U.S. patent application number 17/114432 was filed with the patent office on 2021-06-24 for oncolytic rhabdovirus.
This patent application is currently assigned to TURNSTONE LIMITED PARTNERSHIP. The applicant listed for this patent is TURNSTONE LIMITED PARTNERSHIP. Invention is credited to John BELL, David F. STOJDL.
Application Number | 20210187048 17/114432 |
Document ID | / |
Family ID | 1000005448000 |
Filed Date | 2021-06-24 |
United States Patent
Application |
20210187048 |
Kind Code |
A1 |
STOJDL; David F. ; et
al. |
June 24, 2021 |
ONCOLYTIC RHABDOVIRUS
Abstract
Embodiments of the invention include compositions and methods
related to non-VSV rhabdoviruses and their use as anti-cancer
therapeutics. Such rhabdoviruses possess tumor cell killing
properties in vitro and in vivo.
Inventors: |
STOJDL; David F.; (Ottawa,
CA) ; BELL; John; (Ottawa, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
TURNSTONE LIMITED PARTNERSHIP |
Toronto |
|
CA |
|
|
Assignee: |
TURNSTONE LIMITED
PARTNERSHIP
Toronto
CA
|
Family ID: |
1000005448000 |
Appl. No.: |
17/114432 |
Filed: |
December 7, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16884967 |
May 27, 2020 |
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17114432 |
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16101265 |
Aug 10, 2018 |
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16884967 |
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15436520 |
Feb 17, 2017 |
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16101265 |
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13937043 |
Jul 8, 2013 |
9572883 |
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15436520 |
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12441494 |
Oct 21, 2010 |
8481023 |
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PCT/IB2007/004701 |
Sep 17, 2007 |
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13937043 |
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60844726 |
Sep 15, 2006 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2760/20221
20130101; C12N 2760/20232 20130101; C12N 2760/20045 20130101; A61K
35/768 20130101; C12N 7/00 20130101; A61K 35/763 20130101; A61K
45/06 20130101; C12N 2810/6081 20130101; A61K 35/761 20130101; C12N
2760/20271 20130101; C12N 2760/20222 20130101; A61K 35/766
20130101; C12N 2760/20032 20130101 |
International
Class: |
A61K 35/766 20060101
A61K035/766; C12N 7/00 20060101 C12N007/00; A61K 35/761 20060101
A61K035/761; A61K 35/763 20060101 A61K035/763; A61K 35/768 20060101
A61K035/768; A61K 45/06 20060101 A61K045/06 |
Claims
1. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier and an oncolytic recombinant rhabdovirus
encoding a G protein from a first rhabdovirus and M, P, N and L
proteins from a second rhabdovirus.
2. The pharmaceutical composition of claim 1, wherein the G protein
has at least about 85% amino acid sequence identity to an Isfahan
virus G protein, a Chandipura virus G protein, a Maraba virus G
protein, a Bahia Grande virus G protein, a Klamath virus G protein,
or a Farmington virus G protein.
3. The pharmaceutical composition of claim 2, wherein the oncolytic
recombinant rhabdovirus encodes a G protein having at least about
85% amino acid sequence identity to an Isfahan virus G protein, a
Chandipura virus G protein, a Maraba virus G protein, or a Muir
Springs virus G protein.
4. The pharmaceutical composition of claim 3, wherein the oncolytic
recombinant rhabdovirus encodes a G protein having at least about
85% amino acid sequence identity to an Isfahan virus G protein, a
Maraba virus G protein, or a Muir Springs virus G protein.
5. The pharmaceutical composition of claim 4, wherein the oncolytic
recombinant rhabdovirus encodes a G protein having at least about
85% amino acid sequence identity to a Maraba virus G protein.
6. The pharmaceutical composition of claim 1, wherein the oncolytic
recombinant rhabdovirus encodes M, P, N and L proteins from
vesicular stomatitis virus (VSV).
7. The pharmaceutical composition of claim 2, wherein the oncolytic
recombinant rhabdovirus encodes M, P, N and L proteins from
vesicular stomatitis virus (VSV).
8. The pharmaceutical composition of claim 3, wherein the oncolytic
recombinant rhabdovirus encodes M, P, N and L proteins from
vesicular stomatitis virus (VSV).
9. The pharmaceutical composition of claim 4, wherein the oncolytic
recombinant rhabdovirus encodes M, P, N, and L proteins from
vesicular stomatitis virus (VSV).
10. The pharmaceutical composition of claim 5, wherein the
oncolytic recombinant rhabdovirus encodes M, P, N, and L proteins
from vesicular stomatitis virus (VSV).
11. The pharmaceutical composition of claim 1, wherein said
composition comprises 10.sup.3 to 10.sup.13 plaque forming units
(pfu) of the oncolytic recombinant rhabdovirus.
12. A method for treating cancer in a subject comprising
administering to the subject an effective amount of the
pharmaceutical composition of claim 1.
13. The method of claim 12, wherein the cancer is selected from the
group consisting of lung cancer, head and neck cancer, breast
cancer, cancer of the central nervous system, pancreatic cancer,
prostate cancer, renal cancer, bone cancer, testicular cancer,
cervical cancer, ovarian cancer, gastrointestinal cancer, lymphoma,
liver cancer, colon cancer, melanoma, and bladder cancer.
14. The method of claim 12, wherein the cancer is metastatic.
15. The method of claim 12, wherein the subject is a human.
16. The method of claim 12, wherein the composition is administered
by intraperitoneal, intravascular, intramuscular, intratumoral,
subcutaneous or intranasal administration.
17. The method of claim 16, wherein the composition is administered
by intratumoral or intravascular administration.
18. The method of claim 12, wherein the composition is administered
multiple times.
19. The method of claim 12, further comprising administering an
additional anti-cancer therapy selected from the group consisting
of chemotherapy, radiotherapy, and immunotherapy.
20. A method for treating cancer in a subject comprising
administering to the subject an effective amount of an oncolytic
recombinant rhabdovirus encoding a G protein from a first
rhabdovirus and M, P, N and L proteins from a second
rhabdovirus.
21. The method of claim 20, wherein the G protein has at least
about 85% amino acid sequence identity to an Isfahan virus G
protein, a Chandipura virus G protein, a Maraba virus G protein, a
Bahia Grande virus G protein, a Klamath virus G protein, or a
Farmington virus G protein.
22. The method of claim 21, wherein the oncolytic recombinant
rhabdovirus encodes M, P, N and L proteins from vesicular
stomatitis virus (VSV).
23. The method of claim 22, wherein the G protein has at least
about 85% amino acid sequence identity to a Maraba virus G
protein.
24. The method of claim 20, wherein the cancer is selected from the
group consisting of lung cancer, head and neck cancer, breast
cancer, cancer of the central nervous system, pancreatic cancer,
prostate cancer, renal cancer, bone cancer, testicular cancer,
cervical cancer, ovarian cancer, gastrointestinal cancer, lymphoma,
liver cancer, lung cancer, colon cancer, melanoma, and bladder
cancer.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 16/884,967, filed May 27, 2020, which is a continuation of U.S.
application Ser. No. 16/101,265, filed Aug. 10, 2018, now
abandoned, which is a continuation of U.S. application Ser. No.
15/436,520, filed Feb. 17, 2017, now abandoned, which is a
continuation of U.S. application Ser. No. 13/937,043, filed Jul. 8,
2013, now U.S. Pat. No. 9,572,883, which is a continuation of U.S.
application Ser. No. 12/441,494 filed Oct. 21, 2010, now U.S. Pat.
No. 8,481,023, which is a U.S. national stage of International
Patent Application No. PCT/IB2007/004701, filed Sep. 17, 2007,
which claims the benefit of U.S. Provisional Application No.
60/844,726 filed Sep. 15, 2006, each of which is hereby
incorporated by reference herein in its entirety.
SEQUENCE LISTING
[0002] This application incorporate by reference in its entirety
the Computer Readable Form ("CRF") of a Sequence Listing in ASCII
text format submitted via EFS-Web. The Sequence Listing text file
submitted via EFS-Web is entitled
"14596-063-999_Substitute_Seqlisting.txt," was created on Feb. 9,
2021 and is 174,245 bytes in size.
I. FIELD OF THE INVENTION
[0003] This invention relates generally to virology and medicine.
In certain aspects the invention relates to oncolytic viruses,
particularly non-VSV oncolytic rhabdoviruses and oncolytic
rhabdoviruses comprising a non-VSV glycoprotein.
II. BACKGROUND
[0004] A number of viruses have been shown to replicate in and kill
a wide variety of tumor cells in vitro (Sindbis virus (Unno et al.,
2005); Sendai virus (Kinoh et al., 2004); Coxackie virus (Shafren
et al., 2004); Herpes simplex virus (Mineta et al., 1995);
Parvovirus (Abschuetz et al., 2006); Adenovirus (Heise et al.,
2000); Polio virus (Gromeier et al., 2000); Newcastle disease virus
(Sinkovics and Horvath, 2000); Vesicular stomatitis virus (Stojdl
et al., 2000); Measles virus (Grote et al., 2001); Reovirus (Coffey
et al., 1998); Retrovirus (Logg et al., 2001); Vaccinia
(Timiryasova et al., 1999); and Influenza (Bergmann et al., 2001)).
In addition, such viruses have demonstrated efficacy in treating
animal models of cancer.
[0005] Vesicular stomatitis virus (VSV), a well known and well
studied rhabdovirus, has been shown to kill tumor cell lines in
cell culture experiments, and has demonstrated efficacy in a
variety of rodent cancer models (Stojdl et al., 2000; Stojdl et
al., 2003). However, VSV does not kill all cancer cells.
SUMMARY OF THE INVENTION
[0006] Several newly identified rhabdoviruses are much more
efficient at killing particular cancers or cancer cell lines than
VSV. Also, VSV and attenuated mutants of VSV are neurovirulent and
cause CNS pathology in rodents and primates. Several rhabdoviruses
do not infect the CNS (i.e., Muir Springs and Bahia Grande:
Kerschner et al., 1986), and demonstrate a more acceptable safety
profile. In addition, therapies based on the novel rhabdoviruses
can be used to treat cancers of the CNS, both primary and
secondary. The rhabdoviruses of the invention (and/or other
oncolytic agents) can be used in succession to bypass the host
immune response against a particular therapeutic virus(es). This
would allow prolonged therapy and improve efficacy.
[0007] Embodiments of the invention include compositions and
methods related to non-VSV rhabdoviruses and their use as
anti-cancer therapeutics. Such rhabdoviruses possess tumor cell
killing properties in vitro and in vivo.
[0008] As used herein, a non-VSV rhabdovirus will include one or
more of the following viruses or variants thereof: Arajas virus,
Chandipura virus, Cocal virus, Isfahan virus, Maraba virus, Piry
virus, Vesicular stomatitis Alagoas virus, BeAn 157575 virus,
Boteke virus, Calchaqui virus, Eel virus American, Gray Lodge
virus, Jurona virus, Klamath virus, Kwatta virus, La Joya virus,
Malpais Spring virus, Mount Elgon bat virus, Perinet virus, Tupaia
virus, Farmington, Bahia Grande virus, Muir Springs virus, Reed
Ranch virus, Hart Park virus, Flanders virus, Kamese virus,
Mosqueiro virus, Mossuril virus, Barur virus, Fukuoka virus, Kem
Canyon virus, Nkolbisson virus, Le Dantec virus, Keuraliba virus,
Connecticut virus, New Minto virus, Sawgrass virus, Chaco virus,
Sena Madureira virus, Timbo virus, Almpiwar virus, Aruac virus,
Bangoran virus, Bimbo virus, Bivens Arm virus, Blue crab virus,
Charleville virus, Coastal Plains virus, DakArK 7292 virus,
Entamoeba virus, Garba virus, Gossas virus, Humpty Doo virus,
Joinjakaka virus, Kannamangalam virus, Kolongo virus, Koolpinyah
virus, Kotonkon virus, Landjia virus, Manitoba virus, Marco virus,
Nasoule virus, Navarro virus, Ngaingan virus, Oak-Vale virus,
Obodhiang virus, Oita virus, Ouango virus, Parry Creek virus, Rio
Grande cichlid virus, Sandjimba virus, Sigma virus, Sripur virus,
Sweetwater Branch virus, Tibrogargan virus, Xiburema virus, Yata
virus, Rhode Island, Adelaide River virus, Berrimah virus,
Kimberley virus, or Bovine ephemeral fever virus. In certain
aspects, non-VSV rhabdovirus can refer to the supergroup of
Dimarhabdovirus (defined as rhabdovirus capable of infecting both
insect and mammalian cells). In specific embodiments, the
rhabdovirus is not VSV. In particular aspects the non-VSV
rhabdovirus is a Carajas virus, Maraba virus, Farmington, Muir
Springs virus, and/or Bahia grande virus, including variants
thereof.
[0009] One embodiment of the invention includes methods and
compositions comprising an oncolytic non-VSV rhabdovirus or a
recombinant oncolytic non-VSV rhabdovirus encoding one or more of
rhabdoviral N, P, M, G and/or L protein, or variant thereof
(including chimeras and fusion proteins thereof), having an amino
acid identity of at least or at most 20, 30, 40, 50, 60, 65, 70,
75, 80, 85, 90, 92, 94, 96, 98, 99, 100%, including all ranges and
percentages there between, to the N, P, M, G and/or L protein of
Arajas virus, Chandipura virus, Cocal virus, Isfahan virus, Maraba
virus, Piry virus, Vesicular stomatitis Alagoas virus, BeAn 157575
virus, Boteke virus, Calchaqui virus, Eel virus American, Gray
Lodge virus, Jurona virus, Klamath virus, Kwatta virus, La Joya
virus, Malpais Spring virus, Mount Elgon bat virus, Perinet virus,
Tupaia virus, Farmington, Bahia Grande virus, Muir Springs virus,
Reed Ranch virus, Hart Park virus, Flanders virus, Kamese virus,
Mosqueiro virus, Mossuril virus, Barur virus, Fukuoka virus, Kern
Canyon virus, Nkolbisson virus, Le Dantec virus, Keuraliba virus,
Connecticut virus, New Minto virus, Sawgrass virus, Chaco virus,
Sena Madureira virus, Timbo virus, Almpiwar virus, Aruac virus,
Bangoran virus, Bimbo virus, Bivens Arm virus, Blue crab virus,
Charleville virus, Coastal Plains virus, DakArK 7292 virus,
Entamoeba virus, Garba virus, Gossas virus, Humpty Doo virus,
Joinjakaka virus, Kannamangalam virus, Kolongo virus, Koolpinyah
virus, Kotonkon virus, Landjia virus, Manitoba virus, Marco virus,
Nasoule virus, Navarro virus, Ngaingan virus, Oak-Vale virus,
Obodhiang virus, Oita virus, Ouango virus, Parry Creek virus, Rio
Grande cichlid virus, Sandjimba virus, Sigma virus, Sripur virus,
Sweetwater Branch virus, Tibrogargan virus, Xiburema virus, Yata
virus, Rhode Island, Adelaide River virus, Berrimah virus,
Kimberley virus, or Bovine ephemeral fever virus. Any 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11 12 13, 14, 15, 20, 25, 30, 35, 40, 45, 50,
55, 60, 65, 70, 75, 80, 85 or more, including all integers or
ranges there between, of these virus can be specifically excluded
from the claim scope. VSV or any non-VSV rhabdovirus can be the
background sequence into which a variant G-protein or other viral
protein can be integrated.
[0010] In another aspect of the invention, a non-VSV rhabdovirus,
or a recombinant there of, can comprise a nucleic acid segment
encoding at least or at most 10, 20, 30, 40, 45, 50, 60, 65, 70,
80, 90, 100, 125, 175, 250 or more contiguous amino acids,
including all value and ranges there between, of N, P, M, G or L
protein of one or more non-VSV rhabdovirus, including chimeras and
fusion proteins thereof. In certain embodiments a chimeric G
protein will include a cytoplasmic, transmembrane, or both
cytoplasmic and transmembrane portions of a VSV or non-VSV G
protein.
[0011] Methods and compositions of the invention can include a
second therapeutic virus, such as an oncolytic or replication
defective virus. Oncolytic typically refers to an agent that is
capable of killing, lysing, or halting the growth of a cancer cell.
In terms of an oncolytic virus the term refers to a virus that can
replicate to some degree in a cancer cell, cause the death, lysis,
or cessation of cancer cell growth and typically have minimal toxic
effects on non-cancer cells. A second virus includes, but is not
limited to an adenovirus, a vaccinia virus, a Newcastle disease
virus, an alphavirus, a parvovirus, a herpes virus, a rhabdovirus,
a non-VSV rhabdovirus and the like. In other aspects, the
composition is a pharmaceutically acceptable composition. The
composition may also include a second anti-cancer agent, such as a
chemotherapeutic, radiotherapeutic, or immunotherapeutic.
[0012] Further embodiments of the invention include methods of
killing a hyperproliferative cell comprising contacting the cell
with an isolated oncolytic rhabdovirus composition; or
[0013] Still further methods include the treatment of a cancer
patient comprising administering an effective amount of an
oncolytic rhabdovirus composition.
[0014] In certain aspects of the invention, a cell may be comprised
in a patient and may be a hyperproliferative, neoplastic,
pre-cancerous, cancerous, metastatic, or metastasized cell. A
non-VSV rhabdovirus can be administered to a patient having a cell
susceptible to killing by at least one non-VSV rhabdovirus or a
therapeutic regime or composition including a non-VSV rhabdovirus.
Administration of therapeutic compositions may be done 1, 2, 3, 4,
5, 6, 7, 8, 9, 10 or more times with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10
or more non-VSV rhabdovirus or recombinant non-VSV rhabdovirus,
alone or in various combinations. The composition administered can
have 10, 100, 10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6, 10.sup.7,
10.sup.8, 10.sup.9, 10.sup.10, 10.sup.11, 10.sup.12, 10.sup.13,
10.sup.14, or more viral particles or plaque forming units (pfu).
Administration can be by intraperitoneal, intravenous,
intra-arterial, intramuscular, intradermal, subcutaneous, or
intranasal administration. In certain aspects, the compositions are
administered systemically, particularly by intravascular
administration, which includes injection, perfusion and the like.
The methods of invention can further comprise administering a
second anti-cancer therapy, such as a second therapeutic virus. In
particular aspects a therapeutic virus can be an oncolytic virus,
more particularly a non-VSV rhabdovirus. In other aspects, a second
anti-cancer agent is a chemotherapeutic, a radiotherapeutic, an
immunotherapeutic, surgery or the like.
[0015] Embodiments of the invention include compositions and
methods related to a VSV rhabdoviruses comprising a heterologous G
protein and their use as anti-cancer therapeutics. Such
rhabdoviruses possess tumor cell killing properties in vitro and in
vivo.
[0016] As used herein, a heterologous G protein includes non-VSV
rhabdovirus. Non-VSV rhabdoviruses will include one or more of the
following viruses or variants thereof: Arajas virus, Chandipura
virus, Cocal virus, Isfahan virus, Maraba virus, Piry virus,
Vesicular stomatitis Alagoas virus, BeAn 157575 virus, Boteke
virus, Calchaqui virus, Eel virus American, Gray Lodge virus,
Jurona virus, Klamath virus, Kwatta virus, La Joya virus, Malpais
Spring virus, Mount Elgon bat virus, Perinet virus, Tupaia virus,
Farmington, Bahia Grande virus, Muir Springs virus, Reed Ranch
virus, Hart Park virus, Flanders virus, Kamese virus, Mosqueiro
virus, Mossuril virus, Barur virus, Fukuoka virus, Kern Canyon
virus, Nkolbisson virus, Le Dantec virus, Keuraliba virus,
Connecticut virus, New Minto virus, Sawgrass virus, Chaco virus,
Sena Madureira virus, Timbo virus, Almpiwar virus, Aruac virus,
Bangoran virus, Bimbo virus, Bivens Arm virus, Blue crab virus,
Charleville virus, Coastal Plains virus, DakArK 7292 virus,
Entamoeba virus, Garba virus, Gossas virus, Humpty Doo virus,
Joinjakaka virus, Kannamangalam virus, Kolongo virus, Koolpinyah
virus, Kotonkon virus, Landjia virus, Manitoba virus, Marco virus,
Nasoule virus, Navarro virus, Ngaingan virus, Oak-Vale virus,
Obodhiang virus, Oita virus, Ouango virus, Parry Creek virus, Rio
Grande cichlid virus, Sandjimba virus, Sigma virus, Sripur virus,
Sweetwater Branch virus, Tibrogargan virus, Xiburema virus, Yata
virus, Rhode Island, Adelaide River virus, Berrimah virus,
Kimberley virus, or Bovine ephemeral fever virus. In certain
aspects, non-VSV rhabdovirus can refer to the supergroup of
Dimarhabdovirus (defined as rhabdovirus capable of infection both
insect and mammalian cells). In particular aspects the non-VSV
rhabdovirus is a Carajas virus, Maraba virus, Muir Springs virus,
and/or Bahia grande virus, including variants thereof.
[0017] One embodiment of the invention includes methods and
compositions comprising a oncolytic VSV rhabdovirus comprising a
heterologous G protein or a recombinant oncolytic VSV rhabdovirus
encoding one or more of non-VSV rhabdoviral N, P, M, G and/or L
protein, or variant thereof (including chimeras and fusion proteins
thereof), having an amino acid identity of at least or at most 20,
30, 40, 50, 60, 65, 70, 75, 80, 85, 90, 92, 94, 96, 98, 99, 100%,
including all ranges and percentages there between, to the N, P, M,
G, and/or L protein of a non-VSV rhabdovirus.
[0018] In another aspect of the invention, a VSV rhabdovirus
comprising a heterologous G protein or recombinant thereof, can
comprise a nucleic acid comprising a nucleic acid segment encoding
at least or at most 10, 20, 30, 40, 45, 50, 60, 65, 70, 80, 90,
100, 125, 175, 250 or more contiguous amino acids, including all
value and ranges there between, of N, P, M, G, or L protein of a
non-VSV rhabdovirus, including chimeras and fusion proteins
thereof. In certain aspects, a chimeric G protein may comprise a
cytoplasmic, transmembrane, or both a cytoplasmic and transmembrane
portion of VSV or a second non-VSV virus or non-VSV
rhabdovirus.
[0019] Methods and compositions of the invention can include a
second therapeutic virus, such as an oncolytic or replication
defective virus. A second virus includes, but is not limited to an
adenovirus, a vaccinia virus, a Newcastle disease virus, a herpes
virus, a rhabdovirus, a non-VSV rhabdovirus and the like. In other
aspects, the composition is a pharmaceutically acceptable
composition. The composition may also include a second anti-cancer
agent, such as a chemotherapeutic, radiotherapeutic, or
immunotherapeutic.
[0020] Further embodiments of the invention include methods of
killing a hyperproliferative cell comprising contacting the cell
with an isolated oncolytic rhabdovirus, VSV comprising a
heterologous G protein molecule, or a non-VSV rhabdovirus
composition. Still further methods include the treatment of a
cancer patient comprising administering an effective amount of such
a viral composition.
[0021] In certain aspects of the invention, a cell may be comprised
in a patient and may be a hyperproliferative, neoplastic,
pre-cancerous, cancerous, metastatic, or metastasized cell. A virus
of the invention can be administered to a patient having a cell
susceptible to killing by at least one virus or a therapeutic
regime or composition including a virus. Administration of
therapeutic compositions may be done 1, 2, 3, 4, 5, 6, 7, 8, 9, 10
or more times with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more virus,
alone or in various combinations. The composition administered can
have 10, 100, 10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6, 10.sup.7,
10.sup.8, 10.sup.9, 10.sup.10, 10.sup.11, 10.sup.12, 10.sup.13,
10.sup.14, or more viral particles or plaque forming units (pfu).
Administration can be by intraperitoneal, intravenous,
intra-arterial, intramuscular, intradermal, subcutaneous, or
intranasal administration. In certain aspects, the compositions are
administered systemically, particularly by intravascular
administration, which includes injection, perfusion and the like.
The methods of invention can further comprise administering a
second anti-cancer therapy, such as a second therapeutic virus. In
particular aspects a therapeutic virus can be an oncolytic virus
such as a VSV comprising a heterologous G protein, more
particularly a non-VSV rhabdovirus. In other aspects, a second
anti-cancer agent is a chemotherapeutic, a radiotherapeutic, an
immunotherapeutic, surgery or the like.
[0022] Other embodiments of the invention are discussed throughout
this application. Any embodiment discussed with respect to one
aspect of the invention applies to other aspects of the invention
as well, and vice versa. The embodiments in the Detailed
Description and Example sections are understood to be non-limiting
embodiments of the invention that are applicable to all aspects of
the invention.
[0023] The terms "inhibiting," "reducing," or "preventing," or any
variation of these terms, when used in the claims and/or the
specification includes any measurable decrease or complete
inhibition to achieve a desired result. Desired results include but
are not limited to palliation, reduction, slowing, or eradication
of a cancerous or hyperproliferative condition, as well as an
improved quality or extension of life.
[0024] The use of the word "a" or "an" when used in conjunction
with the term "comprising" in the claims and/or the specification
may mean "one," but it is also consistent with the meaning of "one
or more," "at least one," and "one or more than one."
[0025] Throughout this application, the term "about" is used to
indicate that a value includes the standard deviation of error for
the device or method being employed to determine the value.
[0026] The use of the term "or" in the claims is used to mean
"and/or" unless explicitly indicated to refer to alternatives only
or the alternatives are mutually exclusive, although the disclosure
supports a definition that refers to only alternatives and
"and/or."
[0027] As used in this specification and claim(s), the words
"comprising" (and any form of comprising, such as "comprise" and
"comprises"), "having" (and any form of having, such as "have" and
"has"), "including" (and any form of including, such as "includes"
and "include") or "containing" (and any form of containing, such as
"contains" and "contain") are inclusive or open-ended and do not
exclude additional, unrecited elements or method steps.
[0028] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and the specific examples, while indicating specific
embodiments of the invention, are given by way of illustration
only, since various changes and modifications within the spirit and
scope of the invention will become apparent to those skilled in the
art from this detailed description.
DESCRIPTION OF THE DRAWINGS
[0029] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0030] FIG. 1. Phylogenetic relationships between rhabdoviruses
based on a GDE alignment of a relatively conserved region of the N
protein (119 amino acids), and using the paramyxovirus Human
parainfluenza virus 1 (HPIV-1) as the outgroup. The tree was
generated by the neighbor-joining method and bootstrap values
(indicated for each branch node) were estimated using 1000 tree
replicas. Branch lengths are proportional to genetic distances. The
scale bar corresponds to substitutions per amino acid site Courtesy
of H. Badrane and P. J. Walker).
[0031] FIG. 2. Summary of in vitro tumor cell killing assay. Cells
from the NCI 60 cell panel were infected for 96 h with a series of
dilution of various viruses. Cell viability was assayed using
crystal violet staining to detect residual viable cells. The
EC.sub.50 was calculated from the resulting cell killing curves and
summarized in table format. For clarity, the EC.sub.50 values have
been converted to a value from 1-7 as described in the legend. In
addition, the shading has been used to indicate the EC.sub.50 range
(i.e., darkest to lightest represents highest EC.sub.50 to lowest
EC.sub.50 values). Viruses are abbreviated as follows: MS=Muir
Springs, BG=Bahia Grande, NGG=Ngaingan, TIB=Tibrogargan,
FMT=Farmington, MRB=Maraba, CRJ=Carajas, VSVHR=Vesicular Stomatitis
Virus HR strain and VV=Vaccinia virus JX-963. This data
demonstrates that not all rhabdoviruses are equally oncolytic, in
fact closely related rhabdoviruses behave very differently on the
same tumor cell lines. Thus there is currently no method to predict
which rhabdoviruses have oncolytic potential. Empirical testing is
required to identify good oncolytic candidate viruses.
[0032] FIGS. 3A-3B. Rhabdovirus productivity on tumor cell lines.
SNB19 human glioblastoma and NCI H226 human lung carcinoma cell
lines were infected with various rhabdoviruses (MOI=3) and
monitored over time for virus production by plaque assay. The data
shows that not all rhabdoviruses have the same ability to replicate
in these tumor cell lines. NCIH226 cell reveal a great disparity in
virus productivity with Bahia Grande not producing virus at all
while Maraba virus is able to produce copious infectious
virions.
[0033] FIG. 4. Schematic of rescue system to recover recombinant
rhabdoviruses from plasmid DNA form. In this example, the Maraba
virus has been cloned into a DNA plasmid between the T7 promoter
and a rybozyme sequence from Hepatitis D virus. A549 cells are
infected with T7 expressing vaccinia virus and then subsequently
transfected with a Maraba genome vector engineered to express GFP.
The rescued virions are purified and then used to infect Vero cells
for 24 hours, resulting in GFP expression in these cells when
visualized by fluorescence microscopy.
[0034] FIG. 5. Bioselecting improved strains of oncolytic
rhabdoviruses. Rhabdoviruses are quasi-species. Bahia Grande is not
neuropathogenic but has the ability to kill human glioblastoma
cells. The inventors contemplated improving its virulence while
maintaining its selectivity for cancer cells. To improve the
virulence of a rhabdovirus for a tumor cell, the inventors selected
virus mutants with increased replication capacity in a human
glioblastoma cell line. Briefly, 5.times.10.sup.5 SNB19 cells were
infected with 2.5.times.10.sup.6 viral particles, giving an MOI of
5. The initial inoculum had a volume of 200 .mu.l and was allowed 1
hour to infect before the cells were washed 10 times with PBS. The
last wash was analyzed for viral particles by plaque assay to
ensure proper removal of input virus. At increasing time points,
the entire supernatant was collected and replaced with fresh media.
The collected media was used to infect new cells for amplification
and was analyzed by plaque assay for the presence of viral
particles. For the first passage, collections occurred at 4, 8, 12
and 24 hpi (hours post infection) until the initial time for viral
release was determined. Viruses from the earliest time point were
amplified back to a population of 10.sup.6 and then re-passed.
[0035] FIG. 6. Bioselecting improved strains of oncolytic
rhabdoviruses. In this example, Bahia Grande virus underwent up to
6 iterative cycles of bioselection. The parental strain (WT) along
with passages 4-6 were monitored for virus production in SNB19
cells at 4, 6 and 8 hours post infection. A clear and progressive
improvement in speed of initial virus replication is evident during
increasing rounds of bioselection. MRB=Maraba is included as, an
exemplar of rapid and desirable virus replication in the cancer
cell line.
[0036] FIG. 7. Bahia Grande P13 underwent 13 rounds of
bioselection. This virus demonstrated improved virus replication
not only in the human glioblastoma used during the bioselection
protocol, but on an unrelated human glioblastoma and a human
ovarian carcinoma cell line. This demonstrates that rhabdoviruses
can be bioselected to improve their oncolytic properties and these
improvements are effective on other disparate cancers.
[0037] FIG. 8. Balb/C mice were infected intracranially with the
indicated viruses and monitored for morbidity and/or mortality.
Both wild type VSV (HR strain) and the delta M51 mutant strain of
VSV were extremely neurotoxic, demonstrating hind limb paralysis
within days of infection, while Bahia Grande and Muir Springs
viruses showed no neurotoxicity. Bahia Grande P6 is a bioselected
strain of Bahia Grande with improved replication in human
glioblastoma cells. This strain also showed no neurotoxicity,
demonstrating that rhabdoviruses can be bioselected for improved
virulence on tumor cells, while maintaining their safety profile in
normal healthy tissue.
[0038] FIG. 9. In vivo efficacy of Maraba and Carajas rhabdoviruses
compared to Chandripura and WT VSV and delta 51 VSV 4T1 tumors
(firefly luciferase expressing) were established in 5-8 week old
Balb/C female mice by injecting 10.sup.6 tumor cells in the left,
rear mammary gland. After one week, mice were injected
intravenously on day 1 & 2 (each dose=10.sup.7 pfu WT VSV,
.DELTA.51 GFP VSV, Maraba or Chandipura; or 10.sup.8 pfu Carajas).
Tumor responses were measured by bioluminescence imaging using an
IVIS 200 (Xenogen) (measured as photons/s/cm.sup.2).
[0039] FIG. 10. Infectivity of G-less VSV pseudotyped with Isfahan
G and VSV G protein.
[0040] FIG. 11. A one step growth curve of VSV WT, Isfahan and RVR
IsfG1 viruses.
[0041] FIG. 12. RVR comprising an Isfahan G protein remains
oncolytic. The cytotoxicity of Isfahan virus, VSV d51 and RVR IsfG1
were assessed on various cancer cell lines.
[0042] FIGS. 13A-13C. RVR comprising Isf G1 is a able to escape
immune response to VSV in vivo. In vivo luciferase detection was
used to determine the amount of virus in mice inoculated with RVR
IsfG1 or VSV. FIG. 13A, in vivo detection of recombinant virus
injected into naive mice. FIG. 13B, in vivo detection of VSV
injected into mice immunized with VSV. FIG. 13C, in vivo detection
of recombinant RVR IsfG1 virus injected into mice immunized with
VSV.
[0043] FIG. 14. Virus yields from infected tumors. Tumors were
infected with recombinant virus or VSV in the presence or absence
of immunization with VSV (as indicated). Graphed data shows the
amount virus resulting from the infection of the tumor.
[0044] FIG. 15. A one step growth curve of VSV WT, chandipura virus
and RVR.sub.ChaG.sup.1. Results show that the recombinant produces
the same amount of virus as VSV.
[0045] FIG. 16. Cytotoxicity of VSV WT, chandipura virus and
RVR.sub.ChaG.sup.1. Results show that the recombinant is as
cytotoxic as VSV.
[0046] FIG. 17. A one step growth curve of VSV WT, Maraba virus and
RVR.sub.MarG.sup.1. Results show that recombinant virus titer was
greater than VSV at 48 and 72 h.
[0047] FIG. 18. Cytotoxicity of VSV WT, Maraba virus and
RVR.sub.MarG.sup.1. Results show that both maraba and the
RVR.sub.MarG.sup.1 are cytotoxic in tumor cells lines and that they
are generally more cytotoxic to tumor cells that VSV WT.
DETAILED DESCRIPTION OF THE INVENTION
[0048] Aspects of the invention are based on the killing by non-VSV
rhabdovirus or pseudotyped rhabdovirus of several kinds or types
cancer cells, which are resistant to killing by VSV. Some of the
advantages of these oncolytic rhabdoviruses and recombinant
rhabdoviruses include the following: (1) Antibodies to the
inventive rhabdoviruses will be rare to non-existent in most
populations of the world. (2) rhabdoviruses replicate more quickly
than other oncolytic viruses such as adenovirus, reovirus, measles,
parvovirus, retrovirus, and HSV. (3) Rhabdovirus grow to high
titers and are filterable through 0.2 micron filter. (4) The
oncolytic rhabdoviruses and recombinants thereof have a broad host
range, capable of infecting many different types of cancer cells
and are not limited by receptors on a particular cell (e.g.,
coxsackie, measles, adenovirus). (5) The rhabdovirus of the
invention are amenable to genetic manipulation. (6) The rhabdovirus
also has a cytoplasmic life cycle and do not integrate in the
genetic material a host cell, which imparts a more favorable safety
profile.
[0049] Embodiments of the invention include compositions and
methods related to non-VSV rhabdoviruses or pseudotyped
rhabdoviruses and their use as anti-cancer therapeutics.
I. FAMILY RHABDOVIRIDAE (RHABDOVIRUS)
[0050] The archetypal rhabdoviruses are rabies and vesicular
stomatitis virus (VSV), the most studied of this virus family.
Although these viruses share similar morphologies, they are very
different in their life cycle, host range, and pathology.
Rhabdovirus is a family of bullet shaped viruses having
non-segmented (-)sense RNA genomes. There are greater than 250
Rhabdoviruses known that infect mammals, fish, insects, and plants.
The family is split into at least 5 genera: (1) Lyssavirus:
including Rabies virus, other mammalian viruses, some insect
viruses; (2) Vesiculovirus: including Vesicular Stomatitis Virus
(VSV); (3) Ephemerovirus: including Bovine ephemeral fever virus
(vertebrates); (4) Cytorhabdovirus: including Lettuce necrotic
yellows virus (plants); and (5) Nucleorhabdovirus: including Potato
yellow dwarf virus (plants). It has also been suggested that there
is a supergroup of rhabdovirus denoted Dimarhabdovirus that include
a variety of rhabdoviruses that infect both mammals and
insects.
[0051] The family Rhabdovirus includes, but is not limited to:
Arajas virus, Chandipura virus (AF128868/gi:4583436,
AJ810083/gi:57833891, AY871800/gi:62861470, AY871799/gi:62861468,
AY871798/gi:62861466, AY871797/gi:62861464, AY871796/gi:62861462,
AY871795/gi:62861460, AY871794/gi:62861459, AY871793/gi:62861457,
AY871792/gi:62861455, AY871791/gi:62861453), Cocal virus
(AF045556/gi:2865658), Isfahan virus (AJ810084/gi:57834038), Maraba
virus (SEQ ID NO:1-6), Carajas virus (SEQ ID NO:7-12,
AY335185/gi:33578037), Piry virus (D26175/gi:442480,
Z15093/gi:61405), Vesicular stomatitis Alagoas virus, BeAn 157575
virus, Boteke virus, Calchaqui virus, Eel virus American, Gray
Lodge virus, Jurona virus, Klamath virus, Kwatta virus, La Joya
virus, Malpais Spring virus, Mount Elgon bat virus
(DQ457103/gi|91984805), Perinet virus (AY854652/gi:71842381),
Tupaia virus (NC_007020/gi:66508427), Farmington, Bahia Grande
virus (SEQ ID NO:13-18), Muir Springs virus, Reed Ranch virus, Hart
Park virus, Flanders virus (AF523199/gi:25140635,
AF523197/gi:25140634, AF523196/gi:25140633, AF523195/gi:25140632,
AF523194/gi:25140631, AF1012179/gi:25140630), Kamese virus,
Mosqueiro virus, Mossuril virus, Barur virus, Fukuoka virus
(AY854651/gi:71842379), Kern Canyon virus, Nkolbisson virus, Le
Dantec virus (AY854650/gi:71842377), Keuraliba virus, Connecticut
virus, New Minto virus, Sawgrass virus, Chaco virus, Sena Madureira
virus, Timbo virus, Almpiwar virus (AY854645/gi:71842367), Aruac
virus, Bangoran virus, Bimbo virus, Bivens Ann virus, Blue crab
virus, Charleville virus, Coastal Plains virus, DakArK 7292 virus,
Entamoeba virus, Garba virus, Gossas virus, Humpty Doo virus
(AY854643/gi:71842363), Joinjakaka virus, Kannamangalam virus,
Kolongo virus (DQ457100/gi|91984799 nucleoprotein (N) mRNA, partial
cds); Koolpinyah virus, Kotonkon virus (DQ457099/gi|91984797,
AY854638/gi:71842354); Landjia virus, Manitoba virus, Marco virus,
Nasoule virus, Navarro virus, Ngaingan virus
(AY854649/gi:71842375), Oak-Vale virus (AY854670/gi:71842417),
Obodhiang virus (DQ457098/gi|91984795), Oita virus
(AB116386/gi:46020027), Ouango virus, Parry Creek virus
(AY854647/gi:71842371), Rio Grande cichlid virus, Sandjimba virus
(DQ457102/gi|91984803), Sigma virus (AH004209/gi:1680545,
AH004208/gi:1680544, AH004206/gi:1680542), Sripur virus, Sweetwater
Branch virus, Tibrogargan virus (AY854646/gi:71842369), Xiburema
virus, Yata virus, Rhode Island, Adelaide River virus
(U10363/gi:600151, AF234998/gi:10443747, AF234534/gi:9971785,
AY854635/gi:71842348), Berrimah virus (AY854636/gi:71842350]),
Kimberley virus (AY854637/gi:71842352), or Bovine ephemeral fever
virus (NC_002526/gi:10086561).
[0052] Certain unassigned serotypes include (1) Bahia Grande group
(Bahia Grande virus (BGV), Muir Springs virus (MSV), Reed Ranch
virus (RRV); (2) Hart Park group (Flanders virus (FLAV), Hart Park
virus (HPV), Kamese virus (KAMV), Mosqueiro virus (MQOV), Mossuril
virus (MOSV); (3) Kern Canyon group (Barur virus (BARV), Fukuoka
virus (FUKAV), Kern Canyon virus (KCV), Nkolbisson virus (NKOV);
(4) Le Dantec group (Le Dantec virus (LDV), Keuraliba virus (KEUV),
(5) Sawgrass group (Connecticut virus (CNTV), New Minto virus
(NMV), Sawgrass virus (SAWV); (6) Timbo group (Chaco virus (CHOV),
Sena Madureira virus (SMV), Timbo virus (TIMV); and (7) other
unassigned viruses (Almpiwar virus (ALMV), Aruac virus (ARUV),
Bangoran virus (BGNV), Bimbo virus (BBOV), Bivens Arm virus (SAV),
Blue crab virus (BCV), Charleville virus (CHVV), Coastal Plains
virus (CPV), DakArK 7292 virus (DAKV-7292), Entamoeba virus (ENTV),
Garba virus (GARV), Gossas virus (GOSV), Humpty Doo virus (HDOOV),
Joinjakaka virus (JOIV), Kannamangalam virus (KANV), Kolongo virus
(KOLV), Koolpinyah virus (KOOLV), Kotonkon virus (KOTV), Landjia
virus (LJAV), Manitoba virus (MNTBV), Marco virus (MCOV), Ngaingan,
Nasoule virus (NASV), Navarro virus (NAVV), Ngaingan virus (NGAV),
Oak-Vale virus (OVRV), Obodhiang virus (OBOV), Oita virus (OITAV),
Ouango virus (OUAV), Parry Creek virus (PCRV), Rio Grande cichlid
virus (RGRCV), Sandjimba virus (SJAV), Sigma virus [X91062]
(SIGMAV), Sripur virus (SRIV), Sweetwater Branch virus (SWBV),
Tibrogargan virus (TIBV), Xiburema virus (XIBV), Yata virus
(YATAV).
[0053] Aspects of the invention may include, but is not limited to
selecting non-VSV rhabdovirus or pseudotyped rhabdovirus based on
growth in mammalian cell lines, lack of or minimal toxicity in
adult mice (animals), lack of or minimal toxicity in suckling mice
(animals).
[0054] A. Rhabdoviral Genome
[0055] Typically the rhabdovirus genome is approximately 11-15 kb
with an approximately 50 nucleotide 3' leader and an approximately
60 nucleotide non-translated 5' region of a (-) sense viral RNA
(vRNA). Typically, rhabdovirus vRNA has 5 genes encoding 5
proteins. Rhabdoviruses have a conserved polyadenylation signal at
the end of each gene and a short intergenic region between each of
the 5 genes. All Rhabdoviruses contain five genes which encode the
nucleocapsid protein (N), Phosphoprotein (P, also designated NS),
matrix protein (M), glycoprotein (G), and large protein (L).
Typically these genes are ordered on negative sense vRNA as
follows: 3'-N-P-M-G-(X)-L-5'. The order of the genes is important
as it dictates the proportion of proteins synthesized. Any
manipulations of a Rhabdovirus genome will typically include at
least five transcription domains to maintain ability to infect and
replicate at high levels. Rhabdoviruses have an endogenous RNA
polymerase for transcription of plus sense messenger RNA (mRNA).
The X gene does not occur in all Rhabdoviruses. The X gene encodes
a nonstructural protein found in the fish infectious hematopoietic
necrosis virus (GenBank DQ164103/gi|76262981; DQ164102/gi|76262979;
DQ164101/gi|76262977; DQ164100/gi|76262975; DQ164099/gi|76262973;
AB250935/gi|112821165; AB250934/gi|112821163;
AB250933/gi|112821161; AB250932/gi|112821159;
AB250931/gi|112821157; AB250930/gi|112821155; AB250929/gi|1128211
53; AB250928/gi|112821151; AB250927/gi|112821149, describing the G
protein encoding nucleotide sequence), a nonstructural glycoprotein
in the bovine ephemeral fever virus and a pseudogene in the rabies
virus. The extra (X) gene has been found in different locations on
the Rhabdovirus genome. Synthesis of the M protein in infected
cells is cytopathic to the cell, and will eventually result in cell
death.
[0056] Transmission of rhabdovirus varies depending on virus/host,
but most are transmitted by direct contact--e.g., transmission of
rabies by animal bites or insect vector. There is a long incubation
period in vivo, but this is not reflected in the kinetics of virus
replication in culture. The G protein spikes bind to receptors on
the surface of host cells and the viruses enters the cell by
endocytosis and fusion with the membrane of the vesicle, mediated
by the G protein.
[0057] With no intent to be limited to a particular theory, the
receptor molecules for rhabdoviruses are believed to be
phospholipids rather than specific proteins. Rhabdoviral
replication occurs in the cytoplasm--both the L and NS proteins are
necessary for transcription--neither function alone. Five
monocistronic mRNAs are produced, capped at the 5' end and
polyadenylated at the 3' end and each containing the leader
sequence from the 3' end of the vRNA at the 5' end of the message.
These mRNAs are made by sequential transcription of the ORFs in the
virus genome and it has been shown that the intergenic sequence is
responsible for termination and re-initiation of transcription by
the polymerase between each gene, thus producing separate
transcripts.
[0058] Progeny vRNA is made from a (+)sense intermediate. The
genome is replicated by the L+P polymerase complex (as in
transcription), but additional host cell factors are also required.
It is characteristic of Rhabdoviruses that these events all occur
in a portion of the cytoplasm which acts as a virus `factory` and
appears as a characteristic cytoplasmic inclusion body.
[0059] B. Viral Protein Variants
[0060] In certain embodiments, a rhabdovirus or a non-VSV
rhabdovirus will comprise a variant of one or more of the N, P, M,
G, and/or L proteins. In certain aspects of the invention these
viral protein variants can be comprised in a proteinaceous
composition, which is further defined below. Proteinaceous
compositions include viral particles and other compositions having
one or more viral protein components. These polypeptide variant(s)
can be engineered or selected for a modification in one or more
physiological or biological characteristics, such as host cell
range, host cell specificity, toxicity to non-target cells or
organs, replication, cytotoxicity to a target cell, killing of
cancer cells, stasis of cancer cells, infectivity, manufacturing
parameters, size of virus particle, stability of viral particles,
in vivo clearance, immunoreactivity, and the like. These
polypeptide variant can be engineered by using a variety of
methodology know in the art, including various mutagenesis
techniques described see below. In certain aspects, the N, P, M, G,
and/or L proteins can be heterologous to a virus (e.g., a VSV may
comprise a Isfahan G protein or variant thereof).
[0061] C. Recombinant Rhabdoviruses
[0062] Recombinant rhabdovirus can be produced (1) entirely using
cDNAs or (2) a combination of cDNAs transfected into a helper cell,
or (3) cDNAs transfected into a cell, which is further infected
with a minivirus providing in trans the remaining components or
activities needed to produce either an infectious or non-infectious
recombinant rhabdovirus. Using any of these methods (e.g.,
minivirus, helper cell line, or cDNA transfection only), the
minimum components required are an RNA molecule containing the
cis-acting signals for (1) encapsidation of the genomic (or
antigenomic) RNA by the Rhabdovirus N protein, and (2) replication
of a genomic or antigenomic (replicative intermediate) RNA
equivalent.
