U.S. patent application number 12/277282 was filed with the patent office on 2010-05-27 for replication stable and rnase resistant chimeras of pestivirus with insertion in 3' nontranslated region (3'ntr).
Invention is credited to Sven-Erik Behrens, Erhard Ralf Schoenbrunner.
Application Number | 20100129902 12/277282 |
Document ID | / |
Family ID | 41698403 |
Filed Date | 2010-05-27 |
United States Patent
Application |
20100129902 |
Kind Code |
A1 |
Schoenbrunner; Erhard Ralf ;
et al. |
May 27, 2010 |
Replication Stable and RNase Resistant Chimeras of Pestivirus with
Insertion in 3' Nontranslated Region (3'NTR)
Abstract
The invention relates to the field of nucleic acid
amplification, particularly to quality control materials for use in
viral RNA assays. It specifically relates to the construction of a
recombinant Pestivirus by the identification of a region in the
3'NTR of the viral RNA genome where additional sequence elements
can be stably inserted. Chimeric Pestivirus with sequence
insertions in the 3' nontranslated region (3'NTR) of the viral RNA
genome were stable in replication and capable of forming
infectious, RNase resistant virus particles. This chimeric
Pestivirus with a 3'NTR insertion can be utilized as a quality
control material in analytical assays for RNA targets, including
external, internal controls, quantitative standards in PCR and NAT
nucleic acid assays.
Inventors: |
Schoenbrunner; Erhard Ralf;
(Moraga, CA) ; Behrens; Sven-Erik; (Halle/Saale,
DE) |
Correspondence
Address: |
ORRICK, HERRINGTON & SUTCLIFFE, LLP;IP PROSECUTION DEPARTMENT
4 PARK PLAZA, SUITE 1600
IRVINE
CA
92614-2558
US
|
Family ID: |
41698403 |
Appl. No.: |
12/277282 |
Filed: |
November 24, 2008 |
Current U.S.
Class: |
435/325 ;
435/320.1; 536/23.1 |
Current CPC
Class: |
C12N 2770/24211
20130101; C12N 2770/24311 20130101; C12N 7/00 20130101; C12N 15/86
20130101; C12N 2770/24322 20130101; C07H 21/02 20130101; C12N
2770/24222 20130101 |
Class at
Publication: |
435/325 ;
536/23.1; 435/320.1 |
International
Class: |
C12N 5/06 20060101
C12N005/06; C07H 21/02 20060101 C07H021/02; C12N 15/63 20060101
C12N015/63 |
Claims
1. A chimeric viral RNA sequence comprising: first virus RNA
sequence from Pestivirus, and second RNA sequence inserted within a
3' NTR of the first Pestivirus RNA sequence, wherein the chimeric
viral RNA sequence is stable in RNA replication and forms a
ribonuclease resistant viral particle.
2. The chimeric RNA viral sequence of claim 1, wherein the first
virus RNA sequence is bovine viral diarrhea virus (BVDV).
3. The chimeric RNA viral sequence of claim 2, wherein the first
virus RNA sequence is a non-CP7 clone of the bovine viral diarrhea
virus (BVDV).
4. The chimeric RNA viral sequence of claim 1, wherein the second
RNA sequence is inserted in a variable region of the 3' NTR of the
first Pestivirus RNA sequence.
5. The chimeric RNA viral sequence of claim 1, wherein the second
RNA sequence is inserted between a SL.sub.STOP stem-loop structure
and a SLII stem-loop structure in the 3'NTR of the first Pestivirus
RNA sequence.
6. The chimeric RNA viral sequence of claim 5, wherein the second
RNA sequence is inserted between a UGA.sub.pos.cons. box and the
SLII stem-loop structure in the 3'V of the 3'NTR.
7. The chimeric RNA viral sequence of claim 1, wherein the second
RNA sequence is a portion of an HCV virus.
8. The chimeric RNA viral sequence of claim 7, wherein the portion
of the HCV virus is comprised of at least 8 nucleotides.
9. The chimeric RNA viral sequence of claim 7, wherein the second
RNA sequence is a 5'NTR region of the HCV virus.
10. The chimeric RNA viral sequence of claim 1, wherein the second
RNA sequence is a multiplex sequence comprising sequences from more
than one RNA virus.
11. The chimeric RNA viral sequence of claim 1, wherein the second
RNA sequence is selected from the group consisting of HCV, HCV
genotypes 1-7, HIV, HIV-1, HIV-2, HTLV-1, HTLV-2, hepatitis G, an
enterovirus, a respiratory virus and a blood borne virus.
12. A vector comprising: DNA sequence of a chimeric viral RNA
sequence comprising: vector DNA sequence, first DNA sequence
derived from a first Pestivirus RNA sequence, and second RNA
sequence inserted within a 3' NTR of the first Pestivirus RNA
sequence, wherein the sequence of the chimeric RNA virus derived
from the DNA vector is stable in replication, and forms a RNase
resistant viral particle.
13. The chimeric RNA viral sequence of claim 12, wherein the second
RNA sequence is inserted in a variable region of the 3' NTR of the
first Pestivirus RNA sequence.
14. The chimeric RNA viral sequence of claim 12, wherein the second
RNA sequence is inserted between a SL.sub.STOP stem-loop structure
and a SLII stem-loop structure in the 3'NTR of the first Pestivirus
RNA sequence.
15. The chimeric RNA viral sequence of claim 12, wherein the second
RNA sequence is at least a portion of an HCV virus.
16. The chimeric RNA viral sequence of claim 15, wherein the second
RNA sequence is at least 8 nucleotides of the HCV virus.
17. The chimeric RNA viral sequence of claim 15, wherein the second
RNA sequence is from a 5' NTR region of the HCV virus.
