt 8460cagttacaga
ccagagagtc gcnnnnngct tacttgctac cttaannnnn nnncagcttg
8520cacgcgcgnn nnnnttagac tcaattggct tactaaatta aaggagatgg
ctccctcatc 8580gtgagagaaa gttgagctaa ggaaaaattg atgagttcag
caacagcact tgatgnnnnn 8640nnnnnnnnnn nnnnnntgtc aggtgtctgg
ggaccnnnnn nnnnnnnnnn nnnnnnnnnn 8700nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nntgcgtcgc atgctcgnnn nnnnnnnnnn 8760nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnna ctgggtacca tcggtagcta
8820tgttgtcatg tatcccactg cggccgtgac tgtctacgtg ctattgcccc
tgttgtgcat 8880gctcatacga aatgagccat cacagacggg gacacttgtg
acgctgacgg tccacggtaa 8940cagtgtgagc gtgccagtgt ggctgcttcc
aaccatnnnn nnnnnnnnnn nnnnnnnnnn 9000nnnnnnnnnn nnnnnnngtc
atagttgcag ctgactccat ggcggaactg tnnnnnnnnn 9060nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
9120nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 9180nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 9240nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9300nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9360nnnnnnnnnn
nnnnnnnnnn nnnaggagga ggtgaagtca gnnnnnnnnn nnnnnnnnnn
9420nnnnnnnnnn nnnnnnnnnn nnnnnnnnng agttgggcaa ggtannnnnn
nnnnnntgag 9480accccgcccc cccagatcta caccagagag cacgggcaag
gcagctaggn nnnnnnnnnn 9540nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9600nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9660nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnn
97183559353DNAHuman Pegivirus 2misc_feature(1)..(233)N = A, C, T,
or Gmisc_feature(249)..(484)N = A, C, T, or
Gmisc_feature(500)..(696)N = A, C, T, or
Gmisc_feature(936)..(1172)N = A, C, T, or
Gmisc_feature(1250)..(1409)N = A, C, T, or
Gmisc_feature(1250)..(1803)N = A, C, T, or
Gmisc_feature(1819)..(1956)N = A, C, T, or
Gmisc_feature(2003)..(2079)N = A, C, T, or
Gmisc_feature(2221)..(2290)N = A, C, T, or
Gmisc_feature(2319)..(3339)N = A, C, T, or
Gmisc_feature(3358)..(4492)N = A, C, T, or
Gmisc_feature(4509)..(4587)N = A, C, T, or
Gmisc_feature(4613)..(4707)N = A, C, T, or
Gmisc_feature(4723)..(4755)N = A, C, T, or
Gmisc_feature(4781)..(5066)N = A, C, T, or
Gmisc_feature(5083)..(6122)N = A, C, T, or
Gmisc_feature(6153)..(6414)N = A, C, T, or
Gmisc_feature(6440)..(6573)N = A, C, T, or
Gmisc_feature(6592)..(6593)N = A, C, T, or
Gmisc_feature(6613)..(7289)N = A, C, T, or
Gmisc_feature(7305)..(7309)N = A, C, T, or
Gmisc_feature(7340)..(7882)N = A, C, T, or
Gmisc_feature(7907)..(9353)N = A, C, T, or G 355nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
120nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 180nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnatttgtt 240ctccagtcnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 300nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 360nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
420nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 480nnnnttttgc tttcttttcn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 540nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 600nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
660nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnggct cgggctgtta
gctgagactc 720ttggtatgtc cgagatctat gcagctgttc tttgcttcgg
atttattgct tggtatggct 780ggggtatacc taaaacgttg gtgtgcacag
tctgccctgc agtgaacatt tctccctata 840gcttcttatc tccagatact
atcgcatttg gtacgtggat actacaacta cctggtcttt 900tgtggcaaat
gtttgtcagc tttcctatac tttacnnnnn nnnnnnnnnn nnnnnnnnnn
960nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 1020nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nntgaaccat
aaccaatact nnnnnnnnnn 1080nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1140nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnacatttat ctgttcatgg gggtcgtggt 1200ggtggtttgc
tgagaacatc acacgtccat actcggacgt tggcatgccn nnnnnnnnnn
1260nnnnnnnnnn nngctatatc tattctaann nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 1320nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 1380nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1440nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1500nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
1560nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 1620nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 1680nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1740nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1800nnntgagctt
gctacagann nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
1860nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 1920nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnacac
tgcatcttta ctatagcttt 1980gcactggact ttgaatttgc ttnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2040nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnna tatgcttcct gttatttaag 2100ctgtgattgg
gctgtttgta atgatgcgtt ctgtttcacg tctggcactt gtgctacctt
2160caatgacgtc ttgtgtctgc cggttgcggc gcgcatatcg tcctgtggcc
atgctgtgcc 2220nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 2280nnnnnnnnnn ttagatgtct tttccttcaa
ttcagctann nnnnnnnnnn nnnnnnnnnn 2340nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2400nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
2460nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 2520nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 2580nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2640nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnntttg atcagtgctc 2700tacctccagg
ttcnnnnngt gcgggcttgg ccgnnnnnnn nnnnnnnnnn nnnnnnnnnn
2760nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 2820nnnnnnnnnn nnnnnnnnnn tcttggttgg tggagtnnnn
nnnnnnnnnn nnnnnnnnnn 2880nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2940nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3000nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
3060nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 3120nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 3180nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3240nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3300nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnng cggttgcttg gaacaagnnn
3360nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 3420nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 3480nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3540nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3600nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
3660nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 3720nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 3780nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3840nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3900nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
3960nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 4020nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 4080nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnntggttct ggtaagtcaa 4140nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4200nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
4260nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 4320nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 4380nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4440nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nntgctggca 4500ttcaagtann
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
4560nnnnnnnnnn nnnnnnnnnn nnnnnnnaca acatcagaac tggtaggtca
tgnnnnnnnn 4620nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 4680nnnnnnnnnn nnnnnnnnnn nnnnnnnatc
aaaaccattc ccnnnnnnnn nnnnnnnnnn 4740nnnnnnnnnn nnnnngcatt
tcaccgctac acaggaaact nnnnnnnnnn nnnnnnnnnn 4800nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
4860nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 4920nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 4980nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5040nnnnnnnnnn nnnnnnnnnn
nnnnnntgat ttggggtgca cannnnnnnn nnnnnnnnnn 5100nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
5160nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 5220nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 5280nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5340nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5400nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
5460nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 5520nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 5580nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5640nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5700nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
5760nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 5820nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 5880nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5940nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6000nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
6060nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 6120nntgtttcct taagcaattc aagtgtaacc acnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 6180nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6240nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6300nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
6360nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnngcggtg 6420gtgcgggcgc tgcgcctggn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 6480nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6540nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnncgatacg acaggcactg gnnccttacg 6600gccggacacg
tgannnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
6660nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 6720nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 6780nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6840nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6900nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
6960nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 7020nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 7080nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 7140nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 7200nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
7260nnnnnnnnnn nnnnnnnnnn nnnnnnnnna ttcataatat aatcnnnnna
gagtgtcata 7320ttgtgctacg ctgtaccggn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 7380nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 7440nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 7500nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
7560nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 7620nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 7680nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 7740nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 7800nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
7860nnnnnnnnnn nnnnnnnnnn nnttctgcct tgccaaggca gaagacnnnn