[0063] By a replicating element or replicon, the inventors mean a
strand of RNA minimally containing at the 5' and 3' ends the leader
sequence and the trailer sequence of a rhabdovirus. In the genomic
sense, the leader is at the 3' end and the trailer is at the 5'
end. Any RNA-placed between these two replication signals will in
turn be replicated. The leader and trailer regions further must
contain the minimal cis-acting elements for purposes of
encapsidation by the N protein and for polymerase binding which are
necessary to initiate transcription and replication.
[0064] For preparing engineered rhabdoviruses a minivirus
containing the G gene would also contain a leader region, a trailer
region and a G gene with the appropriate initiation and termination
signals for producing a G protein mRNA. If the minivirus further
comprises a M gene, the appropriate initiation and termination
signals for producing the M protein mRNA must also present.
[0065] For any gene contained within the engineered rhabdovirus
genome, the gene would be flanked by the appropriate transcription
initiation and termination signals which will allow expression of
those genes and production of the protein products. Particularly a
heterologous gene, which is a gene that is typically not encoded by
a rhabdovirus as isolated from nature or contains a rhabdovirus
coding region in a position, form or context that it typically is
not found, e.g., a chimeric G-protein.
[0066] To produce "non-infectious" engineered Rhabdovirus, the
engineered Rhabdovirus must have the minimal replicon elements and
the N, P, and L proteins and it must contain the M gene (one
example is the .DELTA.G or G-less construct, which is missing the
coding region for the G protein). This produces virus particles
that are budded from the cell, but are non-infectious particles. To
produce "infectious" particles, the virus particles must
additionally comprise proteins that can mediate virus particle
binding and fusion, such as through the use of an attachment
protein or receptor ligand. The native receptor ligand of
rhabdoviruses is the G protein.
[0067] A "suitable cell" or "host cell" means any cell that would
permit assembly of the recombinant rhabdovirus.
[0068] To prepare infectious virus particles, an appropriate cell
line (e.g., BHK cells) is first infected with vaccinia virus vTF7-3
(Fuerst et al., 1986) or equivalent which encodes a T7 RNA
polymerase or other suitable bacteriophage polymerase such as the
T3 or SP6 polymerases (see Usdin et al., 1993 or Rodriguez et al.,
1990). The cells are then transfected with individual cDNA
containing the genes encoding the G, N, P, L and M Rhabdovirus
proteins. These cDNAs will provide the proteins for building a
recombinant Rhabdovirus particle. Cells can be transfected by any
method known in the art (e.g., liposomes, electroporation,
etc.).
[0069] Also transfected into the cell line is a "polycistronic
cDNA" containing the rhabdovirus genomic RNA equivalent. If the
infectious, recombinant rhabdovirus particle is intended to be
lytic in an infected cell, then the genes encoding for the N, P, M
and L proteins must be present as well as any heterologous nucleic
acid segment. If the infectious, recombinant rhabdovirus particle
is not intended to be lytic, then the gene encoding the M protein
is not included in the polycistronic DNA. By "polycistronic cDNA"
it is meant a cDNA comprising at least transcription units
containing the genes which encode the N, P and L proteins. The
recombinant rhabdovirus polycistronic DNA may also contain a gene
encoding a protein variant or polypeptide fragment thereof, or a
therapeutic nucleic acid. Alternatively, any protein to be
initially associated with the viral particle first produced or
fragment thereof may be supplied in trans.
[0070] Another embodiment contemplated is a polycistronic cDNA
comprising a gene encoding a reporter protein or fluorescent
protein (e.g., green fluorescent protein and its derivatives,
.beta.-galactosidase, alkaline phosphatase, luciferase,
chloramphenicol acetyltransferase, etc.), the N-P-L or N-P-L-M
genes, and/or a fusion protein or a therapeutic nucleic acid.
Another polycistronic DNA contemplated may contain a gene encoding
a protein variant, a gene encoding a reporter, a therapeutic
nucleic acid, and/or either the N-P-L genes or the N-P-L-M
genes.
[0071] The first step in generating a recombinant rhabdovirus is
expression of an RNA that is a genomic or antigenomic equivalent
from a cDNA. Then that RNA is packaged by the N protein and then
replicated by the P/L proteins. The virus thus produced can be
recovered. If the G protein is absent from the recombinant RNA
genome, then it is typically supplied in trans. If both the G and
the M proteins are absent, then both are supplied in trans.
[0072] For preparing "non-infectious rhabdovirus" particles, the
procedure may be the same as above, except that the polycistronic
cDNA transfected into the cells would contain the N, P and L genes
of the Rhabdovirus only. The polycistronic cDNA of non-infectious
rhabdovirus particles may additionally contain a gene encoding a
reporter protein or a therapeutic nucleic acid. For additional
description regarding methods of producing a recombinant
rhabdovirus lacking the gene encoding the G protein, see Takada et
al. (1997).
[0073] 1. Culturing of Cells to Produce Virus
[0074] Transfected cells are usually incubated for at least 24 hr
at the desired temperature, usually about 37.degree. C. For
non-infectious virus particles, the supernatant is collected and
the virus particles isolated. For infectious virus particles, the
supernatant containing virus is harvested and transferred to fresh
cells. The fresh cells are incubated for approximately 48 hours,
and the supernatant is collected.
[0075] 2. Purification of the Recombinant Rhabdovirus
[0076] The terms "isolation" or "isolating" a Rhabdovirus means the
process of culturing and purifying the virus particles such that
very little cellular debris remains. One example would be to take
the virion containing supernatant and pass them through a 0.1-0.2
micron pore size filter (e.g., Millex-GS, Millipore) to remove the
virus and cellular debris. Alternatively, virions can be purified
using a gradient, such as a sucrose gradient. Recombinant
rhabdovirus particles can then be pelleted and resuspended in
whatever excipient or carrier is desired. Titers can be determined
by indirect immunofluorescence using antibodies specific for
particular proteins.
[0077] 3. Methods of Making Recombinant Rhabdoviruses Using cDNAs
and a Minivirus or a Helper Cell Line
[0078] Both "miniviruses" and "helper cells" (also known as "helper
cell lines") provide the same thing: to provide a source of
rhabdovirus proteins for rhabdovirus virion assembly. One example
of a rhabdovirus minivirus is the VSV minivirus which expresses
only the G and M protein, as reported by Stillman et al., (1995).
Helper viruses and miniviruses are used as methods of providing
rhabdovirus proteins that are not produced from transfected DNA
encoding the genes for rhabdovirus proteins.
[0079] When using a minivirus, cells are infected with vaccinia
virus as described above for purposes of providing T7 RNA
polymerase. The desired polycistronic RNA, and plasmids containing
the N, P and L genes are transfected into cells. The transfection
mix is removed after approximately 3 hrs, and cells are infected
with the minivirus at a multiplicity of infection (m.o.i.) of about
1. The minivirus supplies the missing G and/or M proteins. The
polycistronic RNA transfected into the cell will depend on whether
an infectious or non-infectious recombinant rhabdovirus is
wanted.
[0080] Alternatively, a minivirus could be used to provide the N,
P, and L genes. The minivirus could also be used to produce the M
protein in addition to N, P, and L. The minivirus also can produce
the G protein.
[0081] When using a helper cell line, the genes encoding the
missing rhabdovirus proteins are produced by the helper cell line.
The helper cell line has N, P, L, and G proteins for production of
recombinant rhabdovirus particles which does not encode wild-type G
protein. The proteins are expressed from genes or DNAs that are not
part of the recombinant virus genome. These plasmids or other
vector system is stably incorporated into the genome of the cell
line. The proteins are then produced from the cell's genome and not
from a replicon in the cytoplasm. The helper cell line can then be
transfected with a polycistronic DNA and plasmid cDNAs containing
the other rhabdovirus genes not expressed by the helper virus. The
polycistronic RNA used will depend on whether an infectious or
non-infectious recombinant rhabdovirus is desired. Otherwise,
supply of missing gene products (e.g., G and/or M) would be
accomplished as described above.
II. VIRAL COMPOSITIONS
[0082] The present invention concerns rhabdoviruses that are
advantageous in the study and treatment of hyperproliferative or
neoplastic cells (e.g., cancer cells) and hyperproliferative or
neoplastic conditions (e.g., cancer) in a patient. It may concern,
but is not limited to, rhabdoviruses with a reduced neurovirulence,
e.g., non-VSV rhabdoviruses. In certain aspects rhabdovirus that
encode or contain one or more protein components (N, P, M, G,
and/or L proteins) or a nucleic acid genome distinct from those of
VSV (i.e., at least or at most 10, 20, 40, 50, 60, 70, 80%
identical at the amino acid or nucleotide level), and/or that have
been constructed with one or more mutations or variations as
compared to a wild-type virus or viral proteins such that the virus
has desirable properties for use against cancer cells, while being
less toxic or non-toxic to non-cancer cells than the virus as
originally isolated or VSV. The teachings described below provide
various examples of protocols for implementing methods and
compositions of the invention. They provide background for
generating mutated or variant viruses through the use of
bioselection or recombinant DNA or nucleic acid technology.
[0083] A. Proteinaceous Compositions
[0084] Proteinaceous compositions of the invention include viral
particles and compositions including the viral particles, as well
as isolated polypeptides. In certain embodiments, the present
invention concerns generating or isolating pseudotyped or non-VSV
oncolytic rhabdoviruses (rhabdoviruses that lyse, kill, or retard
growth of cancer cells). In certain embodiments, rhabdoviruses will
be engineered to include polypeptide variants of rhabdovirus
proteins (N, P, M, G, and/or L) and/or therapeutic nucleic acids
that encode therapeutic polypeptides. Other aspects of the
invention include the isolation of rhabdoviruses that lack one or
more functional polypeptides or proteins. In other embodiments, the
present invention concerns rhabdoviruses and their use in
combination with or included within proteinaceous compositions as
part of a pharmaceutically acceptable formulation.
[0085] As used herein, a "protein" or "polypeptide" refers to a
molecule comprising polymer of amino acid residues. In some
embodiments, a wild-type version of a protein or polypeptide are
employed, however, in many embodiments of the invention, all or
part of a viral protein or polypeptide is absent or altered so as
to render the virus more useful for the treatment of a patient. The
terms described above may be used interchangeably herein. A
"modified protein" or "modified polypeptide" or "variant protein"
or "variant polypeptide" refers to a protein or polypeptide whose
chemical structure or amino acid sequence is altered with respect
to the wild-type or a reference protein or polypeptide. In some
embodiments, a modified protein or polypeptide has at least one
modified activity or function (recognizing that proteins or
polypeptides may have multiple activities or functions). The
modified activity or function may be reduced, diminished,
eliminated, enhanced, improved, or altered in some other way (such
as infection specificity) with respect to that activity or function
in a wild-type protein or polypeptide, or the characteristics of
virus containing such a polypeptide. It is contemplated that a
modified protein or polypeptide may be altered with respect to one
activity or function yet retain wild-type or unaltered activity or
function in other respects. Alternatively, a modified protein may
be completely nonfunctional or its cognate nucleic acid sequence
may have been altered so that the polypeptide is no longer
expressed at all, is truncated, or expresses a different amino acid
sequence as a result of a frameshift or other modification.
[0086] In certain embodiments the size of a recombinant protein or
polypeptide may comprise, but is not limited to, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,
45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,
62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,
79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,
96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,
200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425,
450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750,
775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1100, 1200,
1300, 1400, 1500, 1750, 2000, 2250, 2500 or greater amino molecule
residues, and any range derivable therein. It is contemplated that
polypeptides may be modified by truncation, rendering them shorter
than their corresponding unaltered form or by fusion or domain
shuffling which may render the altered protein longer.
[0087] As used herein, an "amino molecule" refers to any amino
acid, amino acid derivative, or amino acid mimic as would be known
to one of ordinary skill in the art. In certain embodiments, the
residues of the proteinaceous molecule are sequential, without any
non-amino molecule interrupting the sequence of amino molecule
residues. In other embodiments, the sequence may comprise one or
more non-amino molecule moieties. In particular embodiments, the
sequence of residues of the proteinaceous molecule may be
interrupted by one or more non-amino molecule moieties.
Accordingly, the term "proteinaceous composition" encompasses amino
molecule sequences comprising at least one of the 20 common amino
acids in naturally synthesized proteins, or at least one modified
or unusual amino acid.
[0088] Proteinaceous compositions may be made by any technique
known to those of skill in the art, including the expression of
proteins, polypeptides, or peptides through standard molecular
biological techniques, the isolation of proteinaceous compounds
from natural sources, or the chemical synthesis of proteinaceous
materials. The nucleotide and polypeptide sequences for various
rhabdovirus genes or genomes have been previously disclosed, and
may be found at computerized databases known to those of ordinary
skill in the art. One such database is the National Center for
Biotechnology Information's GenBank and GenPept databases, which
can be accessed via the internet at ncbi.nlm.nih.gov/. The coding
regions for these known genes and viruses may be amplified and/or
expressed using the techniques disclosed herein or as would be know
to those of ordinary skill in the art.
[0089] B. Functional Aspects
[0090] When the present application refers to the function or
activity of viral proteins or polypeptides, it is meant to refer to
the activity or function of that viral protein or polypeptide under
physiological conditions, unless otherwise specified. For example,
the G protein is involved in specificity and efficiency of binding
and infection of particular cell types. Determination of which
molecules possess this activity may be achieved using assays
familiar to those of skill in the art, such as infectivity assays,
protein binding assays, plaque assays and the like.
[0091] C. Variants of Viral Polypeptides
[0092] Amino acid sequence variants of the polypeptides of the
present invention can be substitutional, insertional or deletion
variants. A mutation in a gene encoding a viral polypeptide may
affect 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,
52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,
69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,
86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110,
120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240,
250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more
non-contiguous or contiguous amino acids (i.e., segment) of a
polypeptide, as compared to a wild-type or unaltered polypeptide or
other reference polypeptide. Various polypeptides encoded by
rhabdoviruses may be identified by reference to GenBank Accession
Numbers and the related public database entries for each of the
viruses disclosed herein, all GenBank entries related to the family
rhabdoviridae are incorporated herein by reference.
[0093] Deletion variants lack one or more residues of the native,
unaltered or wild-type protein. Individual residues can be deleted,
or all or part of a domain (such as a catalytic or binding domain)
can be deleted. A stop codon may be introduced (by substitution or
insertion) into an encoding nucleic acid sequence to generate a
truncated protein. Insertional mutants typically involve the
addition of material at a non-terminal point in the polypeptide, a
specific type of insert is a chimeric polypeptide that include
homologous or similar portions of a related protein in place of the
related portion of a target protein. This may include the insertion
of an immunoreactive epitope or simply one or more residues.
Terminal additions, typically called fusion proteins, may also be
generated.
[0094] Substitutional variants typically contain the exchange of
one amino acid for another at one or more sites within the protein,
and may be designed to modulate one or more properties of the
polypeptide, with or without the loss of other functions or
properties. Substitutions may be conservative, that is, one amino
acid is replaced with one of similar shape and charge. Conservative
substitutions are well known in the art and include, for example,
the changes of: alanine to serine; arginine to lysine; asparagine
to glutamine or histidine; aspartate to glutamate; cysteine to
serine; glutamine to asparagine; glutamate to aspartate; glycine to
proline; histidine to asparagine or glutamine; isoleucine to
leucine or valine; leucine to valine or isoleucine; lysine to
arginine; methionine to leucine or isoleucine; phenylalanine to
tyrosine, leucine or methionine; serine to threonine; threonine to
serine; tryptophan to tyrosine; tyrosine to tryptophan or
phenylalanine; and valine to isoleucine or leucine. Alternatively,
substitutions may be non-conservative such that a function or
activity of the polypeptide is affected. Non-conservative changes
typically involve substituting a residue with one that is
chemically dissimilar, such as a polar or charged amino acid for a
nonpolar or uncharged amino acid, and vice versa.
[0095] The term "functionally equivalent codon" is used herein to
refer to codons that encode the same amino acid, such as the six
codons for arginine or serine, and also refers to codons that
encode biologically equivalent amino acids (see Table 1,
below).
TABLE-US-00001 TABLE 1 Codon Table Amino Acids Codons Alanine Ala A
GCA GCC GCG GCU Cysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU
Glutamic acid Glu E GAA GAG Phenylalanine Phe F UUC UUU Glycine Gly
G GGA GGC GGG GGU Histidine His H CAC CAU Isoleucine Ile I AUA AUC
AUU Lysine Lys K AAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUU
Methionine Met M AUG Asparagine Asn N AAC AAU Proline Pro P CCA CCC
CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG
CGU Scrine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr T ACA ACC
ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine
Tyr Y UAC UAU
[0096] It also will be understood that amino acid and nucleic acid
sequences may include additional residues, such as additional N- or
C-terminal amino acids or 5' or 3' sequences, and yet still be
essentially as set as forth herein, including having a certain
biological activity. The addition of terminal sequences
particularly applies to nucleic acid sequences that may, for
example, include various non-coding sequences flanking either of
the 5' or 3' portions of the coding region or may include various
internal sequences, i.e., introns, which are known to occur within
genes.
[0097] The following is a discussion based upon changing of the
amino acids of a N, P, L, or G protein to create an equivalent, or
even an improved, molecule. For example, certain amino acids may be
substituted for other amino acids in a protein structure without
appreciable loss of interactive binding capacity with structures
such as, for example, antigen-binding regions of antibodies or
binding sites on substrate molecules. Since it is the interactive
capacity and nature of a protein that defines that protein's
biological functional activity, certain amino acid substitutions
can be made in a protein sequence, and in its underlying DNA coding
sequence, and nevertheless produce a protein with like properties.
It is thus contemplated by the inventors that various changes may
be made in the DNA sequences of rhabdovirus without appreciable
loss of biological utility or activity of interest, as discussed
below.
[0098] In making such changes, the hydropathic index of amino acids
may be considered. The importance of the hydropathic amino acid
index in conferring a biologic function on a protein is generally
understood in the art (Kyte and Doolittle, 1982). It is accepted
that the relative hydropathic character of the amino acid
contributes to the secondary structure of the resultant protein,
which in turn defines the interaction of the protein with other
molecules, for example, enzymes, substrates, receptors, DNA,
antibodies, antigens, and the like.
[0099] It also is understood in the art that the substitution of
like amino acids can be made effectively on the basis of
hydrophilicity. U.S. Pat. No. 4,554,101, incorporated herein by
reference, states that the greatest local average hydrophilicity of
a protein, as governed by the hydrophilicity of its adjacent amino
acids, correlates with a biological property of the protein. As
detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity
values have been assigned to amino acid residues: arginine (+3.0);
lysine (+3.0); aspartate (+3.0.+-.1); glutamate (+3.0.+-.1); serine
(+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine
(-0.4); proline (-0.5.+-.1); alanine (0.5); histidine*-0.5);
cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8);
isoleucine (-1.8); tyrosine (2.3); phenylalanine (-2.5); tryptophan
(-3.4). It is understood that an amino acid can be substituted for
another having a similar hydrophilicity value and still produce a
biologically equivalent and immunologically equivalent protein. In
such changes, the substitution of amino acids whose hydrophilicity
values are within .+-.2 is preferred, those that are within .+-.1
are particularly preferred, and those within .+-.0.5 are even more
particularly preferred.
[0100] As outlined above, amino acid substitutions generally are
based on the relative similarity of the amino acid side-chain
substituents, for example, their hydrophobicity, hydrophilicity,
charge, size, and the like. Exemplary substitutions that take into
consideration the various foregoing characteristics are well known
to those of skill in the art and include: arginine and lysine;
glutamate and aspartate; serine and threonine; glutamine and
asparagine; and valine, leucine and isoleucine.
III. NUCLEIC ACID MOLECULES
[0101] The present invention includes polynucleotides isolatable
from cells that are capable of expressing all or part of a viral
protein or polypeptide. In some embodiments of the invention, it
concerns all or parts of a viral genome that has been specifically
mutated or altered to generate a virus or viral polypeptide, e.g.,
a pseudotyped or non-VSV rhabdoviral polypeptide or virus, with
certain properties and/or characteristics. The polynucleotides may
encode a peptide or polypeptide containing all or part of a viral
or heterologous amino acid sequence or be engineered so they do not
encode such a viral polypeptide or encode a viral polypeptide
having at least one function or activity added, increased, reduced,
added, diminished, or absent. Recombinant proteins can be purified
from expressing cells to yield active proteins. The genome of
rhabdovirus members may be found in GenBank Accession Numbers in
the NCBI database or similar databases, each of which is
incorporated herein by reference.
[0102] A. Polynucleotides Encoding Native or Modified Proteins
[0103] As used herein, the term "RNA, DNA, or nucleic acid segment"
refers to a RNA, DNA, or nucleic acid molecule that has been
isolated free of total genomic DNA or other contaminants.
Therefore, a nucleic acid segment encoding a polypeptide refers to
a nucleic acid segment that contains wild-type, polymorphic, or
mutant polypeptide-coding sequences yet is isolated away from, or
purified free from, genomic nucleic acid(s). Included within the
term "nucleic acid segment" are polynucleotides, nucleic acid
segments smaller than a polynucleotide, and recombinant vectors,
including, for example, plasmids, cosmids, phage, viruses, and the
like.
[0104] As used in this application, the term "rhabdovirus
polynucleotide" can refer to pseudotyped or non-VSV rhabdoviral
nucleic acid molecule encoding at least one non-VSV rhabdovirus
polypeptide. In certain embodiments the polynucleotide has been
isolated free of other nucleic acids. Similarly, a "Maraba virus,
Carajas virus, Muir Springs virus and/or Bahia Grande virus
polynucleotide" refers to a nucleic acid molecule encoding a Maraba
virus, Carajas virus, Muir Springs virus and/or Bahia Grande virus
polypeptide that has been isolated from other nucleic acids. A
"rhabdovirus genome" or a "Maraba virus, Carajas virus, Muir
Springs virus and/or Bahia Grande virus genome" refers to a VSV or
a non-VSV nucleic acid molecule that can be provided to a host cell
to yield a viral particle, in the presence or absence of a helper
virus or complementing coding regions supplying other factors in
trans. The genome may or may have not been recombinantly mutated as
compared to wild-type or an unaltered virus.
[0105] The term "cDNA" is intended to refer to DNA prepared using
RNA as a template. There may be times when the full or partial
genomic sequence is preferred.
[0106] It also is contemplated that a particular polypeptide from a
given species may be represented by natural variants that have
slightly different nucleic acid sequences but, nonetheless, encode
the same protein (see Table 1 above).
[0107] Similarly, a polynucleotide encoding an isolated or purified
wild-type, or modified polypeptide refers to a DNA segment
including wild-type or mutant polypeptide coding sequences and, in
certain aspects, regulatory sequences, isolated substantially away
from other naturally occurring genes or protein encoding sequences.
In this respect, the term "gene" is used for simplicity to refer to
a nucleic acid unit encoding a protein, polypeptide, or peptide
(including any sequences required for proper transcription,
post-translational modification, or localization). As will be
understood by those in the art, this functional term includes
genomic sequences, cDNA sequences, and smaller engineered nucleic
acid segments that express, or may be adapted to express, proteins,
polypeptides, domains, peptides, fusion proteins, and mutants. A
nucleic acid encoding all or part of a native or modified
polypeptide may contain a contiguous nucleic acid of: 10, 20, 30,
40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170,
180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300,
310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430,
440, 441, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550,
560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680,
690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810,
820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940,
950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060,
1070, 1080, 1090, 1095, 1100, 1500, 2000, 2500, 3000, 3500, 4000,
4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 9000, 10000, or
more nucleotides, nucleosides, or base pairs.
[0108] In particular embodiments, the invention concerns isolated
nucleic acid segments and recombinant vectors incorporating nucleic
acid sequences that encode a wild-type or mutant rhabdovirus
polypeptide(s) that includes within its amino acid sequence a
contiguous amino acid sequence in accordance with, or essentially
corresponding to a native polypeptide. The term "recombinant" may
be used in conjunction with a polypeptide or the name of a specific
polypeptide, and this generally refers to a polypeptide produced
from a nucleic acid molecule that has been manipulated in vitro or
that is the replicated product of such a molecule.
[0109] In other embodiments, the invention concerns isolated
nucleic acid segments and recombinant vectors incorporating nucleic
sequences that encode a polypeptide or peptide that includes within
its amino acid sequence a contiguous amino acid sequence in
accordance with, or essentially corresponding to one or more
rhabdovirus polypeptide.
[0110] The nucleic acid segments used in the present invention,
regardless of the length of the coding sequence itself, may be
combined with other nucleic acid sequences, such as promoters,
polyadenylation signals, additional restriction enzyme sites,
multiple cloning sites, other coding segments, and the like, such
that their overall length may vary considerably. It is therefore
contemplated that a nucleic acid fragment of almost any length may
be employed, with the total length preferably being limited by the
ease of preparation and use in the intended recombinant nucleic
acid protocol.
[0111] It is contemplated that the nucleic acid constructs of the
present invention may encode hill-length polypeptide(s) from any
source or encode a truncated or modified version of the
polypeptide(s), for example a truncated rhabdovirus polypeptide,
such that the transcript of the coding region represents the
truncated version. The truncated transcript may then be translated
into a truncated protein. Alternatively, a nucleic acid sequence
may encode a full-length polypeptide sequence with additional
heterologous coding sequences, for example to allow for
purification of the polypeptide, transport, secretion,
post-translational modification, or for therapeutic benefits such
as targeting or efficacy. As discussed above, a tag or other
heterologous polypeptide may be added to the modified
polypeptide-encoding sequence, wherein "heterologous" refers to a
polypeptide or segment thereof that is not the same as the modified
polypeptide or found associated with or encoded by the naturally
occurring virus.
[0112] In a non-limiting example, one or more nucleic acid
construct may be prepared that include a contiguous stretch of
nucleotides identical to or complementary to a particular viral
segment, such as a rhabdovirus N, P, M, G, or L gene. A nucleic
acid construct may be at least 20, 30, 40, 50, 60, 70, 80, 90, 100,
110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 400,
500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000,
7,000, 8,000, 9,000, 10,000, 15,000, 20,000, 30,000, 50,000,
100,000, 250,000, 500,000, 750,000, to at least 1,000,000
nucleotides in length, as well as constructs of greater size, up to
and including chromosomal sizes (including all intermediate lengths
and intermediate ranges). It will be readily understood that
"intermediate lengths" and "intermediate ranges," as used herein,
means any length or range including or between the quoted values
(i.e., all integers including and between such values).
[0113] The nucleic acid segments used in the present invention
encompass modified nucleic acids that encode modified polypeptides.
Such sequences may arise as a consequence of codon redundancy and
functional equivalency that are known to occur naturally within
nucleic acid sequences and the proteins thus encoded.
Alternatively, functionally equivalent proteins or peptides may be
created via the application of recombinant DNA technology, in which
changes in the protein structure may be engineered, based on
considerations of the properties of the amino acids being
exchanged. Changes designed by human may be introduced through the
application of site-directed mutagenesis techniques, e.g., to
introduce improvements to the antigenicity or lack thereof of the
protein, to reduce toxicity effects of the protein in vivo to a
subject given the protein, or to increase the efficacy of any
treatment involving the protein or a virus comprising such
protein.
[0114] In certain other embodiments, the invention concerns
isolated nucleic acid segments and recombinant vectors that include
within their sequence a contiguous nucleic acid sequence from that
shown in sequences identified herein (and/or incorporated by
reference). Such sequences, however, may be mutated to yield a
protein product whose activity is altered with respect to
wild-type.
[0115] It also will be understood that this invention is not
limited to the particular nucleic acid and amino acid sequences of
these identified sequences. Recombinant vectors and isolated
nucleic acid segments may therefore variously include
rhabdovirus-coding regions themselves, coding regions bearing
selected alterations or modifications in the basic coding region,
or they may encode larger polypeptides that nevertheless include
rhabdovirus-coding regions, or may encode biologically functional
equivalent proteins or peptides that have variant amino acids
sequences.
[0116] The nucleic acid segments of the present invention can
encode rhabdovirus proteins and peptides that are the biological
functional equivalent of, or variants or mutants of rhabdovirus
that increase the therapeutic benefit of the virus. Such sequences
may arise as a consequence of codon redundancy and functional
equivalency that are known to occur naturally within nucleic acid
sequences and the proteins thus encoded. Alternatively,
functionally equivalent proteins or peptides may be created via the
application of recombinant DNA technology, in which changes in the
protein structure may be engineered, based on considerations of the
properties of the amino acids being exchanged. Changes designed by
man may be introduced through the application of site directed
mutagenesis techniques, e.g., to introduce improvements in cancer
cell binding of a viral protein.
[0117] B. Mutagenesis of Rhabdovirus Polynucleotides
[0118] In various embodiments, the rhabdovirus polynucleotide may
be altered or mutagenized. Alterations or mutations may include
insertions, deletions, point mutations, inversions, and the like
and may result in the modulation, activation and/or inactivation of
certain proteins or molecular mechanisms, as well as altering the
function, location, or expression of a gene product, in particular
rendering a gene product non-functional. Where employed,
mutagenesis of a polynucleotide encoding all or part of a
rhabdovirus may be accomplished by a variety of standard, mutagenic
procedures (Sambrook et al., 2001). Mutation is the process whereby
changes occur in the quantity or structure of an organism. Mutation
can involve modification of the nucleotide sequence of a single
gene, blocks of genes or whole genomes. Changes in single genes may
be the consequence of point mutations which involve the removal,
addition or substitution of a single nucleotide base within a DNA
sequence, or they may be the consequence of changes involving the
insertion or deletion of large numbers of nucleotides.
[0119] 1. Random Mutagenesis
[0120] a. Insertional Mutagenesis
[0121] Insertional mutagenesis is based on the inactivation of a
gene via insertion of a known nucleic acid fragment. Because it
involves the insertion of some type of nucleic acid fragment, the
mutations generated are generally loss-of-function, rather than
gain-of-function mutations. However, there are several examples of
insertions generating gain-of-function mutations. Insertional
mutagenesis may be accomplished using standard molecular biology
techniques.
[0122] b. Chemical Mutagenesis
[0123] Chemical mutagenesis offers certain advantages, such as the
ability to find a full range of mutations with degrees of
phenotypic severity, and is facile and inexpensive to perform. The
majority of chemical carcinogens produce mutations in DNA.
Benzo[a]pyrene, N-acetoxy-2-acetyl aminofluorene and aflotoxin B1
cause GC to TA transversions in bacteria and mammalian cells.
Benzo[a]pyrene also can produce base substitutions such as AT to
TA. N-nitroso compounds produce GC to AT transitions. Alkylation of
the O4 position of thymine induced by exposure to n-nitrosourea
results in TA to CC transitions.
[0124] c. Radiation Mutagenesis
[0125] Biological molecules are degraded by ionizing radiation.
Adsorption of the incident energy leads to the formation of ions
and free radicals, and breakage of some covalent bonds.
Susceptibility to radiation damage appears quite variable between
molecules, and between different crystalline forms of the same
molecule. It depends on the total accumulated dose, and also on the
dose rate (as once free radicals are present, the molecular damage
they cause depends on their natural diffusion rate and thus upon
real time). Damage is reduced and controlled by making the sample
as cold as possible. Ionizing radiation causes DNA damage,
generally proportional to the dose rate.
[0126] In the present invention, the term "ionizing radiation"
means radiation comprising particles or photons that have
sufficient energy or can produce sufficient energy to produce
ionization (gain or loss of electrons). An exemplary and preferred
ionizing radiation is an x-radiation. The amount of ionizing
radiation needed in a given cell or for a particular molecule
generally depends upon the nature of that cell or molecule and the
nature of the mutation target. Means for determining an effective
amount of radiation are well known in the art.
[0127] d. In Vitro Scanning Mutagenesis
[0128] Random mutagenesis also may be introduced using error prone
PCR. The rate of mutagenesis may be increased by performing PCR in
multiple tubes with dilutions of templates. One particularly useful
mutagenesis technique is alanine scanning mutagenesis in which a
number of residues are substituted individually with the amino acid
alanine so that the effects of losing side-chain interactions can
be determined, while minimizing the risk of large-scale
perturbations in protein conformation (Cunningham el al.,
1989).
[0129] In vitro scanning saturation mutagenesis provides a rapid
method for obtaining a large amount of stricture-function
information including: (i) identification of residues that modulate
ligand binding specificity, (ii) a better understanding of ligand
binding based on the identification of those amino acids that
retain activity and those that abolish activity at a given
location, (iii) an evaluation of the overall plasticity of an
active site or protein subdomain, (iv) identification of amino acid
substitutions that result in increased binding.
[0130] Site-Directed Mutagenesis
[0131] Structure-guided site-specific mutagenesis represents a
powerful tool for the dissection and engineering of protein-ligand
interactions (Wells, 1996; Braisted et al., 1996). The technique
provides for the preparation and testing of sequence variants by
introducing one or more nucleotide sequence changes into a selected
DNA.
[0132] C. Vectors
[0133] To generate mutations in a rhabdovirus genome, native and
modified polypeptides may be encoded by a nucleic acid molecule
comprised in a vector. The term "vector" is used to refer to a
carrier nucleic acid molecule into which an exogenous nucleic acid
sequence can be inserted for introduction into a cell where it can
be replicated. A nucleic acid sequence can be "exogenous," which
means that it is foreign to the cell into which the vector is being
introduced or that the sequence is homologous to a sequence in the
cell but in a position within the host cell nucleic acid in which
the sequence is ordinarily not found. Vectors include plasmids,
cosmids, viruses (bacteriophage, animal viruses, and plant
viruses), and artificial chromosomes (e.g., YACs). One of skill in
the art would be well equipped to construct a vector through
standard recombinant techniques, which are described in Sambrook et
al. (2001) and Ausubel et al. (1994), both incorporated herein by
reference.
[0134] In addition to encoding a modified polypeptide such as
modified N protein, P protein, M protein, G protein, or L protein,
a vector may encode non-modified polypeptide sequences such as a
tag or targeting molecule. Useful vectors encoding such fusion
proteins include pIN vectors (Inouye et al., 1985), vectors
encoding a stretch of histidines, and pGEX vectors, for use in
generating glutathione S-transferase (GST) soluble fusion proteins
for later purification and separation or cleavage. A targeting
molecule is one that directs the modified polypeptide to a
particular organ, tissue, cell, or other location in a subject's
body. Alternatively, the targeting molecule alters the tropism of
an organism, such as rhabdovirus for certain cell types, e.g.,
cancer cells.
[0135] The term "expression vector" refers to a vector containing a
nucleic acid sequence coding for at least part of a gene product
capable of being transcribed. In some cases, RNA molecules are
translated into a protein, polypeptide, or peptide. In other cases,
these sequences are not translated, for example, in the production
of antisense molecules or ribozymes. Expression vectors can contain
a variety of "control sequences," which refer to nucleic acid
sequences necessary for the transcription and possibly translation
of an operably linked coding sequence in a particular host
organism. In addition to control sequences that govern
transcription and translation, vectors and expression vectors may
contain nucleic acid sequences that serve other functions as well
and are described infra.
[0136] 1. Promoters and Enhancers
[0137] A "promoter" is a control sequence that is a region of a
nucleic acid sequence at which initiation and rate of transcription
are controlled. It may contain genetic elements that bind
regulatory proteins and molecules, such as RNA polymerase and other
transcription factors. The phrases "operatively positioned,"
"operatively coupled," "operatively linked," "under control," and
"under transcriptional control" mean that a promoter is in a
correct functional location and/or orientation in relation to a
nucleic acid sequence to control transcriptional initiation and/or
expression of that sequence. A promoter may or may not be used in
conjunction with an "enhancer," which refers to a cis-acting
regulatory sequence involved in the transcriptional activation of a
nucleic acid sequence.
[0138] A promoter may be one naturally associated with a gene or
sequence, as may be obtained by isolating the 5' non-coding
sequences located upstream of the coding segment and/or exon. Such
a promoter can be referred to as "endogenous." Similarly, an
enhancer may be one naturally associated with a nucleic acid
sequence, located either downstream or upstream of that sequence.
Alternatively, certain advantages will be gained by positioning the
coding nucleic acid segment under the control of a recombinant or
heterologous promoter, which refers to a promoter that is not
normally associated with a nucleic acid sequence in its natural
environment. A recombinant or heterologous enhancer refers also to
an enhancer not normally associated with a nucleic acid sequence in
its natural environment. Such promoters or enhancers may include
promoters or enhancers of other genes, and promoters or enhancers
isolated from any other prokaryotic, viral, or eukaryotic cell, and
promoters or enhancers not "naturally occurring," i.e., containing
different elements of different transcriptional regulatory regions,
and/or mutations that alter expression.
[0139] In addition to producing nucleic acid sequences of promoters
and enhancers synthetically, sequences may be produced using
recombinant cloning and/or nucleic acid amplification technology,
including PCR.TM., in connection with the compositions disclosed
herein (see U.S. Pat. Nos. 4,683,202, 5,928,906, each incorporated
herein by reference). Furthermore, it is contemplated the control
sequences that direct transcription and/or expression of sequences
within non-nuclear organelles such as mitochondria, chloroplasts,
and the like, can be employed as well.
[0140] Naturally, it may be important to employ a promoter and/or
enhancer that effectively directs the expression of the DNA segment
in the cell type, organelle, and organism chosen for expression.
Those of skill in the art of molecular biology generally know the
use of promoters, enhancers, and cell type combinations for protein
expression, for example, see Sambrook et al. (2001), incorporated
herein by reference. The promoters employed may be constitutive,
tissue-specific, cell selective (i.e., more active in one cell type
as compared to another), inducible, and/or useful under the
appropriate conditions to direct high level expression of the
introduced nucleic acid segment, such as is advantageous in the
large-scale production of recombinant proteins and/or peptides. The
promoter may be heterologous or endogenous.
[0141] Several elements/promoters that may be employed, in the
context of the present invention, to regulate the expression of a
gene. This list is not intended to be exhaustive of all the
possible elements involved in the promotion of expression but,
merely, to be exemplary thereof. Also provided are examples of
inducible elements, which are regions of a nucleic acid sequence
that can be activated in response to a specific stimulus.
Promoter/Enhancer (References) include: Immunoglobulin Heavy Chain
(Banerji et al., 1983; Gilles et al., 1983; Grosschedl et al.,
1985; Atchinson et al., 1986, 1987; Imler et al., 1987; Weinberger
et al., 1984; Kiledjian et al., 1988; Porton et al.; 1990);
Immunoglobulin Light Chain (Queen et al., 1983; Picard et al.,
1984); T Cell Receptor (Luria et al., 1987; Winoto et al., 1989;
Redondo et al.; 1990); HLA DQ .alpha. and/or DQ .beta. (Sullivan et
al., 1987); .beta. Interferon (Goodbourn et al., 1986; Fujita et
al., 1987; Goodbourn et al., 1988); Interleukin-2 (Greene et al.,
1989); Interleukin-2 Receptor (Greene et al., 1989; Lin et al.,
1990); MHC Class II 5 (Koch et al., 1989); MHC Class II
HLA-DR.alpha. (Sherman el al., 1989); .beta.-Actin (Kawamoto et
al., 1988; Ng et al.; 1989); Muscle Creatine Kinase (MCK) (Jaynes
et al., 1988; Horlick et al., 1989; Johnson et al., 1989);
Prealbumin (Transthyretin) (Costa et al., 1988); Elastase I (Omitz
et al., 1987); Metallothionein (MTII) (Karin et al., 1987; Culotta
et al., 1989); Collagenase (Pinkert et at., 1987; Angel et al.,
1987); Albumin (Pinkert et al., 1987; Tronche et al., 1989, 1990);
.alpha.-Fetoprotein (Godbout et al., 1988; Campere et al., 1989);
.gamma.-Globin (Bodine at al., 1987; Perez-Stable et al., 1990);
.beta.-Globin (Trudel et al., 1987); c-fos (Cohen et al., 1987);
c-HA-ras (Triesman, 1986; Deschamps et al., 1985); Insulin (Edlund
et al., 1985); Neural Cell Adhesion Molecule (NCAM) (Hirsh et al.,
1990); .alpha.1-Antitrypain (Latimer et al., 1990); H2B (TH2B)
Histone (Hwang et al., 1990); Mouse and/or Type I Collagen (Ripe et
al., 1989); Glucose-Regulated Proteins (GRP94 and GRP78) (Chang et
al., 1989); Rat Growth Hormone (Larsen et al., 1986); Human Serum
Amyloid A (SAA) (Edbrooke et al., 1989); Troponin I (TN I) (Yutzey
et al., 1989); Platelet-Derived Growth Factor (PDGF) (Pech et al.,
1989); Duchenne Muscular Dystrophy (Klamut et al., 1990); SV40
(Banerji et al., 1981; Moreau et al., 1981; Sleigh et al., 1985;
Firak et al., 1986; Herr et al., 1986; Imbra et al., 1986; Kadesch
et al., 1986; Wang et al., 1986; Ondek et al., 1987; Kuhl et al.,
1987; Schaffner et at., 1988); Polyoma (Swartzendruber et al.,
1975; Vasseur et al., 1980; Katinka et al., 1980, 1981; Tyndell et
al., 1981; Dandolo et at., 1983; de Villiers et al., 1984; Hen et
al., 1986; Satake et al., 1988; Campbell et al., 1988);
Retroviruses (Kriegler et al., 1982, 1983; Levinson et al., 1982;
Kriegler et al., 1983, 1984a, b, 1988; Bosze et al., 1986; Miksicek
et al., 1986; Celander et al., 1987; Thiesen et al., 1988; Celander
et al., 1988; Chol et al., 1988; Reisman et al., 1989); Papilloma
Virus (Campo et al., 1983; Lusky et al., 1983; Spandidos and
Wilkie, 1983; Spalholz et al., 1985; Lusky et al., 1986; Cripe et
al., 1987; Gloss et al., 1987; Hirochika et al., 1987; Stephens et
al., 1987); Hepatitis B Virus (Bulla et al., 1986; Jameel et al.,
1986; Shaul et al., 1987; Spandau et al., 1988; Vannice et al.,
1988); Human Immunodeficiency Virus (Muesing et al., 1987; Hauber
et al., 1988; Jakobovits et al., 1988; Feng at al., 1988; Takebe et
al, 1988; Rosen et al., 1988; Berkhout et al., 1989; Laspia et al.,
1989; Sharp et al., 1989; Braddock et al., 1989); Cytomegalovirus
(CMV) (Weber et al., 1984; Boshart et al., 1985; Foecking et al.,
1986); and Gibbon Ape Leukemia Virus (Holbrook et al., 1987; Quinn
et al., 1989).
[0142] Inducible Elements (Element/Inducer (References)) include:
MT II/Phorbol Ester (TFA), Heavy metals (Palmiter et al., 1982;
Haslinger et al., 1985; Searle et al., 1985; Stuart et al., 1985;
Imagawa et al., 1987, Karin et al., 1987; Angel et al., 1987b;
McNeall et al., 1989); MMTV (mouse mammary tumor
virus)/Glucocorticoids (Huang et al., 1981; Lee et al., 1981;
Majors et al., 1983; Chandler et al., 1983; Lee et al., 1984; Ponta
et al., 1985; Sakai et al., 1988); .beta.-Interferon/poly(rI)x,
poly(rc) (Tavernier et al., 1983); Adenovirus 5 E2/E1A (Imperiale
et al., 1984); Collagenase/Phorbol Ester (TPA) (Angel et al.,
1987a); Stromelysin/Phorbol Ester (TPA) (Angel et al., 1987b);
SV40/Phorbol Ester (TPA) (Angel et al., 1987b); Murine MX
Gene/Interferon, Newcastle Disease Virus (Hug et al., 1988); GRP78
Gene/A23187 (Resendez et al., 1988); .alpha.-2-Macroglobulin/IL-6
(Kunz et al., 1989); Vimentin/Serum (Riffling et al., 1989); MHC
Class I Gene H-2.kappa.b/Interferon (Blanar et al., 1989);
HSP70/E1A, SV40 Large T Antigen (Taylor et al., 1989, 1990a,
1990b); Proliferin/Phorbol Ester-TPA (Mordacq et al., 1989); Tumor
Necrosis Factor/PMA (Hensel et al., 1989); and Thyroid Stimulating
Hormone .alpha. Gene/Thyroid Hormone (Chatterjee et al., 1989).
[0143] The identity of tissue-specific or tissue-selective (i.e.,
promoters that have a greater activity in one cell as compared to
another) promoters or elements, as well as assays to characterize
their activity, is well known to those of skill in the art.
Examples of such regions include the human LIMK2 gene (Nomoto et
al. 1999), the somatostatin receptor 2 gene (Kraus et al., 1998),
murine epididymal retinoic acid-binding gene (Lareyre et al.,
1999), human CD4 (Zhao-Emonet et al., 1998), mouse alpha2 (XI)
collagen (Tsumaki, et al., 1998), D1A dopamine receptor gene (Lee,
et al., 1997), insulin-like growth factor II (Wu et al., 1997),
human platelet endothelial cell adhesion molecule-1 (Almendro et
al., 1996), and the SM22.alpha. promoter.
[0144] Additional viral promoters, cellular promoters/enhancers and
inducible promoters/enhancers that could be used in combination
with the present invention are listed herein. Additionally any
promoter/enhancer combination (as per the Eukaryotic Promoter Data
Base EPDB) could also be used to drive expression of structural
genes encoding oligosaccharide processing enzymes, protein folding
accessory proteins, selectable marker proteins or a heterologous
protein of interest. Alternatively, a tissue-specific promoter for
cancer gene therapy (Table 2) or the targeting of tumors (Table 3)
may be employed with the nucleic acid molecules of the present
invention.