18. A culture of cells containing RNA virus comprising: host cell
harboring chimeric first and second RNA virus sequences, wherein
the first RNA virus is Pestivirus and a second RNA sequence is
inserted in a 3' NTR of the first Pestivirus virus RNA sequence,
wherein the chimeric RNA viral sequence is stable in RNA
replication, and ribonuclease resistant viral particles comprising
the first and second RNA virus sequences when cultured.
19. The culture of claim 18, wherein the second RNA sequence is
inserted in a variable region of the 3' NTR of the first Pestivirus
RNA sequence.
20. The culture of claim 18, wherein the second RNA sequence is
inserted between a SL.sub.STOP stem-loop structure and a SLII
stem-loop structure in the 3'NTR of the first Pestivirus RNA
sequence.
21. The culture of claim 18, wherein the second RNA sequence is
from an HCV virus.
22. The culture of claim 21, wherein the second RNA sequence is at
least 8 nucleotides of the HCV virus.
23. The culture of claim 21, wherein the second RNA sequence is
from a 5' NTR region of the HCV virus.
24. The culture of claim 18, wherein the viral particles are
infectious.
25. The culture of claim 18, wherein the viral particles are
non-infectious.
Description
BACKGROUND OF THE INVENTION
[0001] An RNA virus has RNA (ribonucleic acid) as its genetic
material, and infects host cells from bacteria, plants or animals,
such as livestock and humans. The major criteria of how RNA viruses
are classified are the sense and organization of the viral genome
that determines the mode of viral RNA replication, including
whether the viral RNA genome has positive (message) or negative
sense, whether it is single or double stranded, and whether it is
non-segmented or segmented.
[0002] Regulatory agencies often require that assays for detection
of nucleic acids utilize quality control materials, including
standards, calibrators and controls (Molecular Diagnostic Methods
for Infectious Diseases; Approved Guideline, 2nd ed., Clinical and
Laboratory Standards Institute, vol 26 (8), 2006). Quality control
materials insure optimum performance and reliability of test
results, including nucleic acid test (NAT) assays. Laboratories are
required to demonstrate that assays for detection of viral RNA
function properly as intended and are not affected by inhibition or
other forms of interference. Controls for qualitative assays
provide assurance of true negative and positive results while
minimizing the chance for false positive and false negative
results. In quantitative assays controls help ensuring accuracy of
results. Validation and verification panels are frequently used to
check if diagnostic assays or systems (hardware, software and
reagents) perform as intended. A panel consists of more than one
vial of material useful to improve or check, verify or validate the
performance or quality of a diagnostic assay. A panel can, for
example, be made from a dilution series that contains different
concentrations of an analyte. Calibrators are used to calibrate and
recalibrate a diagnostic system, usually after an element of the
diagnostic system was changed (e.g. new lot of reagents) or after a
predetermined time interval.
[0003] Ideally, the quality control material is as similar as
possible in structure and morphology to the target analyte so both
behave the same when tested. If NAT controls, calibrators or
standards behave like a patient sample on different diagnostic
systems, they are considered "commutable" amongst these systems.
Commutability is a key property of quality control materials that
is especially important for calibrators and standards. The quality
control material, however, should still be able to generate a
signal that is distinguishable from that of the target analyte.
Typically, viral RNA assays may have external run controls
(positive or negative control) (EC), various types of internal
controls (IC), or internal quantification or quantitation standards
(QS), as well as calibrators. Internal and external control
concepts are further described in CLSI Guideline MM3-A2 and U.S.
Pat. Nos. 7,183,084 B2 and 7,192,745.
[0004] While PCR and other NAT techniques can test both DNA and
RNA, there are technical challenges especially with quality control
for RNA assays: 1) RNA is generally more labile than DNA,
presenting additional technical difficulties for analytical RNA
assays as compared to DNA assays. Naked RNA is sensitive to
degradation through RNases, ubiquitously present RNA digesting
enzymes. RNases can be found almost everywhere in the environment,
however, they are especially prevalent in animal cells and fluids.
In order to fully quality control all steps of an RNA assay, it is
best to protect the RNA of the quality control material from
potential degradation. An intact naturally occurring RNA virus,
which does protect its RNA inside the virus, may be used as a
calibrator or external positive run control, as long as its RNA
sequence contains the primer and probe regions of the target virus.
It may not be combined, however, with the test sample to be used as
an internal control or quantitative standard, because it would
cause a false positive signal. 2) It is desirable that a RNA
quality control material should be capable of monitoring the entire
diagnostic process and serve as a "full process control", including
nucleic acid isolation, reverse transcription, amplification and
detection. 3) The use of materials potentially infectious for
humans is not desirable in a diagnostic kit due to safety concerns
and shipping regulations. 4) Internal Quality Standards (IQS) like
Internal Controls (IC) and internal Quantification or Quantitation
standards (QS) materials often can not be obtained from naturally
occurring sources. The term "QS" is used in the literature as
abbreviation for Quantification Standard, Quantitation Standard,
internal Quantification Standard or internal Quantitation Standard,
essentially all describing the same type of standard (see Clinical
and Laboratory Standards Institute, CLSI Guideline MM3-A2 for
details of QS use). This is particularly true for any IQS used as a
"competitive" control, which utilizes the same primer sequence as
the target RNA, but can be distinguished by a probe sequence
different from the target sequence. Such IQS materials usually need
to be artificially created. 5) RNA itself is not as amenable to
recombinant genetic engineering as DNA and usually requires a DNA
intermediate. While it is known to transcribe RNA sequences from
recombinant DNA sequences, it is difficult to package and protect
these RNA transcript sequences from degradation by RNases.