nnnnnnnnnn 7920nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 7980nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 8040nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 8100nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
8160nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 8220nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 8280nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 8340nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 8400nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
8460nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 8520nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 8580nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 8640nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 8700nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
8760nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 8820nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 8880nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 8940nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9000nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
9060nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 9120nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 9180nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9240nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9300nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnn 93533569818DNAHuman
Pegivirus 2misc_feature(35)..(135)N = A, C, T or
Gmisc_feature(155)..(195)N = A, C, T or Gmisc_feature(211)..(317)N
= A, C, T or Gmisc_feature(333)..(338)N = A, C, T or
Gmisc_feature(357)..(458)N = A, C, T or Gmisc_feature(480)..(513)N
= A, C, T or Gmisc_feature(559)..(625)N = A, C, T or
Gmisc_feature(646)..(671)N = A, C, T or Gmisc_feature(730)..(730)N
= A, C, T or Gmisc_feature(733)..(733)N = A, C, T or
Gmisc_feature(760)..(760)N = A, C, T or
Gmisc_feature(1084)..(1084)N = A, C, T or
Gmisc_feature(1165)..(1165)N = A, C, T or
Gmisc_feature(1210)..(1210)N = A, C, T or
Gmisc_feature(1243)..(1243)N = A, C, T or
Gmisc_feature(1315)..(1315)N = A, C, T or
Gmisc_feature(1338)..(1483)N = A, C, T or
Gmisc_feature(1502)..(1579)N = A, C, T or
Gmisc_feature(1606)..(1630)N = A, C, T or
Gmisc_feature(1722)..(1722)N = A, C, T or
Gmisc_feature(1789)..(1817)N = A, C, T or
Gmisc_feature(2006)..(2006)N = A, C, T or
Gmisc_feature(2065)..(2101)N = A, C, T or
Gmisc_feature(2213)..(2213)N = A, C, T or
Gmisc_feature(2335)..(2335)N = A, C, T or
Gmisc_feature(2447)..(2447)N = A, C, T or
Gmisc_feature(2449)..(2449)N = A, C, T or
Gmisc_feature(2560)..(2560)N = A, C, T or
Gmisc_feature(2582)..(2582)N = A, C, T or
Gmisc_feature(2632)..(2640)N = A, C, T or
Gmisc_feature(2667)..(2668)N = A, C, T or
Gmisc_feature(2705)..(2708)N = A, C, T or
Gmisc_feature(2737)..(2741)N = A, C, T or
Gmisc_feature(2757)..(2863)N = A, C, T or
Gmisc_feature(2880)..(2906)N = A, C, T or
Gmisc_feature(2925)..(2963)N = A, C, T or
Gmisc_feature(2979)..(2987)N = A, C, T or
Gmisc_feature(2979)..(3057)N = A, C, T or
Gmisc_feature(3079)..(3079)N = A, C, T or
Gmisc_feature(3174)..(3290)N = A, C, T or
Gmisc_feature(3295)..(3295)N = A, C, T or
Gmisc_feature(3327)..(3362)N = A, C, T or
Gmisc_feature(3381)..(3385)N = A, C, T or
Gmisc_feature(3401)..(3425)N = A, C, T or
Gmisc_feature(3449)..(3503)N = A, C, T or
Gmisc_feature(3519)..(3523)N = A, C, T or
Gmisc_feature(3539)..(3579)N = A, C, T or
Gmisc_feature(3605)..(3676)N = A, C, T or
Gmisc_feature(3695)..(3709)N = A, C, T or
Gmisc_feature(3728)..(3809)N = A, C, T or
Gmisc_feature(3830)..(3858)N = A, C, T or
Gmisc_feature(3896)..(3963)N = A, C, T or
Gmisc_feature(3986)..(4145)N = A, C, T or
Gmisc_feature(4168)..(4172)N = A, C, T or
Gmisc_feature(4200)..(4234)N = A, C, T or
Gmisc_feature(4251)..(4288)N = A, C, T or
Gmisc_feature(4304)..(4353)N = A, C, T or
Gmisc_feature(4372)..(4380)N = A, C, T or
Gmisc_feature(4390)..(4390)N = A, C, T or
Gmisc_feature(4471)..(4471)N = A, C, T or
Gmisc_feature(4588)..(4610)N = A, C, T or
Gmisc_feature(4635)..(4676)N = A, C, T or
Gmisc_feature(4715)..(4729)N = A, C, T or
Gmisc_feature(4756)..(4756)N = A, C, T or
Gmisc_feature(4782)..(4783)N = A, C, T or
Gmisc_feature(4785)..(4785)N = A, C, T or
Gmisc_feature(4791)..(4791)N = A, C, T or
Gmisc_feature(4806)..(4884)N = A, C, T or
Gmisc_feature(4909)..(4967)N = A, C, T or
Gmisc_feature(5098)..(5098)N = A, C, T or
Gmisc_feature(5362)..(5362)N = A, C, T or
Gmisc_feature(5385)..(5403)N = A, C, T or
Gmisc_feature(5450)..(5455)N = A, C, T or
Gmisc_feature(5482)..(5527)N = A, C, T or
Gmisc_feature(5543)..(5606)N = A, C, T or
Gmisc_feature(5600)..(5649)N = A, C, T or
Gmisc_feature(5668)..(5778)N = A, C, T or
Gmisc_feature(5830)..(5830)N = A, C, T or
Gmisc_feature(5840)..(5843)N = A, C, T or
Gmisc_feature(5868)..(5869)N = A, C, T or
Gmisc_feature(6019)..(6071)N = A, C, T or
Gmisc_feature(6101)..(6101)N = A, C, T or
Gmisc_feature(6105)..(6105)N = A, C, T or
Gmisc_feature(6121)..(6147)N = A, C, T or
Gmisc_feature(6157)..(6157)N = A, C, T or
Gmisc_feature(6181)..(6311)N = A, C, T or
Gmisc_feature(6327)..(6370)N = A, C, T or
Gmisc_feature(6543)..(6543)N = A, C, T or
Gmisc_feature(6619)..(6620)N = A, C, T or
Gmisc_feature(6638)..(6638)N = A, C, T or
Gmisc_feature(6645)..(6648)N = A, C, T or
Gmisc_feature(6679)..(6679)N = A, C, T or
Gmisc_feature(6831)..(6831)N = A, C, T or
Gmisc_feature(6856)..(6890)N = A, C, T or
Gmisc_feature(6926)..(6939)N = A, C, T or
Gmisc_feature(6958)..(6996)N = A, C, T or
Gmisc_feature(7137)..(7233)N = A, C, T or
Gmisc_feature(7288)..(7288)N = A, C, T or
Gmisc_feature(7348)..(7348)N = A, C, T or
Gmisc_feature(7452)..(7463)N = A, C, T or
Gmisc_feature(7901)..(7912)N = A, C, T or
Gmisc_feature(7937)..(7958)N = A, C, T or
Gmisc_feature(7978)..(8105)N = A, C, T or
Gmisc_feature(8131)..(8167)N = A, C, T or
Gmisc_feature(8191)..(8298)N = A, C, T or
Gmisc_feature(8319)..(8349)N = A, C, T or
Gmisc_feature(8373)..(8457)N = A, C, T or
Gmisc_feature(8481)..(8510)N = A, C, T or
Gmisc_feature(8527)..(8531)N = A, C, T or
Gmisc_feature(8537)..(8539)N = A, C, T or
Gmisc_feature(8542)..(8544)N = A, C, T or
Gmisc_feature(8551)..(8555)N = A, C, T or
Gmisc_feature(8574)..(8581)N = A, C, T or
Gmisc_feature(8597)..(8602)N = A, C, T or
Gmisc_feature(8702)..(8722)N = A, C, T or
Gmisc_feature(8741)..(8797)N = A, C, T or
Gmisc_feature(8813)..(8864)N = A, C, T or
Gmisc_feature(9042)..(9082)N = A, C, T or
Gmisc_feature(9113)..(9818)N = A, C, T or G 356gtgtttgaca
atgccatgag ggatcatgac actgnnnnnn nnnnnnnnnn nnnnnnnnnn 60nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
120nnnnnnnnnn nnnnngcaaa gggatgggtc cctgnnnnnn nnnnnnnnnn
nnnnnnnnnn 180nnnnnnnnnn nnnnnggtgg cggcgggccc nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 240nnnnnnnnnn nnnnnnnnnn nnatttgttc
tccagtcnnn nnnnnnnnnn nnnnnnnnnn 300nnnnnnnnnn nnnnnnntaa
atgggagcta ccnnnnnngt ggtggctctt gccggtnnnn 360nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
420nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnngc attgagtcag
ggcaaatatn 480nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnttttgct
ttcttttcta actgttgcaa 540tcaatctgat attatgtgnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 600nnnnnnnnnn nnnnnnnnnn
nnnnnatctg ctgggactat cttggnnnnn nnnnnnnnnn 660nnnnnnnnnn
ngtcgaatgg gtgagggagc tgaagcgctt cttcgcttga tcggcattgc
720aggttggctn ggnctgttag ctgagtctct tggtatgtcn gagatctatg
cagctattct 780ttgctttgga tttattgctt ggtatggctg gggtatacct
aaaacattgg tgtgcacagt 840ctgccctgca gtgaacattt ctccctatag
cttcttatct ccagatacta tcgcatttgg 900tacgtggata ctacaactac
ctggtctttt gtggcaaatg tttgtcagct tccctatact 960ttacagtact
tggattcttt ggttgttgct cagcggcaag actgttgctg tgatagcgat
1020ccttttggct agtcctacgg ttatggcata caagcatcaa tctgaaagct
acctcaaata 1080ctgnaccata accaatactt caactgctat gaactgtgac
tgcccctttg gaactttcac 1140tcgcaatact gagtctggtt tctcnatacc
tagattctgt cctgttaaaa tcaatagctc 1200tacatttatn tgttcatggg
ggtcgtggtg gtggtttgct ganaacatca cacgtccata 1260ctcggacgtt
ggcatgccgc cagcaccgat ttccgctttg tgctatatct attcnaacaa
1320tgacccacct tcttggtnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 1380nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 1440nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnttctata ctaatggtag 1500tnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1560nnnnnnnnnn
nnnnnnnnnc ctaacatctc gaatacgacc attgannnnn nnnnnnnnnn
1620nnnnnnnnnn atcgcatccg gtcttcgggc tgggtatggt tgcagccgag
cgcacggcgt 1680ggtgcactgc tttaagtgtc cttcataccg tgaccttgaa
cngtttggtc ctgggcttgg 1740gaaatgggtg ccattgcctg gcgagcctgt
cccggagttg tgtattaann nnnnnnnnnn 1800nnnnnnnnnn nnnnggntgt
ctaataatcc tttgagcttg ctacagacct tcgttgagga 1860cattttccta
gcgccttttt gcaatccgac gcctggccgt gtacgtgtgt gtaacaatac
1920tgctttctat ccgagaggag gcggctttgt gcagctcatc ggagacgtcc
aggtgctaac 1980ccctaacact gcatctttac actctntgct gactttgata
tcgcttatct tgttggtgtg 2040tgttgtttct ggtgcgcgat tcgtnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2100ngtatatgct tcttgttact
taagctgtga ttgggctgtt tgcaacgatg cgttctgttt 2160cacatctggc
acttgtgcta ccttcaatga cgtcttgtgt ctgccggttg cgncgcgcat
2220atcgtcctgt ggccatgctg tgccacctcc cgaccgtggt tgggaggtgc
ctgcggcgat 2280gtcatgggcg atttcgcgga ctactggctt gacgttcgat
gtcttttcct tcatncagta 2340ccttcctact gtgcctggca acaataccga
tatcatttac tgtggtgaac caaccttctt 2400cggggacatc acgggcatct
attggcctta ctttttgcct ggcgtgntnc tcttgtactt 2460gactcccttc
ctgggtttaa ggttaatgct tgccggcttc aatatagatg gcttgtttcc
2520catacggcat gccacggctg cactgaggtt ctcgacttcn cgtgtgacct
tgtgtgtcgt 2580anttgctttc ctaatctata tattatctca tcctgttaat
gctgcgctca annnnnnnnn 2640cttagcatct gcaaatttag agatganntt
atcttttgat acctatcatg agactgttct 2700ttatnnnntt tgtctattgc
tctacctcca ggtgtcnnnn ngtgcgggct tggccgnnnn 2760nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
2820nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnntcttggt
tggtggagtn 2880nnnnnnnnnn nnnnnnnnnn nnnnnncctg tgtcttctct
ttttnnnnnn nnnnnnnnnn 2940nnnnnnnnnn nnnnnnnnnn nnnactataa
gattcagann nnnnnnnnnn nnnnnnnnnn 3000nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnccg 3060tgcaagtgtg
ggcgtgctnt ggtgtggagc ttgcatgctc tggccccgtg agtgctctga
3120aatcagcctg gtcttcattc tgtgtgctct gacagtggac accatagaca
catnnnnnnn 3180nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 3240nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn cacgnggcac 3300atacatatac aaccacatgg
gccaagnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3360nngcggttgc
ttggaaccag nnnnnttgga ggagcccacc nnnnnnnnnn nnnnnnnnnn
3420nnnnntgagt ttggtgtgtg gacaattgnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 3480nnnnnnnnnn nnnnnnnnnn nnntcaaccc tgaagatcnn
nnncctggtt tccagctgnn 3540nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnng tccttcctta aggcgacact 3600cacannnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3660nnnnnnnnnn
nnnnnngcag tgaatggcac actgnnnnnn nnnnnnnnng gtgccggcgc
3720gcgcggcnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 3780nnnnnnnnnn nnnnnnnnnn nnnnnnnnnt gagttcccta