TABLE-US-00002 TABLE 2 Candidate Tissue-Specific Promoters for
Cancer Gene Therapy Tissue-specific Cancers in which Normal cells
in which promoter promoter is active promoter is active
Carcinoembryonic Most colorectal Colonic mucosa; antigen (CEA)*
carcinomas; 50% of lung gastric mucosa; lung carcinomas; 40-50% of
epithelia; eccrine gastric carcinomas; most sweat glands; cells in
pancreatic carcinomas; testes many breast carcinomas
Prostate-specific Most prostate carcinomas Prostate epithelium
antigen (PSA) Vasoactive Majority of non-small cell Neurons;
lymphocytes; intestinal peptide lung cancers mast cells;
eosinophils (VIP) Surfactant protein Many lung Type II pneumocytes;
A (SP-A) adenocarcinomas cells Clara Human achaete- Most small cell
lung Neuroendocrinc cells in scute homolog cancers lung (hASH)
Mucin-1 Most adenocarcinomas Glandular epithelial (MUC1)**
(originating from any cells in breast and in tissue) respiratory,
gastrointestinal, and genitourinary tracts Alpha-fetoprotein Most
hepatocellular Hepatocytes (under carcinomas; possibly many certain
conditions); testicular cancers testis Albumin Most hepatocellular
Hepatocytcs carcinomas Tyrosinase Most melanomas Melanocytes;
astrocytes; Schwann cells; some neurons Tyrosine-binding Most
melanomas Melanocytes; protein (TRP) astrocytes, Schwann cells;
some neurons Keratin 14 Presumably many Keratinocytes squamous cell
carcinomas (e.g.: Head and neck cancers) EBV LD-2 Many squamous
cell Keratinocytes of upper carcinomas of head and digestive
Keratinocytes neck of upper digestive tract Glial fibrillary Many
astrocytomas Astrocytes acidic protein (GFAP) Myelin basic Many
gliomas Oligodendrocytes protein (MBP) Testis-specific Possibly
many testicular Spermatazoa angiotensin- cancers converting enzyme
(Testis-specific ACE) Osteocalcin Possibly many Osteoblasts
osteosarcomas
TABLE-US-00003 TABLE 3 Candidate Promoters for Use with a
Tissue-Specific Targeting of Tumors Cancers in which Normal cells
in which Promoter Promoter is active Promoter is active
E2F-regulated Almost all cancers Proliferating cells promoter HLA-G
Many colorectal Lymphocytes; carcinomas; many monocytes; melanomas;
possibly spermatocytes; many other cancers trophoblast FasL Most
melanomas; many Activated leukocytes: pancreatic carcinomas;
neurons; endothelial cells; most astrocytomas keratinocytes; cells
in possibly many other immunoprivileged tissues; cancers some cells
in lungs, ovaries, liver, and prostate Myc-regulated Most lung
carcinomas Proliferating cells (only promoter (both small cell and
some cell-types): non-small cell); most mammary epithelial cells
colorectal carcinomas (including non-proliferating) MAGE-1 Many
melanomas; some Testis non-small cell lung carcinomas; some breast
carcinomas VEGF 70% of all cancers Cells at sites of (constitutive
neovascularization overexpression in (but unlike in tumors, many
cancers) expression is transient, less strong, and never
constitutive) bFGF Presumably many Cells at sites of ischemia
different cancers, since (but unlike tumors, bFGF expression is
expression is transient, induced by ischemic less strong, and never
conditions constitutive) COX-2 Most colorectal Cells at sites of
carcinomas; many lung inflammation carcinomas; possibly many other
cancers IL-10 Most colorectal Leukocytes carcinomas; many lung
carcinomas; many squamous cell carcinomas of head and neck;
possibly many other cancers GRP78/BiP Presumably many Cells at
sites of ishemia different cancers, since GRP7S expression is
induced by tumor- specific conditions CarG elements Induced by
ionization Cells exposed to ionizing from Egr-1 radiation, so
conceivably radiation; leukocytes most tumors upon irradiation
[0145] 2. Initiation Signals and Internal Ribosome Binding
Sites
[0146] A specific initiation signal also may be required for
efficient translation of coding sequences. These signals include
the ATG initiation codon or adjacent sequences. Exogenous
translational control signals, including the ATG initiation codon,
may need to be provided. One of ordinary skill in the art would
readily be capable of determining this and providing the necessary
signals. It is well known that the initiation codon must be
"in-frame" with the reading frame of the desired coding sequence to
ensure translation of the entire insert. The exogenous
translational control signals and initiation codons can be either
natural or synthetic. The efficiency of expression may be enhanced
by the inclusion of appropriate transcription enhancer
elements.
[0147] In certain embodiments of the invention, the use of internal
ribosome entry sites (IRES) elements are used to create multigene,
or polycistronic, messages. IRES elements are able to bypass the
ribosome scanning model of 5'.quadrature. methylated Cap dependent
translation and begin translation at internal sites (Pelletier and
Sonenberg, 1988). IRES elements from two members of the
picornavirus family (polio and encephalomyocarditis) have been
described (Pelletier and Sonenberg, 1988), as well an TRES from a
mammalian message (Macejak and Sarnow, 1991). IRES elements can be
linked to heterologous open reading frames. Multiple open reading
frames can be transcribed together, each separated by an IRES,
creating polycistronic messages. By virtue of the IRES element,
each open reading frame is accessible to ribosomes for efficient
translation. Multiple genes can be efficiently expressed using a
single promoter/enhancer to transcribe a single message (see U.S.
Pat. Nos. 5,925,565 and 5,935,819, herein incorporated by
reference).
[0148] 3. Multiple Cloning Sites
[0149] Vectors can include a multiple cloning site (MCS), which is
a nucleic acid region that contains multiple restriction enzyme
sites any of which can be used in conjunction with standard
recombinant technology to digest the vector. (See Carbonelli et
al., 1999, Levenson et al., 1998, and Cocea, 1997, incorporated
herein by reference.) "Restriction enzyme digestion" refers to
catalytic cleavage of a nucleic acid molecule with an enzyme that
functions only at specific locations in a nucleic acid molecule.
Many of these restriction enzymes are commercially available. Use
of such enzymes is widely understood by those of skill in the art.
Frequently, a vector is linearized or fragmented using a
restriction enzyme that cuts within the MCS to enable exogenous
sequences to be ligated to the vector. "Ligation" refers to the
process of forming phosphodiester bonds between two nucleic acid
fragments, which may or may not be contiguous with each other.
Techniques involving restriction enzymes and ligation reactions are
well known to those of skill in the art of recombinant
technology.
[0150] 4. Termination Signals
[0151] The vectors or constructs of the present invention will
generally comprise at least one termination signal. A "termination
signal" or "terminator" is comprised of the RNA sequences involved
in specific termination of an RNA transcript by an RNA polymerase.
Thus, in certain embodiments a termination signal that ends the
production of an RNA transcript is contemplated. A terminator may
be necessary in vivo to achieve desirable message levels.
[0152] In negative sense RNA viruses, including rhabdoviruses,
termination is defined by a RNA motif.
[0153] Terminators contemplated for use in the invention include
any known terminator of transcription described herein or known to
one of ordinary skill in the art, including but not limited to, for
example, the termination sequences of genes, such as for example
the bovine growth hormone terminator or viral termination
sequences, such as for example the SV40 terminator. In certain
embodiments, the termination signal may be a lack of transcribable
or translatable sequence, such as due to a sequence truncation.
[0154] 5. Polyadenylation Signals
[0155] In expression, particularly eukaryotic expression, one will
typically include a polyadenylation signal to effect proper
polyadenylation of the transcript. The nature of the
polyadenylation signal is not believed to be crucial to the
successful practice of the invention, and/or any such sequence may
be employed. Preferred embodiments include the SV40 polyadenylation
signal and/or the bovine growth hormone polyadenylation signal,
convenient and/or known to function well in various target cells.
Polyadenylation may increase the stability of the transcript or may
facilitate cytoplasmic transport.
[0156] 6. Origins of Replication
[0157] In order to propagate a vector in a host cell, it may
contain one or more origins of replication sites (often termed
"ori"), which is a specific nucleic acid sequence at which
replication is initiated. Alternatively an autonomously replicating
sequence (ARS) can be employed if the host cell is yeast.
[0158] 7. Selectable and Screenable Markers
[0159] In certain embodiments of the invention, cells containing a
nucleic acid construct of the present invention may be identified
in vitro or in vivo by including a marker in the expression vector.
Such markers would confer an identifiable change to the cell
permitting easy identification of cells containing the expression
vector. Generally, a selectable marker is one that confers a
property that allows for selection. A positive selectable marker is
one in which the presence of the marker allows for its selection,
while a negative selectable marker is one in which its presence
prevents its selection. An example of a positive selectable marker
is a drug resistance marker.
[0160] Usually the inclusion of a drug selection marker aids in the
cloning and identification of transformants, for example, genes
that confer resistance to neomycin, puromycin, hygromycin, DHFR,
GPT, zeocin and histidinol are useful selectable markers. In
addition to markers conferring a phenotype that allows for the
discrimination of transformants based on the implementation of
conditions, other types of markers including screenable markers
such as GFP, whose basis is colorimetric analysis, are also
contemplated. Alternatively, screenable enzymes such as herpes
simplex virus thymidine kinase (tk) or chloramphenicol
acetyltransferase (CAT) may be utilized. One of skill in the art
would also know how to employ immunologic markers, possibly in
conjunction with FACS analysis. The marker used is not believed to
be important, so long as it is capable of being expressed
simultaneously with the nucleic acid encoding a gene product.
Further examples of selectable and screenable markers are well
known to one of skill in the art.
[0161] D. Host Cells
[0162] As used herein, the terms "cell," "cell line," and "cell
culture" may be used interchangeably. All of these terms also
include their progeny, which is any and all subsequent generations.
It is understood that all progeny may not be identical due to
deliberate or inadvertent mutations. In the context of expressing a
heterologous nucleic acid sequence, "host cell" refers to a
prokaryotic or eukaryotic cell, and it includes any transformable
organisms that is capable of replicating a vector and/or expressing
a heterologous gene encoded by a vector. A host cell can, and has
been, used as a recipient for vectors or viruses (which does not
qualify as a vector if it expresses no exogenous polypeptides). A
host cell may be "transfected" or "transformed," which refers to a
process by which exogenous nucleic acid, such as a modified
protein-encoding sequence, is transferred or introduced into the
host cell. A transformed cell includes the primary subject cell and
its progeny.
[0163] Host cells may be derived from prokaryotes or eukaryotes,
including yeast cells, insect cells, and mammalian cells, depending
upon whether the desired result is replication of the vector or
expression of part or all of the vector-encoded nucleic acid
sequences. Numerous cell lines and cultures are available for use
as a host cell, and they can be obtained through the American Type
Culture Collection (ATCC), which is an organization that serves as
an archive for living cultures and genetic materials
(www.atcc.org). An appropriate host can be determined by one of
skill in the art based on the vector backbone and the desired
result. A plasmid or cosmid, for example, can be introduced into a
prokaryote host cell for replication of many vectors. Bacterial
cells used as host cells for vector replication and/or expression
include DH5.alpha., JM109, and KC8, as well as a number of
commercially available bacterial hosts such as SURE.RTM. Competent
Cells and SOLOPACK.TM. Gold Cells (STRATAGENE.RTM., La Jolla,
Calif.). Alternatively, bacterial cells such as E. coli LE392 could
be used as host cells for phage viruses. Appropriate yeast cells
include Saccharomyces cerevisiae, Saccharomyces pombe, and Pichia
pastoris.
[0164] Examples of eukaryotic host cells for replication and/or
expression of a vector include HeLa, NIH3T3, Jurkat, 293, Cos, CHO,
Saos, and PC12. Many host cells from various cell types and
organisms are available and would be known to one of skill in the
art. Similarly, a viral vector may be used in conjunction with
either a eukaryotic or prokaryotic host cell, particularly one that
is permissive for replication or expression of the vector.
[0165] Some vectors may employ control sequences that allow it to
be replicated and/or expressed in both prokaryotic and eukaryotic
cells. One of skill in the art would further understand the
conditions under which to incubate all of the above described host
cells to maintain them and to permit replication of a vector. Also
understood and known are techniques and conditions that would allow
large-scale production of vectors, as well as production of the
nucleic acids encoded by vectors and their cognate polypeptides,
proteins, or peptides.
[0166] E. Expression Systems
[0167] Numerous expression systems exist that comprise at least all
or part of the compositions discussed above. Prokaryote- and/or
eukaryote-based systems can be employed for use with the present
invention to produce nucleic acid sequences, or their cognate
polypeptides, proteins and peptides. Many such systems are
commercially and widely available.
[0168] The insect cell/baculovirus system can produce a high level
of protein expression of a heterologous nucleic acid segment, such
as described in U.S. Pat. Nos. 5,871,986 and 4,879,236, both herein
incorporated by reference, and which can be bought, for example,
under the name MAXBAC.RTM. 2.0 from INVITROGEN.RTM. and BACPACK.TM.
BACULOVIRUS EXPRESSION SYSTEM FROM CLONTECH.RTM..
[0169] In addition to the disclosed expression systems of the
invention, other examples of expression systems include
STRATAGENE.RTM.'s COMPLETE CONTROL.TM. Inducible Mammalian
Expression System, which involves a synthetic ecdysone-inducible
receptor, or its pET Expression System, an E. coli expression
system. Another example of an inducible expression system is
available from INVITROGEN.RTM., which carries the T-REX.TM.
(tetracycline-regulated expression) System, an inducible mammalian
expression system that uses the full-length CMV promoter.
INVITROGEN.RTM. also provides a yeast expression system called the
Pichia methanolica Expression System, which is designed for
high-level production of recombinant proteins in the methylotrophic
yeast Pichia methanolica. One of skill in the art would know how to
express a vector, such as an expression construct, to produce a
nucleic acid sequence or its cognate polypeptide, protein, or
peptide.
[0170] F. Nucleic Acid Detection
[0171] In addition to their use in directing the expression of
poxvirus proteins, polypeptides and/or peptides, the nucleic acid
sequences disclosed herein have a variety of other uses. For
example, they have utility as probes or primers for embodiments
involving nucleic acid hybridization. They may be used in
diagnostic or screening methods of the present invention. Detection
of nucleic acids encoding rhabdovirus or rhabdovirus polypeptide
modulators are encompassed by the invention.
[0172] 1. Hybridization
[0173] The use of a probe or primer of between 13 and 100
nucleotides, preferably between 17 and 100 nucleotides in length,
or in some aspects of the invention up to 1-2 kilobases or more in
length, allows the formation of a duplex molecule that is both
stable and selective. Molecules having complementary sequences over
contiguous stretches greater than 20 bases in length are generally
preferred, to increase stability and/or selectivity of the hybrid
molecules obtained. One will generally prefer to design. nucleic
acid molecules for hybridization having one or more complementary
sequences of 20 to 30 nucleotides, or even longer where desired.
Such fragments may be readily prepared, for example, by directly
synthesizing the fragment by chemical means or by introducing
selected sequences into recombinant vectors for recombinant
production.
[0174] Accordingly, the nucleotide sequences of the invention may
be used for their ability to selectively form duplex molecules with
complementary stretches of DNAs and/or RNAs or to provide primers
for amplification of DNA or RNA from samples. Depending on the
application envisioned, one would desire to employ varying
conditions of hybridization to achieve varying degrees of
selectivity of the probe or primers for the target sequence,
[0175] For applications requiring high selectivity, one will
typically desire to employ relatively high stringency conditions to
form the hybrids. For example, relatively low salt and/or high
temperature conditions, such as provided by about 0.02 M to about
0.10 M NaCl at temperatures of about 50.degree. C. to about
70.degree. C. Such high stringency conditions tolerate little, if
any, mismatch between the probe or primers and the template or
target strand and would be particularly suitable for isolating
specific genes or for detecting specific mRNA transcripts. It is
generally appreciated that conditions can be rendered more
stringent by the addition of increasing amounts of formamide.
[0176] For certain applications, for example, site-directed
mutagenesis, it is appreciated that lower stringency conditions are
preferred. Under these conditions, hybridization may occur even
though the sequences of the hybridizing strands are not perfectly
complementary, but are mismatched at one or more positions.
Conditions may be rendered less stringent by increasing salt
concentration and/or decreasing temperature. For example, a medium
stringency condition could be provided by about 0.1 to 0.25 M NaCl
at temperatures of about 37.degree. C. to about 55.degree. C.,
while a low stringency condition could be provided by about 0.15 M
to about 0.9 M salt, at temperatures ranging from about 20.degree.
C. to about 55.degree. C. Hybridization conditions can be readily
manipulated depending on the desired results.
[0177] In other embodiments, hybridization may be achieved under
conditions of, for example, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3
mM MgCl.sub.2, 1.0 mM dithiothreitol, at temperatures between
approximately 20.degree. C. to about 37.degree. C. Other
hybridization conditions utilized could include approximately 10 mM
Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl.sub.2, at temperatures
ranging from approximately 40.degree. C. to about 72.degree. C.
[0178] In certain embodiments, it will be advantageous to employ
nucleic acids of defined sequences of the present invention in
combination with an appropriate means, such as a label, for
determining hybridization. A wide variety of appropriate indicator
means are known in the art, including fluorescent, radioactive,
enzymatic or other ligands, such as avidin/biotin, which are
capable of being detected. In preferred embodiments, one may desire
to employ a fluorescent label or an enzyme tag such as urease,
alkaline phosphatase or peroxidase, instead of radioactive or other
environmentally undesirable reagents. In the case of enzyme tags,
colorimetric indicator substrates are known that can be employed to
provide a detection means that is visibly or spectrophotometrically
detectable, to identify specific hybridization with complementary
nucleic acid containing samples.
[0179] In general, it is envisioned that the probes or primers
described herein will be useful as reagents in solution
hybridization, as in PCR.TM., for detection of expression of
corresponding genes, as well as in embodiments employing a solid
phase. In embodiments involving a solid phase, the test DNA (or
RNA) is adsorbed or otherwise affixed to a selected matrix or
surface. This fixed, single-stranded nucleic acid is then subjected
to hybridization with selected probes under desired conditions. The
conditions selected will depend on the particular circumstances
(depending, for example, on the G+C content, type of target nucleic
acid, source of nucleic acid, size of hybridization probe, etc.).
Optimization of hybridization conditions for the particular
application of interest is well known to those of skill in the art.
After washing of the hybridized molecules to remove
non-specifically bound probe molecules, hybridization is detected,
and/or quantified, by determining the amount of bound label.
Representative solid phase hybridization methods are disclosed in
U.S. Pat. Nos. 5,843,663, 5,900,481 and 5,919,626. Other methods of
hybridization that may be used in the practice of the present
invention are disclosed in U.S. Pat. Nos. 5,849,481, 5,849,486 and
5,851,772. The relevant portions of these and other references
identified in this section of the Specification are incorporated
herein by reference.
[0180] 2. Amplification of Nucleic Acids
[0181] Nucleic acids used as a template for amplification may be
isolated from cells, tissues or other samples according to standard
methodologies (Sambrook et al., 2001). In certain embodiments,
analysis is performed on whole cell or tissue homogenates or
biological fluid samples without substantial purification of the
template nucleic acid. The nucleic acid may be genomic DNA or
fractionated or whole cell RNA. Where RNA is used, it may be
desired to first convert the RNA to a complementary DNA.
[0182] The term "primer," as used herein, is meant to encompass any
nucleic acid that is capable of priming the synthesis of a nascent
nucleic acid in a template-dependent process. Typically, primers
are oligonucleotides from ten to twenty and/or thirty base pairs in
length, but longer sequences can be employed. Primers may be
provided in double-stranded and/or single-stranded form, although
the single-stranded form is preferred.
[0183] Pairs of primers designed to selectively hybridize to
nucleic acids corresponding to sequences of genes identified herein
are contacted with the template nucleic acid under conditions that
permit selective hybridization. Depending upon the desired
application, high stringency hybridization conditions may be
selected that will only allow hybridization to sequences that are
completely complementary to the primers. In other embodiments,
hybridization may occur under reduced stringency to allow for
amplification of nucleic acids contain one or more mismatches with
the primer sequences. Once hybridized, the template-primer complex
is contacted with one or more enzymes that facilitate
template-dependent nucleic acid synthesis. Multiple rounds of
amplification, also referred to as "cycles," are conducted until a
sufficient amount of amplification product is produced.
[0184] A number of template dependent processes are available to
amplify the oligonucleotide sequences present in a given template
sample. One of the best known amplification methods is the
polymerase chain reaction (referred to as PCR.TM.) which is
described in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and
4,800,159, and in Innis et al., 1988, each of which is incorporated
herein by reference in their entirety.
[0185] A reverse transcriptase PCR.TM. amplification procedure may
be performed to quantify the amount of mRNA amplified and are well
known (see Sambrook et al., 2001; WO 90/07641; and U.S. Pat. No.
5,882,864).
[0186] Another method for amplification is ligase chain reaction
("LCR"), disclosed in European Application No. 320 308,
incorporated herein by reference in its entirety. U.S. Pat. No.
4,883,750 describes a method similar to LCR for binding probe pairs
to a target sequence. A method based on PCR.TM. and oligonucleotide
ligase assay (OLA), disclosed in U.S. Pat. No. 5,912,148, may also
be used. Alternative methods for amplification of target nucleic
acid sequences that may be used in the practice of the present
invention are disclosed in U.S. Pat. Nos. 5,843,650, 5,846,709,
5,846,783, 5,849,546, 5,849,497, 5,849,547, 5,858,652, 5,866,366,
5,916,776, 5,922,574, 5,928,905, 5,928,906, 5,932,451, 5,935,825,
5,939,291 and 5,942,391, GB Application No. 2 202 328, and in PCT
Application No. PCT/US89/01025, each of which is incorporated
herein by reference in its entirety. Qbeta Replicase, described in
PCT Application No. PCT/US87/00880, may also be used as an
amplification method in the present invention. Isothermal
amplification as described by Walker et al. (1992) can also be
used. As well as Strand Displacement Amplification (SDA), disclosed
in U.S. Pat. No. 5,916,779.
[0187] Other nucleic acid amplification procedures include
transcription-based amplification systems (TAS), including nucleic
acid sequence based amplification (NASBA) and 3SR (Kwoh et al.,
1989; PCT Application WO 88/10315, incorporated herein by reference
in their entirety). European Application No. 329 822 disclose a
nucleic acid amplification process involving cyclically
synthesizing single-stranded RNA ("ssRNA"), ssDNA, and
double-stranded DNA (dsDNA), which may be used in accordance with
the present invention.
[0188] PCT Application WO 89/06700 (incorporated herein by
reference in its entirety) disclose a nucleic acid sequence
amplification scheme based on the hybridization of a promoter
region/primer sequence to a target single-stranded DNA ("ssDNA")
followed by transcription of many RNA copies of the sequence. Other
amplification methods include "RACE" and "one-sided PCR" (Frohman,
1990; Ohara et al., 1989).
[0189] 3. Detection of Nucleic Acids
[0190] Following any amplification, it may be desirable to separate
and/or isolate the amplification product from the template and/or
the excess primer. In one embodiment, amplification products are
separated by agarose, agarose-acrylamide, or polyacrylamide gel
electrophoresis using standard methods (Sambrook et al., 2001).
[0191] Separation of nucleic acids may also be effected by
chromatographic techniques known in art. There are many kinds of
chromatography which may be used in the practice of the present
invention, including adsorption, partition, ion-exchange,
hydroxylapatite, molecular sieve, reverse-phase, column, paper,
thin-layer, and gas chromatography as well as HPLC.
[0192] Typical visualization methods includes staining of a gel
with ethidium bromide and visualization of bands under UV light.
Alternatively, if the amplification products are integrally labeled
with radio- or fluorometrically-labeled nucleotides, the separated
amplification products can be exposed to x-ray film or visualized
under the appropriate excitatory spectra.
[0193] In particular embodiments, detection is by Southern blotting
and hybridization with a labeled probe. The techniques involved in
Southern blotting are well known to those of skill in the art (see
Sambrook et al., 2001). One example of the foregoing is described
in U.S. Pat. No. 5,279,721, incorporated by reference herein, which
discloses an apparatus and method for the automated electrophoresis
and transfer of nucleic acids.
[0194] Other methods of nucleic acid detection that may be used in
the practice of the instant invention are disclosed in U.S. Pat.
Nos. 5,840,873, 5,843,640, 5,843,651, 5,846,708, 5,846,717,
5,846,726, 5,846,729, 5,849,487, 5,853,990, 5,853,992, 5,853,993,
5,856,092, 5,861,244, 5,863,732, 5,863,753, 5,866,331, 5,905,024,
5,910,407, 5,912,124, 5,912,145, 5,919,630, 5,925,517, 5,928,862,
5,928,869, 5,929,227, 5,932,413 and 5,935,791, each of which is
incorporated herein by reference.
[0195] 4. Other Assays
[0196] Other methods for genetic screening may be used within the
scope of the present invention, for example, to detect mutations in
genomic nucleic acids, cDNA and/or RNA samples. Methods used to
detect point mutations include denaturing gradient gel
electrophoresis ("DGGE"), restriction fragment length polymorphism
analysis ("RFLP"), chemical or enzymatic cleavage methods, direct
sequencing of target regions amplified by PCR.TM. (see above),
single-strand conformation polymorphism analysis ("SSCP") and other
methods well known in the art. One method of screening for point
mutations is based on RNase cleavage of base pair mismatches in
RNA/DNA or RNA/RNA heteroduplexes. As used herein, the term
"mismatch" is defined as a region of one or more unpaired or
mispaired nucleotides in a double-stranded RNA/RNA, RNA/DNA or
DNA/DNA molecule. This definition thus includes mismatches due to
insertion/deletion mutations, as well as single or multiple base
point mutations (for example see U.S. Pat. No. 4,946,773.
Alternative methods for detection of deletion, insertion or
substitution mutations that may be used in the practice of the
present invention are disclosed in U.S. Pat. Nos. 5,849,483,
5,851,770, 5,866,337, 5,925,525 and 5,928,870, each of which is
incorporated herein by reference in its entirety.
[0197] G. Methods of Gene Transfer
[0198] Suitable methods for nucleic acid delivery to effect
expression of compositions of the present invention are believed to
include virtually any method by which a nucleic acid (e.g., DNA or
RNA, including viral and nonviral vectors) can be introduced into
an organelle, a cell, a tissue or an organism, as described herein
or as would be known to one of ordinary skill in the art. Such
methods include, but are not limited to, direct delivery of nucleic
acid such as by injection (U.S. Pat. Nos. 5,994,624, 5,981,274,
5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466
and 5,580,859, each incorporated herein by reference), including
microinjection (Harland and Weintraub, 1985; U.S. Pat. No.
5,789,215, incorporated herein by reference); by electroporation
(U.S. Pat. No. 5,384,253, incorporated herein by reference); by
calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen
and Okayama, 1987; Rippe et at, 1990); by using DEAE dextran
followed by polyethylene glycol (Gopal, 1985); by direct sonic
loading (Fechheimer et al., 1987); by liposome mediated
transfection (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau
et al., 1987; Wong et at, 1980; Kaneda et at, 1989; Kato et al.,
1991); by microprojectile bombardment (PCT Application Nos. WO
94/09699 and 95/06128; U.S. Pat. Nos. 5,610,042; 5,322,783
5,563,055, 5,550,318, 5,538,877 and 5,538,880, and each
incorporated herein by reference); by agitation with silicon
carbide fibers (Kaeppler et at, 1990; U.S. Pat. Nos. 5,302,523 and
5,464,765, each incorporated herein by reference); by Agrobacterium
mediated transformation (U.S. Pat. Nos. 5,591,616 and 5,563,055,
each incorporated herein by reference); or by PEG mediated
transformation of protoplasts (Omirulleh et al., 1993; U.S. Pat.
Nos. 4,684,611 and 4,952,500, each incorporated herein by
reference); by desiccation/inhibition mediated DNA uptake (Potrykus
et al., 1985). Through the application of techniques such as these,
organelle(s), cell(s), tissue(s) or organism(s) may be stably or
transiently transformed.
[0199] H. Lipid Components and Moieties
[0200] In certain embodiments, the present invention concerns
compositions comprising one or more lipids associated with a
nucleic acid, an amino acid molecule, such as a peptide, or another
small molecule compound. In any of the embodiments discussed
herein, the molecule may be either a rhabdovirus polypeptide or a
rhabdovirus polypeptide modulator, for example a nucleic acid
encoding all or part of either a rhabdovirus polypeptide, or
alternatively, an amino acid molecule encoding all or part of
rhabdovirus polypeptide modulator. A lipid is a substance that is
characteristically insoluble in water and extractable with an
organic solvent. Compounds other than those specifically described
herein are understood by one of skill in the art as lipids, and are
encompassed by the compositions and methods of the present
invention. A lipid component and a non-lipid may be attached to one
another, either covalently or non-covalently.
[0201] A lipid may be naturally occurring or synthetic (i.e.,
designed or produced by man). However, a lipid is usually a
biological substance. Biological lipids are well known in the art,
and include for example, neutral fats, phospholipids,
phosphoglycerides, steroids, terpenes, lysolipids,
glycosphingolipids, glucolipids, sulphatides, lipids with ether and
ester-linked fatty acids and polymerizable lipids, and combinations
thereof.
[0202] A nucleic acid molecule or amino acid molecule, such as a
peptide, associated with a lipid may be dispersed in a solution
containing a lipid, dissolved with a lipid, emulsified with a
lipid, mixed with a lipid, combined with a lipid, covalently bonded
to a lipid, contained as a suspension in a lipid or otherwise
associated with a lipid. A lipid or lipid/virus-associated
composition of the present invention is not limited to any
particular structure. For example, they may also simply be
interspersed in a solution, possibly forming aggregates which are
not uniform in either size or shape. In another example, they may
be present in a bilayer structure, as micelles, or with a
"collapsed" structure. In another non-limiting example, a
lipofectamine (Gibco BRL)-poxvirus or Superfect (Qiagen)-virus
complex is also contemplated.
[0203] In certain embodiments, a lipid composition may comprise
about 1%, about 2%, about 3%, about 4% about 5%, about 6%, about
7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%,
about 14%, about 15%, about 16%, about 17%, about 18%, about 19%,
about 20%, about 21%, about 22%, about 23%, about 24%, about 25%,
about 26%, about 27%, about 28%, about 29%, about 30%, about 31%,
about 32%, about 33%, about 34%, about 35%, about 36%, about 37%,
about 38%, about 39%, about 40%, about 41%, about 42%, about 43%,
about 44%, about 45%, about 46%, about 47%, about 48%, about 49%,
about 50%, about 51%, about 52%, about 53%, about 54%, about 55%,
about 56%, about 57%, about 58%, about 59%, about 60%, about 61%,
about 62%, about 63%, about 64%, about 65%, about 66%, about 67%,
about 68%, about 69%, about 70%, about 71%, about 72%, about 73%,
about 74%, about 75%, about 76%, about 77%, about 78%, about 79%,
about 80%, about 81%, about 82%, about 83%, about 84%, about 85%,
about 86%, about 87%, about 88%, about 89%, about 90%, about 91%,
about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,
about 98%, about 99%, about 100%, or any range derivable therein,
of a particular lipid, lipid type, or non-lipid component such as a
chug, protein, sugar, nucleic acids or other material disclosed
herein or as would be known to one of skill in the art. In a
non-limiting example, a lipid composition may comprise about 10% to
about 20% neutral lipids, and about 33% to about 34% of a
cerebroside, and about 1% cholesterol. Thus, it is contemplated
that lipid compositions of the present invention may comprise any
of the lipids, lipid types, or other components in any combination
or percentage range.
IV. PHARMACEUTICAL FORMULATIONS AND TREATMENT REGIMENS
[0204] In an embodiment of the present invention, a method of
treatment for a hyperproliferative or neoplastic disease, such as
cancer, by the delivery of a non-VSV rhabdovirus, such as Maraba
virus, Carajas virus, Muir Springs virus, and/or Bahia Grande
virus, is contemplated. Examples of cancer contemplated for
treatment include lung cancer, head and neck cancer, breast cancer,
pancreatic cancer, prostate cancer, renal cancer, bone cancer,
testicular cancer, cervical cancer, gastrointestinal cancer,
lymphomas, pre-neoplastic lesions, pre-neoplastic lesions in the
lung, colon cancer, melanoma, bladder cancer and any other cancers
or tumors that may be treated, including metastatic or systemically
distributed cancers.
[0205] An effective amount of the pharmaceutical composition,
generally, is defined as that amount sufficient to detectably and
repeatedly to slow, ameliorate, reduce, minimize, or limit. the
extent of the disease or its symptoms. More rigorous definitions
may apply, including elimination, eradication, or cure of
disease.
[0206] Preferably, patients will have adequate bone marrow function
(defined as a peripheral absolute granulocyte count of
>2,000/mm.sup.3 and a platelet count of 100,000/mm.sup.3),
adequate liver function (bilirubin <1.5 mg/dl) and adequate
renal function (creatinine <1.5 mg/dl).
[0207] A. Administration
[0208] To kill cells, inhibit cell growth, inhibit metastasis,
decrease tumor or tissue size, and otherwise reverse, stay, or
reduce the malignant phenotype of tumor cells, using the methods
and compositions of the present invention, one would generally
contact a hyperproliferative or neoplastic cell with a therapeutic
composition such as a virus or an expression construct encoding a
polypeptide. The routes of administration will vary, naturally,
with the location and nature of the lesion, and include, e.g.,
intradermal, transdermal, parenteral, intravascular, intravenous,
intramuscular, intranasal, subcutaneous, regional, percutaneous,
intratracheal, intraperitoneal, intraarterial, intravesical,
intratumoral, inhalation, perfusion, lavage, direct injection,
alimentary, and oral administration and formulation.
[0209] To effect a therapeutic benefit with respect to a vascular
condition or disease, one would contact a vascular cell with the
therapeutic compound. Any of the formulations and routes of
administration discussed with respect to the treatment or diagnosis
of cancer may also be employed with respect to vascular diseases
and conditions.
[0210] Intratumoral injection, or injection into the tumor
vasculature is contemplated for discrete, solid, accessible tumors.
Local, regional or systemic administration is also contemplated,
particularly for those cancers that are disseminated or are likely
to disseminated systemically. The viral particles may be
administering by at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10
injections.
[0211] In the case of surgical intervention, the present invention
may be used preoperatively, to render an inoperable tumor subject
to resection. Alternatively, the present invention may be used at
the time of surgery, and/or thereafter, to treat residual or
metastatic disease. For example, a resected tumor bed may be
injected or perfused with a formulation comprising a rhabdovirus
polypeptide or a rhabdovirus, which may or may not harbor a
mutation, that is advantageous for treatment of cancer or cancer
cells. The perfusion may be continued post-resection, for example,
by leaving a catheter implanted at the site of the surgery.
Periodic post-surgical treatment also is envisioned.
[0212] Continuous administration also may be applied where
appropriate, for example, where a tumor is excised and the tumor
bed is treated to eliminate residual, microscopic disease. Delivery
via syringe or catherization is preferred. Such continuous
perfusion may take place for a period from about 1-2 hours, to
about 2-6 hours, to about 6-12 hours, to about 12-24 hours, to
about 1-2 days, to about 1-2 wk or longer following the initiation
of treatment. Generally, the dose of the therapeutic composition
via continuous perfusion will be equivalent to that given by a
single or multiple injections, adjusted over a period of time
during which the perfusion occurs. It is further contemplated that
limb perfusion may be used to administer therapeutic compositions
of the present invention, particularly in the treatment of
melanomas and sarcomas.
[0213] Treatment regimens may vary as well, and often depend on
tumor type, tumor location, disease progression, and health and age
of the patient. Obviously, certain types of tumor will require more
aggressive treatment, while at the same time, certain patients
cannot tolerate more taxing protocols. The clinician will be best
suited to make such decisions based on the known efficacy and
toxicity (if any) of the therapeutic formulations.
[0214] In certain embodiments, the tumor being treated may not, at
least initially, be resectable. Treatments with therapeutic viral
constructs may increase the resectability of the tumor due to
shrinkage at the margins or by elimination of certain particularly
invasive portions. Following treatments, resection may be possible.
Additional treatments subsequent to resection will serve to
eliminate microscopic residual disease at the tumor site.
[0215] A typical course of treatment, for a primary tumor or a
post-excision tumor bed, will involve multiple doses. Typical
primary tumor treatment involves a 1, 2, 3, 4, 5, 6 or more dose
application over a 1, 2, 3, 4, 5, 6-week period or more. A two-week
regimen may be repeated one, two, three, four, five, six or more
times. During a course of treatment, the need to complete the
planned dosings may be re-evaluated.
[0216] The treatments may include various "unit doses." Unit dose
is defined as containing a predetermined quantity of the
therapeutic composition. The quantity to be administered, and the
particular route and formulation, are within the skill of those in
the clinical arts. A unit dose need not be administered as a single
injection but may comprise continuous infusion over a set period of
time. Unit dose of the present invention may conveniently be
described in terms of plaque forming units (pfu) or viral particles
for viral constructs. Unit doses range from 10.sup.3, 10.sup.4,
10.sup.5, 10.sup.6, 10.sup.7, 10.sup.8, 10.sup.9, 10.sup.10,
10.sup.11, 10.sup.12, 10.sup.13 pfu or vp and higher.
Alternatively, depending on the kind of virus and the titer
attainable, one will deliver 1 to 100, 10 to 50, 100-1000, or up to
about 1.times.10.sup.4, 1.times.10.sup.5, 1.times.10.sup.6,
1.times.10.sup.7, 1.times.10.sup.8, 1.times.10.sup.9,
1.times.10.sup.10, 1.times.10.sup.11, 1.times.10.sup.12,
1.times.10.sup.13, 1.times.10.sup.14, or 1.times.10.sup.15 or
higher infectious viral particles (vp) to the patient or to the
patient's cells.
[0217] B. Injectable Compositions and Formulations
[0218] The preferred method for the delivery of an expression
construct or virus encoding all or part of a rhabdovirus genome to
cancer or tumor cells in the present invention is via intravascular
injection. However, the pharmaceutical compositions disclosed
herein may alternatively be administered intratumorally,
parenterally, intravenously, intrarterially, intradermally,
intramuscularly, transdermally or even intraperitoneally as
described in U.S. Pat. Nos. 5,543,158, 5,641,515 and 5,399,363
(each specifically incorporated herein by reference in its
entirety).
[0219] Injection of nucleic acid constructs may be delivered by
syringe or any other method used for injection of a solution, as
long as the expression construct can pass through the particular
gauge of needle required for injection (for examples see U.S. Pat.
Nos. 5,846,233 and 5,846,225).
[0220] Solutions of the active compounds as free base or
pharmacologically acceptable salts may be prepared in water
suitably mixed with a surfactant, such as hydroxypropylcellulose.
Dispersions may also be prepared in glycerol, liquid polyethylene
glycols, and mixtures thereof and in oils. Under ordinary
conditions of storage and use, these preparations contain a
preservative to prevent the growth of microorganisms. The
pharmaceutical forms suitable for injectable use include sterile
aqueous solutions or dispersions and sterile powders for the
extemporaneous preparation of sterile injectable solutions or
dispersions (U.S. Pat. No. 5,466,468, specifically incorporated
herein by reference in its entirety). In all cases the form must be
sterile and must be fluid to the extent that easy syringability
exists. It must be stable under the conditions of manufacture and
storage and must be preserved against the contaminating action of
microorganisms, such as bacteria and fungi. The carrier can be a
solvent or dispersion medium containing, for example, water,
ethanol, polyol (e.g., glycerol, propylene glycol, and liquid
polyethylene glycol, and the like), suitable mixtures thereof,
and/or vegetable oils. Proper fluidity may be maintained, for
example, by the use of a coating, such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. The prevention of the action of
microorganisms can be brought about by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
sorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars or
sodium chloride. Prolonged absorption of the injectable
compositions can be brought about by the use in the compositions of
agents delaying absorption, for example, aluminum monostearate and
gelatin.
[0221] For parenteral administration in an aqueous solution, for
example, the solution should be suitably buffered if necessary and
the liquid diluent first rendered isotonic with sufficient saline
or glucose. These particular aqueous solutions are especially
suitable for intravenous, intramuscular, subcutaneous,
intratumoral, and intraperitoneal administration. In this
connection, sterile aqueous media that can be employed will be
known to those of skill in the art in light of the present
disclosure. For example, one dosage may be dissolved in 1 ml of
isotonic NaCl solution and either added to 1000 ml of
hypodermoclysis fluid or injected at the proposed site of infusion,
(see for example, "Remington's Pharmaceutical Sciences" 15th
Edition, pages 1035-1038 and 1570-1580). Some variation in dosage
will necessarily occur depending on the condition of the subject
being treated. The person responsible for administration will, in
any event, determine the appropriate dose for the individual
subject. Moreover, for human administration, preparations should
meet sterility, pyrogenicity, general safety and purity standards
required by governments of the countries in which the compositions
are being used.
[0222] The compositions disclosed herein may be formulated in a
neutral or salt form. Pharmaceutically-acceptable salts, include
the acid addition salts (formed with the free amino groups of the
protein) and which are formed with inorganic acids such as, for
example, hydrochloric or phosphoric acids, or such organic acids as
acetic, oxalic, tartaric, mandelic, and the like. Salts formed with
the free carboxyl groups can also be derived from inorganic bases
such as, for example, sodium, potassium, ammonium, calcium, or
ferric hydroxides, and such organic bases as isopropylamine,
trimethylamine, histidine, procaine and the like. Upon formulation,
solutions will be administered in a manner compatible with the
dosage formulation and in such amount as is therapeutically
effective. The formulations are easily administered in a variety of
dosage forms such as injectable solutions, drug release capsules
and the like.
[0223] As used herein, "carrier" includes any and all solvents,
dispersion media, vehicles, coatings, diluents, antibacterial and
antifungal agents, isotonic and absorption delaying agents,
buffers, carrier solutions, suspensions, colloids, and the like.
The use of such media and agents for pharmaceutical active
substances is well known in the art. Except insofar as any
conventional media or agent is incompatible with the active
ingredient, its use in the therapeutic compositions is
contemplated. Supplementary active ingredients can also be
incorporated into the compositions.
[0224] The phrase "pharmaceutically-acceptable" or
"pharmacologically-acceptable" refers to molecular entities and
compositions that do not produce an allergic or similar untoward
reaction when administered to a human. The preparation of an
aqueous composition that contains a protein as an active ingredient
is well understood in the art. Typically, such compositions are
prepared as injectables, either as liquid solutions or suspensions;
solid forms suitable for solution in, or suspension in, liquid
prior to injection can also be prepared.
[0225] C. Combination Treatments
[0226] The compounds and methods of the present invention may be
used in the context of hyperproliferative or neoplastic
diseases/conditions including cancer and atherosclerosis. In order
to increase the effectiveness of a treatment with the compositions
of the present invention, such as rhabdoviruses, it may be
desirable to combine these compositions with other agents effective
in the treatment of those diseases and conditions. For example, the
treatment of a cancer may be implemented with therapeutic compounds
of the present invention and other anti-cancer therapies, such as
anti-cancer agents or surgery.
[0227] Various combinations may be employed; for example, a non-VSV
rhabdovirus, such as Maraba virus, Carajas virus, Muir Springs
virus, and/or Bahia Grande virus, is "A" and the secondary
anti-cancer therapy is "B", which may include a second
rhabdovirus:
TABLE-US-00004 A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B
B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B
A/A/A/B B/A/A/A A/B/A/A A/A/B/A
[0228] Administration of the therapeutic virus or viral constructs
of the present invention to a patient will follow general protocols
for the administration of that particular secondary therapy, taking
into account the toxicity, if any, of the virus treatment. It is
expected that the treatment cycles would be repeated as necessary.
It also is contemplated that various standard therapies, as well as
surgical intervention, may be applied in combination with the
described cancer or tumor cell therapy.
[0229] 1. Anti-Cancer Therapy
[0230] An "anti-cancer" agent is capable of negatively affecting
cancer in a subject, for example, by killing cancer cells, inducing
apoptosis in cancer cells, reducing the growth rate of cancer
cells, reducing the incidence or number of metastases, reducing
tumor size, inhibiting tumor growth, reducing the blood supply to a
tumor or cancer cells, promoting an immune response against cancer
cells or a tumor, preventing or inhibiting the progression of
cancer, or increasing the lifespan of a subject with cancer.
Anti-cancer agents include biological agents (biotherapy),
chemotherapy agents, and radiotherapy agents. More generally, these
other compositions would be provided in a combined amount effective
to kill or inhibit proliferation of the cell. This process may
involve contacting the cells with virus or viral construct and the
agent(s) or multiple factor(s) at the same time. This may be
achieved by contacting the cell with a single composition or
pharmacological formulation that includes both agents, or by
contacting the cell with two distinct compositions or formulations,
at the same time, wherein one composition includes the virus and
the other includes the second agent(s).
[0231] Tumor cell resistance to chemotherapy and radiotherapy
agents represents a major problem in clinical oncology. One goal of
current cancer research is to find ways to improve the efficacy of
chemo- and radiotherapy by combining it with gene therapy. For
example, the herpes simplex-thymidine kinase (HS-tK) gene, when
delivered to brain tumors by a retroviral vector system,
successfully induced susceptibility to the antiviral agent
ganciclovir (Culver et al., 1992). In the context of the present
invention, it is contemplated that poxvirus therapy could be used
similarly in conjunction with chemotherapeutic, radiotherapeutic,
immunotherapeutic, or other biological intervention, in addition to
other pro-apoptotic or cell cycle regulating agents.
[0232] Alternatively, a viral therapy may precede or follow the
other treatment by intervals ranging from minutes to weeks. In
embodiments where the other agent and virus are applied separately
to the cell, one would generally ensure that a significant period
of time did not expire between the time of each delivery, such that
the agent and virus would still be able to exert an advantageously
combined effect on the cell. In such instances, it is contemplated
that one may contact the cell with both modalities within about
12-24 h of each other and, more preferably, within about 6-12 h of
each other. In some situations, it may be desirable to extend the
time period for treatment significantly, however, where several
days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or
8) lapse between the respective administrations.
[0233] a. Chemotherapy
[0234] Cancer therapies also include a variety of combination
therapies with both chemical and radiation based treatments.
Combination chemotherapies include, for example, cisplatin (CDDP),
carboplatin, procarbazine, mechlorethamine, cyclophosphamide,
camptothecin, ifosfamide, melphalan, chlorambucil, busulfan,
nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin,
plicomycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene,
estrogen receptor binding agents, taxol, gemcitabien, navelbine,
farnesyl-protein transferase inhibitors, transplatinum,
5-fluorouracil, vincristine, vinblastine and methotrexate,
Temazolomide (an aqueous form of DTIC), or any analog or derivative
variant of the foregoing. The combination of chemotherapy with
biological therapy is known as biochemotherapy.
[0235] b. Radiotherapy
[0236] Other factors that cause DNA damage and have been used
extensively include what are commonly known as .gamma.-rays,
X-rays, proton beams, and/or the directed delivery of radioisotopes
to tumor cells. Other forms of DNA damaging factors are also
contemplated such as microwaves and UV-irradiation. It is most
likely that all of these factors effect a broad range of damage on
DNA, on the precursors of DNA, on the replication and repair of
DNA, and on the assembly and maintenance of chromosomes. Dosage
ranges for X-rays range from daily doses of 50 to 200 roentgens for
prolonged periods of time (3 to 4 wk), to single doses of 2000 to
6000 roentgens. Dosage ranges for radioisotopes vary widely, and
depend on the half-life of the isotope, the strength and type of
radiation emitted, and the uptake by the neoplastic cells.
[0237] The terms "contacted" and "exposed," when applied to a cell,
are used herein to describe the process by which a therapeutic
construct and a chemotherapeutic or radiotherapeutic agent are
delivered to a target cell or are placed in direct juxtaposition
with the target cell. To achieve cell killing or stasis, both
agents are delivered to a cell in a combined amount effective to
kill the cell or prevent it from dividing.
[0238] c. Immunotherapy
[0239] Immunotherapeutics, generally, rely on the use of immune
effector cells and molecules to target and destroy cancer cells.
The immune effector may be, for example, an antibody specific for
some marker on the surface of a tumor cell. The antibody alone may
serve as an effector of therapy or it may recruit other cells to
actually effect cell killing. The antibody also may be conjugated
to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain,
cholera toxin, pertussis toxin, etc.) and serve merely as a
targeting agent. Alternatively, the effector may be a lymphocyte
carrying a surface molecule that interacts, either directly or
indirectly, with a tumor cell target. Various effector cells
include cytotoxic T cells and NK cells. The combination of
therapeutic modalities, i.e., direct cytotoxic activity and
inhibition or reduction of certain rhabdovirus or rhabdovirus
polypeptides would provide therapeutic benefit in the treatment of
cancer.