[0005] One approach to solve the issues of RNA instability for
quality control materials for RNA viral testing has been the use of
"armored RNA," recombinant RNA packaged in MS2 bacteriophage (e.g.,
see U.S. Pat. Nos. 5,677,124, and 7,033,749). Armored RNA has a
single strand of MS2 RNA containing a recombinant heterologous RNA
encapsidated by MS2 bacteriophage proteins to form a pseudo-viral
particle.
[0006] Armored RNA offers several advantages over naked RNA as a
quality control material: 1) It is stable, non-infectious and RNase
resistant, and 2) It is useful both as a positive control, an
internal control (IC) and quantification standard (QS), and an
extraction control. Armored RNA has been useful for the
quantification of RNA viruses such as HIV and HCV from human blood.
Armored RNA also is utilized as a high titer HCV surrogate
material, since it is not possible to grow all different HCV
subtypes in culture.
[0007] Armored RNA has several disadvantages for use as a quality
control material in analytical assays. Ideally, quality control
materials should react like the tested analyte in an assay in order
to monitor meaningfully all aspects of the procedure. The structure
and infected host for armored RNA, however, is very different from
that of many animal RNA viruses, e.g. HCV or HIV. A bacteriophage,
which infects bacteria, is genetically distant to animal or other
eukaryotic viruses. MS2 bacteriophage is not detergent sensitive,
because it has a protein coat instead of a lipid bilayer. Many
diagnostically relevant enveloped viruses causing harm to humans
and livestock (e.g. HIV, Pestiviruses, West Nile Virus (WNV) or
HCV) possess detergent sensitive outer envelopes. Because the
armored RNA protein coat is very different biochemically from the
lipid envelope of these animal viruses, the MS2 bacteriophage
particles may behave differently from the targeted animal viral
particles in analytical assays. It is conceivable that an Armored
RNA QS used to monitor a reaction would fail to indicate that HIV
had been destroyed by detergent which contaminated a blood donor
test sample. In this case the result could be a false negative
blood screening result for HIV resulting in the transfusion of HIV
positive CD4 blood cells to several recipients. Armored RNA has
never been shown to be commutable, meaning that it behaves like the
virus it is supposed to mimic as a quality control. 4) Recovery
efficiency of RNA with the most commonly used silica based sample
preparation methods is to a certain degree dependent on the length
of the RNA. Most human RNA viruses, such as HCV, HIV or WNV, have
about three times longer RNA genomes than armored RNA.
[0008] Although armored RNA has been useful as a control in
analytical assays, it would be preferable to have a quality control
material for in vitro diagnostic assays that more closely resembles
the targeted animal or human RNA virus than a bacteriophage derived
pseudo-viral particle. It would be preferable to have quality
control material that is structurally and morphologically similar
to the target RNA virus and thus behaves more like e.g. HCV, West
Nile Virus (WNV) or HIV viruses. Finally, it would be preferable to
have the option of being able to insert heterologous sequences into
replication competent viruses.
[0009] Chimeric RNA viruses that are similar in structure to the
virus being tested would be an ideal quality control material for
RNA assays if they were genetically stable and could be grown in
culture. In this method, a region of a targeted virus is inserted
into the genome of another virus to form a chimeric virus. By
testing for the inserted target region, the chimeric virus can
function as a quality control material.
[0010] In designing a stable viral chimera, it is important to
identify a point of insertion in the compact viral genome that does
not interfere with the viability of the virus. It is known that the
choice of the target region to be inserted, as well as the site for
the insertion can dramatically affect chimeric RNA viral functions,
especially RNA replication, packaging of the RNA genome, virion
stability, and virus infectivity. If the chimeric viral RNA
replicates improperly, spontaneous sequence changes, such as
deletions or frameshifts, may occur during replication in the RNA
sequence of the virus chimera to form useless sequence revertants
or pseudo-revertants. The ultimate genomic sequence of the
revertant virus is unpredictable and may exclude part or parts of
the applied insert. Unstable chimeric RNA virus, therefore, is
usually not useful as a quality control material in an analytical
RNA assay.
[0011] Examples of positive-strand ssRNA chimeras are known that
utilize the 5' nontranslated region (5'NTR) and the open reading
frame (ORF). Martin disclosed chimeric GBV-B/HCV (U.S. Pat. No.
7,141,405; US2006/0160067; US2006/0105365). Ilya et al. disclosed
chimeric Eastern Equine Encephalitis virus and Sindbis virus (WO
2007/002793). Hong et al. describe HCV/BVDV chimeric constructs
where the N.sup.pro protease gene is replaced (U.S. Pat. No.
6,326,137). Nam et al. disclosed HCV/BVDV constructs involving
exchange of structural genes, especially E1, E2 or C. (U.S. Pat.
No. 7,009,044). Rice and Kolykhalov (U.S. Pat. No. 6,127,116)
disclosed that functional HCV clones can be used for the assay of
HCV by constructing chimeric viruses using components of the IRES,
proteases, RNA helicase, polymerase, or 3'NTR to create chimeric
derivatives of BVDV whose productive replication is dependent on
one or more of these HCV elements. None of these examples, however,
disclosed stable Pestivirus RNA chimeras with insertion within the
3'NTR.
[0012] While the general concept of chimeric RNA viral constructs
is known, no functional examples are provided in the prior art that
utilize intentional insertion of heterologous sequences within the
3'NTR of a Pestivirus to obtain a chimeric virus that is
replication stable and forms infectious viral particles at titers
comparable to that of a wild type virus.