gacccctgcn nnnnnnnnnn 3840nnnnnnnnnn nnnnnnnnga ataacaacgg
agggttggtg tgtggcagag tggagnnnnn 3900nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3960nnntttgtgt
gatcacggtc atgcannnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
4020nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 4080nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 4140nnnnnctggt tctggtaagt caacaaannn
nncagtggac ctagtcaaac agggacacan 4200nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnaaaagt atggcccctt nnnnnnnnnn 4260nnnnnnnnnn
nnnnnnnnnn nnnnnnnngc tggcacaggt cctnnnnnnn nnnnnnnnnn
4320nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnccgcttc cttgtggatc
cnnnnnnnnn 4380tctgcggggn tacgctgtag tcatttgcga cgagtgccat
gacacatcat ccaccacgct 4440actcggcatc ggcgcagtgc gcatgtatgc
ngagaaagct ggagtgaaga ccgttgtatt 4500cgccacagcc actcctgctg
gcattcaagt acagtcacat tccaacattg atgaatatct 4560attgactgat
acaggcgacg tggaattnnn nnnnnnnnnn nnnnnnnnnn acaacatcag
4620aactggtaga catgnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnagca 4680gctctccggc cttggcattc gtgcagtgag ttttnnnnnn
nnnnnnnnna tcaaaaccat 4740tcccgcctca gactcnattg ttgtggtggc
aactgatgca tnntncaccg nctacacagg 4800aaactnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4860nnnnnnnnnn
nnnnnnnnnn nnnnccgagt ggtgccatgt actgctgcnn nnnnnnnnnn
4920nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnncta
ctccaggagc 4980agcaccctgc gtcagcgttc ccgatgctaa cgtctggcaa
gcagtggagt cagccatggt 5040cttttatgat tggagtgctg ccaggataca
gcagtgcctg gcggcatacc atgatttngg 5100gtgcacacca cgcatcagct
gtgacccaca cactccagtg cgggtgatgg acacactgag 5160ggcgtatctg
cgcagacctg aggtgacgac tgcagctctc gcaggagagc agtggccgct
5220gctttacggt gtgcagttgt gcatctgcaa agagaccgag gcccacggtc
cagacgatgg 5280catcaagtgg aagtgcttac tcaacaacag taacaaaaca
cccctgttgt atgccttaga 5340caatcctaca ctggaattca cnacccaaca
tgacttgact cgccnnnnnn nnnnnnnnnn 5400nnngagcaca gtgttcgtgg
agacaggcta cggccccatc ctccttgctn nnnnngcttt 5460ggctgcctcc
ttcgcctttg cnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
5520nnnnnnngtc aggcctgctg gcnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 5580nnnnnnnnnn nnnnnnnnnn nnnnnncaga ttcaggaatt
tnnnnnnnnn nnnnnnnnnn 5640nnnnnnnnng gcgctgggca aactggcnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5700nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5760nnnnnnnnnn
nnnnnnnncg gcctgctctg tgctggctgc caggctggcg agtgcttcac
5820tgcgcttgcn gggttggtgn nnngtgctac agctggcttg gggggtgnnc
ataagtggtt 5880gttagctatt gcaggaactt ggctagttag cttgcagact
gggccccgtg gcggcatggt 5940tgcgggcctc tcagttctag caggctgttg
catcggtagt gtcaccgggc ttgacttcct 6000gtttgggtgc cttacaggnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6060nnnnnnnnnn
ncagctgata tgaccactct ggtagatctc ntacntgctc ttttctcccc
6120nnnnnnnnnn nnnnnnnnnn nnnnnnntgt cttcatntta agcaattcaa
gtgtaaccac 6180nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 6240nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6300nnnnnnnnnn ngtgagggat
acacacnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6360nnnnnnnnnn
ctacgtgctg tgtgggattg ggcggccaaa catgtgccac ggttccgtgt
6420gcctatgatt ggctgctcac ctgcgtggtg cgggcgctgg cttggtactg
gcaccttgtt 6480gaccacctgt gggtgtggag aacgtgtatc ccttcagtgc
ctttgctcaa catctgaccc 6540acnactcagt gtgggccgtt ggtgttggtg
tagttggagt gttgggttcc cattcaaccc 6600gacgacgaca ggcactggnn
ccttacggcc ggacatcngt gacgnnnnca aattgggttt 6660ccggtatggt
gtcgccgana tcgtggagct agagcggcgg ggcaacaaat ggcatgtctg
6720tgcagcatca tgttgcttgg accgggccag cgttgcatcc gctgtgaagg
ccccaccggt 6780cacggccgat ggcataccta tcagtacctt ttctccacca
caaacttatt ncctatctct 6840ctgttccttt gatacnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn agaccctgag 6900tgtgtgccaa gaggaggcgg
ttgagnnnnn nnnnnnnnna gttgacacag cacaagtnnn 6960nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnngttt gatgcatggc aagttcgcga
7020agcaattcgc gacgagtaca cgcgtttggc agacgaggat gttgacgcga
caacgtcggt 7080gaaacccccg gtggccaggg ctgctgtggg tagctcgacg
ttggatgatg ttagcgnnnn 7140nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 7200nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnacgcacg cgtgtctcgg gtgtgttgga 7260gcgcgtgcgc
atgtgcatgc gcacgccncc aatcaagttt gaggccaccg cagtaccaat
7320tcataatata atcccagaag agtgtcanat tgtgctacgc tgtaccggct
gtaatgacca 7380ggccttgact gttccgtacg gcacttgcac tcagacttta
accaaacatt tgactaacaa 7440acacagtcac tnnnnnnnnn aanagaagat
agaagaagac acagaaatag ctgtcatttg 7500cgccgtacca acaaagcgcg
caagcaaact catcactttc agagcaggtg accgatcagt 7560ctcatgttgt
caccccttgc aaactcctat tagggccctg cttcaaaagt acgggttacc
7620tatcgggaag tggtccgact gcaacgggcc ccttggtgac gatgcccgag
tctgtgacgt 7680caatggagta acaacttatg aaccatgcat gcaatcctac
agttggttcc gaccgattgt 7740ggcaccaaca accccacctt tacctgcaac
ccggagcgtg gctggcattt tacgcgcaga 7800cacatcgcgc gtttacacca
caacggcggt tgacgtctcc gagcggcagg ctaaggtcac 7860aattgatcaa
acatcagcca aggtggatca gtgtttccga nnnnnnnnnn nntgctgcct
7920tgccaaggca aagaccnnnn nnnnnnnnnn nnnnnnnnat gaggatgctg
tgtcaaannn 7980nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 8040nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 8100nnnnntgcgc attattccaa
aacctgaagt nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 8160nnnnnnnttc
atcgttttcc ctgggtgtgc nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
8220nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 8280nnnnnnnnnn nnnnnnnnac tggaacaatg gtggaaccnn
nnnnnnnnnn nnnnnnnnnn 8340nnnnnnnnnt gcagtctgct ttgatagcac
cannnnnnnn nnnnnnnnnn nnnnnnnnnn 8400nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnntta 8460caaaagaagc
cccatgtgtg nnnnnnnnnn nnnnnnnnnn nnnnnnngtn gctgtcgagc
8520ctcaggnncn nttacannnc annnagtggc nnnnngctta cttgctacct
taannnnnnn 8580ncagcttgca cgcgcgnnnn nnttaaacca attggcttac
taattcatgg agatgacacc 8640ctcatcgtca cagaacgttg cgctcaggaa
actctcgatg agttcagcaa cgcacttgat 8700gnnnnnnnnn nnnnnnnnnn
nntgcaggtg tctggggacc nnnnnnnnnn nnnnnnnnnn 8760nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnntgc gtcgcatgct cgnnnnnnnn
8820nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnactggg
taccatcggt 8880agctatgttg tcatgtatcc cactgcggcc gtgactgtct
acgtgctatt gcccctgttg 8940tgcatgctca tacgaaatga gccatcacag
acggggacac ttgtgacgct gacggtccac 9000ggtaacagtg tgagcgtgcc
agtgtggctg cttccaacca tnnnnnnnnn nnnnnnnnnn 9060nnnnnnnnnn
nnnnnnnnnn nngtcacagt gcagcttcca tggcggaact gtnnnnnnnn
9120nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 9180nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 9240nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9300nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9360nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
9420nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 9480nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 9540nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnng gnnnnnnnnn 9600nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 9660nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
9720nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 9780nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnn
981835723DNAArtificial sequenceSynthetic 357tgatcaagty gggcgggtgg
aat 2335821DNAArtificial sequenceSynthetic 358gaacaccaca rcccgaacca
a 2135926DNAArtificial sequenceSynthetic 359cgtccgtacg aaaatttgca
cttgag 2636026DNAArtificial sequenceSynthetic 360cgcaatcgtg
gtgctagtcg cttacg 2636124DNAArtificial sequenceSynthetic
361gctagtgcta tactcgctct gctt 2436219DNAArtificial
sequenceSynthetic 362tgatagggtg gcggcgggc 1936318DNAArtificial
sequenceSynthetic 363atacctcctc gggctgcc 1836421DNAArtificial
sequenceSynthetic 364atgggagcta ccactgcggt g 2136523DNAArtificial
sequenceSynthetic 365gccggtcacc aagtcgtrtg cag 2336624DNAArtificial
sequenceSynthetic 366ggtatgtgtt csatccggtc caaa
2436724DNAArtificial sequenceSynthetic 367atccttctgg ctagtcctac
ggtt 2436823DNAArtificial sequenceSynthetic 368tttatttgtt
catgggggtc gtg 2336923DNAArtificial sequenceSynthetic 369gaaaacatca
cgcgtccata cac 2337019DNAArtificial sequenceSynthetic 370caccagcacc
gatttccgc 1937119DNAArtificial sequenceSynthetic 371ttgtattctt
gaccgccgg 1937219DNAArtificial sequenceSynthetic 372gtaatccgac
gcctggccg 1937318DNAArtificial sequenceSynthetic 373tgtgtctgcc
ggttgcga 1837423DNAArtificial sequenceSynthetic 374tctcaccctg
ttaatgctgc gct 2337524DNAArtificial sequenceSynthetic 375ctgtcggttg
tggtcctctc ggtc 2437623DNAArtificial sequenceSynthetic
376aagtctcgga acgggtggcg caa 2337720DNAArtificial sequenceSynthetic
377tggacaattg cttggaggta 2037820DNAArtificial sequenceSynthetic
378tccttcctta aggcgacact 2037920DNAArtificial sequenceSynthetic
379atcgtggtgt tgtcttccct 2038019DNAArtificial sequenceSynthetic
380cactgtatgc gaccggcca 1938124DNAArtificial sequenceSynthetic
381tagacccctg ctgctgttcg ccga 2438220DNAArtificial
sequenceSynthetic 382ctgtcccacg cacatagatc 2038320DNAArtificial
sequenceSynthetic 383tttgtgtgat cacggtcatg 2038421DNAArtificial
sequenceSynthetic 384ccaagtgtgg ctgtagtcaa a 2138524DNAArtificial
sequenceSynthetic 385gacgaatctg cggggctatg ctgt
2438620DNAArtificial sequenceSynthetic 386gctactcggc attggcgcag
2038720DNAArtificial sequenceSynthetic 387aaagctggag tgaagaccgt
2038823DNAArtificial sequenceSynthetic 388aacaacagta acaaaacacc cct
2338921DNAArtificial sequenceSynthetic 389cttcctgttt gggtgcctta c
2139024DNAArtificial sequenceSynthetic 390ttacaggttg ggaagccgtg
gtcg 2439124DNAArtificial sequenceSynthetic 391ttacaggttg
ggaggccgtg gtyg 2439224DNAArtificial sequenceSynthetic
392ttacgggttg ggaagccgtg gtcg 2439321DNAArtificial
sequenceSynthetic 393wtcgtggaky tagagcgscg g 2139424DNAArtificial
sequenceSynthetic 394gtagttcagg cggcttcacg gttt
2439523DNAArtificial sequenceSynthetic 395agtttgaggc caccgcagta cca
2339621DNAArtificial sequenceSynthetic 396ttgaygtcyc cgagcggcag g
2139723DNAArtificial sequenceSynthetic 397gttgtcgggg tgcgtagctg tcg
2339818DNAArtificial sequenceSynthetic 398ctcaaggttc gcgcagct
1839924DNAArtificial sequenceSynthetic 399aggatgctgt gtcaaagatg
cgcg 2440024DNAArtificial sequenceSynthetic 400tatcctactg
cggctgtgac tgtc 2440121DNAArtificial sequenceSynthetic
401cctcagcgtt ggccttcttt g 2140219DNAArtificial sequenceSynthetic
402cctatccgag ttgggcaag 1940324DNAArtificial sequenceSynthetic
403gtaagaacac cacagcccga acca 2440425DNAArtificial
sequenceSynthetic 404accacttaat ggtcgtaact cgacc
2540519DNAArtificial sequenceSynthetic 405gtcaacggcc cctttcatt
194061044DNAArtificial sequenceSynthetic 406atgagagttc ctgcacaatt
attaggatta ttattattat ggtttcctgg atctaggtgc 60tacaagcacc agagcgagag
ctacctgaag tattgtacaa ttacaaatac atctacaagc 120atgaactgcg
attgcccttt tggcaccttc accaggaata cagagtctag attttctatt
180cctagatttt gtccagtgaa gatcaatagc agcaccttca tctgctcttg
gggatcttgg 240tggtggtttg ctgaaaatat tacaagacct tatacagatg
tgggaatgcc tccagctcca 300atttctgctc tgtgttacat ctacagcaat
aatgatcctc ctccttggta tcataatacc 360accatcattc ctcagaactg
cagaaatagc accgttgatc ctacaacagc tccttgtaga 420gataaatggg