[0240] Immunotherapy could also be used as part of a combined
therapy. The general approach for combined therapy is discussed
below. In one aspect of immunotherapy, the tumor cell must bear
some marker that is amenable to targeting, i.e., is not present on
the majority of other cells. Many tumor markers exist and any of
these may be suitable for targeting in the context of the present
invention. Common tumor markers include carcinoembryonic antigen,
prostate specific antigen, urinary tumor associated antigen, fetal
antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis
Antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb
B and p155. Tumor cell lysates may also be used in an antigenic
composition.
[0241] An alternative aspect of immunotherapy is to combine
anticancer effects with immune stimulatory effects. Immune
stimulating molecules include: cytokines such as IL-2, IL-4, IL-12,
GM-CSF, IFN.gamma., chemokines such as MIP-1, MCP-1, IL-8 and
growth factors such as FLT3 ligand. Combining immune stimulating
molecules, either as proteins or using gene delivery in combination
with a tumor suppressor has been shown to enhance anti-tumor
effects (Ju et al., 2000).
[0242] As discussed earlier, examples of immunotherapies currently
under investigation or in use are immune adjuvants (e.g.,
Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene
and aromatic compounds) (U.S. Pat. Nos. 5,801,005 and 5,739,169;
Hui and Hashimoto, 1998; Christodoulides et al., 1998), cytokine
therapy (e.g., interferons .alpha., .beta. and .gamma.; IL-1,
GM-CSF and TNF) (Bukowski et al., 1998; Davidson et al., 1998;
Hellstrand et al., 1998) gene therapy (e.g., TNF, IL-1, IL-2, p53)
(Qin et al., 1998; Austin-Ward and Villaseca, 1998; U.S. Pat. Nos.
5,830,880 and 5,846,945) and monoclonal antibodies (e.g.,
anti-ganglioside GM2, anti-HER-2, anti-p185) (Pietras et al., 1998;
Hanibuchi et al., 1998; U.S. Pat. No. 5,824,311). Herceptin
(trastuzumab) is a chimeric (mouse-human) monoclonal antibody that
blocks the HER2-neu receptor (Dillman, 1999). Combination therapy
of cancer with herceptin and chemotherapy has been shown to be more
effective than the individual therapies. Thus, it is contemplated
that one or more anti-cancer therapies may be employed with the
rhabdovirus-related therapies described herein.
[0243] (1) Passive Immunotherapy
[0244] A number of different approaches for passive immunotherapy
of cancer exist. They may be broadly categorized into the
following: injection of antibodies alone; injection of antibodies
coupled to toxins or chemotherapeutic agents; injection of
antibodies coupled to radioactive isotopes; injection of
anti-idiotype antibodies; and finally, purging of tumor cells in
bone marrow.
[0245] Preferably, human monoclonal antibodies are employed in
passive immunotherapy, as they produce few or no side effects in
the patient. However, their application is somewhat limited by
their scarcity and have so far only been administered
intralesionally. Human monoclonal antibodies to ganglioside
antigens have been administered intralesionally to patients
suffering from cutaneous recurrent melanoma (Irie and Morton,
1986). Regression was observed in six out of ten patients,
following, daily or weekly, intralesional injections. In another
study, moderate success was achieved from intralesional injections
of two human monoclonal antibodies (Irie et al., 1989).
[0246] It may be favorable to administer more than one monoclonal
antibody directed against two different antigens or even antibodies
with multiple antigen specificity. Treatment protocols also may
include administration of lymphokines or other immune enhancers as
described by Bajorin et al. (1988). The development of human
monoclonal antibodies is described in further detail elsewhere in
the specification.
[0247] (2) Active Immunotherapy
[0248] In active immunotherapy, an antigenic peptide, polypeptide
or protein, or an autologous or allogenic tumor cell composition or
"vaccine" is administered, generally with a distinct bacterial
adjuvant (Ravindranath and Morton, 1991; Morton et al., 1992;
Mitchell et al., 1990; Mitchell et al., 1993). In melanoma
immunotherapy, those patients who elicit high IgM response often
survive better than those who elicit no or low IgM antibodies
(Morton et al., 1992). IgM antibodies are often transient
antibodies and the exception to the rule appears to be anti
ganglioside or anticarbohydrate antibodies.
[0249] (.sup.3) Adoptive Immunotherapy
[0250] In adoptive immunotherapy, the patient's circulating
lymphocytes, or tumor infiltrated lymphocytes, are isolated in
vitro, activated by lymphokines such as IL 2 or transduced with
genes for tumor necrosis, and readministered (Rosenberg et al.,
1988; 1989). To achieve this, one would administer to an animal, or
human patient, an immunologically effective amount of activated
lymphocytes in combination with an adjuvant incorporated antigenic
peptide composition as described herein. The activated lymphocytes
will most preferably be the patient's own cells that were earlier
isolated from a blood or tumor sample and activated (or "expanded")
in vitro. This form of immunotherapy has produced several cases of
regression of melanoma and renal carcinoma, but the percentage of
responders were few compared to those who did not respond.
[0251] d. Genes
[0252] In yet another embodiment, the secondary treatment is a gene
therapy in which a therapeutic polynucleotide is administered
before, after, or at the same time as a rhabdovirus is
administered. Delivery of a rhabdovirus in conjunction with a
vector encoding one of the following gene products will have a
combined anti-cancer effect on target tissues. Alternatively, the
rhabdovirus may be engineered as a viral vector to include the
therapeutic polynucleotide. A variety of proteins are encompassed
within the invention, some of which are described below. Table 4
lists various genes that may be targeted for gene therapy of some
form in combination with the present invention.
[0253] (1) Inducers of Cellular Proliferation
[0254] The proteins that induce cellular proliferation further fall
into various categories dependent on function. The commonality of
all of these proteins is their ability to regulate cellular
proliferation. For example, a form of PDGF, the sis oncogene, is a
secreted growth factor. Oncogenes rarely arise from genes encoding
growth factors, and at the present, sis is the only known
naturally-occurring oncogenic growth factor. In one embodiment of
the present invention, it is contemplated that anti-sense mRNA
directed to a particular inducer of cellular proliferation is used
to prevent expression of the inducer of cellular proliferation.
[0255] (2) Inhibitors of Cellular Proliferation
[0256] The tumor suppressor oncogenes function to inhibit excessive
cellular proliferation. The inactivation of these genes destroys
their inhibitory activity, resulting in unregulated proliferation.
Tumor suppressors include p53, p16 and C-CAM. Other genes that may
be employed according to the present invention include Rb, APC,
DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II, zac1, p73, VHL, MMAC1/PTEN,
DBCCR-1, FCC, rsk-3, p27, p27/p16 fusions, p21/p27 fusions,
anti-thrombotic genes (e.g., COX-1, TFPI), PGS, Dp, E2F, ras, myc,
ncu, raf, erb, fms, trk, ret, gsp, hst, abl, E1A, p300, genes
involved in angiogenesis (e.g., VEGF, FGF, thrombospondin, BAI-1,
GDAIF, or their receptors) and MCC.
[0257] (3) Regulators of Programmed Cell Death
[0258] Apoptosis, or programmed cell death, is an essential process
for normal embryonic development, maintaining homeostasis in adult
tissues, and suppressing carcinogenesis (Kerr et al., 1972). The
Bcl-2 family of proteins and ICE-like proteases have been
demonstrated to be important regulators and effectors of apoptosis
in other systems. The Bcl 2 protein, discovered in association with
follicular lymphoma, plays a prominent role in controlling
apoptosis and enhancing cell survival in response to diverse
apoptotic stimuli (Bakhshi et al., 1985; Cleary and Sklar, 1985;
Cleary et al., 1986; Tsujimoto et al., 1985; Tsujimoto and Croce,
1986). The evolutionarily conserved Bcl-2 protein now is recognized
to be a member of a family of related proteins, which can be
categorized as death agonists or death antagonists.
[0259] Subsequent to its discovery, it was shown that Bcl 2 acts to
suppress cell death triggered by a variety of stimuli. Also, it now
is apparent that there is a family of Bcl-2 cell death regulatory
proteins which share in common structural and sequence homologies.
These different family members have been shown to either possess
similar functions to Bcl 2 (e.g., BclXL, BclW, BclS, Mcl-1, A1,
Bfl-1) or counteract Bcl 2 function and promote cell death (e.g.,
Bax, Bak, Bik, Bim, Bid, Bad, Harakiri).
[0260] c. Surgery
[0261] Approximately 60% of persons with cancer will undergo
surgery of some type, which includes preventative, diagnostic or
staging, curative and palliative surgery. Curative surgery is a
cancer treatment that may be used in conjunction with other
therapies, such as the treatment of the present invention,
chemotherapy, radiotherapy, hormonal therapy, gene therapy,
immunotherapy and/or alternative therapies.
[0262] Curative surgery includes resection in which all or part of
cancerous tissue is physically removed, excised, and/or destroyed.
Tumor resection refers to physical removal of at least part of a
tumor. In addition to tumor resection, treatment by surgery
includes laser surgery, cryosurgery, electrosurgery, and
microscopically controlled surgery (Mohs' surgery). It is further
contemplated that the present invention may be used in conjunction
with removal of superficial cancers, pre-cancers, or incidental
amounts of normal tissue.
[0263] Upon excision of part of all of cancerous cells, tissue, or
tumor, a cavity may be formed in the body. Treatment may be
accomplished by perfusion, direct injection or local application of
the area with an additional anti-cancer therapy. Such treatment may
be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or
every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, or 12 months. These treatments may be of varying dosages as
well.
[0264] f. Other Agents
[0265] It is contemplated that other agents may be used in
combination with the present invention to improve the therapeutic
efficacy of treatment. These additional agents include
immunomodulatory agents, agents that affect the upregulation of
cell surface receptors and GAP junctions, cytostatic and
differentiation agents, inhibitors of cell adhesion, agents that
increase the sensitivity of the hyperproliferative cells to
apoptotic inducers, or other biological agents. Immunomodulatory
agents include tumor necrosis factor; interferon .alpha., .beta.,
and .gamma.; IL-2 and other cytokines; F42K and other cytokine
analogs; or MIP-1, MIP-1.beta., MCP-1, RANTES, and other
chemokines. It is further contemplated that the upregulation of
cell surface receptors or their ligands such as Fas/Fas ligand, DR4
or DR5/TRAIL (Apo-2 ligand) would potentiate the apoptotic inducing
ability of the present invention by establishment of an autocrine
or paracrine effect on hyperproliferative cells. Increases
intercellular signaling by elevating the number of GAP junctions
would increase the anti-hyperproliferative effects on the
neighboring hyperproliferative cell population. In other
embodiments, cytostatic or differentiation agents can be used in
combination with the present invention to improve the
anti-hyperproliferative efficacy of the treatments. Inhibitors of
cell adhesion are contemplated to improve the efficacy of the
present invention. Examples of cell adhesion inhibitors are focal
adhesion kinase (FAKs) inhibitors and Lovastatin. It is further
contemplated that other agents that increase the sensitivity of a
hyperproliferative cell to apoptosis, such as the antibody c225,
could be used in combination with the present invention to improve
the treatment efficacy.
[0266] There have been many advances in the therapy of cancer
following the introduction of cytotoxic chemotherapeutic drugs.
However, one of the consequences of chemotherapy is the
development/acquisition of drug-resistant phenotypes and the
development of multiple drug resistance. The development of drug
resistance remains a major obstacle in the treatment of such tumors
and therefore, there is an obvious need for alternative approaches
such as viral therapy.
[0267] Another form of therapy for use in conjunction with
chemotherapy, radiation therapy or biological therapy includes
hyperthermia, which is a procedure in which a patient's tissue is
exposed to high temperatures (up to 106.degree. F.). External or
internal heating devices may be involved in the application of
local, regional, or whole-body hyperthermia. Local hyperthermia
involves the application of heat to a small area, such as a tumor.
Heat may be generated externally with high-frequency waves
targeting a tumor from a device outside the body. Internal heat may
involve a sterile probe, including thin, heated wires or hollow
tubes filled with warm water, implanted microwave antennae, or
radiofrequency electrodes.
[0268] A patient's organ or a limb is heated for regional therapy,
which is accomplished using devices that produce high energy, such
as magnets. Alternatively, some of the patient's blood may be
removed and heated before being perfused into an area that will be
internally heated. Whole-body heating may also be implemented in
cases where cancer has spread throughout the body. Warm-water
blankets, hot wax, inductive coils, and thermal chambers may be
used for this purpose.
[0269] Hormonal therapy may also be used in conjunction with the
present invention or in combination with any other cancer therapy
previously described. The use of hormones may be employed in the
treatment of certain cancers such as breast, prostate, ovarian, or
cervical cancer to lower the level or block the effects of certain
hormones such as testosterone or estrogen. This treatment is often
used in combination with at least one other cancer therapy as a
treatment
V. EXAMPLES
[0270] The following examples are given for the purpose of
illustrating various embodiments of the invention and are not meant
to limit the present invention in any fashion. One skilled in the
art will appreciate readily that the present invention is well
adapted to carry out the objects and obtain the ends and advantages
mentioned, as well as those objects, ends and advantages inherent
herein. The present examples, along with the methods described
herein are presently representative of preferred embodiments, are
exemplary, and are not intended as limitations on the scope of the
invention. Changes therein and other uses which are encompassed
within the spirit of the invention as defined by the scope of the
claims will occur to those skilled in the art.
Example 1
Screening for Novel Oncolytic Candidate Rhabdoviruses
[0271] In vitro screens. As an initial screen to identify novel
oncolytic viruses, rhabdovirus field isolates were assessed for
their ability to kill human tumor cells from the NCI 60 cell panel.
This has been a fruitful strategy .sup.-for the inventors in the
past to determine the relative effectiveness of a series of VSV
mutants as oncolytic (cancer cell lysing) candidates. Initially,
the inventors have examined 13 novel rhabdoviruses that have been
previously determined to replicate in mammalian cells. It is
contemplated that this procedure will be extended to study
rhabdoviruses for which there is less experience in cell culture.
In an effort to rapidly and efficiently screen through a matrix of
60 cells infected with 13 different viruses, the inventors use a
rapid and inexpensive assay in 96 well format using MTS reduction
to formazan, or crystal violet staining of residual cells, to
measure cell number and viability. The inventors grow cell lines to
80% confluence in 96 well plates and then expose them in parallel
to our rhabdovirus field isolates at increasing MOIs (MOI=0.0001-10
PFUs/cell). At 48 and 96 hours post infection, cells are stained
with aqueous MTS regent (Promega USA) and incubated for 3 hours to
allow sufficient formazan formation. Alternatively, the plates of
infected cells are washed with buffer to remove dead cells, stained
with crystal violet dye, washed to remove residual dye, after which
time the dye is solublized using detergent. These plates are then
read using the integrated multiwell plate reader (Biotek SynergyHT;
USA), the data curve fitted, and the EC.sub.50 determined from this
curve. Typically, assays are performed in sextuplet, with the
highest and lowest EC.sub.50 values removed, and averaging the
remaining four EC.sub.50 to ultimately determine a value and
confidence interval. (For example see FIG. 2)
[0272] As a counter screen to assess whether a particular virus
infects/kills normal human cells in vitro, cultures of normal human
fibroblasts, epithelium and endothelium and neuronal cultures from
the inventors collection and those commercially available (Cambrex,
USA) will be screened. Cultures will be infected with candidate
viruses (0.1 to 20 pfu/cell) for 48 and 96 hours. Cell viability
will be detected by MTS assay, or crystal violet assay, and further
characterized by labeling with activated caspase 3 antibody D175
(Cell Signaling Technologies, USA) and detected using a
FITC-conjugated secondary antibody. Studies will be done in
parallel with known susceptible/resistant human and mouse tumor
cell lines. A combination of untreated cells and cells treated with
TRAIL and cyclohexamide has been used to establish the dynamic
range of the assay, with preliminary z-factor determinations
significantly above 0.5.
[0273] Another contingency is that viruses may replicate and spread
efficiently within cultures without rapidly killing these cells.
These are also potentially interesting viruses, provided their
replication is tumor selective in nature, as their lytic capacity
could subsequently be increased through recombinant engineering. To
detect these viruses, the inventors will infect cells of the NCI 60
cell panel with field isolates at a low MOI (0.1 pfu/cell) in
duplicate wells of a 24 well plate. After 1 hour, wells will be
washed thoroughly to remove free input virus, medium added and the
cultures incubated for a further 72 hours. These culture
supernatants will subsequently be titered on a permissive cell line
(Vero cells) to detect and quantify productive infection. The final
wash from each of these will be titered to control for residual
input virus. Candidate virus hits in this assay will be confirmed
in tissue culture cells using virus-specific antisera and standard
immunofluorescence microscopy.
[0274] Rank based on all parameters. Several properties contribute
to oncolytic killing of tumor cells including: ability to induce
apoptosis, rate of virus production, quantity of virus produced, as
well as special functions such as syncytia formation. Promising
candidates from the initial screen will be characterized further
with respect to apoptosis induction (as determined by TUNEL assay
and immunofluorescence staining for activated caspase-3), and one
step growth curves to compare kinetics and to quantify virus
production. These studies will serve as a guide to improving these
strains. For example: (1) if a virus kills tumor cells well but
shows unacceptable toxicity to normal cells, the inventors will
attenuate this virus using one or more of the strategies outline
below; (2) alternatively, if a virus shows slower killing kinetics
while maintaining a high replication rate, then the inventors may
add a toxic or therapeutic transgene; (3) If a candidate virus
replicates slowly yet is an effective killer, the inventor will
select a variant with increased growth kinetics to boost its
potency.
[0275] From the inventors experience with VSV and other oncolytic
viruses, they have identified three key in vitro gating criteria to
narrow the list of candidates: (1) selective tumor cell killing,
(2) productive replication within tumor cells (independent of
killing), and (3) efficacy on VSV resistant tumor lines (UACC-62
melanoma, A431 and NCI-H226 lung, DU-145 prostate, HL60 leukemia).
Based on these criteria, results from the screening assays
described above will be integrated to pare the list for further
evaluate in preliminary in vivo testing.
[0276] In vivo Toxicity and Biodistribution. The two routes of
administration related to a clinical setting are intravenous (IV)
and intracranial (IC) injections. Lead candidates identified during
in vitro screening for toxicity and biodistribution in mice
following infection will be assessed by these routes. Groups of 3
mice will be infected either by IV at doses of 1.times.10.sup.5 to
1.times.10.sup.9 pfu, or by IC at 1.times.10.sup.2 to
1.times.10.sup.6 pfu. In addition to mortality, morbidity will be
monitored daily for signs of lethargy, dehydration, weight loss and
limb paralysis. Histopathology will be performed on 2 mice from the
minimum lethal dose group (highest dose if no lethal dose is
achieved) from each candidate virus infection. WT VSV and mock
infection will serve as appropriate positive and negative controls
respectively. Organs will be harvested from the remaining mouse in
this group, homogenized and titered as a preliminary assessment of
virus biodistribution.
[0277] For viruses that display an acceptable lethal dose range,
the inventors will subsequently assess biodistribution in tumor
bearing mice to identify viruses compatible with systemic
administration. The inventor will employ three of our existing
cancer models representing very different organ targets of critical
clinical relevance: (1) CT-26 mouse colon carcinoma
(1.times.10.sup.5 cells) injected intravenously to form
disseminated lungs tumors in syngeneic Balb/C mice (2), 4T1 mouse
breast carcinoma (4.times.10.sup.5 cells) injected into the fat pad
of syngeneic Balb/C mice to form a single primary tumor with
spontaneous metastases, and (3) U87 human glioblastoma cells
(1.times.10.sup.5 cells) stereotactically implanted in the cortex
of nude mice. A maximum tolerable dose for each virus and route (IV
or IC) will be determined from the preliminary in vivo toxicity
experiments. This value will serve as an initial therapeutic dose
for biodistribution studies in tumor bearing mice. In groups of 3
mice, tumors will be established for 1 week and then treated IV or
IC with a single dose of each candidate virus at their respective
MTD. Forty-eight hours post treatment, animals will be perfused
with saline to flush any free virus from the circulation, and
tumors and organs will be harvested, homogenized and titered to
quantify infectious virus. In this fashion, the inventors will
determine which viruses can be delivered to tumor sites by systemic
injection, as well as the relative tumor selectivity of virus
replication in vivo.
[0278] Re-Rank. Based on the toxicity, biodistribution, systemic
delivery and tumor selectivity profiles in in vivo studies, the
inventors will select the best candidates to proceed with detailed
characterization and further development.
Example 2
Building Recombinants
[0279] Sequencing and Recombinant System. In order to facilitate
rapid research and development, subsequent production of clinical
material and to ensure the safety and stability of therapeutic
viruses, the inventors will clone and rescue recombinant forms
selected viruses.
[0280] Many negative strand ssRNA viruses have been cloned and
rescued using standard recombinant techniques. The inventors will
employ similar strategies that have been adopted successfully for
reported recombinant -ssRNA viruses. Briefly, the genome of a
candidate virus will be isolated by RNA extraction (Qiagen Corp)
from 1.times.10.sup.9 virus purified particles. The purified
genomic RNA is then primed with random hexamers and reverse
transcribed to cDNA, subsequently rendered double-stranded and
cloned by ligating EcoRI adapters, size fractionated and finally
ligating into an EcoRI digested bacterial plasmid (pT7Blue;
Novagen). The result is a library of genomic fragments that can be
easily sequenced by standard techniques. Because of the random
primed nature of this library, this strategy will not "capture" the
extreme 3' and 5' ends. To do this the inventors ligate oligos to
the 3' or 5' ends of the purified genomic RNA using T4 RNA ligase.
Using primers complementary to the newly ligated oligo flanking the
genome, the inventors PCR amplify and clone the ends of the genome
for subsequent sequencing. This sequence information is then used
to design end-specific primers for amplifying the entire genome,
which is then cloned into a specialized plasmid. This plasmid
flanks the genome with a T7 promoter on one end and a hepatitis
delta self-cleaving ribozyme and T7 terminator sequence on the
opposite flank. When transfected into T7 RNA polymerase expressing
(previously infected with a T7 expressing vaccinia virus) A549
cells, this plasmid generates viral genomes in the cytoplasm. In
parallel, the viruses' coding sequences for N, P and L genes are
cloned into CMV promoter driven expression plasmids.
Co-transfection of the genome construct with the N, P and L
plasmids into these A549 cells reconstitutes the viral replication
complex on the viral genome and results in rescue of infectious
virus. As a proof of principle the inventors have cloned,
genetically manipulated, and rescued Maraba virus using this
method. See FIG. 17 and FIG. 18 for examples of Maraba related
viruses.
Example 3
Optimization/Augmentation
[0281] The non-VSV rhabdoviruses are feral viruses; and as with all
oncolytic viruses reported thus far, including VSV, the inventors
predict that these field isolates will benefit from further
optimization through in vitro selection and/or recombinant
engineering strategies. Some candidates may require attenuation
(e.g., Maraba virus) while some may require augmentation of their
replication and/or tumor killing kinetics (e.g., Muir Springs
virus). The following is a summary of several strategies the
inventors will employ to maximize the effectiveness of newly
identified therapeutic viruses.
[0282] Engineered Mutations. VSV blocks nuclear/cytoplasmic mRNA
transport as a means to defeat host cell innate immunity. The
inventors have previously described engineering mutations into the
M protein of VSV to disable this activity and thereby selectively
attenuate this virus in normal cells. Given that other members of
the vesiculoviruses genus have also demonstrated this ability
(Chandipura, and spring viremia of carp) and that most
vesiculoviruses sequenced thus far (VSV, Chandripura, Piry, Cocal,
spring viremia of carp, Maraba) have the critical sequence motif
required by VSV for this function, the inventors contemplate
attenuate of non-VSV rhabdovirus in an analogous fashion to that
used for VSV. However, other rhabdoviruses such as rabies and
bovine ephemeral fever virus do not have this motif and do not
block nuclear cytoplasmic mRNA transport and perhaps will not be
amenable to this strategy of attenuation. As more information
becomes available regarding rhabdovirus/host interaction from
consortium labs and others, additional structure/functioned-guided
manipulations to attenuate theses viruses will be possible.
[0283] Transgenes. There are now several reports of "arming"
oncolytic viruses with suicide genes or immune mediators to
increase their potency. The inventors will focus on adding
transgenes to increase the cytotoxicity of candidate viruses that
show efficient replication, but insufficient tumor killing. The
inventors have a priority-weighted list of transgenes that are
currently being engineered into Maraba virus. At present the
ranking consists of: (1) Apoptosis Inducing Factor (AIF)--an
oxido-reductase homolog responsible for chromatin collapse and
degradation in a caspase-independent manner. (2) HaraKiri--the most
potent of the BH3-only pro-apoptotic member of the Bcl-2 family
responsible for induction of conventional caspase-dependent
apoptosis (Type I PCD). (3) XAF1--a potent tumor suppressor gene
and direct inhibitor of the IAP family. (4) Atg4B--the key protease
responsible for initiating autophagy (Type II PCD).
[0284] Ultimately, members of the intrinsic or extrinsic pathways
of cell death could be engineered with Tat or other protein
transduction domains to be secreted from virus infected cells to
induce bystander killing within the tumor mass. The inventors
remain cognizant that other bystander killing effects maybe
mediated through components of the host immunity to virus and/or
tumor. Thus an alternative strategy would be to engineer a
transgene(s) to draw immune cells to sites of infection. Evidence
indicates that virus infection of CT26 lung tumors induces
neutrophils to infiltrate the tumor and cause a massive apoptotic
bystander killing effect.
[0285] Directed evolution to improve oncolytic Rhabdoviruses. Many
examples of directed evolution have been described where the
replication fitness of a parental virus strain was either increased
or decreased by serial passage in mammalian cell culture.
Rhabdoviruses are particularly amenable to this type of procedure
as they exist not as a single entity, but as a population of
strains called a quasi-species. The members of the quasi-species
represent point mutants of the dominant genome. When an appropriate
selection pressure is applied, the fittest member of the population
is selected for, and becomes the dominant genome. This has
tremendous utility in efforts to build a better oncolytic virus
because it provides one with a ready-made collection of mutants
from which to select a variant with better oncolytic capabilities.
Thus, to attenuate a given candidate, the inventors will select
small plaque mutants on primary fibroblasts and subsequently
amplify this cloned virus on tumor cells to back-select against
non-productive mutations (i.e., mutations which uniformly
debilitate, such as polymerase mutations, as opposed to specific
disabilities in normal cells/tissues). By performing this in
iterative cycles at high MOI (10 pfu/cell), the inventors expect to
isolate a mutant that maintains robust replication in tumor cells,
yet has lost the ability to productively infect healthy normal
cells. Alternatively, the inventors may augment the potency of
non-VSV rhabdoviruses, either by selecting faster replicators, or
more lethal killers. To speed up the replication rate of a
candidate virus the inventors will perform iterative rounds of
infection/replication in tumor cell lines, but at each subsequent
round will decrease the post infection harvest time. This selection
pressure will force viruses to evolve towards rapid replication. If
enhanced cytotoxicity is desirable, the inventors will infect
resistant or recalcitrant tumor cell lines (1.times.10.sup.6 cells)
with candidate viruses (MOI=1). Live cells will subsequently be
stained with JC1 vital dye to detect early apoptosis events by dual
color flow cytometry. Cells undergoing apoptosis will be sorted
onto monolayers of Vero cells to recover the virus replicating
within them. Iterative rounds of this assay, again with decreasing
harvest times, will select for a more rapidly lethal phenotype.
Viruses improved in this way will be sequenced to map the genetic
alterations and contribute to our structure/function analysis
efforts toward better understanding of the biology of rhabdoviruses
and oncolysis. The reverse genetic screen allows for an unbiased
approach to improving rhabdoviruses, and represents a good
complement to efforts to make improvements through recombinant
engineering of transgenes or rational mutations based on
structure/function studies.
Example 4
In Vivo Testing of Novel Recombinant Oncolytic Rhabdovirus(es)
[0286] The inventors have chosen to use orthotopic models of cancer
as they more accurately recapitulate the human clinical disease.
However, unlike subcutaneous tumor models, orthotopic tumors are
not readily accessible and therefore difficult to assess without
sacrificing the experimental animal. To solve this problem, a
multimodal optical imaging technology is adopted that allows
non-invasive imaging, and repeated measure the growth or regression
of the implanted tumors, as well as the development or regression
of distal metastatic lesions. The inventors have a highly sensitive
fully integrated whole animal imaging platform (IVIS 200; Xenogen
Corp) that can detect photons emitted even from within deep tissue.
It can measure fluorescent light emitted by recombinant fluorescent
proteins such as GFP as well as detect luciferase-generated
bioluminescence. By using substrate-specific luciferase reporter
genes, one expressed from the virus and the other expressed from
tumor cells, the inventors can measure the bioluminescence
resulting from virus replication concurrently with tumor
measurements. To do this the inventors have cloned either YFP or a
novel monomeric RFP in frame with either firefly luciferase or a
novel Renilla-like luciferase from the marine copepod Gaussia
princeps. Between these two coding sequences the inventors have
engineered a translation "stop-restart" sequence of 30 amino acids.
This small motif comes from the foot and mouth disease virus and
allows for the stoichiometric expression of two proteins from a
single mRNA, is very small and does not suffer from cell to cell
variability as do IRES motifs. These dual reporter constructs were
cloned into lentivirus vectors, packaged into virus, and used to
establish stable reporter tagged 4T1, CT26 and U87 human
glioblastoma cells. These cells lines are used in three orthotopic
mouse tumor models: U87 human gliomas implanted intracranially into
CD-1 nude mice; 4T1 mouse breast carcinoma cells implanted into the
fat pad of Balb/C females (spontaneous, aggressive metastatic
disease model); CT-26 colon carcinoma injected into the tail vein
of Balb/C mice (disseminated tumors in the lung). The choice of
orthotopic model was predicated on the following criteria:
aggressive, rapidly developing tumor, and therefore challenging to
treat; represent very different organ targets; span both immune
competent and immunocompromised host systems.
[0287] The first studies will be to evaluate dose response
characteristics in our models to identify an optimal dose. From
preliminary toxicity experiments, the inventors will have defined
an MTD for each of our candidate strains in non-tumor bearing
Balb/C animals. Therefore the inventors will test doses from the
MTD, decreasing in half log intervals down to 1.times.10.sup.3 pfu.
Using the IVIS to image replication in the established tumors,
kinetics of initial virus delivery and duration of subsequent
replication will be studied as a function of dose. In parallel
studies, mice will be sacrificed during this time course and
examined using fluorescence microscopy to determine how dose
affects the ability to reach all portions of the tumor and distal
metastatic lesions. Healthy tissue will be examined to assess tumor
specific replication. Finally, safety at each dose will be
determined by monitoring mice for any signs of morbidity such as
weight loss, dehydration, and behavioral changes. Tumor responses
to the viruses in head-to-head comparisons will be assessed
following single dose IV treatment. The sensitivity and
quantitative nature of optical imaging technology make it ideally
suited for this purpose. Thus tumors will be established as
described above and monitor tumor growth or regression following
virus dosing and compare these results to UV inactivated virus
controls. Based on previous work with VSV, it is contemplated that
a single dose may not be sufficient for complete and durable tumor
regressions. This necessitates a series of experiments to determine
the most efficacious number and timing of doses. In a strategy
similar to that described above, the inventors will use tumor
models to develop maximally effective dosing strategies. This will
be done while monitoring for virus deliver to the tumor,
replication, duration of replication at the tumor bed and spread to
distant tumor sites, in concert with tumor growth/regression. In
addition, the inventors will examine immune cell infiltration and
activation in tumor beds and surrounding lymph nodes using flow
cytometry and immunohistochemistry as another parameter of
oncolytic activity. Ultimately, efficacy will be confirmed by
monitoring these mice for overall survival, and/or time to
progression; comparing virus treated groups with those treated with
UV-inactivated virus as controls. An example of the animal model
can be found in FIG. 13.
[0288] Cycle back to Optimization/Augmentation. It may be that
several cycles of optimization and then re-testing will be required
to ultimately develop a maximally effective therapeutic virus.
Therefore, the inventors will use the results from in vivo testing
to guide additional rounds of biological and/or recombinant
optimization and then re-test in tumor models.
TABLE-US-00005 TABLE 4 Rhabdovirus mediated cell killing on the NCI
60 cell panel. Cells from the NCI 60 cell panel were plated in 6
well plates to a confluency of 90%. These cells were infected at
log dilutions with various rhabdoviruses, as indicated. After 48
hours, the monolayers were washed, fixed and stained with crystal
violet to score for viable cells. Values represent the pfu required
to kill 50% of cells within 48 h. Malignancy Cell Line Chandipura
Maraba Carajas Isfahan Klamath Sawgrass VSV HR NSC LUNG A549-ATCC
.ltoreq.10.sup.2 .ltoreq.10.sup.2 10.sup.4 10.sup.5
.gtoreq.10.sup.6 NE .gtoreq.10.sup.6 NSC LUNG EKVX .ltoreq.10.sup.2
10.sup.3 .gtoreq.10.sup.6 10.sup.3 NSC LUNG HOP92 10.sup.3 10.sup.3
10.sup.5 .ltoreq.10.sup.2 NSC LUNG NCI-H226 .gtoreq.10.sup.6
.gtoreq.10.sup.6 10.sup.4 NSC LUNG NCI-H23 .ltoreq.10.sup.2
.ltoreq.10.sup.2 .ltoreq.10.sup.2 10.sup.4 .ltoreq.10.sup.2
MELANOMA LOX IMVI .ltoreq.10.sup.2 103 10.sup.3 .ltoreq.10.sup.2
MELANOMA M 14 10.sup.3 .ltoreq.10.sup.2 10.sup.3 .gtoreq.10.sup.6
10.sup.5 MELANOMA SK-MEL-2 .ltoreq.10.sup.2 10.sup.3
.ltoreq.10.sup.2 MELANOMA MALME 3M 10.sup.3 10.sup.5 10.sup.5
10.sup.3 10.sup.5 MELANOMA UACC-257 .ltoreq.10.sup.2
.ltoreq.10.sup.2 .ltoreq.10.sup.2 10.sup.3 .ltoreq.10.sup.2
MELANOMA UACC-62 .ltoreq.10.sup.2 10.sup.3 .gtoreq.10.sup.6
LEUKEMIA MOLT-4 10.sup.3 .ltoreq.10.sup.2 LEUKEMIA K-562 10.sup.5
OVARIAN OVCAR-3 10.sup.3 .ltoreq.10.sup.2 OVARIAN OVCAR-4 10.sup.3
.ltoreq.10.sup.2 10.sup.5 10.sup.4 .gtoreq.10.sup.6 10.sup.4
10.sup.3 OVARIAN OVCAR-8 NE .gtoreq.10.sup.6 .gtoreq.10.sup.6 NE NE
10.sup.3 OVARIAN SK-OV-3 .ltoreq.10.sup.2 10.sup.5 10.sup.5
.gtoreq.10.sup.6 .gtoreq.10.sup.6 10.sup.4 CNS SF-268
.ltoreq.10.sup.2 10.sup.4 10.sup.4 CNS SF-539 .ltoreq.10.sup.2
.ltoreq.10.sup.2 10.sup.3 10.sup.4 10.sup.5 CNS SNB-19 10.sup.3
10.sup.4 .ltoreq.10.sup.2 .ltoreq.10.sup.2 CNS SNB-75 10.sup.3
10.sup.3 NE 10.sup.5 .gtoreq.10.sup.6 .ltoreq.10.sup.2 COLON HT29
10.sup.4 .gtoreq.10.sup.6 NE NE NE 10.sup.5 COLON COLO 205
.ltoreq.10.sup.2 .ltoreq.10.sup.2 .gtoreq.10.sup.6 10.sup.3 COLON
HCT-15 10.sup.5 10.sup.4 10.sup.5 .gtoreq.10.sup.6 10.sup.3 COLON
SW-620 .ltoreq.10.sup.2 .ltoreq.10.sup.2 10.sup.3 10.sup.5
.ltoreq.10.sup.2 BREAST HS 578T .gtoreq.10.sup.6 .gtoreq.10.sup.6
.gtoreq.10.sup.6 10.sup.4 BREAST MDA-MB-435 .ltoreq.10.sup.2
.ltoreq.10.sup.2 .ltoreq.10.sup.2 10.sup.3 .ltoreq.10.sup.2 RENAL
TK-10 .ltoreq.10.sup.2 10.sup.3 10.sup.4 10.sup.4 RENAL 786-O
10.sup.4 .ltoreq.10.sup.2 10.sup.5 10.sup.5 10.sup.5 RENAL ACHN
10.sup.5 10.sup.3 10.sup.5 .gtoreq.10.sup.6 NE .ltoreq.10.sup.2
RENAL A498 10.sup.5 10.sup.5 .gtoreq.10.sup.6 10.sup.4 PROSTATE
DU-145 .ltoreq.10.sup.2 .gtoreq.10.sup.6 .gtoreq.10.sup.6 PROSTATE
PC-3 .gtoreq.10.sup.6 NE .ltoreq.10.sup.2 MOUSE COLON CT26
.ltoreq.10.sup.2 .ltoreq.10.sup.2 .gtoreq.10.sup.6 NE
.ltoreq.10.sup.2
TABLE-US-00006 TABLE 5 Focused comparison between four
rhabdoviruses. Cells from the NCI 60 cell panel were plated in 6
well plates to a confluency of 90%. These cells were infected at
log dilutions with various rhabdoviruses, as indicated. After 48
hours, the monolayers were washed, fixed and stained with crystal
violet to score for viable cells. Values represent the pfu required
to kill 50% of cells within 48 h. Chandipura Maraba Carajas WT VSV
Lung A549 .ltoreq.10.sup.2 .ltoreq.10.sup.2 10.sup.4
.gtoreq.10.sup.6 H226 .gtoreq.10.sup.6 .gtoreq.10.sup.6 10.sup.4
.ltoreq.10.sup.2 melanoma M14 10.sup.3 .ltoreq.10.sup.2 10.sup.3
10.sup.5 Malme 3M 10.sup.3 10.sup.5 10.sup.5 10.sup.5 UACC-62
.ltoreq.10.sup.2 10.sup.3 .gtoreq.10.sup.6 leukemia K562 10.sup.5
10.sup.3 Ovarian OVCAR4 10.sup.3 .ltoreq.10.sup.2 10.sup.5 10.sup.3
OVCAR8 .gtoreq.10.sup.6 .gtoreq.10.sup.6 10.sup.3 SK-OV-3
.ltoreq.10.sup.2 10.sup.5 10.sup.5 10.sup.4 CNS SF268
.ltoreq.10.sup.2 10.sup.4 10.sup.4 SF539 .ltoreq.10.sup.2
.ltoreq.10.sup.2 10.sup.3 10.sup.5 Colon HCT-15 10.sup.5 10.sup.4
10.sup.5 10.sup.3 Breast HS578T .gtoreq.10.sup.6 .gtoreq.10.sup.6
10.sup.4 Renal 786-O 10.sup.4 .ltoreq.10.sup.2 10.sup.5 10.sup.5
ACHN 10.sup.5 10.sup.3 10.sup.5 .ltoreq.10.sup.2 Prostate DU-145
.ltoreq.10.sup.2 .gtoreq.10.sup.6 PC-3 .gtoreq.10.sup.6
.ltoreq.10.sup.2
[0289] Differences between VSV and other rhabdoviruses on the NCI
60 cell panel include: (1) preferential killing by Maraba virus
compared to VSV of A549 lung, M14 melanoma, UACC-62 melanoma, SF268
CNS, SF539 CNS, 786-O renal, DU-145 prostate; (2) preferential
killing by Carajas virus compared to VSV for M14 melanoma, UACC-62
melanoma, SF539 CNS; preferential killing by VSV for H226 lung,
K562 leukemia, OVCAR-8 ovarian, HCT-15, HS578T breast, and PC-3
prostate. All other cell lines of the 60 cell panel show similar
susceptibilities to VSV, Maraba and Carajas and Chandipura
TABLE-US-00007 TABLE 6 In vitro killing of selected transformed and
immortalized cells by novel rhabdoviruses. Cells were plated in 6
well dishes and allowed reach 75% confluency. These cells were
subsequently infected with each virus at a fixed titer. Cultures
were scored visually for cell death after 96 h. Muir Rio Le
Farmington Springs Grande Ngaingan Tibrogargan Dantec Kwatta Human
293T ++++ ++++ +++ ++ + Mouse 4T1 + + ++ + Human SW620 +++ +++ +++
+ Hamster BHKT7 + +++ +++ +++ +++ Human U2OS ++++ ++ ++++ ++++
monkey Vero +++ ++++ +++ ++++ 4+ = 100% obliterated, 3+ = 75-90%
dead, 2+ = 50% dead, 1+ = <30% dead, -- = no death.
Example 5
Chimeric Rhabdoviruses
[0290] One potential problem with oncolytic viral compositions is
the potential for an immune response in a patient. Such an immune
response may blunt the effectiveness of further applications of
oncolytic virus since a significant portion of the applied virus
may be neutralized by the patient's immune system. To avoid this
problem is would be preferable to have a plurality of oncolytic
viral compositions that are immunologically distinct. In this case
a different oncolytic virus may be applied to a patient for each
subsequent therapy thereby providing sustained oncolytic activity
that is minimally effected by a host immune response. To this end a
number of pseudotyped viral compositions were constructed and
tested for their ability to infect cells.
[0291] To study the possibility of using oncolytic Rhabdoviruses
that comprises various G proteins from a number of Rhabdoviruses
various recombinant viruses were constructed. Each recombinant
included the VSV Indiana wild type backbone (N, P, M and L genes)
unless otherwise specified. Furthermore, recombinants included a
luciferase reporter gene, either Firefly (FL) or Renilla (RL)
between the G and the L gene. The general nomenclature used to
refer to the recombinants is RVR.sub.aG.sup.x, wherein RVR stands
for Rhabdovirus recombinant, (a) denotes the origin to the
G-protein or G-protein-like gene and (x) denotes the version
number.
[0292] RVR with Isfahan G protein. A RVR genome was cloned into the
pXN2VSV vector such that XhoI and NheI restriction sites flanked
the G or G-like genes. The viral stop start sequence was added to
the 3' end of all G or G-like genes which encoded the following
sequence: CTCGAGGGTATGAAAAAAACTAACAGATATCACGGCTAG (SEQ ID NO:25).
Recombinant virus was pseudotyped with the Isfahan G protein which
has a protein sequence identity of 37% compared to VSV G Ind. The
RVR comprising the FL reporter gene was designated RVR.sub.Isf
(Isfahan) G.sup.1 (wherein version 1 indicates the presence of the
FL reporter gene).
[0293] Furthermore antibody neutralization studies showed that
serum comprising antibodies from mice immunized with VSV WT did not
significantly neutralize the activity of RVR Isf G1 in vitro.
[0294] Furthermore, when mice immunized with VSV-WT were injected
with RVR.sub.IsfG.sup.1 the virus with the Isf G polypeptide is
able to evade the immune system. As shown in FIG. 6C,
RVR.sub.IsfG.sup.1 was detectable at various locations in immunized
mice following viral inoculation. The level of RVR.sub.IsfG.sup.1
detect in the immunized mice was similar to the level detected in
naive controls animals (FIG. 6A). On the other hand, no virus was
detected in immunized mice that were inoculated with VSV (FIG. 6B).
Thus, oncolytic viruses comprising the Isf G polypeptide escape
host immune response to previously administered VSV in vivo.
[0295] These results were further confirmed by injecting tumors in
immunized naive mice with VSV or recombinant virus and determined
the virus yield from the infections. As shown in FIG. 7,
recombinant virus injected into tumors of immunized or naive mice
yielded large amounts of progeny virus. On the other hand,
propagation of VSV injected in immunized mice was barely
detectible.
[0296] Two additional RVRs comprising the Isf were also
constructed. RVR.sub.IsfG.sup.2 comprises an RL reporter gene in
place of the FL reporter gene from RVR.sub.IsfG.sup.1. Also,
RVR.sub.IsfG.sup.3 comprises a chimeric VSV-Isf G protein. The
chimeric protein (SEQ ID NO:19) comprises the Isfahan G ectodomain
with VSV G transmembrane domain and cytoplasmic tail.
[0297] RVR with Chandipura G protein. Chandipura G has a protein
sequence homology of 42% with VSV G (Indiana). The same cloning
strategy described above was used to construct RVR.sub.ChaG.sup.1.
A one step growth curve with RVR.sub.ChaG.sup.1 showed that it
produces similar amounts of virus compared to VSV (FIG. 8).
Furthermore, the RVR had similar cytotoxicity as compared to VSV
(FIG. 9).
[0298] RVR with Mamba G protein. Maraba G has a protein sequence
homology 83% to VSV G (Indiana). Tins is the first report of the
sequence of the Maraba G protein provided as a DNA sequence in SEQ
ID NO:20. The same cloning strategy described above was used to
construct RVR.sub.MarG.sup.1. A one step growth curve with
RVR.sub.MarG.sup.1 showed that recombinant virus titer was greater
than VSV at 48 and 72 h. Thus, switching the G protein may
stabilize the virus and thereby enhance yield (FIG. 10).
Furthermore, the RVR.sub.MarG.sup.1 was shown to be cytotoxic (FIG.
11). Furthermore, antibody neutralization assays showed that serum
from mice immunized with VSV WT did not neutralize the activity of
RVR.sub.MarG.sup.1 indicating the RVR is capable of immune
evasion.
[0299] RVR with Muir Springs G protein. Muir Springs G has 25.4%
protein sequence homology to VSV G (Indiana). The Muir Springs G
sequence is provided in SEQ ID NO:21 (amino acid) and SEQ ID NO:22
(DNA). The same cloning strategy described above was used to
construct RVR.sub.MurG.sup.1.
[0300] RVR with Klamath virus G protein. Pseudotyping experiments
confirmed that the Klamath G protein is functional at in a low pH
(6.8) environment, unlike VSV G. This of great importance since it
is known that the tumor core is hypoxic and acidic. Thus, it may be
an advantage to have a virus which can replicate in such an
environment. VSV HRGFP-Klamath pseudotyped were generated such that
the virions contained the genome of one virus but the envelope
proteins of both viruses by co infection into CT26 Cells. 24 hours
after co infection the supernatant was collected and the
pseudotyped particles tittered. Pseudotyped virus was then used
(along with control virus to infect target cells in media of two
different acidity. Results show that the Klamath G protein was
responsible for the ability of the virus to infect at low pH.
[0301] Essentially the same cloning strategy described above was
used to construct RVR.sub.KlaG.sup.2. However, unlike previous
strategies, this recombinant includes the Klamath G in addition to
the original VSV G (Indiana).
[0302] RVR with Farmington (Far) virus G protein. Farmington virus
is a non-vesiculovirus that is non-neurotropic and demonstrates
formation of large syncitia.
[0303] RVR with Bahia Grande (Bah) virus G protein. Bahia Grande
virus is a non-vesiculovirus that is non-neurotropic.