[0013] Rice et al. disclosed a concept of constructing BVDV
chimeras with inserted sequences from HCV (WO 99/55366; see also
Frolov et al., 1998, RNA 4, 1418-1435). No data were given,
however, that showed their chimeric constructs involving the 3'NTR
were genetically stable. In Example 5 (WO 99/55366), Rice et al
described a tandem 3'NTR construct where an HCV 3'NTR insert was
placed downstream of the ORF and immediately followed by the intact
3'NTR of BVDV (FIG. 19). Rice et al. reported this 3'NTR HCV-BVDV
tandem construct replicated poorly and revertants formed, which
showed deletions when sequenced (FIG. 20). Significantly, Rice et
al placed the HCV 3'NTR insert precisely downstream of the stop
codon of the ORF of BVDV, not within the 3'NTR of BVDV. Rice et
al., therefore, did not construct a replication competent BVDV
chimera with insertion within the 3'NTR that was genetically stable
and could be grown in culture.
[0014] Recently, progress has been made in the development of
(copy) cDNA clones of full-length BVDV genomes. These so-called
"infectious BVDV cDNA clones" allow the in vitro transcription of
infectious BVDV RNA genomes (Meyers, et al., J. Virology, 1996, 70:
8606-8613, erratum in J. Virol. 1997, 7 (2): 1735; Vassilev, et
al., J. Virology, 1997, 71: 471-478; U.S. Pat. No. 6,001,613).
[0015] There is a need in RNA viral testing of Flaviviridae and
other RNA viruses to have viral quality control material that is
similar in genomic composition and virion composition to the RNA
virus being tested.
[0016] There is a need in nucleic acid test assays (NAT) for QC
materials, standards, calibrators, reference materials, validation
and verification material that behave like the target material.
[0017] There is a need for such RNA viral quality control materials
to be stable in sequence, to be replication stable and to be
resistant to degradation by RNases.
[0018] It would be useful to have a known region within the 3'NTR
of a Flaviviridae/Pestivirus virus defined that allows the stable
insertion of heterologous sequences.
SUMMARY OF THE INVENTION
[0019] This invention provides the identification of the location
of a region within the 3'NTR of the genomic RNA of a Flaviviridae
member, the Pestivirus BVDV, where a heterologous RNA sequence can
be inserted to generate stable viral RNA chimeras and stable
chimeric virus particles. Similar to the wild-type BVDV virus
particles, the chimeric virus particles with a heterologous
insertion in the 3'NTR of the viral RNA genome are sequence stable,
replication-competent, resistant to RNases, and infectious.
[0020] Useful and stable Pestivirus RNA chimeras are constructed
from an understanding of the viral RNA genome to identify areas
within the 3'NTR for stable insertion of heterologous sequences. In
this invention, a defined area within the 3'NTR of the genomic RNA
of a Flaviviridae member, the Pestivirus BVDV, is used to create a
stable chimeric virus, which was demonstrated to be useful in an
analytical viral assay. A stable chimeric Pestivirus can be
utilized similar to the ways in which armored RNA is used in
diagnostic and analytical assays. Current armored RNA applications
are known, and also have been described in U.S. Pat. Nos.
5,677,124, and 7,033,749 and their dependent patents. Unlike
armored RNA, however, the chimeric Pestivirus is viral quality
control material that is similar in genomic composition and virion
composition to the RNA virus being tested.
[0021] In this invention we used our current understanding of the
detailed "signal" function of the different elements of the 3'NTR
of Pestivirus genomic RNA to generate a stable replication
competent BVDV chimera containing a heterologous sequence within
the 3'NTR of the genomic RNA. In a preferred embodiment, the
heterologous insertion is made within the 3' NTR variable region
(3'V). Most preferably, the insertion is made in a defined sequence
region located downstream of the UGA.sub.pos.cons. box (Isken et
al., 2004). This insertion site is located downstream of the region
that was experimentally shown with the BVDV DI9c replicon RNA
(Isken et al., 2004) to form the SL.sub.stop (SLIII) structure and
that is proposed by RNA folding programs such as mfold 3-1
(http://www.bioinfo.rpi.edu/.about.zukerm/export/) to form
SL.sub.stop (SLIII) with the genomic RNA of all other Pestivirus
members. The insertion site is located upstream of the region that
was experimentally shown with the BVDV DI9c replicon RNA to form
the SLII structure and that is proposed by the RNA folding program
mfold 3-1 to fold SLII with the genomic RNA of all other Pestivirus
members. (Grassmann, C., Yu, H., Isken, O., and Behrens, S.-E.
(2005). Hepatitis C virus and the related bovine viral diarrhea
virus considerably differ in the functional organization of the 5'
non-translated region: implications for the viral life cycle.
Virology 333: 349-366.; Isken, O., Grassmann, C. W., Sarisky, R.
T., Kann, M., Zhang, S., Grosse, F., Kao, P. N., and Behrens, S.-E.
(2003). Members of the NF90/NFAR protein group are involved in the
life cycle of a positive-strand RNA virus. EMBO J. 22: 5655-5665.;
Isken, O., Grassmann, C. W., Yu, H., and Behrens, S.-E. (2004).
Complex signals in the genomic 3' non-translated region of bovine
viral diarrhea virus coordinate translation and replication of the
viral RNA. RNA 10: 1637-52.; Isken, O., Baroth, M., Grassmann, C.
W., Weinlich, S., Ostareck, D. H., Ostareck-Lederer, A. and
Behrens, S.-E. (2007). Nuclear factors are involved in hepatitis C
virus RNA replication. RNA 13: 1675-1692.)
[0022] The location of the UGA.sub.pos.cons. box is conserved in
the genomes of all Pestivirus 3'NTRs (Becher, et al., J. Virology,
1998, 72 (6): 5165-73.; Isken, et al., 2003; Isken, et al., 2004).