gaaatgctac agcttgtatt cttgacagaa gaagcagatt ttgcggcgat
480tgttatggag gatgctttta cacaaatgga agccatgata gatcttggga
tagatgtgga 540atcggctaca gagatggact gattgaattt gttcagttag
gccagattag acccaatatc 600agcaatacaa ccatcgaact gcttgctgga
gcttctttag ttattgcttc tggattaaga 660cctggatttg gatgttctag
agctcatgga gttgtgcact gctatagatg tccttcttac 720agagatttag
agcaatttgg acctggactt ggaaaatggg tgcctttacc tggagaacct
780gttcctgaat tatgtattaa tcctcaatgg gctagaagag gattcagaat
gagcaataac 840cctctgtctc tgctgcagac atttgttgaa gatatctttc
ttgccccttt ctgtaatcct 900acacctggaa gagttagagt gtgcaacaat
acagcctttt atcctagagg aggaggattt 960gttcaactta ttggcgatgt
tcaggttctg acccctaata caggatctgg atctggacat 1020catcatcatc
atcatcatca ctaa 1044407249DNAArtificial sequenceSynthetic
407atggcccttg ttcccagcgc tgtgtggagt gttgaagtcc gccccgcagg
cgtgacgcgc 60cctgatgcca ccgatgaaac cgctgcgtac gctcaacgct tgtatcaggc
ctgcgccgat 120tcaggtatct ttgcgtcact tcaaggaacc gcgagtgcgg
cgttgggcaa gctggcggat 180gcctcgcgtg gcgcgagtca atacctggca
gccgccccac catcacctgc cccactggtg 240caggtatta 24940820DNAArtificial
sequenceSynthetic 408tgttggccct accggtgtta 2040918DNAArtificial
sequenceSynthetic 409ccgtacgtgg gcgtcgtt 1841024DNAArtificial
sequenceSynthetic 410ctcgtcgtta aaccgagccc gtca
2441124DNAArtificial sequenceSynthetic 411ctcgtcgtta aaccgagacc
gtca
2441224DNAArtificial sequenceSynthetic 412cacgccgtta aaccgagacc
gtta 2441322DNAArtificial sequenceSynthetic 413gtgggacacc
tcaaccctga ag 2241424DNAArtificial sequenceSynthetic 414gggaagacaa
caccacgatc tggc 2441523DNAArtificial sequenceSynthetic
415cctggtttcc agctgagtgc tcc 2341621DNAArtificial sequenceSynthetic
416cgctgatcgt gcaaagggat g 2141721DNAArtificial sequenceSynthetic
417gctccacgga cgtcacactg g 2141822DNAArtificial sequenceSynthetic
418gcaccactcc gtacagcctg at 224199864DNAHuman Pegivirus
2misc_feature(9595)..(9635)N = A, C, T, or
Gmisc_feature(9851)..(9864)N = A, C, T, or G 419aactgttgtt
gtagcaatgc gcatattgct acttcggtac gcctaattgg taggcgcccg 60gccgaccggc
cccgcaaggg cctagtagga cgtgtgacaa tgccatgagg gattatgaca
120ctggggtgag cggaggcagc accgaagtcg ggtgaactcg actcccagtg
cgaccacctg 180gcttggtcgt tcatggaggg catgcccacg ggaacgctga
tcgtgcaaag ggatgggtcc 240ctgcactggt gccatgcgcg gcaccactcc
gtacagcctg atagggtggc ggcgggcccc 300cccagtgtga cgtccgtgga
gcgcaacatg gggtgttcaa ctgatcaaac catttgttct 360ccagtcgtgg
gggccgacta taatacctcc tcgggctgcc gggccttaaa tgggagctac
420cactgcggtg gtggctcttg ccggtcacca agtcgtgtgc aggttgcgag
acgagtcttg 480cagctgagcg cattccttgc gttgatcgga tccggtatgt
gttcgatccg gtccaaaact 540gaagggcgca ttgagtcggg gcaaatattg
cagtctcagc gcgcatgttg gactggtgag 600ggttttgctt tcttttctaa
ctgttgcaat caatctgata tcatgtggtg tttgcaccgt 660tggtgtgtga
caagacctgg ctgtttagtg tgcacgggca atgccactca tcctgtctgc
720tgggactatc ttgggtccgg tgtaagccgg cggcctgcgc gtcgaatggg
tgagggagct 780gaagcgcttc ttcgcttgat cggcattgca ggttggctcg
ggctgctagc tgaggctctt 840ggtatgtccg aactctacgc agctattctt
tgctttggat ttattgcttg gtatggttgg 900ggtataccta aaacattggt
gtgcacagtc tgccctgcag taaacatttc tccctatagc 960ttcttatctc
cagacactat cgcatttggt acgtggatac tacaactacc tggtcttttg
1020tggcaaatgt ttgtcagctt tcctatactt tacagcactt ggattctttg
gttgttgctc 1080agcggcaaaa ctgttgctgt gatagcaatt cttttggcga
gtcctacggt tatggcatac 1140aagcatcaat ctgacagcta cctcaaatac
tgtaccataa ccaatgcttc aactgctatg 1200aactgtgact gcccctttgg
aacctttact cgcaatactg agtctcgttt ctctatacct 1260agattctgtc
ctgttaaaat taatagctct acatttatct gctcatgggg gtcgtggtgg
1320tggtttgctg agaacatcac gcgtccatac tcggacgttg gcatgccacc
ggcaccgatt 1380tctgctttgt gctatatcta ttcaaacaat gaccctcctc
cttggtacca taatacaact 1440atcatacctc agaactgtcg caactccacg
gctgatccca ccacagcccc atgccgtgac 1500aagtggggca acgcaactgc
ttgtattctt gaccgccggt cgcggttctg cggggactgc 1560tatggcggct
gcttctatac taatggtagt catgatcgat cctgggatcg atgcgggatt
1620ggttaccgtg atggactcat agagttcgtg cagctcggtc agattcgacc
caacatctcg 1680aatacgacca ttgagctcct cgctggcgcc tcgctagtga
ttgcatccgg tcttcgggct 1740gggtacggtt gcagccgagc gcatggcgtg
gtgcactgct ataagtgtcc ttcataccgt 1800gaccttgaac aatttggtcc
cgggctcggg aaatgggtgc cattgcctgg cgagcctgtc 1860ccggagttgt
gtattaaccc ccagtgggcg aggcgcggct tccgggtgtc taacaatcct
1920ttgcacttga tacagacctt tgttgaggac atcttcctag cacctttttg
cagtccgacg 1980cctggccgtg tacgtgtgtg taacaatact gctttctatc
cgacaggagg tggttttgtg 2040cagctcatcg gagacgtcca ggtgctaacc
cctaacactg catctttaca ctctctgctg 2100actttaatat cccttatctt
gctagtgtgt gttgtttctg gcgcgcggtt catcccatta 2160atcatcatat
ttttctggag cgtgcgccac gtatatgctt cttgttactt aagctgtgat
2220tgggctgttt gcaacgatgc gttctgtttc acatctggca cttgtgctac
cttcaatgac 2280gtcttgtgtc tgccggttgc ggcgcgcata tcgtcctgtg
gccacgctgt gccacctccc 2340gaccgtggtt gggaggtgcc tgcggcgatg
tcatgggcga tttcgcggac tactggtttg 2400acgttcgatg tcttctcctt
tattcagtat ttccctacag tgcctggcaa caacaccgat 2460atcatttact
gtggtgaacc aaccttcttc ggggacatca caggcatcta ttggccttac
2520tttttgcctg gcttgttgct cttgtacttg actcctctac tgggttttag
gttaatgctt 2580gccggcttca atatagatgg cttgtttccc atacggcatg
ccacggctgc gctgaggttc 2640tcgacctcac gtgcgaccat gtgtgtcgta
tctgctttcc taatctatat attatctcat 2700cctgttaatg ctgcgctcaa
tagaatgttc ctagcatctg caaacttaga gatgatctta 2760tcttttgata
cctatcatga gactatcctt tacatcgctt gtctattgct ctacctccag
2820gtgtcgcccc gcgcgggctt ggccgctatg gtggccatca agctgtctcg
aggcctgcta 2880ttcgctgtgg tgttggcgca cggcgtgtgc cgacctgggc
gggtatttgg tcttgaggtt 2940tgcgcggaca tctcatggtt ggtggagttt
actggcaact gcacttggta catgtcctgt 3000gtcttctctt tttggtgcgc
agtgtttgcc ttcaccagtc cacttggacg acactataag 3060cttcagatct
accggtactg ggcgcaggtc tatgccagac tcatccttgc tgtcggttgt
3120ggtcctctcg gacgagagtt ccatttccgt gcaagcgtgg gtgtgctttg
gtgtggtgct 3180tgcatgctct ggccccgtga gtgctctgaa atcagcttgg
tcttcattct gtgtgctctg 3240acagtggaca ccatagacac atggttagta
gcgtgcttgt ccgcagggcc gagtgcgcga 3300acccttgcaa cactggccga
tgacatggcg cgctttggtg accaccgggc gttgcgcgcc 3360gtgttgcgtt
gctttggatc acgtggcaca tacatataca accacatggg ccaggtctca
3420gaacgggtgg cgcaagcagt cagggatttc ggcggttgct tggaaccagt
cgtgttggag 3480gagcccacct ttactgaggt cgtggatgac acaatgaatt
tagtgtgtgg acaattgctt 3540ggaggtaagc ctgtggtggc ccgctgcggc
acgcgtgtct tggtgggaca cctcaaccct 3600gaagatctgc cacctggttt
ccagctgagt gctccggtca ttatcaccaa accaagcatt 3660ggtacgtggt
ccttccttaa ggcgacactc acagggcgtg ctgaaacacc gggatccggc
3720cagatcgtgg tgttgtcttc cctgacaggt cggtcaatgg gtactgcagt
gagtggcaca 3780ctgtatgcga ccggccatgg tgctggtgcg cgcggcctag
ccacgtgcgc cggtttgagg 3840acgccacttt acacggcatt atctgatgat
gtcgtggcct actcttgcct cccgggcatg 3900agttccctag agccctgccg
ctgtgcgccg agccgggttt gggtgatgaa caacaatgga 3960gggttggtgt
gtggcagagt ggagaatgag gacgtctgtt tggactgtcc cacgcacata
4020gatcaactgc ggggtgcttc gggatcgccg gtcttgtgtg atcacggtca
tgcatacgcg 4080ctgatgctcg gtggttactc taccagtggt atttgtgcgc
gtgtccggat agtccggcca 4140tggcagaacg cctattcctc ctcagggggg
caaggcggga tgcaggcgcc agctgtgaca 4200ccaacatact ctgaaatcac
ctactatgcc cctactggtt ctggtaagtc aacaaaatat 4260ccagtggacc
tggtcaaaca gggacacaaa gtattggtcc ttataccaag tgtggctgtc
4320gtcaaaagca tggcccctta cattaaggag acatataaga ttagacctga
aattagagct 4380ggcacaggcc ctgacggtgt gacggtcatc actggtgaga
acttggcgta catgacctat 4440ggccgcttcc ttgtggatcc ggagacgaat
ctgcagggtt atgccgtagt catttgcgac 4500gagtgccacg acacatcatc
caccacgcta ctcggcattg gcgcagtgcg catgtatgcc 4560gagaaagctg
gagtgaggac cgttgtattc gccacagcca ctcctgctgg cattcaagta
4620cagccacacc ccaacattga tgaatattta ttgactaatg aaggcgacgt
ggacttctac 4680ggcgccaaaa tcaaattgga caacatcaga actggtagac
atgttatctt ttgtcactcg 4740aaggccaggt gcgcggaact aacgcagcag
ctctccggcc ttggcgttca tgcagtgagt 4800ttttggcgcg gctgtgacat
caaaaccatt cccgcctcag gctctattgt tgtagtggca 4860actgatgcat
tgtccacagg cttcacagga aattttgatt cggtcatcga ctgcgggtgt
4920tgcgtagagc aaactgtgac aattgacatg gaccccacgt tctccatctc
ggcccgagtg 4980gtgccatgta ctgctgcatt gcgtatgcag cggcgcggac
gcaccggtcg tggcagaagg 5040ggagcgtact acacaaccac tccaggagca
gcaccctgcg tcagcgttcc cgatgctaac 5100gtctggcaag cagcggagag
cgccatggtc ttttatgatt ggaatgctgc taggatacag 5160cagtgcctgg
cggcatacca tgacttaggg tgcacaccac gcatcggttg tgacccacac
5220actccagtgc gggtgatgga cacactgagg gcgtacctgc gcagacctga
ggtgacgact 5280gcagctctcg cgggagagca gtggccgctg ctttatggtg
tgcagttgtg catctgcaaa 5340gagaccgagg cccacggtcc agatgatggc
atcaagtgga agtgcttact caacaatact 5400aataaaacac ccctgttgta
tgccttagac aatcctacac tggaattcac tacccaacat 5460gacttgactc
gccgtatagc cggcgcttta tcgagcacag tgttcgtgga agcaggctac
5520ggccccatcc tccttgctgg cgccgctttg gcagcctcct tcgcctttgc
gggcgccact 5580ggagctttag tgccgtcggc cgtttggagc gttgacaacg
ggcctgctgg cgtgacccgt 5640cccgacgcga cagacgagac tgcggcctac
gcgcagcgct tgtaccaagc ctgcgcagat 5700tcaggaattc tcgccagctt
gcagggcaca gcgtgtgcgg cgctgagcaa actggccgat 5760gccagtaggg
gtgctagtca atatctggca gccgcgcctc cttcgcccgc ccccctggtg
5820caggtgctgc agttcctcga gactaacttt agctccattg catccttcgg
tctgctctgt 5880gctggttgtc aggctggcga gtgcttcacc gcacttgccg
ggttggtgtc cggcgctaca 5940gcaggcttgg gaggtgccca taagtggttg
ttggctattg caggaacttg gctggttagc 6000ctgcagaccg ggccccgtgg
cggcatggtt gcgggcctct cggttctagc aggctgttgc 6060atcggcagtg
tcaccgggct tgacttcctg tttgggtgcc ttacgggttg ggaggccgtg
6120gtcggtgctg cggttgcgac acagaaaatc ttgtctggtt cggctgacat
gaccactctg 6180gtagatctct tacctgctat cttctctcct ggtgccggca
tagccggcgt cgtgctcgtc 6240tttattctaa gcaactcaag tgtaaccacg
tgggctaatc ggctattgtc catgtgtgca 6300aaacaaacca tttgtgaaaa
ttacttcttg actgagagat ttggccaaca attaagcaaa 6360ctttccctgt
ggcgcgctgt gtaccattgg gcacaggcgc gcgagggata cacacagtgc
6420ggtgtggtcg gcgggatctg gagctttgcc ttgtgcgtcc tgcgagctgt
gtgggattgg 6480gcggctaggc atgtgccacg gttccgtgtg cccatgattg
gctgctcacc tgcatggtgc 6540gggcgctggc ttggtaccgg caccttgttg
accacctgtg ggtgtggaga acgtgtgtcc 6600cttcagtgcc tttgctcgac
gtctgaccca acactcagtg tgggccgttg gtgtcggtgc 6660agttggagtg
ttgggttccc attcaacccg actacgacag ccaccggcac tttacggccg
6720gacattagcg acgccactaa attgggtttc cggtatggtg ttgctgagat
tgtggagcta 6780gagtggcggg acaacaaatg gcacgtctgc gcagcatcat
gttgcgtgga ccgagctagt 6840gttgcatctg ccgtgaaggc cccaccggtc
acagccaatg gcatacctat cagtactttt 6900tctccaccag aaacttacaa
actctctctc tgttcttttg attcagtctg catgtctaac 6960tcaagtaacc
cagctaagac cctgagtgtg tgctcacagg aggctgttga gctgctggaa
7020gaaacagttg atacagcaca agcagtgatg tgtaagaatc tggaggcgcg
aagacgcgct 7080gaatatgatg catggcaggt tcgtcaagca gttggcgacg
agtacacgcg cttggctgac 7140gaggatgttg acacaacagc gtcggtgaaa
cccccggtgg ccagggctgc tgtgggtagc 7200tcaacgttgg atgatgttaa
cgtgctgact gtcttgcgcg agctcggtga ccaatgccaa 7260aatgctatca
aatttgtagt ccaggcggct tcacggtttg ttccaccagt gcccaggccg
7320cgcacgcgtg tctcgggtgt gttggagcgc gtgcgcatgt gcatgcgcac
gccaccaatc 7380aagtttgagg ccaccgcagt accaattcat aacataatcc
cagaagagtg tcacattgtg 7440ctacgctgta ccggctgtag tgaccaggcc
ttgactgttc cgtacggcac ttgcactcag 7500actttaacca aacatttgac
taacaaacac agccattaca ttccaaaaca gaagatagaa 7560gaagacacag
aagtaactgt tatctgcgcc gtaccaacaa cgcgcgcatc taaactcatc
7620actttcagag caggtgatcg atcagtctct tgttgtcacc ccttacaaac
tcctattagg 7680gccctgcttc taaagtacgg gttacctatc gggaagtggt