[0304] RVR with JSR retroviral Env protein. Since VSV has a known
neurotoxicity, a strategy whereby a VSV recombinant would not
infect neurons would be advantageous. JSR Env is originally from
the JSRV retrovirus (a non-neurotropic virus) envelope (Env) gene
non-neurotropic. A chimera comprising JSRV Env ectodomain with VSV
G transmembrane domain and cytoplasmic tail is generated (DNA
sequence provided as SEQ ID NO:23).
[0305] RVR with Ebola G protein. Ebola is a non-neurotropic virus
with a glycoprotein that functions to bind receptor and mediate
membrane fusion. The G protein contains a furin Cleavage site at
amino acid position 497-501. The products of cleavage (GP1 &
GP2) are linked by disulfide bonds and thought to act as a possible
decoy for neutralizing antibodies or immunomodulator. However, the
furin cleavage site not required for infection or tropism. The
Ebola G protein DNA sequence is provided as SEQ ID NO:24.
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Sequence CWU 1
1
28111068DNAMaraba Virus 1ctgttacagt caagagagtc attgatgatt
cactcatcac ccccaaattg cctgcgaatg 60aggaccctgt ggagtaccct gctgattatt
tcaaaaagtc ccgtgatatt ccggtgtaca 120taaacacgac caaaagtttg
tctgatttgc ggggctatgt ttatcaaggc ctaaagtcag 180gcaacatctc
tataattcat gtcaacagtt atctgtatgc agcattaaaa gagatcagag
240gaaaattgga cagagattgg atcacctttg gtatccaaat cggaaaaaca
ggagatagcg 300tggggatatt cgatttactg accctaaaac ctctagatgg
tgttttacca gatggggtgt 360ctgatgctac tcgaactagc tcagacgatg
catggcttcc actgtatcta ttggggttat 420acagagttgg tcgaacacag
atgccagaat acaggaagaa gctgatggat ggtctgatta 480atcaatgtaa
gatgatcaat gagcagtttg aaccactgtt gccagaagga agagatgtct
540ttgatgtctg gggaaatgac agcaattaca caaagattgt ggccgctgta
gatatgttct 600tccatatgtt caaaaagcat gagaaggcct ctttcaggta
tggcacaata gtgtcaagat 660ttaaggattg tgcagcattg gctacatttg
gtcatctgtg taagatcact ggtatgtcca 720ctgaagatgt gacaacttgg
attctaaaca gggaggtggc tgatgagatg gttcaaatga 780tgtacccagg
acaggagata gataaggctg attcttacat gccttatcta atcgacttag
840gtctgtcctc aaaatctcca tatccatcag ttaaaaatcc agctttccat
ttttggggtc 900aattgaccgc attgttactg agatcaacca gagccagaaa
tgcacgtcag ccggatgaca 960tcgagtatac atccctgacc actgctgggc
tgttgtatgc atatgccgtt ggttcgtctg 1020cagacctggc tcaacaattc
tacgttgggg acaacaagta tgtgccagaa actggagatg 1080gaggattaac
caccaatgca ccgccacaag ggcgagatgt ggtcgagtgg cttagttggt
1140ttgaagatca aaacagaaaa cctaccccag acatgctcat gtatgctaag
agagctgtca 1200gtgctttaca aggattgagg gagaagacga ttggcaagta
cgccaagtca gagtttgaca 1260aatgacaact cactcaccat atgtattact
acctttgctt catatgaaaa aaactaacag 1320cgatcatgga tcagctatca
aaggtcaagg aattccttaa gacttacgcg cagttggatc 1380aagcagtaca
agagatggat gacattgagt ctcagagaga ggaaaagact aattttgatt
1440tgtttcagga agaaggattg gagattaagg agaagccttc ctattatcgg
gcagatgaag 1500aagagattga ttcagatgaa gacagcgtgg atgatgcaca
agacttaggg atacgtacat 1560caacaagtcc catcgagggg tatgtggatg
aggagcagga tgattatgag gatgaggaag 1620tgaacgtggt gtttacatcg
gactggaaac agcctgagct ggaatccgac ggggatggga 1680aaactctccg
attgacgata ccagatggat tgactgggga gcagaagtcg caatggcttg
1740ccacgattaa ggcagttgtt cagagtgcta aatattggaa catctcagaa
tgttcatttg 1800agagttatga gcaaggggtt ttgattagag agagacaaat
gactcctgat gtctacaaag 1860tcactcctgt tttaaatgct ccaccggttc
aaatgacagc taatcaagat gtttggtctc 1920tcagcagcac tccatttaca
tttttgccca agaaacaagg tgtgactcca ttgaccatgt 1980ccttagaaga
actcttcaac acccgaggtg aattcatatc tctgggagga aacgggaaaa
2040tgagtcaccg ggaggccatc attctagggt tgagacacaa gaagctctat
aatcaagcca 2100gactaaagta taacttagct tgaatatgaa aaaaactaac
agatatcaaa agatatctct 2160aactcagtcc attgtgttca gttcaatcat
gagctctctc aagaaaattt tgggtattaa 2220agggaaaggg aagaaatcta
agaaattagg tatggctccc ccaccctatg aagaagagac 2280tccaatggaa
tattctccaa gtgcacctta tgataagtca ttgtttggag tcgaagatat
2340ggatttccat gatcaacgtc aactccgata tgagaaattt cacttctcat
tgaagatgac 2400tgtgagatca aacaaaccat ttcgaaatta tgatgacgtt
gcagcagcgg tgtccaattg 2460ggatcatatg tacatcggca tggcaggaaa
acgtcctttt tataagatat tagcattcat 2520gggttctact ctattgaagg
ctacaccagc tgtcttggct gaccaaggac agccagaata 2580tcatgctcac
tgtgagggac gagcttactt gccgcatcgg ttagggccga cccctccgat
2640gttgaatgtc cctgaacatt ttcgccgtcc atttaacatc ggattattca
gagggacaat 2700cgacataacc ctggtacttt tcgatgatga atctgtagat
tctgccccgg tcatatggga 2760tcattttaat gcatccagat tgagcagctt
cagagaaaag gctttgttgt ttggtttgat 2820tctagaaaag aaagccactg
ggaattgggt attggactct attagtcatt tcaagtaatt 2880atcacaagtg
ttgaggtgat gggcagacta tgaaaaaaac taacagggtt caaacactct
2940tgatcgaggt acccagttat atttgttaca acaatgttga gactttttct
cttttgtttc 3000ttggccttag gagcccactc caaatttact atagtattcc
ctcatcatca aaaagggaat 3060tggaagaatg tgccttccac atatcattat
tgcccttcta gttctgacca gaattggcat 3120aatgatttga ctggagttag
tcttcatgtg aaaattccca aaagtcacaa agctatacaa 3180gcagatggct
ggatgtgcca cgctgctaaa tgggtgacta cttgtgactt cagatggtac
3240ggacccaaat acatcacgca ttccatacac tctatgtcac ccaccctaga
acagtgcaag 3300accagtattg agcagacaaa gcaaggagtt tggattaatc
caggctttcc ccctcaaagc 3360tgcggatatg ctacagtgac ggatgcagag
gtggttgttg tacaagcaac acctcatcat 3420gtgttggttg atgagtacac
aggagaatgg attgactcac aattggtggg gggcaaatgt 3480tccaaggagg
tttgtcaaac ggttcacaac tcgaccgtgt ggcatgctga ttacaagatt
3540acagggctgt gcgagtcaaa tctggcatca gtggatatca ccttcttctc
tgaggatggt 3600caaaagacgt ctttgggaaa accgaacact ggattcagga
gtaattactt tgcttacgaa 3660agtggagaga aggcatgccg tatgcagtac
tgcacacaat gggggatccg actaccttct 3720ggagtatggt ttgaattagt
ggacaaagat ctcttccagg cggcaaaatt gcctgaatgt 3780cctagaggat
ccagtatctc agctccttct cagacttctg tggatgttag tttgatacaa
3840gacgtagaga ggatcttaga ttactctcta tgccaggaga cgtggagtaa
gatacgagcc 3900aagcttcctg tatctccagt agatctgagt tatctcgccc
caaaaaatcc agggagcgga 3960ccggccttca ctatcattaa tggcactttg
aaatatttcg aaacaagata catcagagtt 4020gacataagta atcccatcat
ccctcacatg gtgggaacaa tgagtggaac cacgactgag 4080cgtgaattgt
ggaatgattg gtatccatat gaagacgtag agattggtcc aaatggggtg
4140ttgaaaactc ccactggttt caagtttccg ctgtacatga ttgggcacgg
aatgttggat 4200tccgatctcc acaaatcctc ccaggctcaa gtcttcgaac
atccacacgc aaaggacgct 4260gcatcacagc ttcctgatga tgagacttta
ttttttggtg acacaggact atcaaaaaac 4320ccagtagagt tagtagaagg
ctggttcagt agctggaaga gcacattggc atcgttcttt 4380ctgattatag
gcttgggggt tgcattaatc ttcatcattc gaattattgt tgcgattcgc
4440tataaataca aggggaggaa gacccaaaaa atttacaatg atgtcgagat
gagtcgattg 4500ggaaataaat aacagatgac gcatgagggt cagatcagat
ttacagcgta agtgtgatat 4560ttaggattat aaaggttcct tcattttaat
ttgttacaga ctgtatgaaa aaaactcatc 4620aacagccatc atggatgtta
acgattttga gttgcatgag gactttgcat tgtctgaaga 4680tgactttgtc
acttcagaat ttctcaatcc ggaagaccaa atgacatacc tgaatcatgc
4740cgattataat ttgaattctc ccttaatcag cgatgatatt gatttcctga
tcaagaaata 4800taatcatgag caaattccga aaatgtggga tgtaaagaat
tgggagggag tgttagagat 4860gttgacagcc tggcaagcca gtccaatttt
atctagcact atgcataagt gggtgggaaa 4920gtggctcatg tctgatgatc
atgacgcaag ccaaggcttc agttttcttc atgaagtgga 4980caaagaagct
gatctgacgt ttgaggtggt ggagacattc attagaggat ggggaggtcg
5040agaattgcag tacaagagga aagacacatt tccggactcc tttagagttg
cagcctcatt 5100gtgtcaaaaa ttccttgatt tgcacaaact cactctgata
atgaattcag tctctgaagt 5160cgaacttacc aacctagcaa agaattttaa
aggaaaaaac aggaaagcaa aaagcggaaa 5220tctgataacc agattgaggg
ttcccagttt aggtcctgct tttgtgactc agggatgggt 5280gtacatgaag
aagttggaaa tgattatgga tcggaatttt ttgttgatgt tgaaagacgt
5340tatcatcggg aggatgcaga cgatcctgtc catgatctca agagatgata
atctcttctc 5400cgagtctgat atctttactg tattaaagat ataccggata
ggggataaga tattagaaag 5460gcaagggaca aagggttacg acttgatcaa
aatgattgag cctatttgta acttaaagat 5520gatgaatctg gcacgtaaat
atcgtcctct catccctaca tttcctcatt ttgaaaaaca 5580tattgctgac
tctgttaagg aaggatcgaa aatagacaaa gggattgagt ttatatatga
5640tcacattatg tcaatccctg gtgtggactt gaccttagtt atttacggat
catttcggca 5700ctggggtcat ccttttatca actactatga gggcttagag
aagctacaca agcaggttac 5760aatgcccaag actattgaca gagaatatgc
agaatgtctt gctagtgatc tggcaagaat 5820cgttcttcag caacaattca
atgaacataa gaaatggttt gttgatgtag ataaagtccc 5880acaatcccat
cctttcaaaa gccatatgaa agagaatact tggcctactg cagcccaagt
5940tcaggattac ggcgatcgct ggcatcagct cccactcatc aaatgcttcg
aaatcccaga 6000tttgttagat ccatcgatca tctactcaga caaaagtcat
tccatgaacc ggtctgaagt 6060actacgacat gtaagactta cacctcatgt
gcccattcca agcaggaaag tattgcagac 6120aatgttggag actaaggcaa
cagactggaa agagttttta aagaaaattg acgaagaggg 6180gttagaggat
gatgatcttg tcataggact caaagggaaa gagagagaat taaaaattgc
6240gggaagattc ttttctttga tgtcctggaa gctcagagag tattttgtca
tcactgagta 6300tttgattaag acgcactttg tcccgatgtt taaagggttg
accatggcgg atgacttgac 6360agcggtgata aagaagatga tggacacatc
ttcaggacaa ggcttagata attatgaatc 6420catttgtata gccaaccata
ttgactatga gaagtggaac aatcatcaaa gaaaagagtc 6480gaacgggccc
gtgttcaagg tgatgggtca attcttggga tatccacgtc tgattgagag
6540aactcatgaa ttttttgaga agagtctgat atattacaat ggacgaccag
atctgatgcg 6600ggttcgagga aattctctag tcaacgcctc atctttaaat
gtctgctggg agggtcaagc 6660tgggggatta gaaggactgc gacagaaggg
atggagtatt ctaaatttgc ttgtcattca 6720gagagaagca aaaataagga
acaccgccgt gaaagtgcta gctcaaggtg acaatcaggt 6780gatatgtact
cagtataaaa cgaagaaatc ccggaatgat attgagctta aggcagctct
6840aacacagatg gtatctaata atgagatgat tatgtctgcg attaaatcag
gcaccgagaa 6900actgggtctt ttgattaatg atgatgagac aatgcaatct
gctgattacc tcaattacgg 6960gaaggttccc attttcagag gagtaatcag
aggccttgag acaaaaagat ggtcacgcgt 7020gacctgtgtg acaaatgatc
agattccaac gtgtgcgaac attatgagct ctgtgtcaac 7080taatgcatta
actgtagccc attttgccga gaatccagtc aatgccatca ttcagtataa
7140ctactttgga acatttgcaa ggctactgct gatgatgcat gaccccgctc
tgaggatctc 7200tctgtatgaa gtccaatcaa aaattccagg acttcacagt
ttgacattta aatattctat 7260gttgtatctg gatccttcga taggaggagt
ctccggaatg tcactctcga gattcctcat 7320aagatcattt ccagatccag
tgacagaaag tttggcgttc tggaaattta tccactctca 7380tgcaagaagc
gattcattaa aggagatatg tgcagttttt ggaaatcctg aaattgcaag
7440atttcggcta actcatgtcg ataaattggt ggaagaccca acctcattga
acatagctat 7500gggaatgagt cctgctaatc tattaaagac agaggtaaaa
aaatgtctac tggaatcaag 7560gcagagcatc aagaaccaga ttgtaagaga
tgctactatt tacctacacc atgaggaaga 7620caaacttcgt agtttcttat
ggtccataac accactgttc cctcggttct tgagtgaatt 7680caaatctggg
acattcatcg gagtagcaga tggcctgatc agcttatttc agaactctag
7740gactattcga aattctttta aaaagcgtta tcacagggaa cttgatgatt
taataatcaa 7800gagcgaagtt tcctcactta tgcatttggg taagctacat
ttgaggcgag gctcagttcg 7860tatgtggact tgctcttcta ctcaggctga
tcttctccga ttccggtcat ggggaagatc 7920tgttatagga accacagtcc
ctcatccctt agagatgtta ggacaacatt ttaaaaagga 7980gactccttgc
agtgcttgca acatatccgg attagactat gtatctgtcc actgtccgaa
8040tgggattcat gacgtttttg aatcacgtgg tccactccct gcatatttgg
gttctaaaac 8100atccgaatca acttcgatct tgcagccgtg ggagagagag
agtaaagtac cgttgattaa 8160gcgtgccaca aggcttcgtg atgcaatttc
atggtttgtg tctcccgact ctaacttggc 8220ctcaactatc cttaagaaca
taaatgcatt aacaggagaa gaatggtcaa agaagcagca 8280tggatttaaa
aggacgggat cggcgttaca caggttctcc acatccagga tgagtcatgg
8340tggttttgct tctcagagta cggctgcctt gactagattg atggcaacta
ctgacactat 8400gagagatctg ggagaacaga actatgattt cctgtttcag
gcgacattat tgtatgctca 8460aataaccaca actgtagtca ggaatggatc
atttcatagc tgcacggacc attaccatat 8520aacctgcaaa tcttgtctga
gggccattga tgagattacc ttggattcag cgatggaata 8580tagccctcca
gatgtatcat cagttttaca atcttggagg aatggagaag gctcttgggg
8640acatgaagtg aaacaaatat acccagttga aggtgactgg aggggactat
ctcctgttga 8700acaatcttat caagtcggac gctgtatcgg gtttctgttc
ggtgatctgg cgtatagaaa 8760atcatcccat gcagatgata gctccatgtt
tccgttatct atacaaaaca aagtcagagg 8820aagaggcttt ttaaaagggc
ttatggatgg gttaatgaga gccagttgtt gccaggtgat 8880ccatcgtcga
agcttagccc atctgaagag accggctaat gcagtctatg gagggctgat
8940ttatttgata gacaaattga gtgcatctgc cccttttctt tcactgacga
gacatggacc 9000tttaagggaa gaattagaaa ctgttccaca taagataccg
acttcttatc ctacgagcaa 9060ccgagatatg ggggtgatag ttcgtaatta
ttttaaatat cagtgcagac tggtagaaaa 9120aggtcggtac aagacacatt
atcctcaatt gtggcttttc tcagatgtgc tgtccattga 9180tttcttagga
cccctgtcta tatcttcaac tctattgggt attctgtata aacagacgtt
9240atcttctcga gacaaaaatg agttgagaga actcgctaac ttgtcttcat
tgttgagatc 9300aggagaagga tgggaagata tccatgtcaa attcttctct
aaggacactt tactctgccc 9360tgaagagatc cgacatgcgt gcaaatttgg
gattgctaag gaatccgctg ttttaagcta 9420ttatcctcct tggtctcaag
agtcttatgg aggcatcacc tcgatccccg tatatttttc 9480gaccaggaag
tatcccaaaa ttttagatgt ccctcctcgg gttcaaaacc cattggtctc
9540gggtctacga ttggggcaac tccctactgg agcacattat aagattagga
gcattgtaaa 9600gaacaagaac cttcgttata gagatttcct tagttgtggg
gatggatctg gggggatgac 9660cgcggcacta ttgagagaaa acagacaaag
taggggaatc ttcaacagcc tgttagagtt 9720agccggatct cttatgagag
gagcatctcc agagcctcca agtgcactgg agacgctcgg 9780gcaagaacga
tctaggtgtg tgaatggaag cacatgttgg gagtactcat ctgacctaag
9840ccaaaaagag acatgggatt acttcttaag attgaagaga ggcctgggtt
tgaccgtgga 9900cttaatcacc atggacatgg aggtcagaga ccctaataca
agtttgatga tagaaaagaa 9960cctcaaagtt tatctgcatc agatattaga
accaactggt gtcttaatat ataaaacata 10020cgggacccat attgcgacac
aaacagataa tatcctgacg ataatcggtc ctttctttga 10080gacggttgac
ctagtccagt ccgaatacag cagctcacaa acgtccgagg tctattttgt
10140aggacgaggc ttgcgctctc atgttgacga accctgggtg gactggccat
ccttaatgga 10200caattggaga tccatttatg cttttcatga tcctactaca
gaatttatca gagcaaaaaa 10260agtctgtgaa attgacagtc ttataggcat
tccggctcaa ttcattccag acccatttgt 10320aaatctcgag accatgctac
agatagttgg tgttccaaca ggagtttcgc atgccgcagc 10380tctattatca
tcacaatatc caaatcaatt ggtcacaacg tcaatatttt atatgacact
10440cgtgtcttat tataatgtaa accatattcg aagaagcccc aagcctttct
ctcctccgtc 10500tgatggagtc tcacagaaca ttggttcagc catagtcgga
ctaagttttt gggtgagttt 10560gatggagaat gatctcggat tatacaaaca
ggctctaggt gcaataaaga cgtcattccc 10620tattagatgg tcctctgtcc
agaccaagga tgggtttaca caagaatgga gaactaaagg 10680aaacggaatt
cctaaagatt gtcgtctctc agactctttg gctcagatag gaaactggat
10740cagagcgatg gaattggtta ggaacaaaac gaggcaatca ggattttctg
aaaccctatt 10800tgatcaattc tgcggacttg cagaccatca cctcaaatgg
cggaagttgg gaaacagaac 10860aggaattatt gattggctaa ataatagaat
ttcatccatt gacaaatcca tcttggtgac 10920caaaagtgat ctgcatgacg
agaactcatg gagggagtga agatgtattc ttccacctct 10980cattgggtga
tacccatata tgaaaaaaac tataagtact ttaaactctc tttgtttttt
11040aatgtatatc tggttttgtt gtttccgt 110682422PRTMaraba Virus 2Met
Ser Val Thr Val Lys Arg Val Ile Asp Asp Ser Leu Ile Thr Pro1 5 10
15Lys Leu Pro Ala Asn Glu Asp Pro Val Glu Tyr Pro Ala Asp Tyr Phe
20 25 30Lys Lys Ser Arg Asp Ile Pro Val Tyr Ile Asn Thr Thr Lys Ser
Leu 35 40 45Ser Asp Leu Arg Gly Tyr Val Tyr Gln Gly Leu Lys Ser Gly
Asn Ile 50 55 60Ser Ile Ile His Val Asn Ser Tyr Leu Tyr Ala Ala Leu
Lys Glu Ile65 70 75 80Arg Gly Lys Leu Asp Arg Asp Trp Ile Thr Phe
Gly Ile Gln Ile Gly 85 90 95Lys Thr Gly Asp Ser Val Gly Ile Phe Asp
Leu Leu Thr Leu Lys Pro 100 105 110Leu Asp Gly Val Leu Pro Asp Gly
Val Ser Asp Ala Thr Arg Thr Ser 115 120 125Ser Asp Asp Ala Trp Leu
Pro Leu Tyr Leu Leu Gly Leu Tyr Arg Val 130 135 140Gly Arg Thr Gln
Met Pro Glu Tyr Arg Lys Lys Leu Met Asp Gly Leu145 150 155 160Ile
Asn Gln Cys Lys Met Ile Asn Glu Gln Phe Glu Pro Leu Leu Pro 165 170
175Glu Gly Arg Asp Val Phe Asp Val Trp Gly Asn Asp Ser Asn Tyr Thr
180 185 190Lys Ile Val Ala Ala Val Asp Met Phe Phe His Met Phe Lys
Lys His 195 200 205Glu Lys Ala Ser Phe Arg Tyr Gly Thr Ile Val Ser
Arg Phe Lys Asp 210 215 220Cys Ala Ala Leu Ala Thr Phe Gly His Leu
Cys Lys Ile Thr Gly Met225 230 235 240Ser Thr Glu Asp Val Thr Thr
Trp Ile Leu Asn Arg Glu Val Ala Asp 245 250 255Glu Met Val Gln Met
Met Tyr Pro Gly Gln Glu Ile Asp Lys Ala Asp 260 265 270Ser Tyr Met
Pro Tyr Leu Ile Asp Leu Gly Leu Ser Ser Lys Ser Pro 275 280 285Tyr
Pro Ser Val Lys Asn Pro Ala Phe His Phe Trp Gly Gln Leu Thr 290 295
300Ala Leu Leu Leu Arg Ser Thr Arg Ala Arg Asn Ala Arg Gln Pro
Asp305 310 315 320Asp Ile Glu Tyr Thr Ser Leu Thr Thr Ala Gly Leu
Leu Tyr Ala Tyr 325 330 335Ala Val Gly Ser Ser Ala Asp Leu Ala Gln
Gln Phe Tyr Val Gly Asp 340 345 350Asn Lys Tyr Val Pro Glu Thr Gly
Asp Gly Gly Leu Thr Thr Asn Ala 355 360 365Pro Pro Gln Gly Arg Asp
Val Val Glu Trp Leu Ser Trp Phe Glu Asp 370 375 380Gln Asn Arg Lys
Pro Thr Pro Asp Met Leu Met Tyr Ala Lys Arg Ala385 390 395 400Val
Ser Ala Leu Gln Gly Leu Arg Glu Lys Thr Ile Gly Lys Tyr Ala 405 410
415Lys Ser Glu Phe Asp Lys 4203265PRTMaraba Virus 3Met Asp Gln Leu
Ser Lys Val Lys Glu Phe Leu Lys Thr Tyr Ala Gln1 5 10 15Leu Asp Gln
Ala Val Gln Glu Met Asp Asp Ile Glu Ser Gln Arg Glu 20 25 30Glu Lys
Thr Asn Phe Asp Leu Phe Gln Glu Glu Gly Leu Glu Ile Lys 35 40 45Glu
Lys Pro Ser Tyr Tyr Arg Ala Asp Glu Glu Glu Ile Asp Ser Asp 50 55
60Glu Asp Ser Val Asp Asp Ala Gln Asp Leu Gly Ile Arg Thr Ser Thr65
70 75 80Ser Pro Ile Glu Gly Tyr Val Asp Glu Glu Gln Asp Asp Tyr Glu
Asp 85 90 95Glu Glu Val Asn Val Val Phe Thr Ser Asp Trp Lys Gln Pro
Glu Leu 100 105 110Glu Ser Asp Gly Asp Gly Lys Thr Leu Arg Leu Thr
Ile Pro Asp Gly 115 120 125Leu Thr Gly Glu Gln Lys Ser Gln Trp Leu
Ala Thr Ile Lys Ala Val 130 135 140Val Gln Ser Ala Lys Tyr Trp Asn
Ile Ser Glu Cys Ser Phe Glu Ser145 150 155 160Tyr Glu Gln Gly Val
Leu Ile Arg Glu Arg Gln Met Thr Pro Asp Val 165 170 175Tyr Lys Val
Thr Pro Val Leu Asn Ala Pro Pro Val Gln Met Thr Ala 180 185 190Asn
Gln Asp Val Trp Ser Leu Ser Ser Thr Pro Phe Thr Phe Leu Pro 195 200
205Lys Lys Gln Gly Val Thr Pro Leu Thr Met Ser Leu Glu Glu Leu Phe
210
215 220Asn Thr Arg Gly Glu Phe Ile Ser Leu Gly Gly Asn Gly Lys Met
Ser225 230 235 240His Arg Glu Ala Ile Ile Leu Gly Leu Arg His Lys
Lys Leu Tyr Asn 245 250 255Gln Ala Arg Leu Lys Tyr Asn Leu Ala 260
2654229PRTMaraba Virus 4Met Ser Ser Leu Lys Lys Ile Leu Gly Ile Lys
Gly Lys Gly Lys Lys1 5 10 15Ser Lys Lys Leu Gly Met Ala Pro Pro Pro
Tyr Glu Glu Glu Thr Pro 20 25 30Met Glu Tyr Ser Pro Ser Ala Pro Tyr
Asp Lys Ser Leu Phe Gly Val 35 40 45Glu Asp Met Asp Phe His Asp Gln
Arg Gln Leu Arg Tyr Glu Lys Phe 50 55 60His Phe Ser Leu Lys Met Thr
Val Arg Ser Asn Lys Pro Phe Arg Asn65 70 75 80Tyr Asp Asp Val Ala
Ala Ala Val Ser Asn Trp Asp His Met Tyr Ile 85 90 95Gly Met Ala Gly
Lys Arg Pro Phe Tyr Lys Ile Leu Ala Phe Met Gly 100 105 110Ser Thr
Leu Leu Lys Ala Thr Pro Ala Val Leu Ala Asp Gln Gly Gln 115 120
125Pro Glu Tyr His Ala His Cys Glu Gly Arg Ala Tyr Leu Pro His Arg
130 135 140Leu Gly Pro Thr Pro Pro Met Leu Asn Val Pro Glu His Phe
Arg Arg145 150 155 160Pro Phe Asn Ile Gly Leu Phe Arg Gly Thr Ile
Asp Ile Thr Leu Val 165 170 175Leu Phe Asp Asp Glu Ser Val Asp Ser
Ala Pro Val Ile Trp Asp His 180 185 190Phe Asn Ala Ser Arg Leu Ser
Ser Phe Arg Glu Lys Ala Leu Leu Phe 195 200 205Gly Leu Ile Leu Glu
Lys Lys Ala Thr Gly Asn Trp Val Leu Asp Ser 210 215 220Ile Ser His
Phe Lys2255512PRTMaraba Virus 5Met Leu Arg Leu Phe Leu Phe Cys Phe
Leu Ala Leu Gly Ala His Ser1 5 10 15Lys Phe Thr Ile Val Phe Pro His
His Gln Lys Gly Asn Trp Lys Asn 20 25 30Val Pro Ser Thr Tyr His Tyr
Cys Pro Ser Ser Ser Asp Gln Asn Trp 35 40 45His Asn Asp Leu Thr Gly
Val Ser Leu His Val Lys Ile Pro Lys Ser 50 55 60His Lys Ala Ile Gln
Ala Asp Gly Trp Met Cys His Ala Ala Lys Trp65 70 75 80Val Thr Thr
Cys Asp Phe Arg Trp Tyr Gly Pro Lys Tyr Ile Thr His 85 90 95Ser Ile
His Ser Met Ser Pro Thr Leu Glu Gln Cys Lys Thr Ser Ile 100 105
110Glu Gln Thr Lys Gln Gly Val Trp Ile Asn Pro Gly Phe Pro Pro Gln
115 120 125Ser Cys Gly Tyr Ala Thr Val Thr Asp Ala Glu Val Val Val
Val Gln 130 135 140Ala Thr Pro His His Val Leu Val Asp Glu Tyr Thr
Gly Glu Trp Ile145 150 155 160Asp Ser Gln Leu Val Gly Gly Lys Cys
Ser Lys Glu Val Cys Gln Thr 165 170 175Val His Asn Ser Thr Val Trp
His Ala Asp Tyr Lys Ile Thr Gly Leu 180 185 190Cys Glu Ser Asn Leu
Ala Ser Val Asp Ile Thr Phe Phe Ser Glu Asp 195 200 205Gly Gln Lys
Thr Ser Leu Gly Lys Pro Asn Thr Gly Phe Arg Ser Asn 210 215 220Tyr
Phe Ala Tyr Glu Ser Gly Glu Lys Ala Cys Arg Met Gln Tyr Cys225 230
235 240Thr Gln Trp Gly Ile Arg Leu Pro Ser Gly Val Trp Phe Glu Leu
Val 245 250 255Asp Lys Asp Leu Phe Gln Ala Ala Lys Leu Pro Glu Cys
Pro Arg Gly 260 265 270Ser Ser Ile Ser Ala Pro Ser Gln Thr Ser Val
Asp Val Ser Leu Ile 275 280 285Gln Asp Val Glu Arg Ile Leu Asp Tyr
Ser Leu Cys Gln Glu Thr Trp 290 295 300Ser Lys Ile Arg Ala Lys Leu
Pro Val Ser Pro Val Asp Leu Ser Tyr305 310 315 320Leu Ala Pro Lys
Asn Pro Gly Ser Gly Pro Ala Phe Thr Ile Ile Asn 325 330 335Gly Thr
Leu Lys Tyr Phe Glu Thr Arg Tyr Ile Arg Val Asp Ile Ser 340 345
350Asn Pro Ile Ile Pro His Met Val Gly Thr Met Ser Gly Thr Thr Thr
355 360 365Glu Arg Glu Leu Trp Asn Asp Trp Tyr Pro Tyr Glu Asp Val
Glu Ile 370 375 380Gly Pro Asn Gly Val Leu Lys Thr Pro Thr Gly Phe
Lys Phe Pro Leu385 390 395 400Tyr Met Ile Gly His Gly Met Leu Asp
Ser Asp Leu His Lys Ser Ser 405 410 415Gln Ala Gln Val Phe Glu His
Pro His Ala Lys Asp Ala Ala Ser Gln 420 425 430Leu Pro Asp Asp Glu
Thr Leu Phe Phe Gly Asp Thr Gly Leu Ser Lys 435 440 445Asn Pro Val
Glu Leu Val Glu Gly Trp Phe Ser Ser Trp Lys Ser Thr 450 455 460Leu
Ala Ser Phe Phe Leu Ile Ile Gly Leu Gly Val Ala Leu Ile Phe465 470
475 480Ile Ile Arg Ile Ile Val Ala Ile Arg Tyr Lys Tyr Lys Gly Arg
Lys 485 490 495Thr Gln Lys Ile Tyr Asn Asp Val Glu Met Ser Arg Leu
Gly Asn Lys 500 505 51062109PRTMaraba Virus 6Met Asp Val Asn Asp
Phe Glu Leu His Glu Asp Phe Ala Leu Ser Glu1 5 10 15Asp Asp Phe Val
Thr Ser Glu Phe Leu Asn Pro Glu Asp Gln Met Thr 20 25 30Tyr Leu Asn
His Ala Asp Tyr Asn Leu Asn Ser Pro Leu Ile Ser Asp 35 40 45Asp Ile
Asp Phe Leu Ile Lys Lys Tyr Asn His Glu Gln Ile Pro Lys 50 55 60Met
Trp Asp Val Lys Asn Trp Glu Gly Val Leu Glu Met Leu Thr Ala65 70 75
80Trp Gln Ala Ser Pro Ile Leu Ser Ser Thr Met His Lys Trp Val Gly
85 90 95Lys Trp Leu Met Ser Asp Asp His Asp Ala Ser Gln Gly Phe Ser
Phe 100 105 110Leu His Glu Val Asp Lys Glu Ala Asp Leu Thr Phe Glu
Val Val Glu 115 120 125Thr Phe Ile Arg Gly Trp Gly Gly Arg Glu Leu
Gln Tyr Lys Arg Lys 130 135 140Asp Thr Phe Pro Asp Ser Phe Arg Val
Ala Ala Ser Leu Cys Gln Lys145 150 155 160Phe Leu Asp Leu His Lys
Leu Thr Leu Ile Met Asn Ser Val Ser Glu 165 170 175Val Glu Leu Thr
Asn Leu Ala Lys Asn Phe Lys Gly Lys Asn Arg Lys 180 185 190Ala Lys
Ser Gly Asn Leu Ile Thr Arg Leu Arg Val Pro Ser Leu Gly 195 200
205Pro Ala Phe Val Thr Gln Gly Trp Val Tyr Met Lys Lys Leu Glu Met
210 215 220Ile Met Asp Arg Asn Phe Leu Leu Met Leu Lys Asp Val Ile
Ile Gly225 230 235 240Arg Met Gln Thr Ile Leu Ser Met Ile Ser Arg
Asp Asp Asn Leu Phe 245 250 255Ser Glu Ser Asp Ile Phe Thr Val Leu
Lys Ile Tyr Arg Ile Gly Asp 260 265 270Lys Ile Leu Glu Arg Gln Gly
Thr Lys Gly Tyr Asp Leu Ile Lys Met 275 280 285Ile Glu Pro Ile Cys
Asn Leu Lys Met Met Asn Leu Ala Arg Lys Tyr 290 295 300Arg Pro Leu
Ile Pro Thr Phe Pro His Phe Glu Lys His Ile Ala Asp305 310 315
320Ser Val Lys Glu Gly Ser Lys Ile Asp Lys Gly Ile Glu Phe Ile Tyr
325 330 335Asp His Ile Met Ser Ile Pro Gly Val Asp Leu Thr Leu Val
Ile Tyr 340 345 350Gly Ser Phe Arg His Trp Gly His Pro Phe Ile Asn
Tyr Tyr Glu Gly 355 360 365Leu Glu Lys Leu His Lys Gln Val Thr Met
Pro Lys Thr Ile Asp Arg 370 375 380Glu Tyr Ala Glu Cys Leu Ala Ser
Asp Leu Ala Arg Ile Val Leu Gln385 390 395 400Gln Gln Phe Asn Glu
His Lys Lys Trp Phe Val Asp Val Asp Lys Val 405 410 415Pro Gln Ser
His Pro Phe Lys Ser His Met Lys Glu Asn Thr Trp Pro 420 425 430Thr
Ala Ala Gln Val Gln Asp Tyr Gly Asp Arg Trp His Gln Leu Pro 435 440
445Leu Ile Lys Cys Phe Glu Ile Pro Asp Leu Leu Asp Pro Ser Ile Ile
450 455 460Tyr Ser Asp Lys Ser His Ser Met Asn Arg Ser Glu Val Leu
Arg His465 470 475 480Val Arg Leu Thr Pro His Val Pro Ile Pro Ser
Arg Lys Val Leu Gln 485 490 495Thr Met Leu Glu Thr Lys Ala Thr Asp
Trp Lys Glu Phe Leu Lys Lys 500 505 510Ile Asp Glu Glu Gly Leu Glu
Asp Asp Asp Leu Val Ile Gly Leu Lys 515 520 525Gly Lys Glu Arg Glu
Leu Lys Ile Ala Gly Arg Phe Phe Ser Leu Met 530 535 540Ser Trp Lys
Leu Arg Glu Tyr Phe Val Ile Thr Glu Tyr Leu Ile Lys545 550 555
560Thr His Phe Val Pro Met Phe Lys Gly Leu Thr Met Ala Asp Asp Leu
565 570 575Thr Ala Val Ile Lys Lys Met Met Asp Thr Ser Ser Gly Gln
Gly Leu 580 585 590Asp Asn Tyr Glu Ser Ile Cys Ile Ala Asn His Ile
Asp Tyr Glu Lys 595 600 605Trp Asn Asn His Gln Arg Lys Glu Ser Asn
Gly Pro Val Phe Lys Val 610 615 620Met Gly Gln Phe Leu Gly Tyr Pro
Arg Leu Ile Glu Arg Thr His Glu625 630 635 640Phe Phe Glu Lys Ser
Leu Ile Tyr Tyr Asn Gly Arg Pro Asp Leu Met 645 650 655Arg Val Arg
Gly Asn Ser Leu Val Asn Ala Ser Ser Leu Asn Val Cys 660 665 670Trp
Glu Gly Gln Ala Gly Gly Leu Glu Gly Leu Arg Gln Lys Gly Trp 675 680
685Ser Ile Leu Asn Leu Leu Val Ile Gln Arg Glu Ala Lys Ile Arg Asn
690 695 700Thr Ala Val Lys Val Leu Ala Gln Gly Asp Asn Gln Val Ile
Cys Thr705 710 715 720Gln Tyr Lys Thr Lys Lys Ser Arg Asn Asp Ile
Glu Leu Lys Ala Ala 725 730 735Leu Thr Gln Met Val Ser Asn Asn Glu
Met Ile Met Ser Ala Ile Lys 740 745 750Ser Gly Thr Glu Lys Leu Gly
Leu Leu Ile Asn Asp Asp Glu Thr Met 755 760 765Gln Ser Ala Asp Tyr
Leu Asn Tyr Gly Lys Val Pro Ile Phe Arg Gly 770 775 780Val Ile Arg
Gly Leu Glu Thr Lys Arg Trp Ser Arg Val Thr Cys Val785 790 795
800Thr Asn Asp Gln Ile Pro Thr Cys Ala Asn Ile Met Ser Ser Val Ser
805 810 815Thr Asn Ala Leu Thr Val Ala His Phe Ala Glu Asn Pro Val
Asn Ala 820 825 830Ile Ile Gln Tyr Asn Tyr Phe Gly Thr Phe Ala Arg
Leu Leu Leu Met 835 840 845Met His Asp Pro Ala Leu Arg Ile Ser Leu
Tyr Glu Val Gln Ser Lys 850 855 860Ile Pro Gly Leu His Ser Leu Thr
Phe Lys Tyr Ser Met Leu Tyr Leu865 870 875 880Asp Pro Ser Ile Gly
Gly Val Ser Gly Met Ser Leu Ser Arg Phe Leu 885 890 895Ile Arg Ser
Phe Pro Asp Pro Val Thr Glu Ser Leu Ala Phe Trp Lys 900 905 910Phe
Ile His Ser His Ala Arg Ser Asp Ser Leu Lys Glu Ile Cys Ala 915 920
925Val Phe Gly Asn Pro Glu Ile Ala Arg Phe Arg Leu Thr His Val Asp
930 935 940Lys Leu Val Glu Asp Pro Thr Ser Leu Asn Ile Ala Met Gly
Met Ser945 950 955 960Pro Ala Asn Leu Leu Lys Thr Glu Val Lys Lys
Cys Leu Leu Glu Ser 965 970 975Arg Gln Ser Ile Lys Asn Gln Ile Val
Arg Asp Ala Thr Ile Tyr Leu 980 985 990His His Glu Glu Asp Lys Leu
Arg Ser Phe Leu Trp Ser Ile Thr Pro 995 1000 1005Leu Phe Pro Arg
Phe Leu Ser Glu Phe Lys Ser Gly Thr Phe Ile 1010 1015 1020Gly Val
Ala Asp Gly Leu Ile Ser Leu Phe Gln Asn Ser Arg Thr 1025 1030
1035Ile Arg Asn Ser Phe Lys Lys Arg Tyr His Arg Glu Leu Asp Asp
1040 1045 1050Leu Ile Ile Lys Ser Glu Val Ser Ser Leu Met His Leu
Gly Lys 1055 1060 1065Leu His Leu Arg Arg Gly Ser Val Arg Met Trp
Thr Cys Ser Ser 1070 1075 1080Thr Gln Ala Asp Leu Leu Arg Phe Arg
Ser Trp Gly Arg Ser Val 1085 1090 1095Ile Gly Thr Thr Val Pro His
Pro Leu Glu Met Leu Gly Gln His 1100 1105 1110Phe Lys Lys Glu Thr
Pro Cys Ser Ala Cys Asn Ile Ser Gly Leu 1115 1120 1125Asp Tyr Val
Ser Val His Cys Pro Asn Gly Ile His Asp Val Phe 1130 1135 1140Glu
Ser Arg Gly Pro Leu Pro Ala Tyr Leu Gly Ser Lys Thr Ser 1145 1150
1155Glu Ser Thr Ser Ile Leu Gln Pro Trp Glu Arg Glu Ser Lys Val
1160 1165 1170Pro Leu Ile Lys Arg Ala Thr Arg Leu Arg Asp Ala Ile
Ser Trp 1175 1180 1185Phe Val Ser Pro Asp Ser Asn Leu Ala Ser Thr
Ile Leu Lys Asn 1190 1195 1200Ile Asn Ala Leu Thr Gly Glu Glu Trp
Ser Lys Lys Gln His Gly 1205 1210 1215Phe Lys Arg Thr Gly Ser Ala
Leu His Arg Phe Ser Thr Ser Arg 1220 1225 1230Met Ser His Gly Gly
Phe Ala Ser Gln Ser Thr Ala Ala Leu Thr 1235 1240 1245Arg Leu Met
Ala Thr Thr Asp Thr Met Arg Asp Leu Gly Glu Gln 1250 1255 1260Asn
Tyr Asp Phe Leu Phe Gln Ala Thr Leu Leu Tyr Ala Gln Ile 1265 1270
1275Thr Thr Thr Val Val Arg Asn Gly Ser Phe His Ser Cys Thr Asp
1280 1285 1290His Tyr His Ile Thr Cys Lys Ser Cys Leu Arg Ala Ile
Asp Glu 1295 1300 1305Ile Thr Leu Asp Ser Ala Met Glu Tyr Ser Pro
Pro Asp Val Ser 1310 1315 1320Ser Val Leu Gln Ser Trp Arg Asn Gly
Glu Gly Ser Trp Gly His 1325 1330 1335Glu Val Lys Gln Ile Tyr Pro
Val Glu Gly Asp Trp Arg Gly Leu 1340 1345 1350Ser Pro Val Glu Gln
Ser Tyr Gln Val Gly Arg Cys Ile Gly Phe 1355 1360 1365Leu Phe Gly
Asp Leu Ala Tyr Arg Lys Ser Ser His Ala Asp Asp 1370 1375 1380Ser
Ser Met Phe Pro Leu Ser Ile Gln Asn Lys Val Arg Gly Arg 1385 1390
1395Gly Phe Leu Lys Gly Leu Met Asp Gly Leu Met Arg Ala Ser Cys
1400 1405 1410Cys Gln Val Ile His Arg Arg Ser Leu Ala His Leu Lys
Arg Pro 1415 1420 1425Ala Asn Ala Val Tyr Gly Gly Leu Ile Tyr Leu
Ile Asp Lys Leu 1430 1435 1440Ser Ala Ser Ala Pro Phe Leu Ser Leu
Thr Arg His Gly Pro Leu 1445 1450 1455Arg Glu Glu Leu Glu Thr Val
Pro His Lys Ile Pro Thr Ser Tyr 1460 1465 1470Pro Thr Ser Asn Arg
Asp Met Gly Val Ile Val Arg Asn Tyr Phe 1475 1480 1485Lys Tyr Gln
Cys Arg Leu Val Glu Lys Gly Arg Tyr Lys Thr His 1490 1495 1500Tyr
Pro Gln Leu Trp Leu Phe Ser Asp Val Leu Ser Ile Asp Phe 1505 1510
1515Leu Gly Pro Leu Ser Ile Ser Ser Thr Leu Leu Gly Ile Leu Tyr
1520 1525 1530Lys Gln Thr Leu Ser Ser Arg Asp Lys Asn Glu Leu Arg
Glu Leu 1535 1540 1545Ala Asn Leu Ser Ser Leu Leu Arg Ser Gly Glu
Gly Trp Glu Asp 1550 1555 1560Ile His Val Lys Phe Phe Ser Lys Asp
Thr Leu Leu Cys Pro Glu 1565 1570 1575Glu Ile Arg His Ala Cys Lys
Phe Gly Ile Ala Lys Glu Ser Ala 1580 1585 1590Val Leu Ser Tyr Tyr
Pro Pro Trp Ser Gln Glu Ser Tyr Gly Gly 1595 1600 1605Ile Thr Ser
Ile Pro Val Tyr Phe Ser Thr Arg Lys Tyr Pro Lys 1610 1615 1620Ile
Leu Asp Val Pro Pro Arg Val Gln Asn Pro Leu Val Ser Gly 1625 1630
1635Leu Arg Leu Gly Gln Leu Pro Thr Gly Ala His Tyr Lys Ile Arg
1640 1645 1650Ser Ile Val Lys Asn Lys Asn Leu Arg Tyr Arg Asp Phe
Leu Ser 1655 1660 1665Cys Gly Asp Gly Ser Gly
Gly Met Thr Ala Ala Leu Leu Arg Glu 1670 1675 1680Asn Arg Gln Ser
Arg Gly Ile Phe Asn Ser Leu Leu Glu Leu Ala 1685 1690 1695Gly Ser
Leu Met Arg Gly Ala Ser Pro Glu Pro Pro Ser Ala Leu 1700 1705
1710Glu Thr Leu Gly Gln Glu Arg Ser Arg Cys Val Asn Gly Ser Thr