Given the general structural alignment of the UGA.sub.pos.cons.
box, the stem-loop structures SL.sub.stop (SLIII), SLII and SLI,
and other structural elements within the 3'NTR of Pestivirus, the
construction of chimeric virus is not only applicable to the
Pestivirus BVDV but to all Pestivirus members.
[0023] This invention provides RNA viral chimeras with insertions
in the 3'NTR as quality control material, including use as
controls, standards and calibrators in RNA viral analytical NAT
assays.
BRIEF DESCRIPTION OF THE FIGURES AND TABLES
[0024] FIG. 1 shows a comparison of the genomic organization of
Hepacivirus HCV and Pestivirus BVDV viruses. The 5' NTR (IRES) and
3'NTRs are indicated as single lines, and the open reading frame
(ORF) for the viral polyprotein is schematized as boxes, with the
different viral proteins indicated within the boxes. The enzymatic
activities that proteolytically mature the viral polyprotein are
designated as autoprotease (A); cellular signalase (closed oval),
and viral protease (arrow).
[0025] FIG. 2 is a schematic diagram of the secondary structures of
the HCV and BVDV NTRs flanking the ORF, which is shown as a box.
The area of the HCV 5'NTR is enclosed with a long-dashed line, and
the area of the BVDV 3'NTR is indicated with a dotted line. An
arrow indicates that the HCV 5'NTR was inserted within the region
of the 3'NTR of BVDV.
[0026] FIG. 3 shows a schematic of the secondary structure of the
3'NTR of Pestivirus BVDV and of proposed functions of this region
during viral replication. The sequence (taken from the BVDV
replicon construct DI9c; Behrens et al., 1998; Behrens, S.-E.,
Grassmann, C. W., Thiel, H.-J., Meyers, G., and Tautz, N. 1998
Characterization of an autonomous RNA replicon of a Pestivirus. J.
Virol. 72: 2364-2372) reads 5' to 3' in direction. The UGA
translational stop codon and the so-called "pseudo-stop codons" are
boxed with a thin line, and the UGA boxes found in this isolate are
boxed with a heavy line. The stem-loop structures, which were
determined experimentally, are labeled SL.sub.stop, SLII and SLI
(Isken et al., 2004, Yu et al., 1999; Yu, H., Grassmann, C. W., and
Behrens, S.-E. 1999 Sequence and structural elements at the 3'
terminus of the bovine viral diarrhea virus: functional role during
RNA replication. J. Virol. 73: 3638-3648). The 3' variable (V)
region and the 3' constant (C) region are indicated; the border is
indicated by an arrow (according to sequence alignments by Deng and
Brock, 1993 (Deng R, Brock K V. Nucleic Acids Res. 1993 Apr.
25:21(8):1949-57). A translating ribosome is indicated by a double
structure at the 5' end of the 3'NTR, indicating that ribosomes
terminate at the position of the translation termination codon. The
following double line indicates that the 3'V region is involved in
efficient termination of translation at the translational stop
codon (Isken et al., 2004). The NFAR protein binding site is
indicated by a group of circles, and the site where the viral
replication complex presumably forms is indicated by a large oval
(Isken et al., 2004. The heterologous RNA sequence may be inserted
within the 3'NTR. A preferred site of insertion of the HCV 5'NTR
within the 3'NTR is indicated by a thick arrow.
[0027] FIG. 4 is a schematic diagram of the 3'NTR of Pestivirus
BVDV. As in FIG. 3, the sequence and secondary structure
information are derived from the BVDV D19c replicon (Behrens et
al., 1998; Yu et al., 1999). The 3' variable [3'V (1-90 nt)] and
the 3' constant [3'C regions (91-192 nt)] are indicated. The
stem-loop structures: SL.sub.STOP, SLII and SLI are labeled. The
UGA translational stop codon and the pseudo-stop codons are boxed.
The UGA box motifs within the 3'V are shown boxed with a heavy
line, including the 5'UGA box, the UGA.sub.pos.cons. box and the
3'UGA-like box (Isken et al., 2003; Isken et al., 2004). A
preferred site of insertion of the HCV 5'NTR within the 3'NTR is
indicated by a thick arrow.
[0028] FIG. 5A shows a schematic sequence alignment of the 3'NTRs
of various Pestivirus strains, including BDV X818, CSFV C-strain,
CSFV Alfort-T, BVDV type 1 NADL, BVDV type 1 Osloss and BVDV type 2
890. For all these Pestivirus strains, the positions that form the
stem-loop structures SL.sub.stop (SLIII) and SLII are indicated.
Nucleotides that are 100% conserved between the different strains
are indicated with an asterisk (*). The UGA.sub.pos.cons. box is
boxed. A thin arrow indicates a preferred site between the
SL.sub.stop (SLIII) and SLII stem-loop structures for the insertion
of heterologous RNA sequence within the 3'NTR. A thick arrow
indicates the border between the 3'V and 3'C regions within the
3'NTRs (Deng and Brock, 1993). FIG. 5B shows a sequence alignment
of UGA box sequences in the 3'NTRs of various Pestivirus strains
(Isken et al., 2003). 100% conserved nucleotides are boxed. The
consensus sequence for the twelve nucleotides from this sequence
alignment of the UGA boxes is indicated.
[0029] FIG. 6 shows the cDNA sequence of the BVDV-non-CP7-HCV 5'NTR
chimera with the restriction endonuclease sites for ClaI, part of
SmaI, SnaBI and PacI indicated. The nucleotide exchanges (c changed
into t) are also indicated.
[0030] FIG. 7 shows the cDNA sequence of the BVDV-non-CP7+ cloning
site chimera with the restriction endonuclease sites for ClaI, part
of SmaI, SnaBI and PacI indicated.