ctgactgcaa cggccccctt 7740ggtgacgacg cccgagtctg tgacgtcaat
ggagtaacaa cttatgaacc atgcatgcaa 7800tcctacagtt ggttccgacc
gattgtggca ccaacaaccc cacctttgcc tgcaacccgg 7860agcgtggctg
gcattttacg cgcagacaca tcgcgcgttt acaccacaac ggcggttgac
7920gtctccgaga ggcaggctaa ggtcacaatc gatcaaacat cagccaaggt
ggatcagtgt 7980ttccgagaca cctacaattg ttgccttgct aaggcaaaga
ccttcaaaca atctggcatg 8040tcatatgagg atgctgtgtc aaagatgcgc
gcaaacacca cgcgtgatca taaccatggc 8100actacttatt cagatttggt
ctctggacgc gcaaaacctg tcgttcagaa aattgtaaat 8160caaatgcgcg
ctggagtgta cgacgctcca atgcgcatta tcccaaaacc tgaggtgttc
8220cctcgagaca aggaaacacg gaagccacca cggttcattg tttttcctgg
gtgcgccgca 8280cgagtcgcgg agaaaatgat cctgggcgat cctggtgcga
taaccaagca cgtgctaggt 8340gatgcctacg ggtttgccac tccgccccac
gagcgcgcgc gcctgttgga acaatggtgg 8400aaccgcgcaa cagagccaca
agctatcgcg gttgatgcga tctgctttga tagcactatc 8460acggcagagg
acatggatcg tgaagccaac atcgtggctg cagcgcatgc ggaccctgaa
8520ggtgttcacg gcctatacaa ttattacaaa agaagcccca tgtgtgacat
cacaggaaat 8580attgtcgggg tgcgttgctg ccgagcctca ggtacgctta
caacaagcag tggcaacacg 8640cttacttgct acctcaaggt tcgcgcggct
tgcacgcggg ccggcattaa accaattggc 8700ttactaattc atggagatga
caccctcatt atcacagaac gttgcgctca agaaactctc 8760gatgagttca
gcagggcact tgatgactat gggttccccc acaccttcca ggcgtctggg
8820gacctctcgt ctatcgagtg ctgcagcgca cacgtggaca gtgtttgcct
ccggggaggt 8880atgcgtcgca tgctcgtgcc acaagctcga cgtgcgattg
cacgcgttct cggggaaaag 8940ggcgatccac tgggtgtcat cagcagctat
attgtcatgt atcctactgc ggccgtgact 9000gtctatgtgc tattgcctct
gttgtgcatg ctcattcgaa atgagccatc gcagacgggg 9060acatttgtga
cgttgacggt ccacggcaac agtgtgagcg tgccagtgtg gctgcttcca
9120accatcattg taaatttaca tggtcgtgac gcactacaag tagtccgtca
cactgcagct 9180tccatggcgg agctgtcctc agcgttggcc ttctttggca
tgagagggtt gaactgctgg 9240aggcggagac gccgtgccat cagggctgat
atgatcaagt tgggcgggtg gaatgcgaac 9300ttcgcgcaga tgttactgtg
gtcaccggag gtgagaacac cacagcccga accaaagggc 9360atgtgtctct
taccaccgga actatgggag cgtccgtacg aaaatttgca tttgagcacg
9420atcgaccgca atcgtggtgc tagtcgctta cggttttggt tggttgctag
tgctatactc 9480gctctgcttt gcttgtaaat cctaaatcaa tgtagtacca
ggactacaag gcaggaggtg 9540aagtcagctg tacccacggc tggctgaaac
cggggcttga cgaccccccc tatcnnnnnn 9600nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnccccc atgtcgcgcg taagcgcacg 9660ggcaaggcag
ctaggctgag agtctgggca actctcccgt accccacccg aggctacgcc
9720tcgtcctggc gaggaccgta aacatacgtc gtcagcgtgg tgacctgacg
tatcttgtta 9780accacttaat ggtcgtaact cgacccccgt gctggggatc
taagcgcggc accgcgatga 9840gaagggtcaa nnnnnnnnnn nnnn
986442079PRTHuman Pegivirus 2 420Met Gly Cys Ser Thr Asp Gln Thr
Ile Cys Ser Pro Val Val Gly Ala1 5 10 15Asp Tyr Asn Thr Ser Ser Gly
Cys Arg Ala Leu Asn Gly Ser Tyr His 20 25 30Cys Gly Gly Gly Ser Cys
Arg Ser Pro Ser Arg Val Gln Val Ala Arg 35 40 45Arg Val Leu Gln Leu
Ser Ala Phe Leu Ala Leu Ile Gly Ser Gly Met 50 55 60Cys Ser Ile Arg
Ser Lys Thr Glu Gly Arg Ile Glu Ser Gly Gln65 70 75421191PRTHuman
Pegivirus 2 421Ile Leu Gln Ser Gln Arg Ala Cys Trp Thr Gly Glu Gly
Phe Ala Phe1 5 10 15Phe Ser Asn Cys Cys Asn Gln Ser Asp Ile Met Trp
Cys Leu His Arg 20 25 30Trp Cys Val Thr Arg Pro Gly Cys Leu Val Cys
Thr Gly Asn Ala Thr 35 40 45His Pro Val Cys Trp Asp Tyr Leu Gly Ser
Gly Val Ser Arg Arg Pro 50 55 60Ala Arg Arg Met Gly Glu Gly Ala Glu
Ala Leu Leu Arg Leu Ile Gly65 70 75 80Ile Ala Gly Trp Leu Gly Leu
Leu Ala Glu Ala Leu Gly Met Ser Glu 85 90 95Leu Tyr Ala Ala Ile Leu
Cys Phe Gly Phe Ile Ala Trp Tyr Gly Trp 100 105 110Gly Ile Pro Lys
Thr Leu Val Cys Thr Val Cys Pro Ala Val Asn Ile 115 120 125Ser Pro
Tyr Ser Phe Leu Ser Pro Asp Thr Ile Ala Phe Gly Thr Trp 130 135
140Ile Leu Gln Leu Pro Gly Leu Leu Trp Gln Met Phe Val Ser Phe
Pro145 150 155 160Ile Leu Tyr Ser Thr Trp Ile Leu Trp Leu Leu Leu
Ser Gly Lys Thr 165 170 175Val Ala Val Ile Ala Ile Leu Leu Ala Ser
Pro Thr Val Met Ala 180 185 190422354PRTHuman Pegivirus 2 422Tyr
Lys His Gln Ser Asp Ser Tyr Leu Lys Tyr Cys Thr Ile Thr Asn1 5 10
15Ala Ser Thr Ala Met Asn Cys Asp Cys Pro Phe Gly Thr Phe Thr Arg
20 25 30Asn Thr Glu Ser Arg Phe Ser Ile Pro Arg Phe Cys Pro Val Lys
Ile 35 40 45Asn Ser Ser Thr Phe Ile Cys Ser Trp Gly Ser Trp Trp Trp
Phe Ala 50 55 60Glu Asn Ile Thr Arg Pro Tyr Ser Asp Val Gly Met Pro
Pro Ala Pro65 70 75 80Ile Ser Ala Leu Cys Tyr Ile Tyr Ser Asn Asn
Asp Pro Pro Pro Trp 85 90 95Tyr His Asn Thr Thr Ile Ile Pro Gln Asn
Cys Arg Asn Ser Thr Ala 100 105 110Asp Pro Thr Thr Ala Pro Cys Arg
Asp Lys Trp Gly Asn Ala Thr Ala 115 120 125Cys Ile Leu Asp Arg Arg
Ser Arg Phe Cys Gly Asp Cys Tyr Gly Gly 130 135 140Cys Phe Tyr Thr
Asn Gly Ser His Asp Arg Ser Trp Asp Arg Cys Gly145 150 155 160Ile
Gly Tyr Arg Asp Gly Leu Ile Glu Phe Val Gln Leu Gly Gln Ile 165 170
175Arg Pro Asn Ile Ser Asn Thr Thr Ile Glu Leu Leu Ala Gly Ala Ser
180 185 190Leu Val Ile Ala Ser Gly Leu Arg Ala Gly Tyr Gly Cys Ser
Arg Ala 195 200 205His Gly Val Val His Cys Tyr Lys Cys Pro Ser Tyr
Arg Asp Leu Glu 210 215 220Gln Phe Gly Pro Gly Leu Gly Lys Trp Val
Pro Leu Pro Gly Glu Pro225 230 235 240Val Pro Glu Leu Cys Ile Asn
Pro Gln Trp Ala Arg Arg Gly Phe Arg 245 250 255Val Ser Asn Asn Pro
Leu His Leu Ile Gln Thr Phe Val Glu Asp Ile 260 265 270Phe Leu Ala
Pro Phe Cys Ser Pro Thr Pro Gly Arg Val Arg Val Cys 275 280 285Asn
Asn Thr Ala Phe Tyr Pro Thr Gly Gly Gly Phe Val Gln Leu Ile 290 295
300Gly Asp Val Gln Val Leu Thr Pro Asn Thr Ala Ser Leu His Ser
Leu305 310 315 320Leu Thr Leu Ile Ser Leu Ile Leu Leu Val Cys Val
Val Ser Gly Ala 325 330 335Arg Phe Ile Pro Leu Ile Ile Ile Phe Phe
Trp Ser Val Arg His Val 340 345 350Tyr Ala423237PRTHuman Pegivirus
2 423Ser Cys Tyr Leu Ser Cys Asp Trp Ala Val Cys Asn Asp Ala Phe
Cys1 5 10 15Phe Thr Ser Gly Thr Cys Ala Thr Phe Asn Asp Val Leu Cys
Leu Pro 20 25 30Val Ala Ala Arg Ile Ser Ser Cys Gly His Ala Val Pro
Pro Pro Asp 35 40 45Arg Gly Trp Glu Val Pro Ala Ala Met Ser Trp Ala
Ile Ser Arg Thr 50 55 60Thr Gly Leu Thr Phe Asp Val Phe Ser Phe Ile
Gln Tyr Phe Pro Thr65 70 75
80Val Pro Gly Asn Asn Thr Asp Ile Ile Tyr Cys Gly Glu Pro Thr Phe
85 90 95Phe Gly Asp Ile Thr Gly Ile Tyr Trp Pro Tyr Phe Leu Pro Gly
Leu 100 105 110Leu Leu Leu Tyr Leu Thr Pro Leu Leu Gly Phe Arg Leu
Met Leu Ala 115 120 125Gly Phe Asn Ile Asp Gly Leu Phe Pro Ile Arg
His Ala Thr Ala Ala 130 135 140Leu Arg Phe Ser Thr Ser Arg Ala Thr
Met Cys Val Val Ser Ala Phe145 150 155 160Leu Ile Tyr Ile Leu Ser
His Pro Val Asn Ala Ala Leu Asn Arg Met 165 170 175Phe Leu Ala Ser
Ala Asn Leu Glu Met Ile Leu Ser Phe Asp Thr Tyr 180 185 190His Glu
Thr Ile Leu Tyr Ile Ala Cys Leu Leu Leu Tyr Leu Gln Val 195 200
205Ser Pro Arg Ala Gly Leu Ala Ala Met Val Ala Ile Lys Leu Ser Arg
210 215 220Gly Leu Leu Phe Ala Val Val Leu Ala His Gly Val Cys225
230 235424240PRTHuman Pegivirus 2 424Arg Pro Gly Arg Val Phe Gly
Leu Glu Val Cys Ala Asp Ile Ser Trp1 5 10 15Leu Val Glu Phe Thr Gly
Asn Cys Thr Trp Tyr Met Ser Cys Val Phe 20 25 30Ser Phe Trp Cys Ala
Val Phe Ala Phe Thr Ser Pro Leu Gly Arg His 35 40 45Tyr Lys Leu Gln
Ile Tyr Arg Tyr Trp Ala Gln Val Tyr Ala Arg Leu 50 55 60Ile Leu Ala
Val Gly Cys Gly Pro Leu Gly Arg Glu Phe His Phe Arg65 70 75 80Ala
Ser Val Gly Val Leu Trp Cys Gly Ala Cys Met Leu Trp Pro Arg 85 90
95Glu Cys Ser Glu Ile Ser Leu Val Phe Ile Leu Cys Ala Leu Thr Val
100 105 110Asp Thr Ile Asp Thr Trp Leu Val Ala Cys Leu Ser Ala Gly
Pro Ser 115 120 125Ala Arg Thr Leu Ala Thr Leu Ala Asp Asp Met Ala
Arg Phe Gly Asp 130 135 140His Arg Ala Leu Arg Ala Val Leu Arg Cys
Phe Gly Ser Arg Gly Thr145 150 155 160Tyr Ile Tyr Asn His Met Gly
Gln Val Ser Glu Arg Val Ala Gln Ala 165 170 175Val Arg Asp Phe Gly
Gly Cys Leu Glu Pro Val Val Leu Glu Glu Pro 180 185 190Thr Phe Thr
Glu Val Val Asp Asp Thr Met Asn Leu Val Cys Gly Gln 195 200 205Leu
Leu Gly Gly Lys Pro Val Val Ala Arg Cys Gly Thr Arg Val Leu 210 215
220Val Gly His Leu Asn Pro Glu Asp Leu Pro Pro Gly Phe Gln Leu
Ser225 230 235 240425628PRTHuman Pegivirus 2 425Ala Pro Val Ile Ile
Thr Lys Pro Ser Ile Gly Thr Trp Ser Phe Leu1 5 10 15Lys Ala Thr Leu
Thr Gly Arg Ala Glu Thr Pro Gly Ser Gly Gln Ile 20 25 30Val Val Leu
Ser Ser Leu Thr Gly Arg Ser Met Gly Thr Ala Val Ser 35 40 45Gly Thr
Leu Tyr Ala Thr Gly His Gly Ala Gly Ala Arg Gly Leu Ala 50 55 60Thr
Cys Ala Gly Leu Arg Thr Pro Leu Tyr Thr Ala Leu Ser Asp Asp65 70 75
80Val Val Ala Tyr Ser Cys Leu Pro Gly Met Ser Ser Leu Glu Pro Cys
85 90 95Arg Cys Ala Pro Ser Arg Val Trp Val Met Asn Asn Asn Gly Gly
Leu 100 105 110Val Cys Gly Arg Val Glu Asn Glu Asp Val Cys Leu Asp
Cys Pro Thr 115 120 125His Ile Asp Gln Leu Arg Gly Ala Ser Gly Ser
Pro Val Leu Cys Asp 130 135 140His Gly His Ala Tyr Ala Leu Met Leu
Gly Gly Tyr Ser Thr Ser Gly145 150 155 160Ile Cys Ala Arg Val Arg
Ile Val Arg Pro Trp Gln Asn Ala Tyr Ser 165 170 175Ser Ser Gly Gly
Gln Gly Gly Met Gln Ala Pro Ala Val Thr Pro Thr 180 185 190Tyr Ser
Glu Ile Thr Tyr Tyr Ala Pro Thr Gly Ser Gly Lys Ser Thr 195 200
205Lys Tyr Pro Val Asp Leu Val Lys Gln Gly His Lys Val Leu Val Leu
210 215 220Ile Pro Ser Val Ala Val Val Lys Ser Met Ala Pro Tyr Ile
Lys Glu225 230 235 240Thr Tyr Lys Ile Arg Pro Glu Ile Arg Ala Gly
Thr Gly Pro Asp Gly 245 250 255Val Thr Val Ile Thr Gly Glu Asn Leu
Ala Tyr Met Thr Tyr Gly Arg 260 265 270Phe Leu Val Asp Pro Glu Thr
Asn Leu Gln Gly Tyr Ala Val Val Ile 275 280 285Cys Asp Glu Cys His
Asp Thr Ser Ser Thr Thr Leu Leu Gly Ile Gly 290 295 300Ala Val Arg
Met Tyr Ala Glu Lys Ala Gly Val Arg Thr Val Val Phe305 310 315
320Ala Thr Ala Thr Pro Ala Gly Ile Gln Val Gln Pro His Pro Asn Ile
325 330 335Asp Glu Tyr Leu Leu Thr Asn Glu Gly Asp Val Asp Phe Tyr
Gly Ala 340 345 350Lys Ile Lys Leu Asp Asn Ile Arg Thr Gly Arg His
Val Ile Phe Cys 355 360 365His Ser Lys Ala Arg Cys Ala Glu Leu Thr
Gln Gln Leu Ser Gly Leu 370 375 380Gly Val His Ala Val Ser Phe Trp
Arg Gly Cys Asp Ile Lys Thr Ile385 390 395 400Pro Ala Ser Gly Ser
Ile Val Val Val Ala Thr Asp Ala Leu Ser Thr 405 410 415Gly Phe Thr
Gly Asn Phe Asp Ser Val Ile Asp Cys Gly Cys Cys Val 420 425 430Glu
Gln Thr Val Thr Ile Asp Met Asp Pro Thr Phe Ser Ile Ser Ala 435 440
445Arg Val Val Pro Cys Thr Ala Ala Leu Arg Met Gln Arg Arg Gly Arg
450 455 460Thr Gly Arg Gly Arg Arg Gly Ala Tyr Tyr Thr Thr Thr Pro
Gly Ala465 470 475 480Ala Pro Cys Val Ser Val Pro Asp Ala Asn Val
Trp Gln Ala Ala Glu 485 490 495Ser Ala Met Val Phe Tyr Asp Trp Asn
Ala Ala Arg Ile Gln Gln Cys 500 505 510Leu Ala Ala Tyr His Asp Leu
Gly Cys Thr Pro Arg Ile Gly Cys Asp 515 520 525Pro His Thr Pro Val
Arg Val Met Asp Thr Leu Arg Ala Tyr Leu Arg 530 535 540Arg Pro Glu
Val Thr Thr Ala Ala Leu Ala Gly Glu Gln Trp Pro Leu545 550 555
560Leu Tyr Gly Val Gln Leu Cys Ile Cys Lys Glu Thr Glu Ala His Gly
565 570 575Pro Asp Asp Gly Ile Lys Trp Lys Cys Leu Leu Asn Asn Thr
Asn Lys 580 585 590Thr Pro Leu Leu Tyr Ala Leu Asp Asn Pro Thr Leu
Glu Phe Thr Thr 595 600 605Gln His Asp Leu Thr Arg Arg Ile Ala Gly
Ala Leu Ser Ser Thr Val 610 615 620Phe Val Glu Ala62542641PRTHuman
Pegivirus 2 426Gly Tyr Gly Pro Ile Leu Leu Ala Gly Ala Ala Leu Ala