1715 1720 1725Cys Trp Glu Tyr Ser Ser Asp Leu Ser Gln Lys Glu Thr
Trp Asp 1730 1735 1740Tyr Phe Leu Arg Leu Lys Arg Gly Leu Gly Leu
Thr Val Asp Leu 1745 1750 1755Ile Thr Met Asp Met Glu Val Arg Asp
Pro Asn Thr Ser Leu Met 1760 1765 1770Ile Glu Lys Asn Leu Lys Val
Tyr Leu His Gln Ile Leu Glu Pro 1775 1780 1785Thr Gly Val Leu Ile
Tyr Lys Thr Tyr Gly Thr His Ile Ala Thr 1790 1795 1800Gln Thr Asp
Asn Ile Leu Thr Ile Ile Gly Pro Phe Phe Glu Thr 1805 1810 1815Val
Asp Leu Val Gln Ser Glu Tyr Ser Ser Ser Gln Thr Ser Glu 1820 1825
1830Val Tyr Phe Val Gly Arg Gly Leu Arg Ser His Val Asp Glu Pro
1835 1840 1845Trp Val Asp Trp Pro Ser Leu Met Asp Asn Trp Arg Ser
Ile Tyr 1850 1855 1860Ala Phe His Asp Pro Thr Thr Glu Phe Ile Arg
Ala Lys Lys Val 1865 1870 1875Cys Glu Ile Asp Ser Leu Ile Gly Ile
Pro Ala Gln Phe Ile Pro 1880 1885 1890Asp Pro Phe Val Asn Leu Glu
Thr Met Leu Gln Ile Val Gly Val 1895 1900 1905Pro Thr Gly Val Ser
His Ala Ala Ala Leu Leu Ser Ser Gln Tyr 1910 1915 1920Pro Asn Gln
Leu Val Thr Thr Ser Ile Phe Tyr Met Thr Leu Val 1925 1930 1935Ser
Tyr Tyr Asn Val Asn His Ile Arg Arg Ser Pro Lys Pro Phe 1940 1945
1950Ser Pro Pro Ser Asp Gly Val Ser Gln Asn Ile Gly Ser Ala Ile
1955 1960 1965Val Gly Leu Ser Phe Trp Val Ser Leu Met Glu Asn Asp
Leu Gly 1970 1975 1980Leu Tyr Lys Gln Ala Leu Gly Ala Ile Lys Thr
Ser Phe Pro Ile 1985 1990 1995Arg Trp Ser Ser Val Gln Thr Lys Asp
Gly Phe Thr Gln Glu Trp 2000 2005 2010Arg Thr Lys Gly Asn Gly Ile
Pro Lys Asp Cys Arg Leu Ser Asp 2015 2020 2025Ser Leu Ala Gln Ile
Gly Asn Trp Ile Arg Ala Met Glu Leu Val 2030 2035 2040Arg Asn Lys
Thr Arg Gln Ser Gly Phe Ser Glu Thr Leu Phe Asp 2045 2050 2055Gln
Phe Cys Gly Leu Ala Asp His His Leu Lys Trp Arg Lys Leu 2060 2065
2070Gly Asn Arg Thr Gly Ile Ile Asp Trp Leu Asn Asn Arg Ile Ser
2075 2080 2085Ser Ile Asp Lys Ser Ile Leu Val Thr Lys Ser Asp Leu
His Asp 2090 2095 2100Glu Asn Ser Trp Arg Glu 2105710716DNACarajas
Virus 7cggccggtcg acgctgccta tttacttact gggtctttac cgtgttggaa
kaacaaaact 60gccggaatac cgaaagaagt tgatggaggg gttggaaatg cagtgtaaaa
tcatgtatcc 120tgactttgta ccaatcgttc cggaaggaat ggacttcttt
gatgtgtggg gaaatgatag 180taatttcacc aaaatagtcg ccgcagtgga
tatgtttttc catatgttca aaaagcatga 240gagagcatcc ctcagatatg
gaacaattgt ctccagattc aaggattgtg ctgcattggc 300tacatttggc
catgtatgta aagtttccgg aatgtccaca gaggaggtca ccacttgggt
360gctgaatagg gaagtggcag acgaattatg ccagatgatg ttccctggac
aggaaataga 420ccgagcggac tcatacatgc cgtatatgat agatttcggg
ttgtctcaga aatcgccata 480ttcctctgtc aaaaatccgt cttttcactt
ttgggggcaa cttgcagcac tactgctcag 540atcaaccagg gcaaaaaatg
ccagacaacc tgatgacatt gaatacacat cactgactac 600agcaggtcta
cttcttgcgt atgctgtagg gtcatctgca gacatctctc aacagttcta
660catgggagat gagaaatata tctcagaccc aagtgcgggt ggattaacct
ccaatgcacc 720tccgaaagga aggaatgtag ttgactggct cgggtggttt
gaggatcaag gaggaaatat 780cactccagat atgtacactt cgctaaaagg
gctgtttgct ctttgcaagg gctgcgagat 840aagaccattg gaaagtatgc
caagggagag tttgacaagt gactccattc agatcaaatg 900ctttactaca
tgctgtatta tatataacta tgaaaaaaac taacagagat catggataat
960ctctcgaaac ttaaggagta tatggggact tacacccatc tagactctgc
attgcaagat 1020gcaaatgaat cagaagaatc tcgagatgaa aagagcaatt
ttgatctttt cgatgaggaa 1080agtaaggagg ttgcaagacc ttcttattat
tctgcaattg atgaggagtc tgaccaggag 1140gaaactgaat ccgatgatcc
agatgaggag ctgaatgact caaatgccca tggggcggtg 1200gatggatggg
acgagacgtt gaacgagaat tctcagcctg acgacaatgt ctctgttgag
1260ttcgctcgta catggtcaac accggtgatg gaatcttcgt cagagggaaa
gactttgcat 1320ttggctatgc cagatggact gaatccagat caagtcgcac
agtggctgca gactgtcaag 1380gctttgtttg agagtgccaa atattggaat
ctgtccgaat gcaggatgga agtgctgctt 1440gagggagtat taatcaaaga
gagacaaatg actccagatc ttcagaaggt cacaccgaag 1500ccgaacaatc
ctcctccaga aagtatgcca tgcgatcctc tccctcccgc tatggacgtg
1560tgggaggccg cgtctcaggt gtatacacta gagcccaagc gggcaaacct
ggccccaatg 1620gatgtaaagc tgaaagatct gttttcatct agggccgaat
ttctctcagt cggaggatct 1680ccccagatga gctggaaaga ggccattata
ttgggtctaa gatacaagaa attgtataat 1740caagctcgcc taaaatattc
cctatagggt ataccccata tgaaaaaaac taacagaatt 1800caaaatgagt
tctctcaaga aaatactcgg cctgaaaggc aagaaggagg aaaagtccaa
1860aaagttggga cttcctcctc cttacgagat gccagcaaac aatgagttcg
agccaaatgc 1920tcctttagat cctgacatgt tcggggcgga acatttggag
attgaaagca agtctgccat 1980gcgttatgag aaatttaagt tctctgtcaa
gatcaccctt aggaccaatc gacctttgag 2040aacttatgat gatgtgtgcc
agattctatc caaatgggat gcaatgtatg tcggcatgat 2100gggtaagcga
ccgttctaca aggtattggt cttgatcgga tccagccact tgcaggctac
2160acctgctata ctctcagatc gtggtcaacc agaatatcat atgtacttgg
aagatagagg 2220attcatcgca cacaggttgg ggttgacacc gccaatgtta
agtgggccgg aaagttttag 2280aagacctttc catgtcggtc tttacagagg
gacaattgac attacagtaa atctcatgga 2340cgacgaatca acggaatcag
caccacaggt ttgggatcac ttcaatacca gatatgtgaa 2400tcatttcctt
gagcatgcaa agaggttcgg attggtcctg tccaagaaac caggtggcgg
2460ctggatatta gatcaagcgg tctgtgcata atgcgaatat aatcatagtc
tcatcagacg 2520attatttata cattattcta ttctctctct tagttggtgg
tagctatgaa aaaaactaac 2580agagttcaaa actctacatc tcaactgcaa
aggctatttt tcttaaaaaa accttttaat 2640acagagtcat cattcaaaaa
tgaagatgaa aatggtcata gcaggattaa tcctttgtat 2700agggatttta
ccggctattg ggaaaataac aatttctttc ccacaaagct tgaaaggaga
2760ttggaggcct gtacctaagg gatacaatta ttgtcctaca agtgcggata
aaaatctcca 2820tggtgatttg attgacatag gtctcagact tcgggcccct
aagagcttca aagggatctc 2880cgcagatgga tggatgtgcc atgcggcaag
atggatcacc acctgtgatt tcagatggta 2940tggacccaag tacatcaccc
actcaattca ctctttcagg ccgagcaatg accaatgcaa 3000agaagcaatc
cggctgacta atgaagggaa ttggattaat ccaggtttcc ctccgcaatc
3060ttgcggatat gcttctgtaa ccgactcaga atccgttgtc gtaaccgtga
ccaagcacca 3120ggtcctagta gatgagtact ccggctcatg gatcgatagt
caattccccg gaggaagttg 3180cacatccccc atttgcgata cagtgcacaa
ctcgacactt tggcacgcgg accacaccct 3240ggacagtatc tgtgaccaag
aattcgtggc aatggacgca gttctgttca cagagagtgg 3300caaatttgaa
gagttcggaa aaccgaactc cggcatcagg agcaactatt ttccttatga
3360gagtctgaaa gatgtatgtc agatggattt ctgcaagagg aaaggattca
agctcccatc 3420cggtgtctgg tttgaaatcg aggatgcaga gaaatctcac
aaggcccagg ttgaattgaa 3480aataaaacgg tgccctcatg gagcagtaat
ctcagctcct aatcagaatg cagcagatat 3540caatctgatc atggatgtgg
aacgaattct agactactcc ctttgccaag caacttggag 3600caaaatccaa
aacaaggaag cgttgacccc catcgatatc agttatcttg gtccgaaaaa
3660cccaggacca ggcccagcct tcaccataat aaatggaaca ctgcactact
tcaatactag 3720atacattcga gtggatattg cagggcctgt taccaaagag
attacaggat ttgtttcggg 3780aacatctaca tctagggtgc tgtgggatca
gtggtcccat atggagagaa ttccattgga 3840cccaatggct tgctgaaaac
cgccagcgga tacaaatatc cattgttcat ggttggtaca 3900ggtgtgctgg
atgcggacat ccacaagctg ggagaagcaa ccgtgattga acatccacat
3960gccaaagagg ctcagaaggt agttgatgac agtgaggtta tattttttgg
tgacaccgga 4020gtctccaaga atccagtgga ggtagtcgaa ggatggttta
gcggatggag aagctctttg 4080atgagcatat ttggcataat tttgttgatt
gtttgtttag tcttgattgt tcgaatcctt 4140atagccctta aatactgttg
tgttagacac aaaaagagaa ctatttacaa agaggacctt 4200gaaatgggtc
gaattcctcg gagggcttaa ttacttataa ttacggactt taaatgtatg
4260aaaaaaacta taacagaagt caaaatggac ttcttacccg ttgaacaaga
ggaggactgg 4320ggttatgcag aagatgattt ctctagctca gattatctag
attttgaaga acgaatgaca 4380tatttaaatc aggctgatta taatctaaac
tcaccattga tatctgatga catttattac 4440ctgagtcgaa aattccactc
atatggcatc ccccccatgt ggaacctcaa agaatgggat 4500ggaccattgg
agatgttaaa atcatgtcaa gcagacccga ttccacatga tctgatgcac
4560aaatggtttg gaacttggtt agaagacttt gatcacgact ctgcacaagg
gatagtgttt 4620ttaagggaag tagacaaaga ggcctccgag acctatgatt
tagtggatac ctttttgaaa 4680aattgggcag ggaaatccta tccttacaaa
gcaaaggaga gatacttaga tcagatgaag 4740atcattggcc ctttgtgtca
aaagttcctt gatttgcaca agctgacatt gatcctcaat 4800gctgttggtc
ctgaagagtt gaaaaacctg ttacgaacat ttaagggaag aacgagagat
4860ttatcgacca aagatccatg cactcggcta cgtgttccca gccttgggcc
cgtattcata 4920tgcaaaggct gggtctatat ccacaagcac aaaattttga
tggaccgaaa tttcctgctt 4980atgtgtaaag atgtcataat aggacgcatg
cagaccctat tgtctatgat aggtagatct 5040gacgatgcat tcactcagca
agacttcttc acccttgtaa atatctacag gacaggagat 5100atcatcttac
aagagaaagg aaatctggcc tatgacttaa tcaagatggt ggagcctatc
5160tgcaatctga aattgatgaa attggcgaga gaatacagac cactgattcc
cccttttcca 5220cattttgaaa atcatgttaa aaatgcagtg gacgaacaat
ctaaggtctc gaggaggatc 5280aaagttctct ttgagctgat tatgggaatc
aaaaatgtgg atcttgtcct ggtgatctat 5340ggatcattta ggcattgggg
gcatccattc atagattatt tcgaaggatt aaacaagcta 5400cataagcagg
taaccatgtc gaaggagatt gacacggagt atgcaaatgc tctggcaagt
5460gatttggcta gaatcgttct gactaaacag tttgactctg ttaagaagtg
gtttgtagac 5520aagacaaaaa tcccctctgc ccatcccttt ttcaagcata
tcatggataa cacatggccc 5580actgccgccc agatccaaga ctttggagac
cactggcatg aactgccgtt aatcaagtgt 5640tatgagatac ctgacctcat
cgatccatct atcatctatt cagacaagag ccactcaatg 5700aaccgatctg
aggtgcttgg acatgtgagg agatcccctc atttgccaat accgagcaaa
5760aaggtactcc agactatgct tgataccagg gcgacaaact gggttgagtt
tctagaaatg 5820gtagacaaac atggtcttga aaaggatgat ttgataattg
gactcaaggg gaaagaacgt 5880gagttaaaat tagcaggtag atttttttca
ttgatgtcct ggaagttgag agaatacttc 5940gttatcacgg aatatcttat
aaaaacacat tttgtaccct tgtttaaggg gctgacgatg 6000gcagatgatt
taacttccgt catcaaaaag atgttggata gttcttccgg acagggaata
6060gacgactact cttcagtgtg ttttgccaat catatagatt acgagaagtg
gaataatcac 6120cagagaaagg aatcaaacgg accagtgttt cgggtgatgg
gccaattttt gggataccca 6180cgtttgattg aacgaaccca tgagttcttt
gagaaaagtc tcatttatta taacaacaga 6240ccggatctaa tgtgggtcaa
tgaagacaca ctgattaatc gtacacaaca gcgagtatgt 6300tgggaaggtc
aggctggagg ccttgagggg ttgaggcaaa agggttggag tattctcaat
6360cttcttgtga ttcagagaga ggcaaaaatt cgaaacacag cagtcaaggt
attggcacaa 6420ggggacaatc aggtcatctg tactcaatat aagacgaaga
aatccagaga tcagagtgaa 6480ctcatcaatg cattagatca aatggtgaaa
aacaacaaca aaattatgga ggaaataaag 6540aagggaacga gcaaactggg
actattgatt aacgatgatg agaccatgca atcggctgat 6600tatttgaatt
acggtaaagt tccaatattc cgtggggtaa ttagagggtt agagacaaaa
6660agatggtccc gggtcacatg tgtgacaaat gatcaaattc caacgtgtgc
caatctgatg 6720gcttctgtct caactaatgc actaacagta gctcattttg
cgtctaaccc aatcaattca 6780atgatacagt acaattactt cggtaacttt
tcccgactac tgttgtttat gcatgaccca 6840gcactgcgaa gatcacttta
cgatgtgcag aatgaaatac cgggattgca cagtaagact 6900ttcaaatatg
caatgctata tttggaccca tctattggcg gcgtttcagg gatggcattg
6960agtagattcc ttatacgtgc attcccggac cctgtaactg aaagcttatc
tttctggaaa 7020tttattcatg accatactga tgatgaatac ctcaaaagct
tatcaattgc ctttgggaat 7080cctgatatag cgaaattccg actagagcat
atcagtaaac tgcttgagga tccaacttcc 7140ctcaatatat ctatgggaat
gagtccttca aatcttttga aaaccgaagt taaaaaatgt 7200ctcattgaaa
atagaacatc tatcaggaac gatattatca aagatgccac catctatttg
7260aaccaagagg aagcaaaatt gaaaagcttc ttatggtcta tcaatccact
gtttcctaga 7320tttttgagtg agttcaaatc tggcaccttc ctgggagtat
ccgaaggatt aatcagtcta 7380ttccaaaatt ctcggaccat ccgaaattcc
ttcaagggta agtatcggaa agagctggat 7440cacttgatcg tgaagagtga
aatttcttct ctcaaacatc tgggcggcat tcacttcaaa 7500ttggggaatg
ggaaaatttg gggatgctcg tcatcccaat cagatttgct tagatacaga
7560tcctggggaa gaaaactggt gggaactaca attcctcatc ctttggaaat
gcacggagca 7620gcgagtccta aagaggctcc ttgcaccttg tgtaactgct
ctggcctgac ttacatctct 7680gttcattgcc cgaaaggaat tacagaggta
ttttccagaa gaggaccctt accggcgtac 7740ctgggttcta agacatcgga
gaccacttca attcttcagc cttgggaaaa agaaagtaag 7800gttcctattg
taagacgagc tactagactg agagatgcca tctcatggtt catagaccca
7860gattctacac ttgctcaatc tattcttgac aacattaaat ctttgacagg
ggaagagtgg 7920ggaggaagac agcatgggta taagagaact ggctctgcat
tgcatagatt ttctacctca 7980cgtatgagca atggagggtt tgcttctcaa
agtcccgcgg ctttgacccg attgattgct 8040acgactgaca ccatgcacga
ttatggagac aagaattatg atttcatgtt ccaggcctct 8100ttgttatacg
cacagatgac tacatctata tccagatggg ggcatgtcgg ggcttgcaca
8160gatcattacc atgtccgttg tgacagctgc attcgagaaa tacaagagat
tgaattgaac 8220actggagtcc agtactctcc ccccgatgtg tcttatgttt
tgacaaaatg gcggaacggc 8280tcaggttctt ggggtactgt caccaaacaa
ctcatcccga aagaaggaaa ctggaccgta 8340ctctcgcctg cagaacaatc
ctatcaagtt ggacggtgta tcggatttct gtacggagat 8400ctagtacata
agaaatcaca tcaagcggac gacagttcat tatttccgtt aagcatacaa
8460cacaaagtga gagggagagg ttttcttgaa ggtcttttag atggaataat
gagagctagc 8520tgttgtcaag tcattcacag gagaagtgtc gcaaccttaa
agcgtccggc aaatgctgtg 8580tatgggggag tcatattctt gattgacaaa
ttgagtatgt cagccccatt cttgtcttta 8640acccgtactg gtcctatcag
ggaagaacta gaaaatgtcc ctcacaaaat gccagcgtcc 8700tacccaacta
ataatcgaga tttggggatg accgtcagaa actacttcaa gtatcaatgt
8760cgaatcattg agagaggaca gtataaatcc cattatccca caatttggtt
attttccgat 8820gtcttatcgg tggactttat tggtcctatg tccttgtcat
ctggacttat gagattgtta 8880tacaagaaca gtctcagtaa gaaagacaaa
aatgagctcc gagacttggc aaatctttca 8940tctcttctca gatcaggaga
agaatgggat gatatacatg tcaaattttt ctctcaagac 9000ttactctttt
gttctcagga gatacgacat gcctgtaaat tcgggattat acgagacaaa
9060gtaagtctag aagtggatca tgggtggggg aaagaagcat atggaggatg
tacagtgctt 9120ccagtgttct acaggtctca gatttataag aaaagtttga
ctgtaccccc acgaattcaa 9180aaccctatca tatctggact ccgcttgggg
caacttccta caggagctca ttataagatc 9240agatcaatca tcatgactct
aaagatcaat tatcaggact tcctgtcatg tggagacggt 9300tcagggggga
tgactgcctg cttgctccgg ttaaacccta atagtcgggg aattttcaat
9360agtttgctag aattagatgg agcattaatg agaggatcat cccccgagcc
acccagtgcg 9420ctagagacgt tggggagcca aagaactcga tgtgtaaacg
gaggaacatg ttgggaacat 9480ccctctgact tgagcgaccc caatacttgg
aagtatttta ttggattgaa gagaggatta 9540ggcttgcaga tcaatctgat
tactatggat atggaagttc gagatccagt gatctcacac 9600aaaattgaag
caaacatccg agcatttctc tatgatcttt tagacccgga gggaaccctt
9660atatacaaaa cgtatggcac atatctggca gaagaggaaa ggaatattct
gacagaagta 9720ggtcctttgt ttcacactac tgacttggtg caaactattt
acagtagtgc ccagacttcg 9780gaggtttact gtgtatgcag acggttaaag
aaatatgctg atcaacaaca tgtggattgg 9840tcattgttga ctgatggatg
gtctcggtta tatgcgtttt ctgtgaatcg attggaattc 9900caaagggctc
agagtcttcg gaaactggac acactgcaag gaattccaag ctttttcata
9960ccagatcctt ttgtcaatgc ggagacttta ttgcaaattg caggtgttcc
aacagggatt 10020tctcacacag ccgtattaca tggatcgtta cattctgaac
aattgataac gcttggtatt 10080ttcttctgtg cgctaatctc tcaccataca
atgaacatca tacgaatatc acctgtcccc 10140ccgtctcctc catccgatgg
gtcaataagt agaatgtgtt ctgcaatcac agggatccta 10200ttttgggtct
ccttagtgga gaaggacttg actctataca actcattgtt gtcaataata
10260cagagatcct ttccaatccg atggtacaaa aataaggaga aaaacggatg
gtcccaatgt 10320tggggggcaa atggagacgg gatacccaaa gatactcgac
taaatgattc gatggcgaac 10380ataggaaact ggataagggc tatggagttg
ctttgcaata agaccgctca gatgcccttc 10440tctcccaagt tgttcaatcg
attggccgca caatatgaca gagaattaac atggaagaag 10500gtgttggcta
aaacaggact tgcagattta ctaacaggac aaatttcaca aattgatcga
10560tcagttgcga atgtccggag cgagccgagt aatgagaact cttggcaaga
ttagagcgat 10620ccacaagtat gaaaaaaact aatcccatag ccattttaaa
ttattgaaat tgatgaaatt 10680ggcgtcgacc ggccgcgatt ctggakccga tgcgta
107168442PRTCarajas Virus 8Met Asn Ser Ile Val Lys Lys Val Ile Asp
Asp Thr Val Ile Gln Pro1 5 10 15Lys Leu Pro Ala Asn Glu Asp Pro Val
Glu Tyr Pro Ala Asp Tyr Phe 20 25 30Lys Thr Ser Lys Gln Ile Pro Leu
Tyr Ile Asn Thr Asp Lys Thr Leu 35 40 45Ala Glu Leu Arg Ala Phe Val
Tyr Gln Gly Leu Lys Ala Gly Asn Pro 50 55 60Ser Ile Ile His Val Asn
Ser Tyr Leu Tyr Leu Ala Leu Lys Asp Ile65 70 75 80Lys Ala Thr Leu
Glu Arg Asp Trp Thr Ser Phe Ser Ile Thr Ile Gly 85 90 95Lys Gln Gly
Glu Glu Ile Thr Ile Phe Asn Leu Val Ser Val Arg Pro 100 105 110Leu
Val Ile Thr Val Pro Asp Gly Arg Thr Asp Pro Asp Arg Ser Pro 115 120
125Asn Asp Asp Lys Trp Leu Pro Ile Tyr Leu Leu Gly Leu Tyr Arg Val
130 135 140Gly Arg Thr Lys Leu Pro Glu Tyr Arg Lys Lys Leu Met Glu
Gly Leu145 150 155 160Glu Met Gln Cys Lys Ile Met Tyr Pro Asp Phe
Val Pro Ile Val Pro 165 170 175Glu Gly Met Asp Phe Phe Asp Val Trp
Gly Asn Asp Ser Asn Phe Thr 180 185 190Lys Ile Val Ala Ala Val Asp
Met Phe Phe His Met Phe Lys Lys His 195 200 205Glu Arg Ala Ser Leu
Arg Tyr Gly Thr Ile Val Ser Arg Phe Lys Asp 210 215 220Cys Ala Ala
Leu Ala Thr Phe Gly His Val Cys Lys Val Ser Gly Met225 230 235
240Ser Thr Glu Glu Val Thr Thr Trp Val Leu Asn Arg Glu Val Ala Asp
245 250 255Glu Leu Cys Gln Met
Met Phe Pro Gly Gln Glu Ile Asp Arg Ala Asp 260 265 270Ser Tyr Met
Pro Tyr Met Ile Asp Phe Gly Leu Ser Gln Lys Ser Pro 275 280 285Tyr
Ser Ser Val Lys Asn Pro Ser Phe His Phe Trp Gly Gln Leu Ala 290 295
300Ala Leu Leu Leu Arg Ser Thr Arg Ala Lys Asn Ala Arg Gln Pro
Asp305 310 315 320Asp Ile Glu Tyr Thr Ser Leu Thr Thr Ala Gly Leu
Leu Leu Ala Tyr 325 330 335Ala Val Gly Ser Ser Ala Asp Ile Ser Gln
Gln Phe Tyr Met Gly Asp 340 345 350Glu Lys Tyr Ile Ser Asp Pro Ser
Ala Gly Gly Leu Thr Ser Asn Ala 355 360 365Pro Pro Lys Gly Arg Asn
Val Val Asp Trp Leu Gly Trp Phe Glu Asp 370 375 380Gln Gly Gly Asn
Ile Thr Pro Asp Met Tyr Thr Ser Leu Lys Gly Leu385 390 395 400Phe
Ala Leu Cys Lys Gly Cys Glu Ile Arg Pro Leu Glu Ser Met Pro 405 410
415Arg Glu Ser Leu Thr Ser Asp Ser Ile Gln Ile Lys Cys Phe Thr Thr
420 425 430Cys Cys Ile Ile Tyr Asn Tyr Glu Lys Asn 435
4409261PRTCarajas Virus 9Met Gly Thr Tyr Thr His Leu Asp Ser Ala
Leu Gln Asp Ala Asn Glu1 5 10 15Ser Glu Glu Ser Arg Asp Glu Lys Ser
Asn Phe Asp Leu Phe Asp Glu 20 25 30Glu Ser Lys Glu Val Ala Arg Pro
Ser Tyr Tyr Ser Ala Ile Asp Glu 35 40 45Glu Ser Asp Gln Glu Glu Thr
Glu Ser Asp Asp Pro Asp Glu Glu Leu 50 55 60Asn Asp Ser Asn Ala His
Gly Ala Val Asp Gly Trp Asp Glu Thr Leu65 70 75 80Asn Glu Asn Ser
Gln Pro Asp Asp Asn Val Ser Val Glu Phe Ala Arg 85 90 95Thr Trp Ser
Thr Pro Val Met Glu Ser Ser Ser Glu Gly Lys Thr Leu 100 105 110His
Leu Ala Met Pro Asp Gly Leu Asn Pro Asp Gln Val Ala Gln Trp 115 120
125Leu Gln Thr Val Lys Ala Leu Phe Glu Ser Ala Lys Tyr Trp Asn Leu
130 135 140Ser Glu Cys Arg Met Glu Val Leu Leu Glu Gly Val Leu Ile
Lys Glu145 150 155 160Arg Gln Met Thr Pro Asp Leu Gln Lys Val Thr
Pro Lys Pro Asn Asn 165 170 175Pro Pro Pro Glu Ser Met Pro Cys Asp
Pro Leu Pro Pro Ala Met Asp 180 185 190Val Trp Glu Ala Ala Ser Gln
Val Tyr Thr Leu Glu Pro Lys Arg Ala 195 200 205Asn Leu Ala Pro Met
Asp Val Lys Leu Lys Asp Leu Phe Ser Ser Arg 210 215 220Ala Glu Phe
Leu Ser Val Gly Gly Ser Pro Gln Met Ser Trp Lys Glu225 230 235
240Ala Ile Ile Leu Gly Leu Arg Tyr Lys Lys Leu Tyr Asn Gln Ala Arg
245 250 255Leu Lys Tyr Ser Leu 26010228PRTCarajas Virus 10Met Ser
Ser Leu Lys Lys Ile Leu Gly Leu Lys Gly Lys Lys Glu Glu1 5 10 15Lys
Ser Lys Lys Leu Gly Leu Pro Pro Pro Tyr Glu Met Pro Ala Asn 20 25
30Asn Glu Phe Glu Pro Asn Ala Pro Leu Asp Pro Asp Met Phe Gly Ala
35 40 45Glu His Leu Glu Ile Glu Ser Lys Ser Ala Met Arg Tyr Glu Lys
Phe 50 55 60Lys Phe Ser Val Lys Ile Thr Leu Arg Thr Asn Arg Pro Leu
Arg Thr65 70 75 80Tyr Asp Asp Val Cys Gln Ile Leu Ser Lys Trp Asp
Ala Met Tyr Val 85 90 95Gly Met Met Gly Lys Arg Pro Phe Tyr Lys Val
Leu Val Leu Ile Gly 100 105 110Ser Ser His Leu Gln Ala Thr Pro Ala
Ile Leu Ser Asp Arg Gly Gln 115 120 125Pro Glu Tyr His Met Tyr Leu
Glu Asp Arg Gly Phe Ile Ala His Arg 130 135 140Leu Gly Leu Thr Pro
Pro Met Leu Ser Gly Pro Glu Ser Phe Arg Arg145 150 155 160Pro Phe
His Val Gly Leu Tyr Arg Gly Thr Ile Asp Ile Thr Val Asn 165 170
175Leu Met Asp Asp Glu Ser Thr Glu Ser Ala Pro Gln Val Trp Asp His
180 185 190Phe Asn Thr Arg Tyr Val Asn His Phe Leu Glu His Ala Lys
Arg Phe 195 200 205Gly Leu Val Leu Ser Lys Lys Pro Gly Gly Gly Trp
Ile Leu Asp Gln 210 215 220Ala Val Cys Ala22511519PRTCarajas Virus
11Met Val Ile Ala Gly Leu Ile Leu Cys Ile Gly Ile Leu Pro Ala Ile1
5 10 15Gly Lys Ile Thr Ile Ser Phe Pro Gln Ser Leu Lys Gly Asp Trp
Arg 20 25 30Pro Val Pro Lys Gly Tyr Asn Tyr Cys Pro Thr Ser Ala Asp
Lys Asn 35 40 45Leu His Gly Asp Leu Ile Asp Ile Gly Leu Arg Leu Arg
Ala Pro Lys 50 55 60Ser Phe Lys Gly Ile Ser Ala Asp Gly Trp Met Cys
His Ala Ala Arg65 70 75 80Trp Ile Thr Thr Cys Asp Phe Arg Trp Tyr
Gly Pro Lys Tyr Ile Thr 85 90 95His Ser Ile His Ser Phe Arg Pro Ser
Asn Asp Gln Cys Lys Glu Ala 100 105 110Ile Arg Leu Thr Asn Glu Gly
Asn Trp Ile Asn Pro Gly Phe Pro Pro 115 120 125Gln Ser Cys Gly Tyr
Ala Ser Val Thr Asp Ser Glu Ser Val Val Val 130 135 140Thr Val Thr
Lys His Gln Val Leu Val Asp Glu Tyr Ser Gly Ser Trp145 150 155
160Ile Asp Ser Gln Phe Pro Gly Gly Ser Cys Thr Ser Pro Ile Cys Asp
165 170 175Thr Val His Asn Ser Thr Leu Trp His Ala Asp His Thr Leu
Asp Ser 180 185 190Ile Cys Asp Gln Glu Phe Val Ala Met Asp Ala Val
Leu Phe Thr Glu 195 200 205Ser Gly Lys Phe Glu Glu Phe Gly Lys Pro
Asn Ser Gly Ile Arg Ser 210 215 220Asn Tyr Phe Pro Tyr Glu Ser Leu
Lys Asp Val Cys Gln Met Asp Phe225 230 235 240Cys Lys Arg Lys Gly
Phe Lys Leu Pro Ser Gly Val Trp Phe Glu Ile 245 250 255Glu Asp Ala
Glu Lys Ser His Lys Ala Gln Val Glu Leu Lys Ile Lys 260 265 270Arg
Cys Pro His Gly Ala Val Ile Ser Ala Pro Asn Gln Asn Ala Ala 275 280
285Asp Ile Asn Leu Ile Met Asp Val Glu Arg Ile Leu Asp Tyr Ser Leu
290 295 300Cys Gln Ala Thr Trp Ser Lys Ile Gln Asn Lys Glu Ala Leu
Thr Pro305 310 315 320Ile Asp Ile Ser Tyr Leu Gly Pro Lys Asn Pro
Gly Pro Gly Pro Ala 325 330 335Phe Thr Ile Ile Asn Gly Thr Leu His
Tyr Phe Asn Thr Arg Tyr Ile 340 345 350Arg Val Asp Ile Ala Gly Pro
Val Thr Lys Glu Ile Thr Gly Phe Val 355 360 365Ser Gly Thr Ser Thr
Ser Arg Val Leu Trp Asp Gln Trp Phe Pro Tyr 370 375 380Gly Glu Asn
Ser Ile Gly Pro Asn Gly Leu Leu Lys Thr Ala Ser Gly385 390 395
400Tyr Lys Tyr Pro Leu Phe Met Val Gly Thr Gly Val Leu Asp Ala Asp
405 410 415Ile His Lys Leu Gly Glu Ala Thr Val Ile Glu His Pro His
Ala Lys 420 425 430Glu Ala Gln Lys Val Val Asp Asp Ser Glu Val Ile
Phe Phe Gly Asp 435 440 445Thr Gly Val Ser Lys Asn Pro Val Glu Val
Val Glu Gly Trp Phe Ser 450 455 460Gly Trp Arg Ser Ser Leu Met Ser
Ile Phe Gly Ile Ile Leu Leu Ile465 470 475 480Val Cys Leu Val Leu
Ile Val Arg Ile Leu Ile Ala Leu Lys Tyr Cys 485 490 495Cys Val Arg
His Lys Lys Arg Thr Ile Tyr Lys Glu Asp Leu Glu Met 500 505 510Gly
Arg Ile Pro Arg Arg Ala 515122109PRTCarajas Virus 12Met Asp Phe Leu
Pro Val Glu Gln Glu Glu Asp Trp Gly Tyr Ala Glu1 5 10 15Asp Asp Phe
Ser Ser Ser Asp Tyr Leu Asp Phe Glu Glu Arg Met Thr 20 25 30Tyr Leu
Asn Gln Ala Asp Tyr Asn Leu Asn Ser Pro Leu Ile Ser Asp 35 40 45Asp
Ile Tyr Tyr Leu Ser Arg Lys Phe His Ser Tyr Gly Ile Pro Pro 50 55
60Met Trp Asn Leu Lys Glu Trp Asp Gly Pro Leu Glu Met Leu Lys Ser65
70 75 80Cys Gln Ala Asp Pro Ile Pro His Asp Leu Met His Lys Trp Phe
Gly 85 90 95Thr Trp Leu Glu Asp Phe Asp His Asp Ser Ala Gln Gly Ile
Val Phe 100 105 110Leu Arg Glu Val Asp Lys Glu Ala Ser Glu Thr Tyr
Asp Leu Val Asp 115 120 125Thr Phe Leu Lys Asn Trp Ala Gly Lys Ser
Tyr Pro Tyr Lys Ala Lys 130 135 140Glu Arg Tyr Leu Asp Gln Met Lys
Ile Ile Gly Pro Leu Cys Gln Lys145 150 155 160Phe Leu Asp Leu His
Lys Leu Thr Leu Ile Leu Asn Ala Val Gly Pro 165 170 175Glu Glu Leu
Lys Asn Leu Leu Arg Thr Phe Lys Gly Arg Thr Arg Asp 180 185 190Leu
Ser Thr Lys Asp Pro Cys Thr Arg Leu Arg Val Pro Ser Leu Gly 195 200
205Pro Val Phe Ile Cys Lys Gly Trp Val Tyr Ile His Lys His Lys Ile
210 215 220Leu Met Asp Arg Asn Phe Leu Leu Met Cys Lys Asp Val Ile
Ile Gly225 230 235 240Arg Met Gln Thr Leu Leu Ser Met Ile Gly Arg
Ser Asp Asp Ala Phe 245 250 255Thr Gln Gln Asp Phe Phe Thr Leu Val
Asn Ile Tyr Arg Thr Gly Asp 260 265 270Ile Ile Leu Gln Glu Lys Gly
Asn Leu Ala Tyr Asp Leu Ile Lys Met 275 280 285Val Glu Pro Ile Cys
Asn Leu Lys Leu Met Lys Leu Ala Arg Glu Tyr 290 295 300Arg Pro Leu
Ile Pro Pro Phe Pro His Phe Glu Asn His Val Lys Asn305 310 315
320Ala Val Asp Glu Gln Ser Lys Val Ser Arg Arg Ile Lys Val Leu Phe
325 330 335Glu Leu Ile Met Gly Ile Lys Asn Val Asp Leu Val Leu Val
Ile Tyr 340 345 350Gly Ser Phe Arg His Trp Gly His Pro Phe Ile Asp
Tyr Phe Glu Gly 355 360 365Leu Asn Lys Leu His Lys Gln Val Thr Met
Ser Lys Glu Ile Asp Thr 370 375 380Glu Tyr Ala Asn Ala Leu Ala Ser
Asp Leu Ala Arg Ile Val Leu Thr385 390 395 400Lys Gln Phe Asp Ser
Val Lys Lys Trp Phe Val Asp Lys Thr Lys Ile 405 410 415Pro Ser Ala
His Pro Phe Phe Lys His Ile Met Asp Asn Thr Trp Pro 420 425 430Thr
Ala Ala Gln Ile Gln Asp Phe Gly Asp His Trp His Glu Leu Pro 435 440
445Leu Ile Lys Cys Tyr Glu Ile Pro Asp Leu Ile Asp Pro Ser Ile Ile
450 455 460Tyr Ser Asp Lys Ser His Ser Met Asn Arg Ser Glu Val Leu
Gly His465 470 475 480Val Arg Arg Ser Pro His Leu Pro Ile Pro Ser
Lys Lys Val Leu Gln 485 490 495Thr Met Leu Asp Thr Arg Ala Thr Asn
Trp Val Glu Phe Leu Glu Met 500 505 510Val Asp Lys His Gly Leu Glu
Lys Asp Asp Leu Ile Ile Gly Leu Lys 515 520 525Gly Lys Glu Arg Glu
Leu Lys Leu Ala Gly Arg Phe Phe Ser Leu Met 530 535 540Ser Trp Lys
Leu Arg Glu Tyr Phe Val Ile Thr Glu Tyr Leu Ile Lys545 550 555
560Thr His Phe Val Pro Leu Phe Lys Gly Leu Thr Met Ala Asp Asp Leu
565 570 575Thr Ser Val Ile Lys Lys Met Leu Asp Ser Ser Ser Gly Gln
Gly Ile 580 585 590Asp Asp Tyr Ser Ser Val Cys Phe Ala Asn His Ile
Asp Tyr Glu Lys 595 600 605Trp Asn Asn His Gln Arg Lys Glu Ser Asn
Gly Pro Val Phe Arg Val 610 615 620Met Gly Gln Phe Leu Gly Tyr Pro
Arg Leu Ile Glu Arg Thr His Glu625 630 635 640Phe Phe Glu Lys Ser
Leu Ile Tyr Tyr Asn Asn Arg Pro Asp Leu Met 645 650 655Trp Val Asn
Glu Asp Thr Leu Ile Asn Arg Thr Gln Gln Arg Val Cys 660 665 670Trp
Glu Gly Gln Ala Gly Gly Leu Glu Gly Leu Arg Gln Lys Gly Trp 675 680
685Ser Ile Leu Asn Leu Leu Val Ile Gln Arg Glu Ala Lys Ile Arg Asn
690 695 700Thr Ala Val Lys Val Leu Ala Gln Gly Asp Asn Gln Val Ile
Cys Thr705 710 715 720Gln Tyr Lys Thr Lys Lys Ser Arg Asp Gln Ser
Glu Leu Ile Asn Ala 725 730 735Leu Asp Gln Met Val Lys Asn Asn Asn
Lys Ile Met Glu Glu Ile Lys 740 745 750Lys Gly Thr Ser Lys Leu Gly
Leu Leu Ile Asn Asp Asp Glu Thr Met 755 760 765Gln Ser Ala Asp Tyr
Leu Asn Tyr Gly Lys Val Pro Ile Phe Arg Gly 770 775 780Val Ile Arg
Gly Leu Glu Thr Lys Arg Trp Ser Arg Val Thr Cys Val785 790 795
800Thr Asn Asp Gln Ile Pro Thr Cys Ala Asn Leu Met Ala Ser Val Ser
805 810 815Thr Asn Ala Leu Thr Val Ala His Phe Ala Ser Asn Pro Ile
Asn Ser 820 825 830Met Ile Gln Tyr Asn Tyr Phe Gly Asn Phe Ser Arg
Leu Leu Leu Phe 835 840 845Met His Asp Pro Ala Leu Arg Arg Ser Leu
Tyr Asp Val Gln Asn Glu 850 855 860Ile Pro Gly Leu His Ser Lys Thr
Phe Lys Tyr Ala Met Leu Tyr Leu865 870 875 880Asp Pro Ser Ile Gly
Gly Val Ser Gly Met Ala Leu Ser Arg Phe Leu 885 890 895Ile Arg Ala
Phe Pro Asp Pro Val Thr Glu Ser Leu Ser Phe Trp Lys 900 905 910Phe
Ile His Asp His Thr Asp Asp Glu Tyr Leu Lys Ser Leu Ser Ile 915 920
925Ala Phe Gly Asn Pro Asp Ile Ala Lys Phe Arg Leu Glu His Ile Ser
930 935 940Lys Leu Leu Glu Asp Pro Thr Ser Leu Asn Ile Ser Met Gly
Met Ser945 950 955 960Pro Ser Asn Leu Leu Lys Thr Glu Val Lys Lys
Cys Leu Ile Glu Asn 965 970 975Arg Thr Ser Ile Arg Asn Asp Ile Ile
Lys Asp Ala Thr Ile Tyr Leu 980 985 990Asn Gln Glu Glu Ala Lys Leu
Lys Ser Phe Leu Trp Ser Ile Asn Pro 995 1000 1005Leu Phe Pro Arg
Phe Leu Ser Glu Phe Lys Ser Gly Thr Phe Leu 1010 1015 1020Gly Val
Ser Glu Gly Leu Ile Ser Leu Phe Gln Asn Ser Arg Thr 1025 1030
1035Ile Arg Asn Ser Phe Lys Gly Lys Tyr Arg Lys Glu Leu Asp His
1040 1045 1050Leu Ile Val Lys Ser Glu Ile Ser Ser Leu Lys His Leu
Gly Gly 1055 1060 1065Ile His Phe Lys Leu Gly Asn Gly Lys Ile Trp
Gly Cys Ser Ser 1070 1075 1080Ser Gln Ser Asp Leu Leu Arg Tyr Arg
Ser Trp Gly Arg Lys Leu 1085 1090 1095Val Gly Thr Thr Ile Pro His
Pro Leu Glu Met His Gly Ala Ala 1100 1105 1110Ser Pro Lys Glu Ala
Pro Cys Thr Leu Cys Asn Cys Ser Gly Leu 1115 1120 1125Thr Tyr Ile
Ser Val His Cys Pro Lys Gly Ile Thr Glu Val Phe 1130 1135 1140Ser
Arg Arg Gly Pro Leu Pro Ala Tyr Leu Gly Ser Lys Thr Ser 1145 1150
1155Glu Thr Thr Ser Ile Leu Gln Pro Trp Glu Lys Glu Ser Lys Val
1160 1165 1170Pro Ile Val Arg Arg Ala Thr Arg Leu Arg Asp Ala Ile
Ser Trp 1175 1180 1185Phe Ile Asp Pro Asp Ser Thr Leu Ala Gln Ser
Ile Leu Asp Asn 1190 1195 1200Ile Lys Ser Leu Thr Gly Glu Glu Trp
Gly Gly Arg Gln His Gly 1205 1210 1215Tyr Lys Arg Thr Gly Ser Ala
Leu His Arg Phe Ser Thr Ser Arg 1220 1225 1230Met Ser Asn Gly Gly
Phe Ala Ser Gln Ser Pro Ala Ala Leu Thr 1235 1240 1245Arg Leu Ile
Ala Thr Thr Asp Thr Met His Asp Tyr Gly Asp Lys 1250 1255 1260Asn
Tyr Asp Phe Met Phe Gln Ala Ser Leu Leu Tyr Ala Gln Met 1265
1270 1275Thr Thr Ser Ile Ser Arg Trp Gly His Val Gly Ala Cys Thr
Asp 1280 1285 1290His Tyr His Val Arg Cys Asp Ser Cys Ile Arg Glu
Ile Gln Glu 1295 1300 1305Ile Glu Leu Asn Thr Gly Val Gln Tyr Ser
Pro Pro Asp Val Ser 1310 1315 1320Tyr Val Leu Thr Lys Trp Arg Asn
Gly Ser Gly Ser Trp Gly Thr 1325 1330 1335Val Thr Lys Gln Leu Ile
Pro Lys Glu Gly Asn Trp Thr Val Leu 1340 1345 1350Ser Pro Ala Glu
Gln Ser Tyr Gln Val Gly Arg Cys Ile Gly Phe 1355 1360 1365Leu Tyr
Gly Asp Leu Val His Lys Lys Ser His Gln Ala Asp Asp 1370 1375
1380Ser Ser Leu Phe Pro Leu Ser Ile Gln His Lys Val Arg Gly Arg
1385 1390 1395Gly Phe Leu Glu Gly Leu Leu Asp Gly Ile Met Arg Ala
Ser Cys 1400 1405 1410Cys Gln Val Ile His Arg Arg Ser Val Ala Thr
Leu Lys Arg Pro 1415 1420 1425Ala Asn Ala Val Tyr Gly Gly Val Ile
Phe Leu Ile Asp Lys Leu 1430 1435 1440Ser Met Ser Ala Pro Phe Leu
Ser Leu Thr Arg Thr Gly Pro Ile 1445 1450 1455Arg Glu Glu Leu Glu
Asn Val Pro His Lys Met Pro Ala Ser Tyr 1460 1465 1470Pro Thr Asn
Asn Arg Asp Leu Gly Met Thr Val Arg Asn Tyr Phe 1475 1480 1485Lys
Tyr Gln Cys Arg Ile Ile Glu Arg Gly Gln Tyr Lys Ser His 1490 1495
1500Tyr Pro Thr Ile Trp Leu Phe Ser Asp Val Leu Ser Val Asp Phe
1505 1510 1515Ile Gly Pro Met Ser Leu Ser Ser Gly Leu Met Arg Leu
Leu Tyr 1520 1525 1530Lys Asn Ser Leu Ser Lys Lys Asp Lys Asn Glu
Leu Arg Asp Leu 1535 1540 1545Ala Asn Leu Ser Ser Leu Leu Arg Ser
Gly Glu Glu Trp Asp Asp 1550 1555 1560Ile His Val Lys Phe Phe Ser
Gln Asp Leu Leu Phe Cys Ser Gln 1565 1570 1575Glu Ile Arg His Ala
Cys Lys Phe Gly Ile Ile Arg Asp Lys Val 1580 1585 1590Ser Leu Glu
Val Asp His Gly Trp Gly Lys Glu Ala Tyr Gly Gly 1595 1600 1605Cys