[0031] FIG. 8 shows the sequence compositions of the 3'NTR for: (A)
Pestivirus BVDV-non-CP7, (B) the BVDV-non-CP7+ cloning site
chimera, and (C) BVDV-non-CP7-HCV 5'NTR chimera. Provided are the
sequences of the 3' non-translated regions (3'NTRs) of: the BVDV
non-CP7 cDNA (starting material; wild type) (FIG. 8A), the BVDV
non-CP7 cDNA+ cloning site chimera (FIG. 8B), which is a
recombinant cDNA containing the inserted SnaBI and PacI restriction
sites at pos 12134-12148), and) the recombinant cDNA of the
Pestivirus BVDV-non-CP7-HCV 5'NTR chimera (FIG. 8C) containing the
inserted HCV 5'NTR (HCV sequence subtype Ib (Lohmann et al.,
Science 1999 Jul. 2: 285 (5424): 110-3) plus SnaBI and PacI sites
inserted at pos. 12134-12492. Note that two SmaI sites in the HCV
insert were inactivated by two site-directed nucleotide exchanges
(c to t exchanges at positions 12269 and 12456 of the
BVDV-non-CP7-HCV 5'NTR chimera sequence; see SEQ ID NO: 2 for the
cDNA of HCV 5'NTR). This was done to enable linearization of the
chimeric BVDV cDNA construct by a single SmaI site at the 3'-end of
the cDNA for run-off in vitro transcription by SP6 RNA polymerase.
The translation stop codon (tga) is boxed; pseudo-stop codons are
underlined; restriction endonuclease sites are bracketed, the
restriction endonuclease sites SnaBI (tacgta) and PacI (ttaattaa)
are indicated; the UGA-box motif sequences are shown in bold and
boxed with dashed lines; the 5'NTR HCV insert is shown in a large
boxed region. Sites that were added or modified, such as the taa
site following the Pac site, are indicated in italics.
[0032] FIG. 9 shows growth curves obtained for the BVDV-non-CP7
(open diamonds) and the BVDV-non-CP7-HCV 5'NTR chimera (open
circles).
[0033] FIG. 10 shows growth curve data in bar graph format for the
BVDV-non-CP7 (open bar) and the BVDV-nonCP7-HCV-5'NTR chimera
(cross-hatched bar).
[0034] FIG. 11 shows sequence information obtained for five viral
isolates following seven passages of re-infections. Following
RT-PCR of total viral RNA isolated from infected cells after seven
(7) passages of re-infections, the PCR fragment was cloned using
BamHI and XbaI. Five clones were sequenced. The details of the
sequence changes observed in these five clones are shown in FIGS.
11A-11E.
[0035] FIG. 12 shows the signals obtained for the use of Pestivirus
BVDV-non-CP7-HCV 5'NTR chimera and a known HCV control as a
calibrator. The measured Ct values for both calibration panels were
plotted against the log of the expected international units (IU).
The equations of the linear regression fit lines were equivalent
within the measurement error: y=-3.80 x+44.0 for HCV-BVDV;
y=-3.82x+44.3 for OptiQuant HCV.
[0036] FIG. 13 shows the results of linear regression analysis of a
West Nile Virus (WNV assay) with HCV-BVDV chimeric RNA as an
internal quantification standard (QS). A slope of 2.891 and a
coefficient of determination of 0.996 was obtained.
[0037] Table 1 shows virus titers obtained for BVDV-non-CP7 and
BVDV-non-CP7+ cloning site chimera at three days post infection
(1st re-infection).
[0038] Table 2 shows virus titers obtained for BVDV-non-CP7 and the
BVDV-non-CP7-HCV 5'NTR chimera at three days post transfection of
viral RNA into MDBK cells. Moreover, virus titers are shown at
three days post infection of MDBK cells measured in the course of
several re-infection experiments with virus-containing supernatant
as indicated. Up to seven re-infection experiments were
performed.
[0039] Table 3 shows the data for growth curves shown in FIGS. 10
and 11.
[0040] Table 4 shows the non-infectivity data obtained for the
BVDV-non-CP7-HCV-5'NTR chimera at various times and at three
different temperatures of treatment with indicated concentrations
of .beta.-propiolactone. Immunofluorescence data (IF) measuring
viral replication are indicated as + (viral replication detectable)
and - (no viral replication detectable due to inactivation by the
indicated concentration of .beta.-propiolactone).
[0041] Table 5 shows the determination of the titer of the HCV
signal of a Pestivirus BVDV-non-CP7-HCV 5'NTR chimera culture.
Positive HCV signal was obtained using real-time PCR amplification
for two Pestivirus RNA chimeras (BVDV-nonCP7-HCV 5'NTR, K4 and K8)
and an HCV control. K4 and K8 Pestivirus RNA chimeras both showed
an early Ct high titer HCV signal, indicating successful
integration of the HCV 5'NTR into BVDV.
[0042] Table 6 shows the HCV signal obtained using real-time PCR
amplification for two Pestivirus RNA chimeras and an HCV control
with and without reverse transcriptase (RT) Results of a reverse
transcriptase (RT) based PCR demonstrated that Pestivirus RNA
chimera HCV signal was derived from RNA, not DNA.
[0043] Table 7 shows the resistance of the Pestivirus chimera
BVDV-non-CP7-HCV 5'NTR to ribonuclease (RNase) digestion. Ct values
are shown for two Pestivirus RNA chimeras and an HCV control
without RNase treatment, and when RNase was present before or after
extraction.
DETAILED DESCRIPTION OF THE INVENTION
[0044] RNA viruses are prevalent human and animal pathogens.