Ala Ser Phe1 5 10 15Ala Phe Ala Gly Ala Thr Gly Ala Leu Val Pro Ser
Ala Val Trp Ser 20 25 30Val Asp Asn Gly Pro Ala Gly Val Thr 35
40427262PRTHuman Pegivirus 2 427Arg Pro Asp Ala Thr Asp Glu Thr Ala
Ala Tyr Ala Gln Arg Leu Tyr1 5 10 15Gln Ala Cys Ala Asp Ser Gly Ile
Leu Ala Ser Leu Gln Gly Thr Ala 20 25 30Cys Ala Ala Leu Ser Lys Leu
Ala Asp Ala Ser Arg Gly Ala Ser Gln 35 40 45Tyr Leu Ala Ala Ala Pro
Pro Ser Pro Ala Pro Leu Val Gln Val Leu 50 55 60Gln Phe Leu Glu Thr
Asn Phe Ser Ser Ile Ala Ser Phe Gly Leu Leu65 70 75 80Cys Ala Gly
Cys Gln Ala Gly Glu Cys Phe Thr Ala Leu Ala Gly Leu 85 90 95Val Ser
Gly Ala Thr Ala Gly Leu Gly Gly Ala His Lys Trp Leu Leu 100 105
110Ala Ile Ala Gly Thr Trp Leu Val Ser Leu Gln Thr Gly Pro Arg Gly
115 120 125Gly Met Val Ala Gly Leu Ser Val Leu Ala Gly Cys Cys Ile
Gly Ser 130 135 140Val Thr Gly Leu Asp Phe Leu Phe Gly Cys Leu Thr
Gly Trp Glu Ala145 150 155 160Val Val Gly Ala Ala Val Ala Thr Gln
Lys Ile Leu Ser Gly Ser Ala 165 170 175Asp Met Thr Thr Leu Val Asp
Leu Leu Pro Ala Ile Phe Ser Pro Gly 180 185 190Ala Gly Ile Ala Gly
Val Val Leu Val Phe Ile Leu Ser Asn Ser Ser 195 200 205Val Thr Thr
Trp Ala Asn Arg Leu Leu Ser Met Cys Ala Lys Gln Thr 210 215 220Ile
Cys Glu Asn Tyr Phe Leu Thr Glu Arg Phe Gly Gln Gln Leu Ser225 230
235 240Lys Leu Ser Leu Trp Arg Ala Val Tyr His Trp Ala Gln Ala Arg
Glu 245 250 255Gly Tyr Thr Gln Cys Gly 260428457PRTHuman Pegivirus
2 428Val Val Gly Gly Ile Trp Ser Phe Ala Leu Cys Val Leu Arg Ala
Val1 5 10 15Trp Asp Trp Ala Ala Arg His Val Pro Arg Phe Arg Val Pro
Met Ile 20 25 30Gly Cys Ser Pro Ala Trp Cys Gly Arg Trp Leu Gly Thr
Gly Thr Leu 35 40 45Leu Thr Thr Cys Gly Cys Gly Glu Arg Val Ser Leu
Gln Cys Leu Cys 50 55 60Ser Thr Ser Asp Pro Thr Leu Ser Val Gly Arg
Trp Cys Arg Cys Ser65 70 75 80Trp Ser Val Gly Phe Pro Phe Asn Pro
Thr Thr Thr Ala Thr Gly Thr 85 90 95Leu Arg Pro Asp Ile Ser Asp Ala
Thr Lys Leu Gly Phe Arg Tyr Gly 100 105 110Val Ala Glu Ile Val Glu
Leu Glu Trp Arg Asp Asn Lys Trp His Val 115 120 125Cys Ala Ala Ser
Cys Cys Val Asp Arg Ala Ser Val Ala Ser Ala Val 130 135 140Lys Ala
Pro Pro Val Thr Ala Asn Gly Ile Pro Ile Ser Thr Phe Ser145 150 155
160Pro Pro Glu Thr Tyr Lys Leu Ser Leu Cys Ser Phe Asp Ser Val Cys
165 170 175Met Ser Asn Ser Ser Asn Pro Ala Lys Thr Leu Ser Val Cys
Ser Gln 180 185 190Glu Ala Val Glu Leu Leu Glu Glu Thr Val Asp Thr
Ala Gln Ala Val 195 200 205Met Cys Lys Asn Leu Glu Ala Arg Arg Arg
Ala Glu Tyr Asp Ala Trp 210 215 220Gln Val Arg Gln Ala Val Gly Asp
Glu Tyr Thr Arg Leu Ala Asp Glu225 230 235 240Asp Val Asp Thr Thr
Ala Ser Val Lys Pro Pro Val Ala Arg Ala Ala 245 250 255Val Gly Ser
Ser Thr Leu Asp Asp Val Asn Val Leu Thr Val Leu Arg 260 265 270Glu
Leu Gly Asp Gln Cys Gln Asn Ala Ile Lys Phe Val Val Gln Ala 275 280
285Ala Ser Arg Phe Val Pro Pro Val Pro Arg Pro Arg Thr Arg Val Ser
290 295 300Gly Val Leu Glu Arg Val Arg Met Cys Met Arg Thr Pro Pro
Ile Lys305 310 315 320Phe Glu Ala Thr Ala Val Pro Ile His Asn Ile
Ile Pro Glu Glu Cys 325 330 335His Ile Val Leu Arg Cys Thr Gly Cys
Ser Asp Gln Ala Leu Thr Val 340 345 350Pro Tyr Gly Thr Cys Thr Gln
Thr Leu Thr Lys His Leu Thr Asn Lys 355 360 365His Ser His Tyr Ile
Pro Lys Gln Lys Ile Glu Glu Asp Thr Glu Val 370 375 380Thr Val Ile
Cys Ala Val Pro Thr Thr Arg Ala Ser Lys Leu Ile Thr385 390 395
400Phe Arg Ala Gly Asp Arg Ser Val Ser Cys Cys His Pro Leu Gln Thr
405 410 415Pro Ile Arg Ala Leu Leu Leu Lys Tyr Gly Leu Pro Ile Gly
Lys Trp 420 425 430Ser Asp Cys Asn Gly Pro Leu Gly Asp Asp Ala Arg
Val Cys Asp Val 435 440 445Asn Gly Val Thr Thr Tyr Glu Pro Cys 450
455429567PRTHuman Pegivirus 2 429Met Gln Ser Tyr Ser Trp Phe Arg
Pro Ile Val Ala Pro Thr Thr Pro1 5 10 15Pro Leu Pro Ala Thr Arg Ser
Val Ala Gly Ile Leu Arg Ala Asp Thr 20 25 30Ser Arg Val Tyr Thr Thr
Thr Ala Val Asp Val Ser Glu Arg Gln Ala 35 40 45Lys Val Thr Ile Asp
Gln Thr Ser Ala Lys Val Asp Gln Cys Phe Arg 50 55 60Asp Thr Tyr Asn
Cys Cys Leu Ala Lys Ala Lys Thr Phe Lys Gln Ser65 70 75 80Gly Met
Ser Tyr Glu Asp Ala Val Ser Lys Met Arg Ala Asn Thr Thr 85 90 95Arg
Asp His Asn His Gly Thr Thr Tyr Ser Asp Leu Val Ser Gly Arg 100 105
110Ala Lys Pro Val Val Gln Lys Ile Val Asn Gln Met Arg Ala Gly Val
115 120 125Tyr Asp Ala Pro Met Arg Ile Ile Pro Lys Pro Glu Val Phe
Pro Arg 130 135 140Asp Lys Glu Thr Arg Lys Pro Pro Arg Phe Ile Val
Phe Pro Gly Cys145 150 155 160Ala Ala Arg Val Ala Glu Lys Met Ile
Leu Gly Asp Pro Gly Ala Ile 165 170 175Thr Lys His Val Leu Gly Asp
Ala Tyr Gly Phe Ala Thr Pro Pro His 180 185 190Glu Arg Ala Arg Leu
Leu Glu Gln Trp Trp Asn Arg Ala Thr Glu Pro 195 200 205Gln Ala Ile
Ala Val Asp Ala Ile Cys Phe Asp Ser Thr Ile Thr Ala 210 215 220Glu
Asp Met Asp Arg Glu Ala Asn Ile Val Ala Ala Ala His Ala Asp225 230
235 240Pro Glu Gly Val His Gly Leu Tyr Asn Tyr Tyr Lys Arg Ser Pro
Met 245 250 255Cys Asp Ile Thr Gly Asn Ile Val Gly Val Arg Cys Cys
Arg Ala Ser 260 265 270Gly Thr Leu Thr Thr Ser Ser Gly Asn Thr Leu
Thr Cys Tyr Leu Lys 275 280 285Val Arg Ala Ala Cys Thr Arg Ala Gly
Ile Lys Pro Ile Gly Leu Leu 290 295 300Ile His Gly Asp Asp Thr Leu
Ile Ile Thr Glu Arg Cys Ala Gln Glu305 310 315 320Thr Leu Asp Glu
Phe Ser Arg Ala Leu Asp Asp Tyr Gly Phe Pro His 325 330 335Thr Phe
Gln Ala Ser Gly Asp Leu Ser Ser Ile Glu Cys Cys Ser Ala 340 345
350His Val Asp Ser Val Cys Leu Arg Gly Gly Met Arg Arg Met Leu Val
355 360 365Pro Gln Ala Arg Arg Ala Ile Ala Arg Val Leu Gly Glu Lys
Gly Asp 370 375 380Pro Leu Gly Val Ile Ser Ser Tyr Ile Val Met Tyr
Pro Thr Ala Ala385 390 395 400Val Thr Val Tyr Val Leu Leu Pro Leu
Leu Cys Met Leu Ile Arg Asn 405 410 415Glu Pro Ser Gln Thr Gly Thr
Phe Val Thr Leu Thr Val His Gly Asn 420 425 430Ser Val Ser Val Pro
Val Trp Leu Leu Pro Thr Ile Ile Val Asn Leu 435 440 445His Gly Arg
Asp Ala Leu Gln Val Val Arg His Thr Ala Ala Ser Met 450 455 460Ala
Glu Leu Ser Ser Ala Leu Ala Phe Phe Gly Met Arg Gly Leu Asn465 470
475 480Cys Trp Arg Arg Arg Arg Arg Ala Ile Arg Ala Asp Met Ile Lys
Leu 485 490 495Gly Gly Trp Asn Ala Asn Phe Ala Gln Met Leu Leu Trp
Ser Pro Glu 500 505 510Val Arg Thr Pro Gln Pro Glu Pro Lys Gly Met
Cys Leu Leu Pro Pro 515 520 525Glu Leu Trp Glu Arg Pro Tyr Glu Asn
Leu His Leu Ser Thr Ile Asp 530 535 540Arg Asn Arg Gly Ala Ser Arg
Leu Arg Phe Trp Leu Val Ala Ser Ala545 550 555 560Ile Leu Ala Leu
Leu Cys Leu 5654309867DNAHuman Pegivirus 2misc_feature(1)..(15)N =
A, C, T, or Gmisc_feature(3260)..(3360)N = A, C, T, or
Gmisc_feature(9542)..(9636)N = A, C, T, or
Gmisc_feature(9857)..(9867)N = A, C, T, or G 430nnnnnnnnnn
nnnnnaatgc gcatattgct acttcggtac gcctaattgg taggcgcccg 60gccgaccggc
cccgcaaggg cctagtagga cgtgtgacaa tgccatgagg gatcatgaca
120ctggggtgag cggaggcagc accgaagtcg ggtgaactcg actcccagtg
cgaccacctg 180gcttggtcgt tcatggaggg catgcccacg ggaacgctga
tcgtgcaaag ggatgggtcc 240ctgcactggt gccatgcgcg gcaccactcc
gtacagcctg atagggtggc ggcgggcccc 300cccagtgtga cgtccgtgga
gcgcaacatg gggtgttcaa ctgatcaaac catttgttct 360ccagtcgtgg
gggccgacta taatacctcc tcgggctgcc gggccttaaa tgggagctac
420cactgcggtg
gtggctcttg ccggtcacca agtcgtgtgc aggtcgcggg acgagtcttg
480cggctgtgcg cattccttgc gttgatcgga tccggtatgt gttcgatccg
gtccaaaact 540gaagggcgca ttgagtcagg gcaaatattg cagtctcagc
gcgcatgttg gactggtgag 600ggtttcgctt tcttttctaa ctgttgcaat
caatctgata ttatgtggtg tttgcaccgt 660tggtgtgtga caagacctgg
ctgtttagtg tgcacgggca atgccactca tcctatctgc 720tgggactatc
ttgggtccgg tgtaagtcgg cggcctgcac gtcgaatggg tgagggagct
780gaagtgcttc ttcgcttgat cggcattgca ggttggctcg ggctgttagc
tgagactctt 840ggtatgtccg aattctatgc agctattcta tgctttggat
ttattgcttg gtatggctgg 900ggtataccta aaacattggt gtgcacggtc
tgccctgcag tgaacatttc tccctatagc 960ttcttatctc cagatactat
cgcatttggt acgtggatac tacaactacc tggtcttttg 1020tggcaaatgt
ttgtcaactt tcctatactt tacagcactt ggattctttg gttgttgctc
1080agcggcaaga ctgttgctgt gatagcaatc cttttggcta gtcctacggt
tatggcatac 1140aagcatcaat ctgaaagcta cctcaaatac tgtaccataa
ccaatgcttc aactgctatg 1200aattgtgact gcccctttgg aacctttact
cgtaatactg agtctcgttt ctctatacct 1260agattctgtc ctgttaaaat
tgacagctct acatttatct gctcgtgggg gtcgtggtgg 1320tggtttgctg
agaacatcac gcgtccatac tcggacgttg gcatgccgcc agcaccgatc
1380tccgctttgt gctatatcta tgcaaacaat gacccacctc cttggtatca
taacacaact 1440atcatacctc agaactgtcg caactcctcg gctgatccta
ccactgctcc atgccgtgac 1500aagtggggca atgcaactgc ttgtattctt
gaccgccggt cgcggttctg cggggactgc 1560tatggcggtt gtttctatac
taatggcact cacgatcgat cctgggatcg atgcgggatt 1620ggttaccgtg
atggactcat agagtttgtg cagcttggtc agattcgacc caacatctcg
1680aatacgacca ttgaactcct cgctggcgcc tcgcttgtga tcgcatccgg
tcttcggcct 1740ggctacggtt gcagccgtgc gcatggcgtg gtgcactgct
ataggtgtcc ttcatatcgt 1800gaccttgaac agtttggtcc cgggctcggg
aaatgggtgc cactgcctgg cgagcctgtc 1860ccggagttgt gtattaaccc
tcagtgggcg agacgcggct tccgggtatc taacaaccct 1920ttaagcttgc
tgcagacctt cgttgaggac attttcctag cacctttttg caacccgacg
1980cctggccgtg tacgtgtgtg taacaatact gctttctatc cgaagggagg
cggctttgtg 2040cagctcatcg gagacgtcca ggtgttaacc cctaacactg
catctttaca ctctctgctg 2100actttaatat cccttatttt gttagtgtgt
gttgtttctg gcgcgcgatt cgtcccattg 2160tttatcatat ttttctggag
cgtgcgtcac gtatatgctt cttgttactt aagctgtgat 2220tgggctgttt
gcaacgatgc gttctgtttc acatctggca cttgtgctac tttcaatgac
2280gtcttgtgtc tgccggttgc gacgcgcgta tcgtcctgcg ggcatgctgt
accacctccc 2340gaccgtggtt gggaggtgcc tgcggcgatg tcatgggcga
tctcacgaac tactggcttg 2400acgttcgatg tcttttcctt catccagtac
tttcctactg tgcctggcaa caacactgat 2460atcatttact gtggtgaccc
aactttcttc ggggacatca cgggcatcta ttggccttac 2520tttttgcctg
gcatgttgct cttgtacttg actcctttcc tgggtttaag gttaatgctt
2580gctggcttca atatagatgg cttgtttccc atacggcacg ccacggctgc
actgaggttc 2640tcgacttcgc gtgtgacctt gagtgtcgta cttgctttct
taatctatat actatctcac 2700cctgttaatg ctgcgctcaa tagaatgttc
ctagcatctg caaatttaga gatgatctta 2760tcctttgaca cctatcatga
gactattctt tacattcttt gcctgttgct ctacctccag 2820gtgtcgcccc
gcgctgggct ggccgctatg gtggccgtca agctatctcg aggcctgtta
2880ttcgccgtgg tgttggcgca cggagtgtgc cgacccgggc gggtatttgg
tcttgaggtc 2940tgcgcggaca tcacttggtt ggtggagttt actggcaact
gcacttggta catgtcctgt 3000gttttctcat tttggtgcgc agtgttcgcc
ttcaccagtc cacttggacg acggtataag 3060cttcagatct accggtactg
ggcgcaggtc tatgccagaa tcatcctcgc tgtcggttgt 3120ggtcctctcg
gacgggagtt ccatttccgt gcaggcgtgg gtgcgttttg gtgtggtgct
3180tgcatgctct ggccccgtga gtgctctgaa atcagcttgg tcttcattct
gtgtgctctg 3240acgatggaca ccatagacan nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 3300nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3360gtgttgcgtt gctttggatc
acgtggcaca tacatataca accacatggg ccaggtctca 3420gaacgggtgg
cgcaagcagt cagggatttc ggcggttgct tggaaccagt cgtgttggag
3480gagcccacct ttactgaggt cgtggatgat acaatgagtc tagtgtgtgg
acaattgctt 3540ggaggtaaac ctgtggtggc ccgctgcggc acgcgtgtct
tggtgggaca cctcaaccct 3600gaagacttgc cacctggttt ccagctgagt
gctccggtgg ttatcaccaa accaagcatt 3660ggtacgtggt ccttccttaa
ggcgacactc acagggcgtg ctgagacacc gggatccggc 3720cagatcgtgg
tgttgtcttc cctgacaggt cggtcaatgg gtaccgcagt gaatggcaca