Thr Val Leu Pro Val Phe Tyr Arg Ser Gln Ile Tyr Lys Lys 1610 1615
1620Ser Leu Thr Val Pro Pro Arg Ile Gln Asn Pro Ile Ile Ser Gly
1625 1630 1635Leu Arg Leu Gly Gln Leu Pro Thr Gly Ala His Tyr Lys
Ile Arg 1640 1645 1650Ser Ile Ile Met Thr Leu Lys Ile Asn Tyr Gln
Asp Phe Leu Ser 1655 1660 1665Cys Gly Asp Gly Ser Gly Gly Met Thr
Ala Cys Leu Leu Arg Leu 1670 1675 1680Asn Pro Asn Ser Arg Gly Ile
Phe Asn Ser Leu Leu Glu Leu Asp 1685 1690 1695Gly Ala Leu Met Arg
Gly Ser Ser Pro Glu Pro Pro Ser Ala Leu 1700 1705 1710Glu Thr Leu
Gly Ser Gln Arg Thr Arg Cys Val Asn Gly Gly Thr 1715 1720 1725Cys
Trp Glu His Pro Ser Asp Leu Ser Asp Pro Asn Thr Trp Lys 1730 1735
1740Tyr Phe Ile Gly Leu Lys Arg Gly Leu Gly Leu Gln Ile Asn Leu
1745 1750 1755Ile Thr Met Asp Met Glu Val Arg Asp Pro Val Ile Ser
His Lys 1760 1765 1770Ile Glu Ala Asn Ile Arg Ala Phe Leu Tyr Asp
Leu Leu Asp Pro 1775 1780 1785Glu Gly Thr Leu Ile Tyr Lys Thr Tyr
Gly Thr Tyr Leu Ala Glu 1790 1795 1800Glu Glu Arg Asn Ile Leu Thr
Glu Val Gly Pro Leu Phe His Thr 1805 1810 1815Thr Asp Leu Val Gln
Thr Ile Tyr Ser Ser Ala Gln Thr Ser Glu 1820 1825 1830Val Tyr Cys
Val Cys Arg Arg Leu Lys Lys Tyr Ala Asp Gln Gln 1835 1840 1845His
Val Asp Trp Ser Leu Leu Thr Asp Gly Trp Ser Arg Leu Tyr 1850 1855
1860Ala Phe Ser Val Asn Arg Leu Glu Phe Gln Arg Ala Gln Ser Leu
1865 1870 1875Arg Lys Leu Asp Thr Leu Gln Gly Ile Pro Ser Phe Phe
Ile Pro 1880 1885 1890Asp Pro Phe Val Asn Ala Glu Thr Leu Leu Gln
Ile Ala Gly Val 1895 1900 1905Pro Thr Gly Ile Ser His Thr Ala Val
Leu His Gly Ser Leu His 1910 1915 1920Ser Glu Gln Leu Ile Thr Leu
Gly Ile Phe Phe Cys Ala Leu Ile 1925 1930 1935Ser His His Thr Met
Asn Ile Ile Arg Ile Ser Pro Val Pro Pro 1940 1945 1950Ser Pro Pro
Ser Asp Gly Ser Ile Ser Arg Met Cys Ser Ala Ile 1955 1960 1965Thr
Gly Ile Leu Phe Trp Val Ser Leu Val Glu Lys Asp Leu Thr 1970 1975
1980Leu Tyr Asn Ser Leu Leu Ser Ile Ile Gln Arg Ser Phe Pro Ile
1985 1990 1995Arg Trp Tyr Lys Asn Lys Glu Lys Asn Gly Trp Ser Gln
Cys Trp 2000 2005 2010Gly Ala Asn Gly Asp Gly Ile Pro Lys Asp Thr
Arg Leu Asn Asp 2015 2020 2025Ser Met Ala Asn Ile Gly Asn Trp Ile
Arg Ala Met Glu Leu Leu 2030 2035 2040Cys Asn Lys Thr Ala Gln Met
Pro Phe Ser Pro Lys Leu Phe Asn 2045 2050 2055Arg Leu Ala Ala Gln
Tyr Asp Arg Glu Leu Thr Trp Lys Lys Val 2060 2065 2070Leu Ala Lys
Thr Gly Leu Ala Asp Leu Leu Thr Gly Gln Ile Ser 2075 2080 2085Gln
Ile Asp Arg Ser Val Ala Asn Val Arg Ser Glu Pro Ser Asn 2090 2095
2100Glu Asn Ser Trp Gln Asp 21051312416DNABahia Grande virus
13acaatattag ataaactcct ctacttctta actatcgtta gacatggccg ccgcaatact
60tccagtttct cgtaacatgc ctgtcagaga aaggacagtg gcaggaagtg taacagcgcc
120accagttcag tatccaagca cctggttcca agcccatgcc ggacaaaaag
tttcaataac 180tatttatcaa aatactaatg cacgacaagc tttctccaga
attactcaac tcagaaacaa 240cggacaatgg gatgataaat tgatcgctac
tttcatgaaa ggtgtcttgg atgaaaatgc 300tgaatggttc caaagccctc
ccctcattga ggactggatt gtaaatgaag cagtcatcgg 360aagagtagat
gacgtagttg cacccactgc acttgcacag tgggaagagg ttgaaaggcc
420tcaaaacatg gatccagtac ccaatgagga aggagaactg gggactcgga
ggtcattttt 480cttggcatta atcaccatct acaggcaagt actgacaaga
accatcaatg tggactacgg 540ccaagaagtg agcagaagga taatagataa
tttcaaagaa caacctttag gtatgtcaca 600ggatgacata aatgaaatcc
aggggtatga atcaaaagaa aggctaacta caaattatgt 660gaaaatctta
tgcatccttg atatgttctt caataagttt cagacccatg acaaaagcac
720catcaggata gctactttac caacaagata tagaggatgt gctgcattca
cttcatacgg 780agaactagca ataagattgg gaattgaacc cataaagctg
cccagtttga ttcttacagt 840agcagtggcc aaagatttcg ataagatcaa
tgtcaatgga gagcaagcag agcaattaga 900tggatatttt ccatatcaat
tagagttggg attagttaaa aagagtgctt attcagcagg 960aaattgtcca
tctttatact tatggatgca caccatagga acaatgctcc atcaacaaag
1020atcttatcga gccaatgttc ccaaaaatgt accagaccaa atgggaacaa
taaattctgc 1080aattgctgtt gccatgcagt ttgttgctgg gggagagttc
agtatgcaat ttgtagggga 1140tgcacgagtt caagaagcca tgagagaaat
gcaaacagca gaagctgaat tgaatgagtt 1200aagaatggct caggcaagag
aaatgagagc tgcagcaaga ggagatgaag atgaagaagg 1260ctctgaagat
ggacttgatg atgaaaatga tggagaaggg gatgatgagt taccagctga
1320aattgaacaa aatcctgaat atttaaatag agtcaacagg atcagagaat
tacaagaaaa 1380cctccaacaa tacaacgcaa cagtacaaca gcacactaat
gcggtagaaa aagccgcact 1440cagagcactc gcttatcttc aagaaaatgg
aggaattgca gataaggaca agagagactt 1500gggtataaga ttcaggaggt
ttgctgatga agcggaaggt agagtcggta aattattagc 1560cagtttgttc
cctgccccga gataaatatt ctttcaggta tcattttctt atttttaaaa
1620tattttatcc agattttaat ttctttatct actgtattat tttattcaaa
tatgttttca 1680attaattttt tcttctttat atgttatatt ctatacatat
gttaatgttc atgaaaaaaa 1740caacaaatct cataagatac tcgtttaaag
aaatggctta ttcaactggt ttgattaaag 1800gtgaagtgtc ccaaggattg
tctaatgcat ttaaagatgc aggaatacat caaatagaat 1860taaataaaga
atatgacaat ttatcaattt tgggggccaa catgagtgca ttgaataaaa
1920tgtttgacac agaagatgaa gggttatctg atactaatac taactcatca
aaaaactcta 1980ttttacaagc gagtgatatg ttcataggaa atgatgaata
tgaatcagat gactctcatc 2040attttctaag ctcacctagt ccagataaag
gaagcagtga agaaggaagc aacctccaag 2100aattcaattt tcagatacct
agaaacaagg ttggaaaaga aaaggcatac aggaggggag 2160tcattgatgt
attggatttt ctacagagac acagatttat agaagaattc cgtatggaag
2220gacttaatga ggatatagtc tgtatcatcc ctacaagagg aatgatcccc
acaaaaacac 2280cccctaccct ggatgacaaa attcatcttg ctaacgatca
gtcaatagaa aaagaagaaa 2340tcctccaaaa agacaagaca tcaaaaccaa
acaaaggaat caaacagcca aacaagcaag 2400aggcacaacc agtctctgaa
tctcaaacag gaatgaagga agacaaaaaa gaacaaaagc 2460caaagcaaaa
ccaaattccc attaaaaaca aacaggaaaa tgaagactca aaagaagttg
2520ctaagaccaa caaagataaa gaaaataaag tcagcaaagg aagtatgtca
aagaatgaca 2580aactaaaaga aggcaatata actgttccaa aacagggatt
tgaaaagaag aaaacaaaac 2640aaataaatga agaaggccac aaatcatttg
attatgctaa tacatatggg acaaaagtca 2700ctgtgaaaac tataaggtat
tgtaagacat gcaatcctaa tactagaaaa aatgctacag 2760tatatcttga
ccatctttat gaacgccaca gtcatgaggt tgctttgatt aahagcttgg
2820cttaccctct tttattttwt ttwwggttga wttaaattaa ctaattagat
actttyttaa 2880tacatgawaa wwacaacaaa tctaataaat tacattgaaa
caaagatgtc tggtgtgatg 2940agtatattta aaaggaagga caagaaaggg
aatgagggtt ccaaagccct agccatacca 3000gatgaaaaat cagtagtccc
atctgcacct ccagacatct cagctatgga ttatgggagg 3060tttggtttat
tagggaggca aactctatta gaagaagatg aggaagaatc tagatgcatc
3120actattatag atctagaagt cgatctacag atagaggtgt tatctaatag
agaaactcga 3180cttgtaatag acttgattgc tcctttgtgt aatcttcaaa
ctgattacat tggaaaagag 3240aacacaaaag caatttggat aggattaact
gtagtagcag cttttggagt gaaaagaacc 3300attaagacaa aaaatcatca
tgtatataaa gggtgtgtct ccagtggact taggctttta 3360atagactcag
aaaaacaatt tgagctagat aagaggaata aatgstctca gcatctcagt
3420tatctcacca atggtgtaaa aacagagtgg gccataagag gggagatgat
caggacaaga 3480gtaccttacc ttcctcagcc aggaagtgag gatgtgctta
tgtttttagc agggatggga 3540ataagttgtt attcaaatcc agatggtcat
ttagtcctca aagtttgaaa aataacaaaa 3600ttctttagag atcatattca
gtatttatac cttagtaata ttgtggctca gatttaatga 3660tgggagtgcc
taaagtattt caattttggg ttagaatcag gacatgaaaa aaacaacaaa
3720tctaattaac tatcatttag tacttagaac gaacttatct tctgttgaat
catgatttcg 3780aatatgtttt tcttgtttca actctcatta tttctacagt
ttatagcagg agatgagtca 3840ttagaaacaa taacagcccc tgaaactcct
gaccctatac tcttaaaagg agatacaaaa 3900tatctgttct tagtcccttc
ttctgtcaaa aattggaaac cagctgacct gaatgaatta 3960acatgccccc
ccctaatctc gaaaccagat acttctgaaa tgacttattt ttccacagat
4020gtgatggagt tacaaaaaca tcatgaattg gcaccagtag aagggtattt
atgttcgggt 4080ttgcgttaca aagtaatatg ttctgaagga ttttttggac
aaaaaacaat agcaaaaaag 4140attgagaaca ttgaacctga tagtaaacaa
tgccttgatg acttgtcaaa atttaagaat 4200gatgattacc tactcccata
tttcccttct gaagattgta attggatgaa agagactccc 4260acccataaag
attttatagt ttttcaaaaa cattttgtta aatatgaccc atacaataat
4320ggtttttatg atcctttact taaaaaagac tactgtgata ctcaagtctg
tgagacagaa 4380catgatcaaa ctatttggat aacagaaaag agtattgaaa
atgaatgcat cttcaattat 4440ccgattaaaa agcatatatt ccatacagct
gactttggga aaatgataat agattacgaa 4500ttaaatcaat ggacttcagt
ggaagatggg tgtttaatta actattgtgg aagagaggga 4560ataaggttat
ctaatgggat gttctttgta ggtaagttct ataaaaatct caataattta
4620cagacctgta gtgctggaac aaaggtcagt tacaagcctt taacctccaa
gctggaagaa 4680attgaaaatg aaatcattct agatcaggaa agattattat
gtcttgattc aattaggcaa 4740atgacagcaa caaaaaaatt atcattttat
tctttatcct ttctagaacc aaaatcttct 4800agtaggcaca aggtctttag
aattcataat aaaacactag aatataccga aaccgaatgg 4860catccaatca
tgtcgtttaa ttttgatgaa ccaaacaaaa ttggaattga caagaatggt
4920aaatcagttt attggaatga atgggttcct agtggaatat ctgggctgtt
atcagggttc 4980aatggagtct acaaaaaaga aaatgaaact aaagtaacta
ttgcccgatt agaaacaata 5040aaagaagatt atgataggga gatgatgata
gatcacgagt tggtagaggt agaacatcct 5100aaaattgtac acttaaaaag
agagaacatc acaggatcta gagtcgaaat tgttaataaa 5160gaacattctg
atgtgagtgg ttggctgtca tcagtattga gtagtttttg gggaaaaatc
5220atgatgacaa taataagtat aatcttaatc gtaataatag gattagtttt
aataaactgc 5280tgcccaatta tatgcaaatc atgtattaaa cgttataaaa
caaaggaaga atcccgcaat 5340agacatagat tggatagaga agataacggt
agattgagga ggcaacatcg agttattttt 5400aacaatcaat ccaatgatga
agaaaatgcc attgaaatgg tagaatatac tgacactccc 5460aggccattgc
gaccgattcc tgatgccaca acatcagaca ctgagtcaag atcccccaca
5520acagcccata gttttttcaa ccgttaaaaa ggtaggttat attatacttt
tctctatacc 5580tctaatagtc atcatcgtgt tttttgtgtt attagataga
aaacatctca aatatatacc 5640tttaaaggca tggaacactt caataattac
aattaaagaa ccttattaaa attaaaaagt 5700tttctttaaa ataattctcc
taattgattt taatttcatg aaaaaaacat taahaaatct 5760aagtatmact
saaatttagg gtatgcttgg tgtgttaaaa tggatttctc ttatgaacaa
5820ttgctggatc ctatagatgt cttagaagaa gaattatatg aatttgattt
cgaatatgat 5880gattacactg atgatgatca gacaccctta cccaatatta
agtacaaaaa cctagaaggt 5940aaagactata atttaaactc acctctcatc
agcgatgtga tcgattcagg aagagaatac 6000ataattaatt ctaaaaagta
cttttctcat gaaagaacaa atccggagtt ggaacaattt 6060agtaaagctc
taatggctat tgggttttct agatttgatt tacgaaaatc atcagaacat
6120cataggtaca tgagttcata tatatatgga aatgagaaaa aacatatgaa
aatcgaaata 6180atacccagat ggaaagaagt cttagaactg actcgcaatc
ctgtagaagt aacctctcat 6240aagatattgg gatcaaaatc acaatctgat
caagaaggat atataaatag attgcgatat 6300attacagtag atggacctca
tgcaagaaaa acaagattac accaagaatg ggaaaaattc 6360tcaacattac
attatataac gtatattatg aattcaaaag cctttagtga caacaaaaat
6420tgggtgaggg aagtctttga gaccatagaa actagtgaag ttgaccctga
aataattaca 6480ataattggaa caggtttatc aaagaaagaa gtatcctgga
ttatatctga gaactttgca 6540ttaaatgtta gaacaggttt atttgtctcc
aaagatttct tgctgatgat taaagatgtc 6600accttagcta gatgtatgag
caaactgagt atgattaaca gaaagtctcc caacacaact 6660tatgatatga
taaaattttt ggatagtcta tatgaaagtg gtgacaaaat attgacaaga
6720catggaaatt tagcttacaa gcatatcaag ttattggagg cagcttgtct
agagagatgg 6780aatcaattag ggcacaaatt tcgaccattg ataccaatct
cttcaagcat gagtgatcat 6840cttagaactc aattagaaga aaatcaagat
ctctatatgg tgagtaggga attcttcgat 6900ttgattggaa agattgaaga
tccttgggtc gttgctcaag cgtatggaac attcaggcat 6960tggggacatc
catacattga ttatttaaat ggtctaaaag atctagaaaa aagagtaaat
7020gaaaatatca aaattgataa aaattatgca gaaaaattgg ctagcgatct
tgcgtttata 7080gttctaaaag accaatttgg aaaacataaa agatggtttg
ctaaacctaa taaagaattg 7140gatgaaaata atcccatgcg aaaatgcata
gaaaacaatg tgtggcctaa cactaaagtt 7200attttagact tcggagacaa
ttggcataaa ttagaattat taccatgttt tgaaatccct 7260gatgcaatag
acctttctga cctatatagt gataaagctc attccatgca atacagtgaa
7320gtattaaatt atgtaaaata caaaaaatcc aaaaagaata tccctgcctt
acgtgttatc 7380gggacattat tagaaaagga aaatccaaat ataaaagaat
ttttacaaaa aataaacgat 7440gaaggtttag atgatgatga tctgataata
gggctgaaag caaagaaaga gaactgaaag 7500ataaaggaag atttttctct
cttatgagtt ggaatattag gttatatttt ktgattacag 7560aatatttaat
twwwttwcaw ttttktmcca ttgttttctg gcttaacagt agcggatgac
7620ttaaatactg dcmsmmamrr attmttaagt gctacagaag gacaaggtct
agatgactat 7680gaaagggtct acatagcaaa tagtttagat tatgaaaaat
ggaacaacag gcagcgttat 7740gaatctaatg aaccagtatt cacagtaatg
gggaaatttt taggttatcc aaacttaata 7800tcgtatactc ataagatttt
tgaaagatca tttatctatt ataacggaag actagactta 7860atgggagtag
atggttacca tatttataat ttatttgatg ataaaatggt ctgttggcat
7920ggtcaattgg gaggatttga aggtgtaaga caaaagggct ggagtgtttt
aaattactta 7980attttgcgaa gagaagctgc aacacgaaat actgcaccga
aatttttagc ccaaggagac 8040aatcaaattg tcattactca gtatacattg
accagtaaaa gcactcaagc tataattgaa 8100cgagaattga ggaatatttg
ggaaaacaat gctcatataa tgcataggat acaacaagcg 8160acaagtcgaa
ttggattagt cataaataat gatgaagtgt taacttccgc agagttattg
8220gtttacggta aaataccagt atttcgaggg aaattgttac ctttagaaac
aaaaagatgg 8280tctagagtca gtaccgtgac aaatgaacag ataccatcct
tttctaattc attggctagt 8340agtacaacta ctgctttggc ggttaatcaa
cactcagaaa atcctatcga ggttatatct 8400caacatcatt tctttagttc
ttttgctggc acattagtaa catttgttaa tcctatctta 8460ggttttgatc
cgattaaata ttctcaattg tcagagagaa ataagaagtt attcttatta
8520aggcttattt acaaagatcc aagtgttggg ggagtttgtg gaactaattt
attaaggttt 8580tttatatcaa gatttcctga tcctttgaca gagacattga
catggtggaa aatattggtt 8640gagaattcta aagataaaga ggttgttaaa
attgcgctag aatgtggaaa tcctaagttt 8700ggagggatta atgataagac
attagctatg ttactcgaag accctatgtc actaaatata 8760ccaggaggac
tctcaagtga cacgatgata aaaaacaaaa tttatgaagg tcttattcat
8820caaatggggc ttaaattgat caaaaatgaa ttggttgtag aatctctaac
cttctataat 8880gattacaaag cacaatttgt aagatggtta ttctccataa
gaccaatttt cccacgattc 8940attagtgaat tttatacatc tacttatttt
tatataacag aaagtgtcct tgccatattt 9000caaaattcta gaaccattag
aaaagttttc tcaaaaagat ttccgaaaga ggtttatctc 9060acgatagtta
aaggagaaca aatgtctata gatagcttat tgacaaccaa aagagggatt
9120gttagggagg ctatttggaa atgttcagca acgaaagcag atgaaatgag
aaaactatca 9180tggggtagag atatggttgg aataacaaca cctcatccag
ctgaattcac acaagaatta 9240ttatgttcag acgggtgttc agaacctcac
attgtagcca aaaaggttat ttactctgat 9300agaaaattat ggactaaggg
taagatgatg ccttaccttg gtactaaaac caaagagtcc 9360acaagtatac
ttcaaccatg ggaaaaaaga ttagagattc cattattgag gaaagcatgt
9420gatttaagaa aagccattag gtggtttgta gaagataatt caaacttagc
aaaatccatt 9480tataaaaatt tagaaagtat gacaggaatt gatttaagag
aagaacttcg aaactataaa 9540agaactggta gtagcaaaca tagattaaga
aactcgagag tctccaatga aggtaatccc 9600gccataggtt ataataacct
aacgtatgtc acagtaacaa ctgatagttt aggaaatatt 9660aattccgaaa
attatgattt catgtatcaa tctatcttat gctggtgtgg tgtattatcg
9720tccctagcaa ccaatcgata tcgagaccat gagactactc attttcatct
taaatgtaat 9780gattgcttca gattggttaa agaggaaata ttagaggctc
cttcagttta cccatttcct 9840aatgtaagat cctctgtaag gagaatgctt
acacaggata ttaaattaaa atatctgcca 9900cgaatttctg cccctgatga
aaacacctgg gatactctgg atgttgatca aaaaagttgg 9960catattggga
gagctcaagg gtttttgtgg ggattaaatg tatttaccaa aaccactaaa
10020gaggttgagg gtgacatttt cccaacttcc ataacgaaaa aagtcgaacc
agaaaattac 10080atggatggtt tacacagagg gttttgttta ggagctactc
tctcccccat gtacacaaga 10140tatggatcac tcagcaggat ggctagaaga
aaattcgaag gagcatactg ggaaatcgta 10200gatgaagcaa tgaaaactaa
tctaccaaat atgattgatc amaaaaattt caaacctttc 10260ctgagaagga
caggaggtga tctaattaaa tcttatcctg cacgaaagga agagttggta
10320cttgttttaa agaaatggtt cttacataaa atggtctctg aaagaaaaaa
caattccata 10380tgggaaagta aaagagtaat tgcctttgct gacatggaca
ctgaatttgt attgtgtctc 10440ttcagattag cggaaagcat actgaattgt
tatcaaaatg aagctttatc tgctggtcag 10500gctagggtct tagggaatgc
aaaagagaca atagatctga tctcaaaata caataactca 10560aacattaatg
cagatgagat tgagcgattg cagcagatat tgatggcttc tgacctgaaa
10620gatcatgaag ttgtagattc acaagctagg catgctgctt ctgacttacc
tgaattggca 10680aaatcagaaa attacaatga agtgattaaa tatgtagaat
ttagaggtta tggtggtaaa 10740accataagat tagaatatca acctagtgat
ttgatagact ggaagggagg aatggttcaa 10800gacctacaag tacctagatt
gaagaaccct ttaatttctg gagtcagagt agtgcaatat 10860agcacaggag
ctcattataa atataaagat atagaaagag aatttcaaat tgctggtgat
10920ggtatattcg ctggtgatgg ttctggtggt atgggtgcaa accatctgag
attacataaa 10980tcagcccgcg ttatatttaa ctctaaatta gagttagaag
gagaatcttt aaaagggtta 11040gcccctgcag gacctggagc ttacacggtc
tcaggtgaag atgttgtgga aagatgtgtc 11100aattacacaa cttgctggga
agaagcttct gatctgagtg acgaaaaaac ttggaagaat 11160ttttttaggc
tcataaaaga gtactcatta gatatagaag tgttttgctg tgatgctgaa
11220gtccaagacc catatatcac aaacaaaatt gaatctaata tattgaaata
catatctttg 11280atccttaata aaagaactgg aactttaatt tacaaaactt
atttcaatag attattggat 11340cccaatacta taacccactt tttgggaatg
tttttccata gatgttacgg atttctccct 11400actactcaag gatcctttac
ctctgaaatt tacattgtct gtcaatatcc aaagacactt 11460gactctacaa
gcaaaacaga gttaacctat actagtttat ttaatattta tcagaacata
11520agagtgatgg aaacttatca aaatgaattt gatagagcat gtagtttatt
gttttctgat 11580atgacggaag gtcttattga taaaacacca tttttagatc
ctgaagaatt ggctattttc 11640ctgacaacag tgggattgga tacggggtgg
gctttactaa tagcagaaca attacagata 11700tcttgctcaa acaaattaca
tccaataatc atattatgga ttttaggctt tataatttcc 11760agacacttag
tgagtataac atcttggttt cgtagaggaa caaaattccc tccttctatc
11820cagttgcaaa aaatgttagc tgctctattt ggaatctggt atggagtctc
ttatattatg 11880aatgatgcag agagttactc aaggatttct gtattgtaca
atcaagagat ttatttctca 11940ttaggcttga ctaatatggt atataggaaa
aaagatgaca tggaattggg tcaattttca 12000acttggaaga taggacctgg
tgataatagt aaactcatag atataggtcc caaagcgggt 12060ataactcaga
caatgataag agctattgta gtcttgtata aaggagaaca tataacttct
12120attgtgacta aggaagataa agtagaagga gatagaattt taagcttatt
tggaaaagga 12180ttgaatctta aaactttaat ggagcgaaca ggaataaatt
atttgcaaat aggggaaaga 12240aatcctcaag aaattccata tacgttagag
gaagaagtat tggaagaagt ggtagaagaa 12300aatacaggag aatttgatca
atcataaaca gataaaggaa atraaaaaaa aaaaaatata 12360tattgaaata
ataaagctta aagaacaaga tcttgaaatt gtgaactact aagtat
1241614513PRTBahia Grande virus 14Met Ala Ala Ala Ile Leu Pro Val
Ser Arg Asn Met Pro Val Arg Glu1 5 10 15Arg Thr Val Ala Gly Ser Val
Thr Ala Pro Pro Val Gln Tyr Pro Ser 20 25 30Thr Trp Phe Gln Ala His
Ala Gly Gln Lys Val Ser Ile Thr Ile Tyr 35 40 45Gln Asn Thr Asn Ala
Arg Gln Ala Phe Ser Arg Ile Thr Gln Leu Arg 50 55 60Asn Asn Gly Gln
Trp Asp Asp Lys Leu Ile Ala Thr Phe Met Lys Gly65 70 75 80Val Leu
Asp Glu Asn Ala Glu Trp Phe Gln Ser Pro Pro Leu Ile Glu 85 90 95Asp
Trp Ile Val Asn Glu Ala Val Ile Gly Arg Val Asp Asp Val Val 100 105
110Ala Pro Thr Ala Leu Ala Gln Trp Glu Glu Val Glu Arg Pro Gln Asn
115 120 125Met Asp Pro Val Pro Asn Glu Glu Gly Glu Leu Gly Thr Arg
Arg Ser 130 135 140Phe Phe Leu Ala Leu Ile Thr Ile Tyr Arg Gln Val
Leu Thr Arg Thr145 150 155 160Ile Asn Val Asp Tyr Gly Gln Glu Val
Ser Arg Arg Ile Ile Asp Asn 165 170 175Phe Lys Glu Gln Pro Leu Gly
Met Ser Gln Asp Asp Ile Asn Glu Ile 180 185 190Gln Gly Tyr Glu Ser
Lys Glu Arg Leu Thr Thr Asn Tyr Val Lys Ile 195 200 205Leu Cys Ile
Leu Asp Met Phe Phe Asn Lys Phe Gln Thr His Asp Lys 210 215 220Ser
Thr Ile Arg Ile Ala Thr Leu Pro Thr Arg Tyr Arg Gly Cys Ala225 230
235 240Ala Phe Thr Ser Tyr Gly Glu Leu Ala Ile Arg Leu Gly Ile Glu
Pro 245 250 255Ile Lys Leu Pro Ser Leu Ile Leu Thr Val Ala Val Ala
Lys Asp Phe 260 265 270Asp Lys Ile Asn Val Asn Gly Glu Gln Ala Glu
Gln Leu Asp Gly Tyr 275 280 285Phe Pro Tyr Gln Leu Glu Leu Gly Leu
Val Lys Lys Ser Ala Tyr Ser 290 295 300Ala Gly Asn Cys Pro Ser Leu
Tyr Leu Trp Met His Thr Ile Gly Thr305 310 315 320Met Leu His Gln
Gln Arg Ser Tyr Arg Ala Asn Val Pro Lys Asn Val 325 330 335Pro Asp
Gln Met Gly Thr Ile Asn Ser Ala Ile Ala Val Ala Met Gln 340 345
350Phe Val Ala Gly Gly Glu Phe Ser Met Gln Phe Val Gly Asp Ala Arg
355 360 365Val Gln Glu Ala Met Arg Glu Met Gln Thr Ala Glu Ala Glu
Leu Asn 370 375 380Glu Leu Arg Met Ala Gln Ala Arg Glu Met Arg Ala
Ala Ala Arg Gly385 390 395 400Asp Glu Asp Glu Glu Gly Ser Glu Asp
Gly Leu Asp Asp Glu Asn Asp 405 410 415Gly Glu Gly Asp Asp Glu Leu
Pro Ala Glu Ile Glu Gln Asn Pro Glu 420 425 430Tyr Leu Asn Arg Val
Asn Arg Ile Arg Glu Leu Gln Glu Asn Leu Gln 435 440 445Gln Tyr Asn
Ala Thr Val Gln Gln His Thr Asn Ala Val Glu Lys Ala 450 455 460Ala
Leu Arg Ala Leu Ala Tyr Leu Gln Glu Asn Gly Gly Ile Ala Asp465 470
475 480Lys Asp Lys Arg Asp Leu Gly Ile Arg Phe Arg Arg Phe Ala Asp
Glu 485 490 495Ala Glu Gly Arg Val Gly Lys Leu Leu Ala Ser Leu Phe
Pro Ala Pro 500 505 510Arg15353PRTBahia Grande virus 15Met Ala Tyr
Ser Thr Gly Leu Ile Lys Gly Glu Val Ser Gln Gly Leu1 5 10 15Ser Asn
Ala Phe Lys Asp Ala Gly Ile His Gln Ile Glu Leu Asn Lys 20 25 30Glu
Tyr Asp Asn Leu Ser Ile Leu Gly Ala Asn Met Ser Ala Leu Asn 35 40
45Lys Met Phe Asp Thr Glu Asp Glu Gly Leu Ser Asp Thr Asn Thr Asn
50 55 60Ser Ser Lys Asn Ser Ile Leu Gln Ala Ser Asp Met Phe Ile Gly
Asn65 70 75 80Asp Glu Tyr Glu Ser Asp Asp Ser His His Phe Leu Ser
Ser Pro Ser 85 90 95Pro Asp Lys Gly Ser Ser Glu Glu Gly Ser Asn Leu
Gln Glu Phe Asn 100 105 110Phe Gln Ile Pro Arg Asn Lys Val Gly Lys
Glu Lys Ala Tyr Arg Arg 115 120 125Gly Val Ile Asp Val Leu Asp Phe
Leu Gln Arg His Arg Phe Ile Glu 130 135 140Glu Phe Arg Met Glu Gly
Leu Asn Glu Asp Ile Val Cys Ile Ile Pro145 150 155 160Thr Arg Gly
Met Ile Pro Thr Lys Thr Pro Pro Thr Leu Asp Asp Lys 165 170 175Ile
His Leu Ala Asn Asp Gln Ser Ile Glu Lys Glu Glu Ile Leu Gln 180 185
190Lys Asp Lys Thr Ser Lys Pro Asn Lys Gly Ile Lys Gln Pro Asn Lys
195 200 205Gln Glu Ala Gln Pro Val Ser Glu Ser Gln Thr Gly Met Lys
Glu Asp 210 215 220Lys Lys Glu Gln Lys Pro Lys Gln Asn Gln Ile Pro
Ile Lys Asn Lys225 230 235 240Gln Glu Asn Glu Asp Ser Lys Glu Val
Ala Lys Thr Asn Lys Asp Lys 245 250 255Glu Asn Lys Val Ser Lys Gly
Ser Met Ser Lys Asn Asp Lys Leu Lys 260 265 270Glu Gly Asn Ile Thr
Val Pro Lys Gln Gly Phe Glu Lys Lys Lys Thr 275 280 285Lys Gln Ile
Asn Glu Glu Gly His Lys Ser Phe Asp Tyr Ala Asn Thr 290 295 300Tyr
Gly Thr Lys Val Thr Val Lys Thr Ile Arg Tyr Cys Lys Thr Cys305 310
315 320Asn Pro Asn Thr Arg Lys Asn Ala Thr Val Tyr Leu Asp His Leu
Tyr 325 330 335Glu Arg His Ser His Glu Val Ala Leu Ile Lys Ser Leu
Ala Tyr Pro 340 345 350Leu16220PRTBahia Grande
virusmisc_feature(160)..(160)Xaa can be any naturally occurring
amino acid 16Met Ser Gly Val Met Ser Ile Phe Lys Arg Lys Asp Lys
Lys Gly Asn1 5 10 15Glu Gly Ser Lys Ala Leu Ala Ile Pro Asp Glu Lys
Ser Val Val Pro 20 25 30Ser Ala Pro Pro Asp Ile Ser Ala Met Asp Tyr
Gly Arg Phe Gly Leu 35 40 45Leu Gly Arg Gln Thr Leu Leu Glu Glu Asp
Glu Glu Glu Ser Arg Cys 50 55 60Ile Thr Ile Ile Asp Leu Glu Val Asp
Leu Gln Ile Glu Val Leu Ser65 70 75 80Asn Arg Glu Thr Arg Leu Val
Ile Asp Leu Ile Ala Pro Leu Cys Asn 85 90 95Leu Gln Thr Asp Tyr Ile
Gly Lys Glu Asn Thr Lys Ala Ile Trp Ile 100 105 110Gly Leu Thr Val
Val Ala Ala Phe Gly Val Lys Arg Thr Ile Lys Thr 115 120 125Lys Asn
His His Val Tyr Lys Gly Cys Val Ser Ser Gly Leu Arg Leu 130 135
140Leu Ile Asp Ser Glu Lys Gln Phe Glu Leu Asp Lys Arg Asn Lys
Xaa145 150 155 160Ser Gln His Leu Ser Tyr Leu Thr Asn Gly Val Lys
Thr Glu Trp Ala 165 170 175Ile Arg Gly Glu Met Ile Arg Thr Arg Val
Pro Tyr Leu Pro Gln Pro 180 185 190Gly Ser Glu Asp Val Leu Met Phe
Leu Ala Gly Met Gly Ile Ser Cys 195 200 205Tyr Ser Asn Pro Asp Gly
His Leu Val Leu Lys Val 210 215 22017591PRTBahia Grande virus 17Met
Ile Ser Asn Met Phe Phe Leu Phe Gln Leu Ser Leu Phe Leu Gln1 5 10
15Phe Ile Ala Gly Asp Glu Ser Leu Glu Thr Ile Thr Ala Pro Glu Thr
20 25 30Pro Asp Pro Ile Leu Leu Lys Gly Asp Thr Lys Tyr Leu Phe Leu
Val 35 40 45Pro Ser Ser Val Lys Asn Trp Lys Pro Ala Asp Leu Asn Glu
Leu Thr 50 55 60Cys Pro Pro Leu Ile Ser Lys Pro Asp Thr Ser Glu Met
Thr Tyr Phe65 70 75 80Ser Thr Asp Val Met Glu Leu Gln Lys His His
Glu Leu Ala Pro Val 85 90 95Glu Gly Tyr Leu Cys Ser Gly Leu Arg Tyr
Lys Val Ile Cys Ser Glu 100 105 110Gly Phe Phe Gly Gln Lys Thr Ile
Ala Lys Lys Ile Glu Asn Ile Glu 115 120 125Pro Asp Ser Lys Gln Cys
Leu Asp Asp Leu Ser Lys Phe Lys Asn Asp 130 135 140Asp Tyr Leu Leu
Pro Tyr Phe Pro Ser Glu Asp Cys Asn Trp Met Lys145 150 155 160Glu
Thr Pro Thr His Lys Asp Phe Ile Val Phe Gln Lys His Phe Val 165 170
175Lys Tyr Asp Pro Tyr Asn Asn Gly Phe Tyr Asp Pro Leu Leu Lys Lys
180 185 190Asp Tyr Cys Asp Thr Gln Val Cys Glu Thr Glu His Asp Gln
Thr Ile 195 200 205Trp Ile Thr Glu Lys Ser Ile Glu Asn Glu Cys Ile
Phe Asn Tyr Pro 210 215 220Ile Lys Lys His Ile Phe His Thr Ala Asp
Phe Gly Lys Met Ile Ile225 230 235 240Asp Tyr Glu Leu Asn Gln Trp
Thr Ser Val Glu Asp Gly Cys Leu Ile 245 250 255Asn Tyr Cys Gly Arg
Glu Gly Ile Arg Leu Ser Asn Gly Met Phe Phe 260 265 270Val Gly Lys
Phe Tyr Lys Asn Leu Asn Asn Leu Gln Thr Cys Ser Ala 275 280 285Gly
Thr Lys Val Ser Tyr Lys Pro Leu Thr Ser Lys Leu Glu Glu Ile 290 295
300Glu Asn Glu Ile Ile Leu Asp Gln Glu Arg Leu Leu Cys Leu Asp
Ser305 310 315 320Ile Arg Gln Met Thr Ala Thr Lys Lys Leu Ser Phe
Tyr Ser Leu Ser 325 330 335Phe Leu Glu Pro Lys Ser Ser Ser Arg His
Lys Val Phe Arg Ile His 340 345 350Asn Lys Thr Leu Glu Tyr Thr Glu
Thr Glu Trp His Pro Ile Met Ser 355 360 365Phe Asn Phe Asp Glu Pro
Asn Lys Ile Gly Ile Asp Lys Asn Gly Lys 370 375 380Ser Val Tyr Trp
Asn Glu Trp Val Pro Ser Gly Ile Ser Gly Leu Leu385 390 395 400Ser
Gly Phe Asn Gly Val Tyr Lys Lys Glu Asn Glu Thr Lys Val Thr 405 410
415Ile Ala Arg Leu Glu Thr Ile Lys Glu Asp Tyr Asp Arg Glu Met Met
420 425 430Ile Asp His Glu Leu Val Glu Val Glu His Pro Lys Ile Val
His Leu 435 440 445Lys Arg Glu Asn Ile Thr Gly Ser Arg Val Glu Ile
Val Asn Lys Glu 450 455 460His Ser Asp Val Ser Gly Trp Leu Ser Ser
Val Leu Ser Ser Phe Trp465 470 475 480Gly Lys Ile Met Met Thr Ile
Ile Ser Ile Ile Leu Ile Val Ile Ile 485 490 495Gly Leu Val Leu Ile
Asn Cys Cys Pro Ile Ile Cys Lys Ser Cys Ile 500 505 510Lys Arg Tyr
Lys Thr Lys Glu Glu Ser Arg Asn Arg His Arg Leu Asp 515 520 525Arg
Glu Asp Asn Gly Arg Leu Arg Arg Gln His Arg Val Ile Phe Asn 530 535
540Asn Gln Ser Asn Asp Glu Glu Asn Ala Ile Glu Met Val Glu Tyr
Thr545 550 555 560Asp Thr Pro Arg Pro Leu Arg Pro Ile Pro Asp Ala
Thr Thr Ser Asp 565 570 575Thr Glu Ser Arg Ser Pro Thr Thr Ala His
Ser Phe Phe Asn Arg 580 585 590182175PRTBahia Grande virus 18Met
Asp Phe Ser Tyr Glu Gln Leu Leu Asp Pro Ile Asp Val Leu Glu1 5 10
15Glu Glu Leu Tyr Glu Phe Asp Phe Glu Tyr Asp Asp Tyr Thr Asp Asp
20 25 30Asp Gln Thr Pro Leu Pro Asn Ile Lys Tyr Lys Asn Leu Glu Gly
Lys 35 40 45Asp Tyr Asn Leu Asn Ser Pro Leu Ile Ser Asp Val Ile Asp
Ser Gly 50 55 60Arg Glu Tyr Ile Ile Asn Ser Lys Lys Tyr Phe Ser His
Glu Arg Thr65 70 75 80Asn Pro Glu Leu Glu Gln Phe Ser Lys Ala Leu
Met Ala Ile Gly Phe 85 90 95Ser Arg Phe Asp Leu Arg Lys Ser Ser Glu
His His Arg Tyr Met Ser 100 105 110Ser Tyr Ile Tyr Gly Asn Glu Lys
Lys His Met Lys Ile Glu Ile Ile 115 120 125Pro Arg Trp Lys Glu Val
Leu Glu Leu Thr Arg Asn Pro Val Glu Val 130 135 140Thr Ser His Lys
Ile Leu Gly Ser Lys Ser Gln Ser Asp Gln Glu Gly145 150 155 160Tyr
Ile Asn Arg Leu Arg Tyr Ile Thr Val Asp Gly Pro His Ala Arg 165 170
175Lys Thr Arg Leu His Gln Glu Trp Glu Lys Phe Ser Thr Leu His Tyr
180 185 190Ile Thr Tyr Ile Met Asn Ser Lys Ala Phe Ser Asp Asn Lys
Asn Trp 195 200 205Val Arg Glu Val Phe Glu Thr Ile Glu Thr Ser Glu
Val Asp Pro Glu 210 215 220Ile Ile Thr Ile Ile Gly Thr Gly Leu Ser
Lys Lys Glu Val Ser Trp225 230 235 240Ile Ile Ser Glu Asn Phe Ala
Leu Asn Val Arg Thr Gly Leu Phe Val 245 250 255Ser Lys Asp Phe Leu
Leu Met Ile Lys Asp Val Thr Leu Ala Arg Cys 260 265 270Met Ser Lys
Leu Ser Met Ile Asn Arg Lys Ser Pro Asn Thr Thr Tyr 275 280 285Asp
Met Ile Lys Phe Leu Asp Ser Leu Tyr Glu Ser Gly Asp Lys Ile 290 295
300Leu Thr Arg His Gly Asn Leu Ala Tyr Lys His Ile Lys Leu Leu
Glu305 310 315 320Ala Ala Cys Leu Glu Arg Trp Asn Gln Leu Gly His
Lys Phe Arg Pro 325 330 335Leu Ile Pro Ile Ser Ser Ser Met Ser Asp
His Leu Arg Thr Gln Leu 340
345 350Glu Glu Asn Gln Asp Leu Tyr Met Val Ser Arg Glu Phe Phe Asp
Leu 355 360 365Ile Gly Lys Ile Glu Asp Pro Trp Val Val Ala Gln Ala
Tyr Gly Thr 370 375 380Phe Arg His Trp Gly His Pro Tyr Ile Asp Tyr
Leu Asn Gly Leu Lys385 390 395 400Asp Leu Glu Lys Arg Val Asn Glu
Asn Ile Lys Ile Asp Lys Asn Tyr 405 410 415Ala Glu Lys Leu Ala Ser
Asp Leu Ala Phe Ile Val Leu Lys Asp Gln 420 425 430Phe Gly Lys His
Lys Arg Trp Phe Ala Lys Pro Asn Lys Glu Leu Asp 435 440 445Glu Asn
Asn Pro Met Arg Lys Cys Ile Glu Asn Asn Val Trp Pro Asn 450 455
460Thr Lys Val Ile Leu Asp Phe Gly Asp Asn Trp His Lys Leu Glu
Leu465 470 475 480Leu Pro Cys Phe Glu Ile Pro Asp Ala Ile Asp Leu
Ser Asp Leu Tyr 485 490 495Ser Asp Lys Ala His Ser Met Gln Tyr Ser
Glu Val Leu Asn Tyr Val 500 505 510Lys Tyr Lys Lys Ser Lys Lys Asn
Ile Pro Ala Leu Arg Val Ile Gly 515 520 525Thr Leu Leu Glu Lys Glu
Asn Pro Asn Ile Lys Glu Phe Leu Gln Lys 530 535 540Ile Asn Asp Glu
Gly Leu Asp Asp Asp Asp Leu Ile Ile Gly Leu Lys545 550 555 560Ala
Lys Glu Arg Glu Leu Lys Asp Lys Gly Arg Phe Phe Ser Leu Met 565 570
575Ser Trp Asn Ile Arg Leu Tyr Phe Val Ile Thr Glu Tyr Leu Ile Lys
580 585 590Leu His Phe Val Pro Leu Phe Ser Gly Leu Thr Val Ala Asp
Asp Leu 595 600 605Asn Thr Val Thr Lys Lys Leu Leu Ser Ala Thr Glu
Gly Gln Gly Leu 610 615 620Asp Asp Tyr Glu Arg Val Tyr Ile Ala Asn
Ser Leu Asp Tyr Glu Lys625 630 635 640Trp Asn Asn Arg Gln Arg Tyr
Glu Ser Asn Glu Pro Val Phe Thr Val 645 650 655Met Gly Lys Phe Leu
Gly Tyr Pro Asn Leu Ile Ser Tyr Thr His Lys 660 665 670Ile Phe Glu
Arg Ser Phe Ile Tyr Tyr Asn Gly Arg Leu Asp Leu Met 675 680 685Gly
Val Asp Gly Tyr His Ile Tyr Asn Leu Phe Asp Asp Lys Met Val 690 695
700Cys Trp His Gly Gln Leu Gly Gly Phe Glu Gly Val Arg Gln Lys
Gly705 710 715 720Trp Ser Val Leu Asn Tyr Leu Ile Leu Arg Arg Glu
Ala Ala Thr Arg 725 730 735Asn Thr Ala Pro Lys Phe Leu Ala Gln Gly
Asp Asn Gln Ile Val Ile 740 745 750Thr Gln Tyr Thr Leu Thr Ser Lys
Ser Thr Gln Ala Ile Ile Glu Arg 755 760 765Glu Leu Arg Asn Ile Trp
Glu Asn Asn Ala His Ile Met His Arg Ile 770 775 780Gln Gln Ala Thr
Ser Arg Ile Gly Leu Val Ile Asn Asn Asp Glu Val785 790 795 800Leu
Thr Ser Ala Glu Leu Leu Val Tyr Gly Lys Ile Pro Val Phe Arg 805 810
815Gly Lys Leu Leu Pro Leu Glu Thr Lys Arg Trp Ser Arg Val Ser Thr