Analytical NAT assays have been developed to allow detection of RNA
viruses in infected individuals. A large group of RNA viruses are
the positive-strand ssRNA viruses (Baltimore Group IV), which have
their viral genome directly utilized as an mRNA. Positive-strand
ssRNA viruses include many families of viruses that severely impact
health and function in livestock and humans, such as the common
cold virus (Rhinovirus), Poliovirus, the Hepatitis A and C viruses,
the Dengue and Yellow fever viruses, and the SARS virus. Currently,
safe and reliable analytical assays for the detection of RNA
viruses are needed, including quality control materials for these
assays.
[0045] The family Flaviviridae in Group IV includes three known
genera, two of which affect humans. The genus Flavivirus includes,
for example, Yellow fever virus, West Nile virus, Dengue fever
virus, St. Louis encephalitis virus, Tick-borne encephalitis virus,
and Japanese encephalitis virus. The genus Hepacivirus includes the
Hepatitis C viruses (HCVs). The third genus Pestivirus affects
livestock, and includes Classical Swine Fever Virus (CSFV), Border
Disease Virus (BDV) and Bovine Viral Diarrhea Virus (BVDV). While
each Flaviviridae member has a definitive host (cell) tropism and
disease specifics, each also shares a significant degree of
structural relatedness for the family Flaviviridae. In particular,
each such member has a similar virus morphology and high degree of
conservation of the genomic organization.
[0046] Recognition of the genomic structural similarities within
the Flaviviridae family has allowed the opportunity of
cross-utilization of intact Flaviviridae viral genomes for research
and medical diagnosis purposes. For example, BVDV has been widely
accepted as a surrogate virus for HCV inactivation studies. HCV
infects humans, but, so far, only certain genomic subtypes or
hybrid forms of these subtypes grow well in cell culture. The
genome structure of BVDV, however, is generally similar to that of
HCV, and BVDV does not infect humans. The use of a genome of a
surrogate virus as quality control (QC) material in analytical
assays, however, is not always applicable. Quality control
materials for diagnostic RNA nucleic acid tests (target or signal
amplification based) are often based on purified "naked" RNA or
surrogate materials like Armored RNA. It would be preferable to
have a quality control material that is as similar as possible to
the target analyte so that all steps of the analytical process are
mimicked as closely as possible.
[0047] The genome of the members of the Flaviviridae family is a
single stranded, non-segmented RNA ranging in length from about 10
kb to 16 kb. The viral RNA contains a long open reading frame (ORF)
encoding at least three major viral structural proteins and at
least six nonstructural proteins. The ORF is flanked by 5' and
3'-nontranslated regions (5'NTR and 3'NTR) that range in length
from about 100 to several hundred nucleotides (Lindenbach et al.,
Flaviviridae: the viruses and their Replication, in Fields
virology, 5th ed., Knipe et al., eds., Lippincott, 2007, p
1101-52).
[0048] There is complexity in the functions of the Flaviviridae
RNA. The viral RNA genome interacts with the host cell translation
machinery to serve as a messenger RNA for the translation of viral
proteins, as well as functioning as a template for viral RNA
replication. Both the 5'NTR and 3'NTR are rich in defined RNA
structure motifs known to be important for both the viral RNA
translation and viral RNA replication functions (Lindenbach et al.,
see above). Along both processes, the NTRs are believed to
functionally interact with each other as well as with host-cell
encoded regulatory factors, such as the NFAR proteins (Isken et
al., 2003; 2004; 2007). The 3'NTR of Flaviviridae is generally
composed of a variable (less conserved) region (3'V) and a constant
(conserved) region (3'C) (Deng and Brock, 1993). With different
members of each Flaviviridae genus, the 3'C was shown to contain
RNA elements that are essential for viral RNA replication while
parts of the 3'V region were indicated to be dispensable for
replication (reviewed by Isken and Behrens, 2006, in Molecular
Biology of the Flavivirus; Horizonbioscience, p 101-134). Detailed
investigations of the 3'V region of the BVDV genome revealed that
3'V, as a whole, is not dispensable for viral replication and that
it acts as an important modulator of the viral translation and
replication process (Isken et al., 2003; Isken et al. 2004).
[0049] The 3'NTRs of pestiviruses have a length of approximately
190-230 nucleotides and display a high degree of similarity between
the different virus members. The 3'V portion of the Pestivirus
3'NTR has defined recognizable regions. Structurally, while there
is sequence heterogeneity between different Pestivirus strains, the
pestiviral 3'V region generally forms two thermodynamically
unstable stem-loop structures, termed SL.sub.stop (also SLIII) and
SLII. It also contains at least one copy of a 12 nucleotide
consensus sequence designated as a UGA box motif (Isken et al 2003;
2004; Pankratz et al., 2005, J Virol. 79:9119-27). Further, there
is conservation within the pestiviral (and all Flaviviridae) 3'V
regarding the presence of so-called pseudo-stop codons. The
pseudo-stop codons are nucleotide triplets in the 3'V region of the
3'NTR that resemble translational stop codons, which are present
`in frame` (following the translational triplet code) with the
viral open reading frame. Mutational and structural analysis showed
that the integrity of the 3'V region and the presence of UGA boxes
and pseudo-stop codons are important for the binding of the NFAR
host proteins and for accurate termination of translation of the
viral RNA, respectively. In summary, these factors were
demonstrated to represent essential determinants of the viral RNA
replication process (Isken et al, 2003; Isken et al., 2004).