3780ctgtatgcga ccggccatgg tgctggtgcg cgcggcctag ccacgtgcgc
tggcttgagg 3840acgccacttt acacggcact atcagatgat gtcgtggcct
actcttgcct tccgggcatg 3900agttccctgg agccctgctg ctgttcgccg
agccgggttt gggtgatgaa caacaatgga 3960gggttggtgt gtggcagagt
ggagaaggac gacgtctgtt tggactgtcc cacgcacata 4020gatcagctgc
ggggtgcctc ggggtcaccg gttttgtgtg atcacggtca cgcatacgcg
4080ttgatgctcg gtggctactc taccagcggt atttgtgcgc gtgtccggat
agtccggcca 4140tggcagaacg cctattcctc ctcagggggg caaggcggaa
tgcaggcgcc agctgtgaca 4200ccaacatact ctgaaatcac ctactatgcc
cctactggtt ctggtaagtc aacaaaatat 4260ccagtggacc tagtcaaaca
gggacacaag gtcttggttc ttataccaag cgtggctgtc 4320gtcaaaagca
tggcccctta cattaaggag acatataaga ttagacctga aattagagct
4380ggcacagggc ctgacggtgt gacggtcatc actggtgaga acttggcgta
catgacctat 4440ggccgtttcc tcgtggatcc ggagacgaat ctgcggggtt
atgccgtagt catttgcgac 4500gagtgtcacg atacatcatc caccacgcta
ctcggcattg gcgcagtgcg catgtatgcc 4560gagcaagctg gagtgaagac
cgttgtattc gccacagcca ctcctgctgg tatccaagta 4620cagccacatc
caaacattga tgaatattta ttgactgaca caggcgacgt ggatttctac
4680ggcgccaaaa tcaaattgga caacattaga actggtagac atgttatctt
ttgtcactcg 4740aaagccaagt gtgcggaact aacgcagcag ctctccggcc
ttggtgttcg tgcagtgagt 4800ttttggcgcg gctgtgacat caaaaccatt
cccgcctcag actctattgt tgtggtggca 4860actgatgcat tgtccacagg
ctacacagga aattttgatt cggtcattga ctgcgggtgt 4920tgcgtagagc
aaactgtgac aattgacatg gaccccacgt tctccatctc ggcccgagta
4980gtgccatgca ctgctgcatt gcgaatgcag cggcgcggac gcaccggtcg
tggcagaagg 5040ggagcgtact acacaaccac tccaggagca gcaccctgcg
tcaacgttcc cgatgctaac 5100gtctggcaag cagtggagtc agccatggtc
ttctatgatt ggaatgctgc caggatacag 5160cagtgtctgg cggcatacca
tgatttaggg tgtacaccac gcatcagttg tgacccacac 5220actccagtgc
gggtgatgga cacactgagg gcgtacctgc gcagacctga ggtgacgact
5280gcggctctcg caggagagca gtggccgctg ctatacggtg tgcagttgtg
catctgcaaa 5340gagaccgagg cccacggtcc agatgatggc atcaagtgga
aatgtttact caataacaac 5400aataaaacac ccctgttgta tgccttagac
aatcctacac tggaattcac tacccaacat 5460gacttgactc gccgtatagc
cggcgctcta tcgagcacag tgttcgtgga gacaggctat 5520ggccccatcc
tcctcgctgg cgccgctttg gctgcctcct tcgcctttgc gggcgccact
5580ggagctttag tgccgtcggc cgtttggagt gttgacaacg ggcctactgg
cgtgacccgt 5640cccgacgcga cagacgagac cgtggcctac gcgcagcgct
tgtaccacgc ctgcgcagat 5700tcaggaattc tcgccagctt gcagggcacg
gcgtgtgcgg cactgagtaa actggccgat 5760gccagtaggg gtgctagtca
atatctggca accgcgcctc cttcgcccgc ccccctggta 5820caggtgctgc
agttcctcga gaccaacttt agctccattg catctttcgg tctgctctgt
5880gctggttgtc aggctggtga gtgcttcact gcgcttgccg ggttggtgtc
cggtgctaca 5940gctggcttgg gaggtgccca taagtggttg ttagctattg
caggaacttg gctagttagc 6000ctgcagaccg ggccccgtgg cggcatggtt
gcgggtctct cggttctagc gggctgttgc 6060atcggcggtg tcaccgggct
tgacttcctg tttgggtgcc ttacaggttg ggaggccgtg 6120gtcggtgctg
ccgttgcgac acagaagatc ttgtctggtt cggctgatat gaccactctg
6180gtagatctct tacctgctct cttttcccct ggtgctggca tagctggcat
cgtgcttgtc 6240tttattctaa gcaacacaag tgtaaccgca tgggccaatc
ggctattgtc catgtgtgca 6300aaacaaacca tttgtgaaaa ctacttctta
actgagaaat ttggccaaca attaagcaaa 6360ctttccttgt ggcgtgctgt
gtaccattgg gcgcaggcac atgaggggta cacacagtgc 6420ggtgtggttg
gcgggatctg gagctttgtc ctgtgcattc tacgcgctgc gtgggattgg
6480gcggccaagc atgtgccacg gttccgtgtg cctatgattg gctgctcacc
tgcgtggtgc 6540gggcgctggc ttggtactgg caccttgttg accacctgtg
ggtgtggaga acgtgtatcc 6600cttcaatgcc tttgttcaac atctgaccca
atactcagtg tgggccgttg gtgccggtgt 6660agttggagtg ttgggtttcc
attcaacccg accacgacag ccaccggcac tttacggccg 6720gacatcggcg
acgccaccag attgggtttc cggtatggca tcgccgagat cgtggagcta
6780gaacggcggg gcgacaaatg gcatgtctgt gcagcatctt gttgcttgga
ccgagctagc 6840gttgcatccg ctgtgaaggc ccctccggtc acggctaatg
gcatacctat cagtcctttc 6900tctccaccac aaacttacaa actctctctc
tgctcttttg attcagtttg catgtctatc 6960aactcatgta atccatctaa
gatcctgagt gtgtgctcac aggaagccgt tgagctgctg 7020gaagaaacag
tcgacacggc acaaacaatg atgtgtaaaa atctggaggc gcgaagacgc
7080gccgaatttg acgcatggca agtccgccaa gcagttggcg acgagtacac
acgcttggca 7140gatgaggatg tcgacacgat aacgtcggtg aaacccccgg
tggccagggc tgctgtgggt 7200agctcaacgt tggatgatgt tggcgtgctg
actgtcttgc gcgagctcgg cgaccaatgc 7260caaaatgcta tcaaatttgt
agttgaagcg gcctcacggt ttgttccacc agtgcccaag 7320ccgcgcacgc
gtgtctcggg tgtgctggag cgtgtgcgca tgtgcatgcg cacgccacca
7380atgaagtttg aggccgccgc agtaccaatc cacaacataa tcccagaaaa
atgtcacatt 7440gtgctacgct gtaccggctg tagtgaccag gccttgactg
ttccgtacgg cacttgcact 7500cagactttaa gcagccattt gactaacaaa
cacagtcact acattccaaa acagaagata 7560gaagaagaca cagaagtaac
tgtcatttgc gccgtaccaa caaagcgcgc aagcaaactc 7620attactttca
gagtaggtga tcgatcagtc tcatgttgtc accccttgca aactcctgtt
7680agggccctgc ttctaaagta cgggttgcct atcgggaagt ggtccgactg
caacggccca 7740cttggtgacg acgctcgagt ctgtgacgtc aatggagtga
caacttatga accatgcatg 7800caatcctaca gttggtttcg accgattgtg
gcaccaacaa ccccaccttt gcctgtaacc 7860cggagcgtgg ctggcatttt
acgcgcagac acatcgcgcg tttacaccac aacagcggtt 7920gatgtctccg
agcggcaggc taaggtcaca attgatcaaa agtcagccaa ggtggatcag
7980tgtttccgag acacatacaa ctgttgcctt gctaaggcaa agaccttcag
acaatctggc 8040atgtcatatg aggatgctgt gtcaaagatg cgcgcaaaca
ccacgcgtga tcataacact 8100ggcatcactt atacagattt ggtctctgga
cgcgcaaaac ctgctgttca gaaaattgta 8160gatcaaatgc gctctggagt
gtacgacgct ccaatgcgca ttatcccaaa gcctgaagtg 8220tttcctcgag
acaagtcaac acggaagcca ccacggttca tcgttttccc tgggtgcgcc
8280gcacgagtcg cggagaaaat gatcctgggc gatcctggtg cgataaccaa
gcacgtgctg 8340ggtgatgcct acgggtttgc cactccgccg catgagcgtg
cgcgcctatt ggaacaatgg 8400tggaaccgcg cgacggagcc acaggctatc
gcggttgatg cgatctgctt tgatagcacc 8460atcacggcgg aggacatgga
tcgcgaggcc cacatcgtgg ctgcagcgca cgcggaccca 8520gaaggtgttc
atggcctata caattattac aaaagaagcc ccatgtgtga catcacagga
8580aaagttgtcg gggtgcgttg ctgtcgagcc tcaggtacgc ttacaacaag
cagtggcaac 8640acgcttactt gctacctcaa ggttcgcgcg gcttgcacgc
gcgccggcat taaaccaatt 8700ggcttactaa ttcatggaga tgacaccctc
attatcacgg aacgttgcac tcaagaaact 8760ctcgatgagt tcagcaacgc
acttgatgac tacgggtttc ctcacacctt ccaggtgtct 8820ggggacctct
cgtctatcga gtgctgtagt gcacgtgtgg acagcgtttg cctccgagga
8880ggtatgcgtc gcatgcttgt gccacaagct cgacgtgcga tcgcacgcgt
tctcggagaa 8940aagggtgatc cactgggtgt tatcagcagc tatattgtca
tgtaccctac tgcagccgtg 9000actgtctacg tactactgcc cctgttgtgc
atgctcattc ggaatgagcc atcgcagacg 9060gggacattgg tgacgctgac
ggtccacggt aacagtgtga gcgtgccagt gtggctgctt 9120ccaaccatca
ttgcaaattt acatggccgt gacgcactac aggtggtccg ccacagtgca
9180gcttccatgg cggaactgtc gtcagcgttg gccttctttg gcatgagagg
gttgaactgc 9240tggcggcgga gacgccgtgc catcaggact gacatgatca
aattgggcgg gtggaatgcg 9300aatttcgcgc agatgttact gtggtcaccg
gaggtaagaa caccacagcc cgaaccaaag 9360ggcatgtgtc ttttgccacc
ggaactatgg gagcgtccgt acgaaaattt gcacttgagc 9420acgatcgacc
gcgaccgtgg tgctagtcgc ttacggtttt ggttggttgc tagtgctgta
9480ctcgctctgc tttgcttgta aatcctaaat caatgtagta ccaggactac
aaggcaggag 9540gnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 9600nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnncccc cccatgtcgc gcgtaagcgc 9660acgggcaagg cagctaggct
gagagtctgg gcaactctcc cgtaccccac ccgaggctac 9720gcctcgtcct
ggcgaggacc gtaaacatac gtcgtcagcg tggtgacctg acgtatcttg
9780ttaaccactt aatggtcgta actcgacccc cgtgccgggg atctaagcgc
ggcaccgcga 9840tgaggggggt caacggnnnn nnnnnnn 986743179PRTHuman
Pegivirus 2 431Met Gly Cys Ser Thr Asp Gln Thr Ile Cys Ser Pro Val
Val Gly Ala1 5 10 15Asp Tyr Asn Thr Ser Ser Gly Cys Arg Ala Leu Asn
Gly Ser Tyr His 20 25 30Cys Gly Gly Gly Ser Cys Arg Ser Pro Ser Arg
Val Gln Val Ala Gly 35 40 45Arg Val Leu Arg Leu Cys Ala Phe Leu Ala
Leu Ile Gly Ser Gly Met 50 55 60Cys Ser Ile Arg Ser Lys Thr Glu Gly
Arg Ile Glu Ser Gly Gln65 70 75432191PRTHuman Pegivirus 2 432Ile
Leu Gln Ser Gln Arg Ala Cys Trp Thr Gly Glu Gly Phe Ala Phe1 5 10
15Phe Ser Asn Cys Cys Asn Gln Ser Asp Ile Met Trp Cys Leu His Arg
20 25 30Trp Cys Val Thr Arg Pro Gly Cys Leu Val Cys Thr Gly Asn Ala
Thr 35 40 45His Pro Ile Cys Trp Asp Tyr Leu Gly Ser Gly Val Ser Arg
Arg Pro 50 55 60Ala Arg Arg Met Gly Glu Gly Ala Glu Val Leu Leu Arg
Leu Ile Gly65 70 75 80Ile Ala Gly Trp Leu Gly Leu Leu Ala Glu Thr
Leu Gly Met Ser Glu 85 90 95Phe Tyr Ala Ala Ile Leu Cys Phe Gly Phe
Ile Ala Trp Tyr Gly Trp 100 105 110Gly Ile Pro Lys Thr Leu Val Cys
Thr Val Cys Pro Ala Val Asn Ile 115 120 125Ser Pro Tyr Ser Phe Leu
Ser Pro Asp Thr Ile Ala Phe Gly Thr Trp 130 135 140Ile Leu Gln Leu
Pro Gly Leu Leu Trp Gln Met Phe Val Asn Phe Pro145 150 155 160Ile
Leu Tyr Ser Thr Trp Ile Leu Trp Leu Leu Leu Ser Gly Lys Thr 165 170
175Val Ala Val Ile Ala Ile Leu Leu Ala Ser Pro Thr Val Met Ala 180
185 190433354PRTHuman Pegivirus 2 433Tyr Lys His Gln Ser Glu Ser
Tyr Leu Lys Tyr Cys Thr Ile Thr Asn1 5 10 15Ala Ser Thr Ala Met Asn
Cys Asp Cys Pro Phe Gly Thr Phe Thr Arg 20 25 30Asn Thr Glu Ser Arg
Phe Ser Ile Pro Arg Phe Cys Pro Val Lys Ile 35 40 45Asp Ser Ser Thr
Phe Ile Cys Ser Trp Gly Ser Trp Trp Trp Phe Ala 50 55 60Glu Asn Ile
Thr Arg Pro Tyr Ser Asp Val Gly Met Pro Pro Ala Pro65 70 75 80Ile
Ser Ala Leu Cys Tyr Ile Tyr Ala Asn Asn Asp Pro Pro Pro Trp 85 90
95Tyr His Asn Thr Thr Ile Ile Pro Gln Asn Cys Arg Asn Ser Ser Ala
100 105 110Asp Pro Thr Thr Ala Pro Cys Arg Asp Lys Trp Gly Asn Ala
Thr Ala 115 120 125Cys Ile Leu Asp Arg Arg Ser Arg Phe Cys Gly Asp
Cys Tyr Gly Gly 130 135 140Cys Phe Tyr Thr Asn Gly Thr His Asp Arg
Ser Trp Asp Arg Cys Gly145 150 155 160Ile Gly Tyr Arg Asp Gly Leu
Ile Glu Phe Val Gln Leu Gly Gln Ile 165 170 175Arg Pro Asn Ile Ser
Asn Thr Thr Ile Glu Leu Leu Ala Gly Ala Ser 180 185 190Leu Val Ile
Ala Ser Gly Leu Arg Pro Gly Tyr Gly Cys Ser Arg Ala 195 200 205His
Gly Val Val His Cys Tyr Arg Cys Pro Ser Tyr Arg Asp Leu Glu 210 215
220Gln Phe Gly Pro Gly Leu Gly Lys Trp Val Pro Leu Pro Gly Glu
Pro225 230 235 240Val Pro Glu Leu Cys Ile Asn Pro Gln Trp Ala Arg
Arg Gly Phe Arg 245 250 255Val Ser Asn Asn Pro Leu Ser Leu Leu Gln
Thr Phe Val Glu Asp Ile 260 265 270Phe Leu Ala Pro Phe Cys Asn Pro
Thr Pro Gly Arg Val Arg Val Cys 275 280 285Asn Asn Thr Ala Phe Tyr
Pro Lys Gly Gly Gly Phe Val Gln Leu Ile 290 295 300Gly Asp Val Gln
Val Leu Thr Pro Asn Thr Ala Ser Leu His Ser Leu305 310 315 320Leu
Thr Leu Ile Ser Leu Ile Leu Leu Val Cys Val Val Ser Gly Ala 325 330
335Arg Phe Val Pro Leu Phe Ile Ile Phe Phe Trp Ser Val Arg His Val
340 345 350Tyr Ala434237PRTHuman Pegivirus 2 434Ser Cys Tyr Leu Ser
Cys Asp Trp Ala Val Cys Asn Asp Ala Phe Cys1 5 10 15Phe Thr Ser Gly
Thr Cys Ala Thr Phe Asn Asp Val Leu Cys Leu Pro 20 25 30Val Ala Thr
Arg Val Ser Ser Cys Gly His Ala Val Pro Pro Pro Asp 35 40 45Arg Gly
Trp Glu Val Pro Ala Ala Met Ser Trp Ala Ile Ser Arg Thr 50 55 60Thr
Gly Leu Thr Phe Asp Val Phe Ser Phe Ile Gln Tyr Phe Pro Thr65 70 75
80Val Pro Gly Asn Asn Thr Asp Ile Ile Tyr Cys Gly Asp Pro Thr Phe
85 90 95Phe Gly Asp Ile Thr Gly Ile Tyr Trp Pro Tyr Phe Leu Pro Gly
Met 100 105 110Leu Leu Leu Tyr Leu Thr Pro Phe Leu Gly Leu Arg Leu
Met Leu Ala 115 120 125Gly Phe Asn Ile Asp Gly Leu Phe Pro Ile Arg
His Ala Thr Ala Ala 130 135 140Leu Arg Phe Ser Thr Ser Arg Val Thr