820 825 830Val Thr Asn Glu Gln Ile Pro Ser Phe Ser Asn Ser Leu Ala
Ser Ser 835 840 845Thr Thr Thr Ala Leu Ala Val Asn Gln His Ser Glu
Asn Pro Ile Glu 850 855 860Val Ile Ser Gln His His Phe Phe Ser Ser
Phe Ala Gly Thr Leu Val865 870 875 880Thr Phe Val Asn Pro Ile Leu
Gly Phe Asp Pro Ile Lys Tyr Ser Gln 885 890 895Leu Ser Glu Arg Asn
Lys Lys Leu Phe Leu Leu Arg Leu Ile Tyr Lys 900 905 910Asp Pro Ser
Val Gly Gly Val Cys Gly Thr Asn Leu Leu Arg Phe Phe 915 920 925Ile
Ser Arg Phe Pro Asp Pro Leu Thr Glu Thr Leu Thr Trp Trp Lys 930 935
940Ile Leu Val Glu Asn Ser Lys Asp Lys Glu Val Val Lys Ile Ala
Leu945 950 955 960Glu Cys Gly Asn Pro Lys Phe Gly Gly Ile Asn Asp
Lys Thr Leu Ala 965 970 975Met Leu Leu Glu Asp Pro Met Ser Leu Asn
Ile Pro Gly Gly Leu Ser 980 985 990Ser Asp Thr Met Ile Lys Asn Lys
Ile Tyr Glu Gly Leu Ile His Gln 995 1000 1005Met Gly Leu Lys Leu
Ile Lys Asn Glu Leu Val Val Glu Ser Leu 1010 1015 1020Thr Phe Tyr
Asn Asp Tyr Lys Ala Gln Phe Val Arg Trp Leu Phe 1025 1030 1035Ser
Ile Arg Pro Ile Phe Pro Arg Phe Ile Ser Glu Phe Tyr Thr 1040 1045
1050Ser Thr Tyr Phe Tyr Ile Thr Glu Ser Val Leu Ala Ile Phe Gln
1055 1060 1065Asn Ser Arg Thr Ile Arg Lys Val Phe Ser Lys Arg Phe
Pro Lys 1070 1075 1080Glu Val Tyr Leu Thr Ile Val Lys Gly Glu Gln
Met Ser Ile Asp 1085 1090 1095Ser Leu Leu Thr Thr Lys Arg Gly Ile
Val Arg Glu Ala Ile Trp 1100 1105 1110Lys Cys Ser Ala Thr Lys Ala
Asp Glu Met Arg Lys Leu Ser Trp 1115 1120 1125Gly Arg Asp Met Val
Gly Ile Thr Thr Pro His Pro Ala Glu Phe 1130 1135 1140Thr Gln Glu
Leu Leu Cys Ser Asp Gly Cys Ser Glu Pro His Ile 1145 1150 1155Val
Ala Lys Lys Val Ile Tyr Ser Asp Arg Lys Leu Trp Thr Lys 1160 1165
1170Gly Lys Met Met Pro Tyr Leu Gly Thr Lys Thr Lys Glu Ser Thr
1175 1180 1185Ser Ile Leu Gln Pro Trp Glu Lys Arg Leu Glu Ile Pro
Leu Leu 1190 1195 1200Arg Lys Ala Cys Asp Leu Arg Lys Ala Ile Arg
Trp Phe Val Glu 1205 1210 1215Asp Asn Ser Asn Leu Ala Lys Ser Ile
Tyr Lys Asn Leu Glu Ser 1220 1225 1230Met Thr Gly Ile Asp Leu Arg
Glu Glu Leu Arg Asn Tyr Lys Arg 1235 1240 1245Thr Gly Ser Ser Lys
His Arg Leu Arg Asn Ser Arg Val Ser Asn 1250 1255 1260Glu Gly Asn
Pro Ala Ile Gly Tyr Asn Asn Leu Thr Tyr Val Thr 1265 1270 1275Val
Thr Thr Asp Ser Leu Gly Asn Ile Asn Ser Glu Asn Tyr Asp 1280 1285
1290Phe Met Tyr Gln Ser Ile Leu Cys Trp Cys Gly Val Leu Ser Ser
1295 1300 1305Leu Ala Thr Asn Arg Tyr Arg Asp His Glu Thr Thr His
Phe His 1310 1315 1320Leu Lys Cys Asn Asp Cys Phe Arg Leu Val Lys
Glu Glu Ile Leu 1325 1330 1335Glu Ala Pro Ser Val Tyr Pro Phe Pro
Asn Val Arg Ser Ser Val 1340 1345 1350Arg Arg Met Leu Thr Gln Asp
Ile Lys Leu Lys Tyr Leu Pro Arg 1355 1360 1365Ile Ser Ala Pro Asp
Glu Asn Thr Trp Asp Thr Leu Asp Val Asp 1370 1375 1380Gln Lys Ser
Trp His Ile Gly Arg Ala Gln Gly Phe Leu Trp Gly 1385 1390 1395Leu
Asn Val Phe Thr Lys Thr Thr Lys Glu Val Glu Gly Asp Ile 1400 1405
1410Phe Pro Thr Ser Ile Thr Lys Lys Val Glu Pro Glu Asn Tyr Met
1415 1420 1425Asp Gly Leu His Arg Gly Phe Cys Leu Gly Ala Thr Leu
Ser Pro 1430 1435 1440Met Tyr Thr Arg Tyr Gly Ser Leu Ser Arg Met
Ala Arg Arg Lys 1445 1450 1455Phe Glu Gly Ala Tyr Trp Glu Ile Val
Asp Glu Ala Met Lys Thr 1460 1465 1470Asn Leu Pro Asn Met Ile Asp
His Lys Asn Phe Lys Pro Phe Leu 1475 1480 1485Arg Arg Thr Gly Gly
Asp Leu Ile Lys Ser Tyr Pro Ala Arg Lys 1490 1495 1500Glu Glu Leu
Val Leu Val Leu Lys Lys Trp Phe Leu His Lys Met 1505 1510 1515Val
Ser Glu Arg Lys Asn Asn Ser Ile Trp Glu Ser Lys Arg Val 1520 1525
1530Ile Ala Phe Ala Asp Met Asp Thr Glu Phe Val Leu Cys Leu Phe
1535 1540 1545Arg Leu Ala Glu Ser Ile Leu Asn Cys Tyr Gln Asn Glu
Ala Leu 1550 1555 1560Ser Ala Gly Gln Ala Arg Val Leu Gly Asn Ala
Lys Glu Thr Ile 1565 1570 1575Asp Leu Ile Ser Lys Tyr Asn Asn Ser
Asn Ile Asn Ala Asp Glu 1580 1585 1590Ile Glu Arg Leu Gln Gln Ile
Leu Met Ala Ser Asp Leu Lys Asp 1595 1600 1605His Glu Val Val Asp
Ser Gln Ala Arg His Ala Ala Ser Asp Leu 1610 1615 1620Pro Glu Leu
Ala Lys Ser Glu Asn Tyr Asn Glu Val Ile Lys Tyr 1625 1630 1635Val
Glu Phe Arg Gly Tyr Gly Gly Lys Thr Ile Arg Leu Glu Tyr 1640 1645
1650Gln Pro Ser Asp Leu Ile Asp Trp Lys Gly Gly Met Val Gln Asp
1655 1660 1665Leu Gln Val Pro Arg Leu Lys Asn Pro Leu Ile Ser Gly
Val Arg 1670 1675 1680Val Val Gln Tyr Ser Thr Gly Ala His Tyr Lys
Tyr Lys Asp Ile 1685 1690 1695Glu Arg Glu Phe Gln Ile Ala Gly Asp
Gly Ile Phe Ala Gly Asp 1700 1705 1710Gly Ser Gly Gly Met Gly Ala
Asn His Leu Arg Leu His Lys Ser 1715 1720 1725Ala Arg Val Ile Phe
Asn Ser Lys Leu Glu Leu Glu Gly Glu Ser 1730 1735 1740Leu Lys Gly
Leu Ala Pro Ala Gly Pro Gly Ala Tyr Thr Val Ser 1745 1750 1755Gly
Glu Asp Val Val Glu Arg Cys Val Asn Tyr Thr Thr Cys Trp 1760 1765
1770Glu Glu Ala Ser Asp Leu Ser Asp Glu Lys Thr Trp Lys Asn Phe
1775 1780 1785Phe Arg Leu Ile Lys Glu Tyr Ser Leu Asp Ile Glu Val
Phe Cys 1790 1795 1800Cys Asp Ala Glu Val Gln Asp Pro Tyr Ile Thr
Asn Lys Ile Glu 1805 1810 1815Ser Asn Ile Leu Lys Tyr Ile Ser Leu
Ile Leu Asn Lys Arg Thr 1820 1825 1830Gly Thr Leu Ile Tyr Lys Thr
Tyr Phe Asn Arg Leu Leu Asp Pro 1835 1840 1845Asn Thr Ile Thr His
Phe Leu Gly Met Phe Phe His Arg Cys Tyr 1850 1855 1860Gly Phe Leu
Pro Thr Thr Gln Gly Ser Phe Thr Ser Glu Ile Tyr 1865 1870 1875Ile
Val Cys Gln Tyr Pro Lys Thr Leu Asp Ser Thr Ser Lys Thr 1880 1885
1890Glu Leu Thr Tyr Thr Ser Leu Phe Asn Ile Tyr Gln Asn Ile Arg
1895 1900 1905Val Met Glu Thr Tyr Gln Asn Glu Phe Asp Arg Ala Cys
Ser Leu 1910 1915 1920Leu Phe Ser Asp Met Thr Glu Gly Leu Ile Asp
Lys Thr Pro Phe 1925 1930 1935Leu Asp Pro Glu Glu Leu Ala Ile Phe
Leu Thr Thr Val Gly Leu 1940 1945 1950Asp Thr Gly Trp Ala Leu Leu
Ile Ala Glu Gln Leu Gln Ile Ser 1955 1960 1965Cys Ser Asn Lys Leu
His Pro Ile Ile Ile Leu Trp Ile Leu Gly 1970 1975 1980Phe Ile Ile
Ser Arg His Leu Val Ser Ile Thr Ser Trp Phe Arg 1985 1990 1995Arg
Gly Thr Lys Phe Pro Pro Ser Ile Gln Leu Gln Lys Met Leu 2000 2005
2010Ala Ala Leu Phe Gly Ile Trp Tyr Gly Val Ser Tyr Ile Met Asn
2015 2020 2025Asp Ala Glu Ser Tyr Ser Arg Ile Ser Val Leu Tyr Asn
Gln Glu 2030 2035 2040Ile Tyr Phe Ser Leu Gly Leu Thr Asn Met Val
Tyr Arg Lys Lys 2045 2050 2055Asp Asp Met Glu Leu Gly Gln Phe Ser
Thr Trp Lys Ile Gly Pro 2060 2065 2070Gly Asp Asn Ser Lys Leu Ile
Asp Ile Gly Pro Lys Ala Gly Ile 2075 2080 2085Thr Gln Thr Met Ile
Arg Ala Ile Val Val Leu Tyr Lys Gly Glu 2090 2095 2100His Ile Thr
Ser Ile Val Thr Lys Glu Asp Lys Val Glu Gly Asp 2105 2110 2115Arg
Ile Leu Ser Leu Phe Gly Lys Gly Leu Asn Leu Lys Thr Leu 2120 2125
2130Met Glu Arg Thr Gly Ile Asn Tyr Leu Gln Ile Gly Glu Arg Asn
2135 2140 2145Pro Gln Glu Ile Pro Tyr Thr Leu Glu Glu Glu Val Leu
Glu Glu 2150 2155 2160Val Val Glu Glu Asn Thr Gly Glu Phe Asp Gln
Ser 2165 2170 217519519PRTVesicular stomatitis virus 19Met Thr Ser
Val Leu Phe Met Val Gly Val Leu Leu Gly Ala Phe Gly1 5 10 15Ser Thr
His Cys Ser Ile Gln Ile Val Phe Pro Ser Glu Thr Lys Leu 20 25 30Val
Trp Lys Pro Val Leu Lys Gly Thr Arg Tyr Cys Pro Gln Ser Ala 35 40
45Glu Leu Asn Leu Glu Pro Asp Leu Lys Thr Met Ala Phe Asp Ser Lys
50 55 60Val Pro Ile Gly Ile Thr Pro Ser Asn Ser Asp Gly Tyr Leu Cys
His65 70 75 80Ala Ala Lys Trp Val Thr Thr Cys Asp Phe Arg Trp Tyr
Gly Pro Lys 85 90 95Tyr Ile Thr His Ser Val His Ser Leu Arg Pro Thr
Val Ser Asp Cys 100 105 110Lys Ala Ala Val Glu Ala Tyr Asn Ala Gly
Thr Leu Met Tyr Pro Gly 115 120 125Phe Pro Pro Glu Ser Cys Gly Tyr
Ala Ser Ile Thr Asp Ser Glu Phe 130 135 140Tyr Val Met Leu Val Thr
Pro His Pro Val Gly Val Asp Asp Tyr Arg145 150 155 160Gly His Trp
Val Asp Pro Leu Phe Pro Thr Ser Glu Cys Asn Ser Asn 165 170 175Phe
Cys Glu Thr Val His Asn Ala Thr Met Trp Ile Pro Lys Asp Leu 180 185
190Lys Thr His Asp Val Cys Ser Gln Asp Phe Gln Thr Ile Arg Val Ser
195 200 205Val Met Tyr Pro Gln Thr Lys Pro Thr Lys Gly Ala Asp Leu
Thr Leu 210 215 220Lys Ser Lys Phe His Ala His Met Lys Gly Asp Arg
Val Cys Lys Met225 230 235 240Lys Phe Cys Asn Lys Asn Gly Leu Arg
Leu Gly Asn Gly Glu Trp Ile 245 250 255Glu Val Gly Asp Glu Val Met
Leu Asp Asn Ser Lys Leu Leu Ser Leu 260 265 270Phe Pro Asp Cys Leu
Val Gly Ser Val Val Lys Ser Thr Leu Leu Ser 275 280 285Glu Gly Val
Gln Thr Ala Leu Trp Glu Thr Asp Arg Leu Leu Asp Tyr 290 295 300Ser
Leu Cys Gln Asn Thr Trp Glu Lys Ile Asp Arg Lys Glu Pro Leu305 310
315 320Ser Ala Val Asp Leu Ser Tyr Leu Ala Pro Arg Ser Pro Gly Lys
Gly 325 330 335Met Ala Tyr Ile Val Ala Asn Gly Ser Leu Met Ser Ala
Pro Ala Arg 340 345 350Tyr Ile Arg Val Trp Ile Asp Ser Pro Ile Leu
Lys Glu Ile Lys Gly 355 360 365Lys Lys Glu Ser Ala Ser Gly Ile Asp
Thr Val Leu Trp Glu Gln Trp 370 375 380Leu Pro Phe Asn Gly Met Glu
Leu Gly Pro Asn Gly Leu Ile Lys Thr385 390 395 400Lys Ser Gly Tyr
Lys Phe Pro Leu Tyr Leu Leu Gly Met Gly Ile Val 405 410 415Asp Gln
Asp Leu Gln Glu Leu Ser Ser Val Asn Pro Val Asp His Pro 420 425
430His Val Pro Ile Ala Gln Ala Phe Val Ser Glu Gly Glu Glu Val Phe
435 440 445Phe Gly Asp Thr Gly Val Ser Lys Asn Pro Ile Glu Leu Ile
Ser Gly 450 455 460Trp Phe Ser Ser Trp Lys Ser Ser Ile Ala Ser Phe
Phe Phe Thr Ile465 470 475 480Gly Leu Ile Ile Gly Leu Phe Leu Val
Leu Arg Val Gly Ile Tyr Leu 485 490 495Cys Ile Lys Leu Lys His Thr
Lys Lys Arg Gln Ile Tyr Thr Asp Ile 500 505 510Glu Met Asn Arg Leu
Gly Thr 515201662DNAMaraba virus 20atgaaaaaaa ctaacagggt tcaaacactc
ttgatcgagg tattgagact ttttctcttt 60tgtttcttgg ccttaggagc ccactccaaa
tttactatag tattccctca tcatcaaaaa 120gggaattgga agaatgtgcc
ttccacatat cattattgcc cttctagttc tgaccagaat 180tggcataatg
atttgactgg agttagtctt catgtgaaaa ttcccaaaag tcacaaagct
240atacaagcag atggctggat gtgccacgct gctaaatggg tgactacttg
tgacttcaga 300tggtacggac ccaaatacat cacgcattcc atacactcta
tgtcacccac cctagaacag 360tgcaagacca gtattgagca gacaaagcaa
ggagtttgga ttaatccagg ctttccccct 420caaagctgcg gatatgctac
agtgacggat gcagaggtgg ttgttgtaca agcaacacct 480catcatgtgt
tggttgatga gtacacagga gaatggattg actcacaatt ggtggggggc
540aaatgttcca aggaggtttg tcaaacggtt cacaactcga ccgtgtggca
tgctgattac 600aagattacag ggctgtgcga
gtcaaatctg gcatcagtgg atatcacctt cttctctgag 660gatggtcaaa
agacgtcttt gggaaaaccg aacactggat tcaggagtaa ttactttgct
720tacgaaagtg gagagaaggc atgccgtatg cagtactgca cacaatgggg
gatccgacta 780ccttctggag tatggtttga attagtggac aaagatctct
tccaggcggc aaaattgcct 840gaatgtccta gaggatccag tatctcagct
ccttctcaga cttctgtgga tgttagtttg 900atacaagacg tagagaggat
cttagattac tctctatgcc aggagacgtg gagtaagata 960cgagccaagc
ttcctgtatc tccagtagat ctgagttatc tcgccccaaa aaatccaggg
1020agcggaccgg ccttcactat cattaatggc actttgaaat atttcgaaac
aagatacatc 1080agagttgaca taagtaatcc catcatccct cacatggtgg
gaacaatgag tggaaccacg 1140actgagcgtg aattgtggaa tgattggtat
ccatatgaag acgtagagat tggtccaaat 1200ggggtgttga aaactcccac
tggtttcaag tttccgctgt acatgattgg gcacggaatg 1260ttggattccg
atctccacaa atcctcccag gctcaagtct tcgaacatcc acacgcaaag
1320gacgctgcat cacagcttcc tgatgatgag actttatttt ttggtgacac
aggactatca 1380aaaaacccag tagagttagt agaaggctgg ttcagtagct
ggaagagcac attggcatcg 1440ttctttctga ttataggctt gggggttgca
ttaatcttca tcattcgaat tattgttgcg 1500attcgatcac gaattctgga
tccgatacgt aacgctctgc agctgcgggt tgcattaatc 1560ttcatcattc
gaattattgt tgcgattcgc tataaataca aggggaggaa gacccaaaaa
1620atttacaatg atgtcgagat gagtcgattg ggaaataaat aa 166221518PRTMuir
Spring virusmisc_feature(384)..(384)Xaa can be any naturally
occurring amino acidmisc_feature(386)..(386)Xaa can be any
naturally occurring amino acidmisc_feature(389)..(390)Xaa can be
any naturally occurring amino acid 21Met Lys Tyr Pro Val Leu Leu
Leu Tyr Gln Asn Gln Ile Leu Leu Lys1 5 10 15Trp Asn Thr Cys Leu Leu
Met Ser Trp Asn Ser Gln Lys His His Glu 20 25 30Leu Ala Pro Val Gln
Gly Tyr Leu Cys Ser Gly Leu Arg Tyr Lys Val 35 40 45Ile Cys Ser Glu
Gly Phe Phe Gly Gln Lys Thr Ile Thr Lys Lys Ile 50 55 60Glu Asn Leu
Glu Pro Asp Gln Asn Lys Cys Val Gln Asp Leu Glu Lys65 70 75 80Phe
Ile Asn Asp Asp Tyr Leu Leu Pro Tyr Phe Pro Ser Glu Asp Cys 85 90
95Asn Trp Met Lys Glu Thr Pro Val His Gln Asp Phe Ile Val Tyr Gln
100 105 110Lys His Gln Val Lys Tyr Asp Pro Tyr His Asn Gly Phe Tyr
Asp Ala 115 120 125Leu Phe Lys Lys Asp Phe Cys Gln Glu Lys Ile Cys
Glu Thr Glu His 130 135 140Asp Gln Thr Ile Trp Ile Thr Asn Gln Glu
Leu Lys Gln Glu Cys Thr145 150 155 160Phe Asn Tyr Pro Val Lys Lys
His Val Phe Tyr Lys Arg Asp Tyr Ser 165 170 175Lys Met Ile Ile Asp
Tyr Glu Ile Asn Gln Trp Thr Ser Val Glu Asp 180 185 190Gly Cys Leu
Ile Arg Tyr Cys Gly Gln Glu Gly Ile Arg Leu Ser Asn 195 200 205Gly
Met Phe Phe Val Gly Lys Phe Tyr Lys Leu Ile Ser Asn Leu Pro 210 215
220Ile Cys Pro Glu Gly Thr Lys Ile Ser Tyr Lys Pro Ile Lys Ala
Gln225 230 235 240Leu Asp Glu Ile Glu Asn Glu Ile Ile Leu Asn Gln
Glu Arg Leu Leu 245 250 255Cys Leu Asp Ser Ile Arg Gln Met Thr Ala
Ser Lys Lys Leu Ser Phe 260 265 270Tyr Ser Leu Ser Phe Leu Glu Pro
Lys Ser Met Ser Arg His Lys Val 275 280 285Tyr Arg Ile His Asn Asn
Thr Leu Glu Tyr Thr Glu Thr Glu Trp Glu 290 295 300Pro Ile Val Ala
Phe Asn Phe Asn Gly Lys Asn Gln Ile Gly Val Asn305 310 315 320Lys
Glu Gly Lys Glu Val Tyr Trp Asn Glu Trp Val Pro Ser Gly Lys 325 330
335Asp Gly Leu Leu Ser Gly Phe Asn Gly Val Tyr Lys Lys Val Asn Ser
340 345 350Ser Lys Ile Ser Ile Ser Arg Leu Glu Thr Ile Lys Glu Asp
Tyr Glu 355 360 365Arg Glu Met Met Ile Asp His Glu Leu Val Thr Val
Glu His Pro Xaa 370 375 380Ile Xaa His Leu Xaa Xaa Glu Asn Ile Thr
Gly Ser Arg Val Glu Ile385 390 395 400Val Asn Thr Glu His Ser Asp
Val Ser Gly Trp Phe Ser Ser Val Leu 405 410 415Lys Ser Phe Trp Gly
Lys Leu Met Met Thr Val Val Ser Ile Ile Ile 420 425 430Ile Ile Ile
Ile Gly Leu Leu Ile Ile Asn Cys Gly Pro Ile Ile Cys 435 440 445Lys
Thr Cys Ile Ser Ser Tyr Lys Lys Lys Lys Ser Arg Arg Asp Arg 450 455
460Phe Arg Ala Asp Arg Glu Thr Glu Thr Gly Leu Arg Arg Gln His
Arg465 470 475 480Val Val Phe His Asn Asn Glu Thr Asp Asp Glu Arg
Ala Ile Glu Met 485 490 495Thr Gly His His Phe Gly Lys His Val Arg
Ser Glu Leu Arg Pro Arg 500 505 510Arg His Pro Gly Ser Gly
515222248DNAMuir Spring virus 22gcggcggggg ctggccatca ctttggcaag
cacgtgagat ctgattcgcg gccgcgtcga 60cgcccctgaa actcctgatc ctatcctcct
ccaaggagat aaaacttatc tctttttagt 120cccttcagag agcaaaaatt
ggaaacccgc agatcttaat gaagtatcct gtcctcctct 180tatatcaaaa
ccagatactg ctgaaatgga atacatgtct actgatgtca tggaactcgc
240aaaaacatca tgaactcgcg cctgtgcaag ggtatttatg ttctggctta
agatataaag 300ttatttgttc tgaaggattc tttggacaaa aaacaataac
taagaaaatt gaaaatcttg 360aacctgatca gaacaaatgt gttcaagatt
tagaaaagtt tattaatgac gattatttgc 420taccctattt cccatcagaa
gattgtaatt ggatgaaaga aacaccagtt catcaagatt 480tcatagttta
ccaaaaacat caggttaaat atgatccata ccacaatggc ttttacgatg
540ctctgttcaa gaaagatttt tgtcaagaga aaatatgtga gacagagcat
gatcagacaa 600tatggataac taaccaagaa ttaaaacaag aatgcacttt
taattatccg gttaaaaaac 660atgtattcta taagagagat tatagcaaaa
tgatcatcga ttatgaaatc aaccaatgga 720cttcagttga ggatggatgt
ttgataagat attgtggtca ggaaggaatt agattatcta 780atgggatgtt
ctttgtagga aaattttaca aattaatatc gaatctgcca atttgtccag
840aaggaaccaa gatcagctac aagcccatta aagcacaatt agatgaaata
gaaaatgaaa 900taattttaaa tcaagaaaga cttttatgtt tagattctat
acgacaaatg actgcttcta 960aaaaattatc tttttattca ttatccttct
tggagcctaa atccatgagt agacataagg 1020tctatagaat tcacaataat
actttagaat acactgaaac tgaatgggaa cctatagtgg 1080cttttaattt
taatggaaag aatcaaatcg gagtaaataa agaagggaag gaagtttatt
1140ggaatgaatg ggtgcccagt ggaaaagatg gattgctctc aggattcaat
ggagtttata 1200agaaagttaa ttcttccaaa atttcaatat caagattaga
aaccattaaa gaagattatg 1260aaagagaaat gatgatagat catgaattgg
ttacagttga gcatcctama attgkccatc 1320ttaawasaga aaacatmaca
ggttctagag tggagatagt taatactgaa cattcagacg 1380tcagtggttg
gttctcatct gttttaaaga gtttttgggg aaagttgatg atgactgttg
1440tcagtataat aataattatc atcataggcc tattgattat caattgtggt
ccaattatct 1500gtaaaacttg cattagcagc tataaaaaga aaaagagtag
aagagataga tttagagcag 1560atagagaaac tgaaactgga ctgcgtcgac
aacatagagt ggtatttcat aataatgaaa 1620cagatgatga aagagcaata
gagatgactg gccatcactt tggcaagcac gtgagatctg 1680aattgcggcc
gcgtcgacat cctggctcag gatgaacgct ggctgtgtgc ctaatacatg
1740catgtcgagc gaggttcttt tgaacctagc ggcgaatggg tgagtaacac
gtgcttaatc 1800taccctttag attggaatac ccaatggaaa cattggctaa
tgccggatac gcatggaatc 1860gcatgattcc gttgtgaaag gagcctttaa
agctccgcta gaggatgagg gtgcggaaca 1920ttagttagtt ggtagggtaa
tggcctacca agactatgat gtttagccgg gtcgagagac 1980tgaacggcca
cattgggact gagatacggc ccaaactcct acgggaggca gcagtaggga
2040atattccaca atgagcgaaa gcttgatgga gcgacacagc gtgcacgatg
aaggtcttcg 2100gattgtaaag tgctgttata gggaaagaac acctggttga
ggaaatgctt ccaggctgac 2160ggtaccctgt cagaaagcga tggctaacta
tgtgccagca gccgcggtaa tacataggtc 2220gcaagcgtta tccggaatta ttgggcgt
2248231948DNAVesicular stomatitis virus 23acgcgttttc gccaccatgc
cgaagcgccg cgctggattc cggaaaggct ggtacgcgcg 60gcagaggaac tccctgacgc
atcaaatgca acgcatgacg ctgagcgagc ccacgagtga 120gctgcccacc
cagaggcaaa ttgaagcgct aatgcgctac gcctggaatg aggcacatgt
180acaacctccg gtgacaccta ctaacatctt gatcatgtta ttattattgt
tacagcgggt 240acaaaatggg gcagctgcgg ctttttgggc gtacattcct
gatccgccaa tgattcaatc 300cttaggatgg gatagagaaa tagtacccgt
atatgttaat gatacgagcc ttttaggagg 360aaaatcagat attcacattt
cccctcagca agcaaatatc tctttttatg gccttaccac 420tcaatatccc
atgtgctttt cttatcaatc gcagcatcct cattgtatac aggtatcagc
480tgacatatca tatcctcgag tgactatctc aggcattgat gaaaaaactg
ggaaaaaatc 540atacgggaac ggatctggac ccctcgacat tccgttttgt
gacaagcatt taagcattgg 600cataggcata gacactcctt ggactttatg
tcgagcccgg gtcgcgtcag tatataacat 660caataatgcc aatgccacct
ttttatggga ttgggcacct ggaggaacac ctgattttcc 720tgaatatcga
ggacagcatc cgcctatttt ctctgtgaat accgctccaa tataccaaac
780ggaactatgg aaacttttgg ctgcttttgg tcatggcaat agtttatatt
tacagcccaa 840tatcagtgga agcaaatatg gtgatgtagg agttacagga
tttttatatc ctcgagcttg 900cgtgccgtat ccattcatgt tgatacaagg
ccatatggaa ataacactgt cattaaatat 960ttatcatttg aattgttcta
attgcatact gactaattgt atcaggggag tagccaaagg 1020agaacaggtt
ataatagtaa aacagcctgc ctttgtaatg ctgcccgttg aaatagctga
1080agcctggtat gatgaaactg ctttagaatt attacaacgc attaatacgg
ctcttagccg 1140ccctaagaga ggcctgagcc tgattattct gggtatagta
tctttaatca ccctcatagc 1200tacagctgtt acggcttccg tatctttagc
acagtctatt caagctgcgc acacggtaga 1260ctccttatca tataatgtta
ctaaagtgat ggggacccaa gaagatattg ataaaaaaat 1320agaagatagg
ctatcagctc tatatgatgt agtcagagtc ttaggagagc aagttcagag
1380cattaatttt cgcatgaaaa tccaatgtca tgctaactat aaatggattt
gtgttacaaa 1440aaaaccatac aacacttctg attttccatg ggacaaagtg
aagaaacatt tgcaaggaat 1500ttggttcaat actaatctat cgttagacct
tttacaactg cataatgaga ttcttgatat 1560tgaaaattcg ccgaaggcta
cactaaatat agccgatact gttgataatt tcttgcaaaa 1620tttattctct
aatttcccta gtctccattc gctgtggaaa accctgattg gtgtaggaat
1680acttgtgttt attataattg tcgtaatcct tatatttcct tgcctcgtac
gtagtagttg 1740gaagagctct attgcctctt ttttctttac catagggtta
atcattggac tattcttggt 1800tctccgagtt ggtatttatc tttgcattaa
attaaagcac accaagaaaa gacagattta 1860tacagacata gagatgaacc
gacttggaac gtaactcaaa tcctcgaggc taggtatgaa 1920aaaaactaac
agatatcacg gctagcgg 1948242031DNAEbola virus 24atgggcgtta
caggaatatt gcagttacct cgtgatcgat tcaagaggac atcattcttt 60ctttgggtaa
ttatcctttt ccaaagaaca ttttccatcc cacttggagt catccacaat
120agcacattac aggttagtga tgtcgacaaa ctagtttgtc gtgacaaact
gtcatccaca 180aatcaattga gatcagttgg actgaatctc gaagggaatg
gagtggcaac tgacgtgcca 240tctgcaacta aaagatgggg cttcaggtcc
ggtgtcccac caaaggtggt caattatgaa 300gctggtgaat gggctgaaaa
ctgctacaat cttgaaatca aaaaacctga cgggagtgag 360tgtctaccag
cagcgccaga cgggattcgg ggcttccccc ggtgccggta tgtgcacaaa
420gtatcaggaa cgggaccgtg tgccggagac tttgccttcc ataaagaggg
tgctttcttc 480ctgtatgatc gacttgcttc cacagttatc taccgaggaa
cgactttcgc tgaaggtgtc 540gttgcatttc tgatactgcc ccaagctaag
aaggacttct tcagctcaca ccccttgaga 600gagccggtca atgcaacgga
ggacccgtct agtggctact attctaccac aattagatat 660caggctaccg
gttttggaac caatgagaca gagtacttgt tcgaggttga caatttgacc
720tacgtccaac ttgaatcaag attcacacca cagtttctgc tccagctgaa
tgagacaata 780tatacaagtg ggaaaaggag caataccacg ggaaaactaa
tttggaaggt caaccccgaa 840attgatacaa caatcgggga gtgggccttc
tgggaaacta aaaaaaacct cactagaaaa 900attcgcagtg aagagttgtc
tttcacagtt gtatcaaacg gagccaaaaa catcagtggt 960cagagtccgg
cgcgaacttc ttccgaccca gggaccaaca caacaactga agaccacaaa
1020atcatggctt cagaaaattc ctctgcaatg gttcaagtgc acagtcaagg
aagggaagct 1080gcagtgtcgc atctaacaac ccttgccaca atctccacga
gtccccaatc cctcacaacc 1140aaaccaggtc cggacaacag cacccataat
acacccgtgt ataaacttga catctctgag 1200gcaactcaag ttgaacaaca
tcaccgcaga acagacaacg acagcacagc ctccgacact 1260ccctctgcca
cgaccgcagc cggaccccca aaagcagaga acaccaacac gagcaagagc
1320actgacttcc tggaccccgc caccacaaca agtccccaaa accacagcga
gaccgctggc 1380aacaacaaca ctcatcacca agataccgga gaagagagtg
ccagcagcgg gaagctaggc 1440ttaattacca atactattgc tggagtcgca
ggactgatca caggcgggag aagaactcga 1500agagaagcaa ttgtcaatgc
tcaacccaaa tgcaacccta atttacatta ctggactact 1560caggatgaag
gtgctgcaat cggactggcc tggataccat atttcgggcc agcagccgag
1620ggaatttaca tagaggggct aatgcacaat caagatggtt taatctgtgg
gttgagacag 1680ctggccaacg agacgactca agctcttcaa ctgttcctga
gagccacaac tgagctacgc 1740accttttcaa tcctcaaccg taaggcaatt
gatttcttgc tgcagcgatg gggcggcaca 1800tgccacattc tgggaccgga
ctgctgtatc gaaccacatg attggaccaa gaacataaca 1860gacaaaattg
atcagattat tcatgatttt gttgataaaa cccttccgga ccagggggac
1920aatgacaatt ggtggacagg atggagacaa tggataccgg caggtattgg
agttacaggc 1980gttataattg cagttatcgc tttattctgt atatgcaaat
ttgtctttta g 20312539DNAArtificial sequenceSynthetic primer
25ctcgagggta tgaaaaaaac taacagatat cacggctag 3926523PRTIsfahan
virus 26Met Thr Ser Val Leu Phe Met Val Gly Val Leu Leu Gly Ala Phe
Gly1 5 10 15Ser Thr His Cys Ser Ile Gln Ile Val Phe Pro Ser Glu Thr
Lys Leu 20 25 30Val Trp Lys Pro Val Leu Lys Gly Thr Arg Tyr Cys Pro
Gln Ser Ala 35 40 45Glu Leu Asn Leu Glu Pro Asp Leu Lys Thr Met Ala
Phe Asp Ser Lys 50 55 60Val Pro Ile Gly Ile Thr Pro Ser Asn Ser Asp
Gly Tyr Leu Cys His65 70 75 80Ala Ala Lys Trp Val Thr Thr Cys Asp
Phe Arg Trp Tyr Gly Pro Lys 85 90 95Tyr Ile Thr His Ser Val His Ser
Leu Arg Pro Thr Val Ser Asp Cys 100 105 110Lys Ala Ala Val Glu Ala
Tyr Asn Ala Gly Thr Leu Met Tyr Pro Gly 115 120 125Phe Pro Pro Glu
Ser Cys Gly Tyr Ala Ser Ile Thr Asp Ser Glu Phe 130 135 140Tyr Val
Met Leu Val Thr Pro His Pro Val Gly Val Asp Asp Tyr Arg145 150 155
160Gly His Trp Val Asp Pro Leu Phe Pro Thr Ser Glu Cys Asn Ser Asn
165 170 175Phe Cys Glu Thr Val His Asn Ala Thr Met Trp Ile Pro Lys
Asp Leu 180 185 190Lys Thr His Asp Val Cys Ser Gln Asp Phe Gln Thr
Ile Arg Val Ser 195 200 205Val Met Tyr Pro Gln Thr Lys Pro Thr Lys
Gly Ala Asp Leu Thr Leu 210 215 220Lys Ser Lys Phe His Ala His Met
Lys Gly Asp Arg Val Cys Lys Met225 230 235 240Lys Phe Cys Asn Lys
Asn Gly Leu Arg Leu Gly Asn Gly Glu Trp Ile 245 250 255Glu Val Gly
Asp Glu Val Met Leu Asp Asn Ser Lys Leu Leu Ser Leu 260 265 270Phe
Pro Asp Cys Leu Val Gly Ser Val Val Lys Ser Thr Leu Leu Ser 275 280
285Glu Gly Val Gln Thr Ala Leu Trp Glu Thr Asp Arg Leu Leu Asp Tyr
290 295 300Ser Leu Cys Gln Asn Thr Trp Glu Lys Ile Asp Arg Lys Glu
Pro Leu305 310 315 320Ser Ala Val Asp Leu Ser Tyr Leu Ala Pro Arg
Ser Pro Gly Lys Gly 325 330 335Met Ala Tyr Ile Val Ala Asn Gly Ser
Leu Met Ser Ala Pro Ala Arg 340 345 350Tyr Ile Arg Val Trp Ile Asp
Ser Pro Ile Leu Lys Glu Ile Lys Gly 355 360 365Lys Lys Glu Ser Ala
Ser Gly Ile Asp Thr Val Leu Trp Glu Gln Trp 370 375 380Leu Pro Phe
Asn Gly Met Glu Leu Gly Pro Asn Gly Leu Ile Lys Thr385 390 395
400Lys Ser Gly Tyr Lys Phe Pro Leu Tyr Leu Leu Gly Met Gly Ile Val
405 410 415Asp Gln Asp Leu Gln Glu Leu Ser Ser Val Asn Pro Val Asp
His Pro 420 425 430His Val Pro Ile Ala Gln Ala Phe Val Ser Glu Gly
Glu Glu Val Phe 435 440 445Phe Gly Asp Thr Gly Val Ser Lys Asn Pro
Ile Glu Leu Ile Ser Gly 450 455 460Trp Phe Ser Asp Trp Lys Glu Thr
Ala Ala Ala Leu Gly Phe Ala Ala465 470 475 480Ile Ser Val Ile Leu
Ile Ile Gly Leu Met Arg Leu Leu Pro Leu Leu 485 490 495Cys Arg Arg
Arg Lys Gln Lys Lys Val Ile Tyr Lys Asp Val Glu Leu 500 505 510Asn
Ser Phe Asp Pro Arg Gln Ala Phe His Arg 515 52027530PRTChandipura
virus 27Met Thr Ser Ser Val Thr Ile Ser Val Val Leu Leu Ile Ser Phe
Ile1 5 10 15Thr Pro Ser Tyr Ser Ser Leu Ser Ile Ala Phe Pro Glu Asn
Thr Lys 20 25 30Leu Asp Trp Lys Pro Val Thr Lys Asn Thr Arg Tyr Cys
Pro Met Gly 35 40 45Gly Glu Trp Phe Leu Glu Pro Gly Leu Gln Glu Glu
Ser Phe Leu Ser 50 55 60Ser Thr Pro Ile Gly Ala Thr Pro Ser Lys Ser
Asp Gly Phe Leu Cys65 70 75 80His Ala Ala Lys Trp Val Thr Thr Cys
Asp Phe Arg Trp Tyr Gly Pro 85 90 95Lys Tyr Ile Thr His Ser Ile His
Asn Ile Lys Pro Thr Arg Ser Asp 100 105 110Cys Asp Thr Ala Leu Ala
Ser Tyr Lys Ser Gly Thr Leu Val Ser Pro 115 120 125Gly Phe Pro Pro
Glu Ser Cys Gly Tyr Ala Ser Val Thr Asp Ser Glu 130 135 140Phe Leu
Val Ile Met Ile Thr Pro His His Val Gly Val Asp Asp Tyr145 150
155 160Arg Gly His Trp Val Asp Pro Leu Phe Val Gly Gly Glu Cys Asp
Gln 165 170 175Ser Tyr Cys Asp Thr Ile His Asn Ser Ser Val Trp Ile
Pro Ala Asp 180 185 190Gln Thr Lys Lys Asn Ile Cys Gly Gln Ser Phe
Thr Pro Leu Thr Val 195 200 205Thr Val Ala Tyr Val Lys Thr Lys Glu
Ile Ala Ala Gly Ala Ile Val 210 215 220Phe Lys Ser Lys Tyr His Ser
His Met Glu Gly Ala Arg Thr Cys Arg225 230 235 240Leu Ser Tyr Cys
Gly Arg Asn Gly Ile Lys Phe Pro Asn Gly Glu Trp 245 250 255Val Ser
Leu Asp Val Lys Thr Lys Ile Gln Glu Lys Pro Leu Leu Pro 260 265
270Leu Phe Lys Glu Cys Pro Ala Gly Thr Glu Val Arg Ser Thr Leu Gln
275 280 285Ser Asp Gly Ala Gln Val Leu Thr Ser Glu Ile Gln Arg Ile
Leu Asp 290 295 300Tyr Ser Leu Cys Gln Asn Thr Trp Asp Lys Val Glu
Arg Lys Glu Pro305 310 315 320Leu Ser Pro Leu Asp Leu Ser Tyr Leu
Ala Ser Lys Ser Pro Gly Lys 325 330 335Gly Leu Ala Tyr Thr Val Ile
Asn Gly Thr Leu Ser Phe Ala His Thr 340 345 350Arg Tyr Val Arg Met
Trp Ile Asp Gly Pro Val Leu Lys Glu Met Lys 355 360 365Gly Lys Arg
Glu Ser Pro Ser Gly Ile Ser Ser Asp Ile Trp Thr Gln 370 375 380Trp
Phe Lys Tyr Gly Asp Met Glu Ile Gly Pro Asn Gly Leu Leu Lys385 390
395 400Thr Ala Gly Gly Tyr Lys Phe Pro Trp His Leu Ile Gly Met Gly
Ile 405 410 415Val Asp Asn Glu Leu His Glu Leu Ser Glu Ala Asn Pro
Leu Asp His 420 425 430Pro Gln Leu Pro His Ala Gln Ser Ile Ala Asp
Asp Ser Glu Glu Ile 435 440 445Phe Phe Gly Asp Thr Gly Val Ser Lys
Asn Pro Val Glu Leu Val Thr 450 455 460Gly Trp Phe Thr Ser Trp Lys
Glu Ser Leu Ala Ala Gly Val Val Leu465 470 475 480Ile Leu Val Val
Val Leu Ile Tyr Gly Val Leu Arg Cys Phe Pro Val 485 490 495Leu Cys
Thr Thr Cys Arg Lys Pro Lys Trp Lys Lys Gly Val Glu Arg 500 505
510Ser Asp Ser Phe Glu Met Arg Ile Phe Lys Pro Asn Asn Met Arg Ala
515 520 525Arg Val 53028611PRTJaagsietke sheep retrovirus 28Met Pro
Lys Arg Arg Ala Gly Phe Arg Lys Gly Trp Tyr Ala Arg Gln1 5 10 15Arg
Asn Ser Leu Thr His Gln Met Gln Arg Met Thr Leu Ser Glu Pro 20 25
30Thr Ser Glu Leu Pro Thr Gln Arg Gln Ile Glu Ala Leu Met Arg Tyr
35 40 45Ala Trp Asn Glu Ala His Val Gln Pro Pro Val Thr Pro Thr Asn
Ile 50 55 60Leu Ile Met Leu Leu Leu Leu Leu Gln Arg Ile Gln Asn Gly
Ala Ala65 70 75 80Ala Thr Phe Trp Ala Tyr Ile Pro Asp Pro Pro Met
Leu Gln Ser Leu 85 90 95Gly Trp Asp Lys Glu Thr Val Pro Val Tyr Val
Asn Asp Thr Ser Leu 100 105 110Leu Gly Gly Lys Ser Asp Ile His Ile
Ser Pro Gln Gln Ala Asn Ile 115 120 125Ser Phe Tyr Gly Leu Thr Thr
Gln Tyr Pro Met Cys Phe Ser Tyr Gln 130 135 140Ser Gln His Pro His
Cys Ile Gln Val Ser Ala Asp Ile Ser Tyr Pro145 150 155 160Arg Val
Thr Ile Ser Gly Ile Asp Glu Lys Thr Gly Met Arg Ser Tyr 165 170
175Arg Asp Gly Thr Gly Pro Leu Asp Ile Pro Phe Cys Asp Lys His Leu
180 185 190Ser Ile Gly Ile Gly Ile Asp Thr Pro Trp Thr Leu Cys Arg
Ala Arg 195 200 205Ile Ala Ser Val Tyr Asn Ile Asn Asn Ala Asn Thr
Thr Leu Leu Trp 210 215 220Asp Trp Ala Pro Gly Gly Thr Pro Asp Phe
Pro Glu Tyr Arg Gly Gln225 230 235 240His Pro Pro Ile Ser Ser Val
Asn Thr Ala Pro Ile Tyr Gln Thr Glu 245 250 255Leu Trp Lys Leu Leu
Ala Ala Phe Gly His Gly Asn Ser Leu Tyr Leu 260 265 270Gln Pro Asn
Ile Ser Gly Ser Lys Tyr Gly Asp Val Gly Val Thr Gly 275 280 285Phe
Leu Tyr Pro Arg Ala Cys Val Pro Tyr Pro Phe Met Val Ile Gln 290 295
300Gly His Met Glu Ile Thr Pro Ser Leu Asn Ile Tyr Tyr Leu Asn
Cys305 310 315 320Ser Asn Cys Ile Leu Thr Asn Cys Ile Arg Gly Val
Ala Lys Gly Glu 325 330 335Gln Val Ile Ile Val Lys Gln Pro Ala Phe
Val Met Leu Pro Val Glu 340 345 350Ile Thr Glu Glu Trp Tyr Asp Glu
Thr Ala Leu Glu Leu Leu Gln Arg 355 360 365Ile Asn Thr Ala Leu Ser
Arg Pro Lys Arg Gly Leu Ser Leu Ile Ile 370 375 380Leu Gly Ile Val
Ser Leu Ile Thr Leu Ile Ala Thr Ala Val Thr Ala385 390 395 400Ser
Val Ser Leu Ala Gln Ser Ile Gln Val Ala His Thr Val Asp Ser 405 410
415Leu Ser Ser Asn Val Thr Lys Val Met Gly Thr Gln Glu Asn Ile Asp
420 425 430Lys Lys Ile Glu Asp Arg Leu Pro Ala Leu Tyr Asp Val Val
Arg Val 435 440 445Leu Gly Glu Gln Val Gln Ser Ile Asn Phe Arg Met
Lys Ile Gln Cys 450 455 460His Ala Asn Tyr Lys Trp Ile Cys Val Thr
Lys Lys Pro Tyr Asn Thr465 470 475 480Ser Asp Phe Pro Trp Asp Lys
Val Lys Lys His Leu Gln Gly Ile Trp 485 490 495Phe Asn Thr Thr Val
Ser Leu Asp Leu Leu Gln Leu His Asn Glu Ile 500 505 510Leu Asp Ile
Glu Asn Ser Pro Lys Ala Thr Leu Asn Ile Ala Asp Thr 515 520 525Val
Asp Asn Phe Leu Gln Asn Leu Phe Ser Asn Phe Pro Ser Leu His 530 535
540Ser Leu Trp Arg Ser Ile Ile Ala Met Gly Ala Val Leu Thr Phe
Val545 550 555 560Leu Ile Ile Ile Cys Leu Ala Pro Cys Leu Ile Arg
Ser Ile Val Lys 565 570 575Glu Phe Leu His Met Arg Val Leu Ile His
Lys Asn Met Leu Gln His 580 585 590Gln His Leu Met Glu Leu Leu Asn
Asn Lys Glu Arg Gly Ala Ala Gly 595 600 605Asp Asp Pro 610
* * * * *