Leu Ser Val Val Leu Ala Phe145 150 155 160Leu Ile Tyr Ile Leu Ser
His Pro Val Asn Ala Ala Leu Asn Arg Met 165 170 175Phe Leu Ala Ser
Ala Asn Leu Glu Met Ile Leu Ser Phe Asp Thr Tyr 180 185 190His Glu
Thr Ile Leu Tyr Ile Leu Cys Leu Leu Leu Tyr Leu Gln Val 195 200
205Ser Pro Arg Ala Gly Leu Ala Ala Met Val Ala Val Lys Leu Ser Arg
210 215 220Gly Leu Leu Phe Ala Val Val Leu Ala His Gly Val Cys225
230 235435240PRTHuman Pegivirus 2MISC_FEATURE(117)..(150)Xaa = any
amino acid 435Arg Pro Gly Arg Val Phe Gly Leu Glu Val Cys Ala Asp
Ile Thr Trp1 5 10 15Leu Val Glu Phe Thr Gly Asn Cys Thr Trp Tyr Met
Ser Cys Val Phe 20 25 30Ser
Phe Trp Cys Ala Val Phe Ala Phe Thr Ser Pro Leu Gly Arg Arg 35 40
45Tyr Lys Leu Gln Ile Tyr Arg Tyr Trp Ala Gln Val Tyr Ala Arg Ile
50 55 60Ile Leu Ala Val Gly Cys Gly Pro Leu Gly Arg Glu Phe His Phe
Arg65 70 75 80Ala Gly Val Gly Ala Phe Trp Cys Gly Ala Cys Met Leu
Trp Pro Arg 85 90 95Glu Cys Ser Glu Ile Ser Leu Val Phe Ile Leu Cys
Ala Leu Thr Met 100 105 110Asp Thr Ile Asp Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 130 135 140Xaa Xaa Xaa Xaa Xaa Xaa
Val Leu Arg Cys Phe Gly Ser Arg Gly Thr145 150 155 160Tyr Ile Tyr
Asn His Met Gly Gln Val Ser Glu Arg Val Ala Gln Ala 165 170 175Val
Arg Asp Phe Gly Gly Cys Leu Glu Pro Val Val Leu Glu Glu Pro 180 185
190Thr Phe Thr Glu Val Val Asp Asp Thr Met Ser Leu Val Cys Gly Gln
195 200 205Leu Leu Gly Gly Lys Pro Val Val Ala Arg Cys Gly Thr Arg
Val Leu 210 215 220Val Gly His Leu Asn Pro Glu Asp Leu Pro Pro Gly
Phe Gln Leu Ser225 230 235 240436628PRTHuman Pegivirus 2 436Ala Pro
Val Val Ile Thr Lys Pro Ser Ile Gly Thr Trp Ser Phe Leu1 5 10 15Lys
Ala Thr Leu Thr Gly Arg Ala Glu Thr Pro Gly Ser Gly Gln Ile 20 25
30Val Val Leu Ser Ser Leu Thr Gly Arg Ser Met Gly Thr Ala Val Asn
35 40 45Gly Thr Leu Tyr Ala Thr Gly His Gly Ala Gly Ala Arg Gly Leu
Ala 50 55 60Thr Cys Ala Gly Leu Arg Thr Pro Leu Tyr Thr Ala Leu Ser
Asp Asp65 70 75 80Val Val Ala Tyr Ser Cys Leu Pro Gly Met Ser Ser
Leu Glu Pro Cys 85 90 95Cys Cys Ser Pro Ser Arg Val Trp Val Met Asn
Asn Asn Gly Gly Leu 100 105 110Val Cys Gly Arg Val Glu Lys Asp Asp
Val Cys Leu Asp Cys Pro Thr 115 120 125His Ile Asp Gln Leu Arg Gly
Ala Ser Gly Ser Pro Val Leu Cys Asp 130 135 140His Gly His Ala Tyr
Ala Leu Met Leu Gly Gly Tyr Ser Thr Ser Gly145 150 155 160Ile Cys
Ala Arg Val Arg Ile Val Arg Pro Trp Gln Asn Ala Tyr Ser 165 170
175Ser Ser Gly Gly Gln Gly Gly Met Gln Ala Pro Ala Val Thr Pro Thr
180 185 190Tyr Ser Glu Ile Thr Tyr Tyr Ala Pro Thr Gly Ser Gly Lys
Ser Thr 195 200 205Lys Tyr Pro Val Asp Leu Val Lys Gln Gly His Lys
Val Leu Val Leu 210 215 220Ile Pro Ser Val Ala Val Val Lys Ser Met
Ala Pro Tyr Ile Lys Glu225 230 235 240Thr Tyr Lys Ile Arg Pro Glu
Ile Arg Ala Gly Thr Gly Pro Asp Gly 245 250 255Val Thr Val Ile Thr
Gly Glu Asn Leu Ala Tyr Met Thr Tyr Gly Arg 260 265 270Phe Leu Val
Asp Pro Glu Thr Asn Leu Arg Gly Tyr Ala Val Val Ile 275 280 285Cys
Asp Glu Cys His Asp Thr Ser Ser Thr Thr Leu Leu Gly Ile Gly 290 295
300Ala Val Arg Met Tyr Ala Glu Gln Ala Gly Val Lys Thr Val Val
Phe305 310 315 320Ala Thr Ala Thr Pro Ala Gly Ile Gln Val Gln Pro
His Pro Asn Ile 325 330 335Asp Glu Tyr Leu Leu Thr Asp Thr Gly Asp
Val Asp Phe Tyr Gly Ala 340 345 350Lys Ile Lys Leu Asp Asn Ile Arg
Thr Gly Arg His Val Ile Phe Cys 355 360 365His Ser Lys Ala Lys Cys
Ala Glu Leu Thr Gln Gln Leu Ser Gly Leu 370 375 380Gly Val Arg Ala
Val Ser Phe Trp Arg Gly Cys Asp Ile Lys Thr Ile385 390 395 400Pro
Ala Ser Asp Ser Ile Val Val Val Ala Thr Asp Ala Leu Ser Thr 405 410
415Gly Tyr Thr Gly Asn Phe Asp Ser Val Ile Asp Cys Gly Cys Cys Val
420 425 430Glu Gln Thr Val Thr Ile Asp Met Asp Pro Thr Phe Ser Ile
Ser Ala 435 440 445Arg Val Val Pro Cys Thr Ala Ala Leu Arg Met Gln
Arg Arg Gly Arg 450 455 460Thr Gly Arg Gly Arg Arg Gly Ala Tyr Tyr
Thr Thr Thr Pro Gly Ala465 470 475 480Ala Pro Cys Val Asn Val Pro
Asp Ala Asn Val Trp Gln Ala Val Glu 485 490 495Ser Ala Met Val Phe
Tyr Asp Trp Asn Ala Ala Arg Ile Gln Gln Cys 500 505 510Leu Ala Ala
Tyr His Asp Leu Gly Cys Thr Pro Arg Ile Ser Cys Asp 515 520 525Pro
His Thr Pro Val Arg Val Met Asp Thr Leu Arg Ala Tyr Leu Arg 530 535
540Arg Pro Glu Val Thr Thr Ala Ala Leu Ala Gly Glu Gln Trp Pro
Leu545 550 555 560Leu Tyr Gly Val Gln Leu Cys Ile Cys Lys Glu Thr
Glu Ala His Gly 565 570 575Pro Asp Asp Gly Ile Lys Trp Lys Cys Leu
Leu Asn Asn Asn Asn Lys 580 585 590Thr Pro Leu Leu Tyr Ala Leu Asp
Asn Pro Thr Leu Glu Phe Thr Thr 595 600 605Gln His Asp Leu Thr Arg
Arg Ile Ala Gly Ala Leu Ser Ser Thr Val 610 615 620Phe Val Glu
Thr62543741PRTHuman Pegivirus 2 437Gly Tyr Gly Pro Ile Leu Leu Ala
Gly Ala Ala Leu Ala Ala Ser Phe1 5 10 15Ala Phe Ala Gly Ala Thr Gly
Ala Leu Val Pro Ser Ala Val Trp Ser 20 25 30Val Asp Asn Gly Pro Thr
Gly Val Thr 35 40438262PRTHuman Pegivirus 2 438Arg Pro Asp Ala Thr
Asp Glu Thr Val Ala Tyr Ala Gln Arg Leu Tyr1 5 10 15His Ala Cys Ala
Asp Ser Gly Ile Leu Ala Ser Leu Gln Gly Thr Ala 20 25 30Cys Ala Ala
Leu Ser Lys Leu Ala Asp Ala Ser Arg Gly Ala Ser Gln 35 40 45Tyr Leu
Ala Thr Ala Pro Pro Ser Pro Ala Pro Leu Val Gln Val Leu 50 55 60Gln
Phe Leu Glu Thr Asn Phe Ser Ser Ile Ala Ser Phe Gly Leu Leu65 70 75
80Cys Ala Gly Cys Gln Ala Gly Glu Cys Phe Thr Ala Leu Ala Gly Leu
85 90 95Val Ser Gly Ala Thr Ala Gly Leu Gly Gly Ala His Lys Trp Leu
Leu 100 105 110Ala Ile Ala Gly Thr Trp Leu Val Ser Leu Gln Thr Gly
Pro Arg Gly 115 120 125Gly Met Val Ala Gly Leu Ser Val Leu Ala Gly
Cys Cys Ile Gly Gly 130 135 140Val Thr Gly Leu Asp Phe Leu Phe Gly
Cys Leu Thr Gly Trp Glu Ala145 150 155 160Val Val Gly Ala Ala Val
Ala Thr Gln Lys Ile Leu Ser Gly Ser Ala 165 170 175Asp Met Thr Thr
Leu Val Asp Leu Leu Pro Ala Leu Phe Ser Pro Gly 180 185 190Ala Gly
Ile Ala Gly Ile Val Leu Val Phe Ile Leu Ser Asn Thr Ser 195 200
205Val Thr Ala Trp Ala Asn Arg Leu Leu Ser Met Cys Ala Lys Gln Thr
210 215 220Ile Cys Glu Asn Tyr Phe Leu Thr Glu Lys Phe Gly Gln Gln
Leu Ser225 230 235 240Lys Leu Ser Leu Trp Arg Ala Val Tyr His Trp
Ala Gln Ala His Glu 245 250 255Gly Tyr Thr Gln Cys Gly
260439458PRTHuman Pegivirus 2 439Val Val Gly Gly Ile Trp Ser Phe
Val Leu Cys Ile Leu Arg Ala Ala1 5 10 15Trp Asp Trp Ala Ala Lys His
Val Pro Arg Phe Arg Val Pro Met Ile 20 25 30Gly Cys Ser Pro Ala Trp
Cys Gly Arg Trp Leu Gly Thr Gly Thr Leu 35 40 45Leu Thr Thr Cys Gly
Cys Gly Glu Arg Val Ser Leu Gln Cys Leu Cys 50 55 60Ser Thr Ser Asp
Pro Ile Leu Ser Val Gly Arg Trp Cys Arg Cys Ser65 70 75 80Trp Ser
Val Gly Phe Pro Phe Asn Pro Thr Thr Thr Ala Thr Gly Thr 85 90 95Leu
Arg Pro Asp Ile Gly Asp Ala Thr Arg Leu Gly Phe Arg Tyr Gly 100 105
110Ile Ala Glu Ile Val Glu Leu Glu Arg Arg Gly Asp Lys Trp His Val
115 120 125Cys Ala Ala Ser Cys Cys Leu Asp Arg Ala Ser Val Ala Ser
Ala Val 130 135 140Lys Ala Pro Pro Val Thr Ala Asn Gly Ile Pro Ile
Ser Pro Phe Ser145 150 155 160Pro Pro Gln Thr Tyr Lys Leu Ser Leu
Cys Ser Phe Asp Ser Val Cys 165 170 175Met Ser Ile Asn Ser Cys Asn
Pro Ser Lys Ile Leu Ser Val Cys Ser 180 185 190Gln Glu Ala Val Glu
Leu Leu Glu Glu Thr Val Asp Thr Ala Gln Thr 195 200 205Met Met Cys
Lys Asn Leu Glu Ala Arg Arg Arg Ala Glu Phe Asp Ala 210 215 220Trp
Gln Val Arg Gln Ala Val Gly Asp Glu Tyr Thr Arg Leu Ala Asp225 230
235 240Glu Asp Val Asp Thr Ile Thr Ser Val Lys Pro Pro Val Ala Arg
Ala 245 250 255Ala Val Gly Ser Ser Thr Leu Asp Asp Val Gly Val Leu
Thr Val Leu 260 265 270Arg Glu Leu Gly Asp Gln Cys Gln Asn Ala Ile
Lys Phe Val Val Glu 275 280 285Ala Ala Ser Arg Phe Val Pro Pro Val
Pro Lys Pro Arg Thr Arg Val 290 295 300Ser Gly Val Leu Glu Arg Val
Arg Met Cys Met Arg Thr Pro Pro Met305 310 315 320Lys Phe Glu Ala
Ala Ala Val Pro Ile His Asn Ile Ile Pro Glu Lys 325 330 335Cys His
Ile Val Leu Arg Cys Thr Gly Cys Ser Asp Gln Ala Leu Thr 340 345
350Val Pro Tyr Gly Thr Cys Thr Gln Thr Leu Ser Ser His Leu Thr Asn
355 360 365Lys His Ser His Tyr Ile Pro Lys Gln Lys Ile Glu Glu Asp
Thr Glu 370 375 380Val Thr Val Ile Cys Ala Val Pro Thr Lys Arg Ala
Ser Lys Leu Ile385 390 395 400Thr Phe Arg Val Gly Asp Arg Ser Val
Ser Cys Cys His Pro Leu Gln 405 410 415Thr Pro Val Arg Ala Leu Leu
Leu Lys Tyr Gly Leu Pro Ile Gly Lys 420 425 430Trp Ser Asp Cys Asn
Gly Pro Leu Gly Asp Asp Ala Arg Val Cys Asp 435 440 445Val Asn Gly
Val Thr Thr Tyr Glu Pro Cys 450 455440567PRTHuman Pegivirus 2
440Met Gln Ser Tyr Ser Trp Phe Arg Pro Ile Val Ala Pro Thr Thr Pro1
5 10 15Pro Leu Pro Val Thr Arg Ser Val Ala Gly Ile Leu Arg Ala Asp
Thr 20 25 30Ser Arg Val Tyr Thr Thr Thr Ala Val Asp Val Ser Glu Arg
Gln Ala 35 40 45Lys Val Thr Ile Asp Gln Lys Ser Ala Lys Val Asp Gln
Cys Phe Arg 50 55 60Asp Thr Tyr Asn Cys Cys Leu Ala Lys Ala Lys Thr
Phe Arg Gln Ser65 70 75 80Gly Met Ser Tyr Glu Asp Ala Val Ser Lys
Met Arg Ala Asn Thr Thr 85 90 95Arg Asp His Asn Thr Gly Ile Thr Tyr
Thr Asp Leu Val Ser Gly Arg 100 105 110Ala Lys Pro Ala Val Gln Lys
Ile Val Asp Gln Met Arg Ser Gly Val 115 120 125Tyr Asp Ala Pro Met
Arg Ile Ile Pro Lys Pro Glu Val Phe Pro Arg 130 135 140Asp Lys Ser
Thr Arg Lys Pro Pro Arg Phe Ile Val Phe Pro Gly Cys145 150 155
160Ala Ala Arg Val Ala Glu Lys Met Ile Leu Gly Asp Pro Gly Ala Ile
165 170 175Thr Lys His Val Leu Gly Asp Ala Tyr Gly Phe Ala Thr Pro
Pro His 180 185 190Glu Arg Ala Arg Leu Leu Glu Gln Trp Trp Asn Arg
Ala Thr Glu Pro 195 200 205Gln Ala Ile Ala Val Asp Ala Ile Cys Phe
Asp Ser Thr Ile Thr Ala 210 215 220Glu Asp Met Asp Arg Glu Ala His
Ile Val Ala Ala Ala His Ala Asp225 230 235 240Pro Glu Gly Val His
Gly Leu Tyr Asn Tyr Tyr Lys Arg Ser Pro Met 245 250 255Cys Asp Ile
Thr Gly Lys Val Val Gly Val Arg Cys Cys Arg Ala Ser 260 265 270Gly
Thr Leu Thr Thr Ser Ser Gly Asn Thr Leu Thr Cys Tyr Leu Lys 275 280
285Val Arg Ala Ala Cys Thr Arg Ala Gly Ile Lys Pro Ile Gly Leu Leu
290 295 300Ile His Gly Asp Asp Thr Leu Ile Ile Thr Glu Arg Cys Thr
Gln Glu305 310 315 320Thr Leu Asp Glu Phe Ser Asn Ala Leu Asp Asp
Tyr Gly Phe Pro His 325 330 335Thr Phe Gln Val Ser Gly Asp Leu Ser
Ser Ile Glu Cys Cys Ser Ala 340 345 350Arg Val Asp Ser Val Cys Leu
Arg Gly Gly Met Arg Arg Met Leu Val 355 360 365Pro Gln Ala Arg Arg
Ala Ile Ala Arg Val Leu Gly Glu Lys Gly Asp 370 375 380Pro Leu Gly
Val Ile Ser Ser Tyr Ile Val Met Tyr Pro Thr Ala Ala385 390 395
400Val Thr Val Tyr Val Leu Leu Pro Leu Leu Cys Met Leu Ile Arg Asn
405 410 415Glu Pro Ser Gln Thr Gly Thr Leu Val Thr Leu Thr Val His
Gly Asn 420 425 430Ser Val Ser Val Pro Val Trp Leu Leu Pro Thr Ile
Ile Ala Asn Leu 435 440 445His Gly Arg Asp Ala Leu Gln Val Val Arg
His Ser Ala Ala Ser Met 450 455 460Ala Glu Leu Ser Ser Ala Leu Ala
Phe Phe Gly Met Arg Gly Leu Asn465 470 475 480Cys Trp Arg Arg Arg
Arg Arg Ala Ile Arg Thr Asp Met Ile Lys Leu 485 490 495Gly Gly Trp
Asn Ala Asn Phe Ala Gln Met Leu Leu Trp Ser Pro Glu 500 505 510Val
Arg Thr Pro Gln Pro Glu Pro Lys Gly Met Cys Leu Leu Pro Pro 515 520
525Glu Leu Trp Glu Arg Pro Tyr Glu Asn Leu His Leu Ser Thr Ile Asp
530 535 540Arg Asp Arg Gly Ala Ser Arg Leu Arg Phe Trp Leu Val Ala
Ser Ala545 550 555 560Val Leu Ala Leu Leu Cys Leu 565
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