[0050] The structurally complex and conserved 5'NTR and 3'NTR of
Flaviviridae present target regions that may be utilized for the
detection of these RNA viruses in analytical assays. This is
particularly true for analytical methods based on target
amplification, such as polymerase chain reaction (PCR) or
transcript mediated amplification (TMA). The 5' NTR of the genus
Flavivirus has here been commonly used as a target sequence for
assays like HCV (Nolte F S, Green A M, Fiebelkorn K R, Caliendo A
M, Sturchio C, Grunwald A, Healy M. Clinical evaluation of two
methods for genotyping hepatitis C virus based on analysis of the
5' noncoding region. J Clin Microbiol. 2003 April; 41(4):1558-64.)
or West Nile virus (WNV). (Detection, Validation and Quantification
of West Nile Virus RNA by the Alternative NAT WNV Assay V.
Shyamala, S. Pichuantes, B. Jaitner, D. Madriaga, P. Arcangel, J.
Cottrell, S. Nguyen, H. Huang, A. Medina-Selby, D. Coit, C. McCoin,
D. Chien, B. Phelps. AABB Poster 2003
(http://www.chiron.com/docs/library/posters/aabb2003posters/aabb2003poste-
rs.sub.--5.pdf)
[0051] The detection of Group IV animal viruses uses known nucleic
acid amplification techniques, such as polymerase chain reaction
(PCR), Nucleic Acid Sequence Based Amplification (NASBA),
Transcription-Mediated Amplification (TMA), other nucleic acid
amplification technologies. Also signal amplification technologies
like bDNA, can be used in medical research and in clinical
diagnosis. Modern NAT testing allows sensitive detection of small
amounts of RNA or DNA from viruses, bacteria and other cells or
tissues. NAT testing usually consist of a series of sequential
steps: sample preparation (purification and concentration of RNA or
DNA), amplification of DNA (e.g. by PCR or TMA) and detection (e.g.
by using signal generating fluorescence probes, bound free
separation and detection of amplicon or gel band analysis). In the
case of RNA, an additional reverse transcription step converting
RNA into cDNA is required before amplification. Meaningful quality
control of NAT assays requires monitoring of every step of this
process. Typically, external and internal controls are used to
quality control NAT assays.
[0052] One embodiment provides for the formation of stable virus
chimeras by insertion of donor heterologous sequences into the 3'
nontranslated region (3'NTR) of the RNA genome of the Flaviviridae
Pestivirus. The chimeric RNA genomes of the Flaviviridae Pestivirus
are sequence stable, replication competent, infectious and the
genome encoded and assembled chimeric virus particles are RNase
resistant.
[0053] The heterologous insert can be located within the Pestivirus
3'NTR. In a preferred embodiment, the insertion is within the 3'
variable region (3'V) of the 3'NTR. Most preferably, the
heterologous insert of the chimera is located between two stem-loop
structures (SL.sub.STOP and SLII) within the 5'-terminal portion of
the 3'NTR of the Pestivirus RNA. The 3'NTR of the Pestivirus
chimera viral RNA contains pseudo stop-codons. The 3'NTR of the
Pestivirus chimera viral RNA contains binding sites for the NF/NFAR
proteins. The 3'NTR of the Pestivirus chimera viral RNA contains at
least one UGA box motif, preferably the UGA.sub.pos.cons.
motif.
[0054] These Pestivirus chimeras may be utilized as quality control
materials in analytical RNA assays, including use as external
positive controls (EC or PC), internal quality standards (IQS),
internal controls (IC), internal quantification standards (QS),
parallel complementary controls (PCC), calibrators, standards and
in validation and verification panels. IC and QS are also referred
to as IQS, since they can be the same substance. A difference
between IC and QS is the way the signal generated by the substance
is analyzed. The invention is useful for any detection assays
including but not limited to target amplification technologies
(e.g., PCR, TMA, NASBA, etc.) and signal amplification technologies
(e.g., bDNA, etc.).
[0055] The same Pestivirus chimera can be used as EC, PC,
calibrator or standard. This includes but is not limited to:
standards traceable to an SI unit (e.g. mol), international
standards, national standards (e.g. those provided by national
measurement standards laboratories which establish standards for a
country or organization like NIST in the USA or PTB (Physikalisch
Technische Bundesantalt) in Germany) reference standards, certified
reference materials, certified reference standards, JCTLM (Joint
Committee for Traceability in Laboratory Medicine) approved
materials, higher order reference materials and WHO standards (e.g.
a WHO standard for HCV NAT assays). This substance contains target
analyte sequences.
[0056] An RNA assay using e.g. a Pestivirus Chimera positive
control and a Pestivirus Chimera QS would require two different
chimeras. The inserted sequence would be different for the PC
control material and QS control material.
[0057] An external control (EC), e.g. a positive control (PC),
utilizes a composition that is the same or very similar to the
target analyte sequence, but is assayed separately in an
independent reaction from the target analyte sample. The EC or PC
quality controls the same sequence as the target. In PCR, NASBA,
TMA or other amplification technologies, an amplification product
is formed identical or similar to the target, and the measurement
signal generated is identical to the target signal. The external
run control's purpose is to verify the EC test results fall within
a predetermined acceptance criteria. The EC or PC are an integral
part of a nucleic acid detection system (e.g. a PCR based
diagnostic system) quality control and can be supplied as part of a
reagent kit. See e.g. package insert for Roche COBAS
AmpliPrep/COBAS TaqMan HIV-1 Test, Roche COBAS AmpliPrep/COBAS
TaqMan HCV Test, ABBOTT RealTime HIV-1 test (69-6672/R1) or ABBOTT
RealTime HCV test (69-6675/R1). These type of controls are,
however, not necessarily part of a kit and can also be supplied as
independent external run controls (e.g. OptiQual controls by
AcroMetrix). Substances useful as external run control can also be
used as calibrators for quantitative assays. While positive and
negative controls indicate the general performance of the assay and
allow a decision on reliability, calibrators help to measure the
correct quantity of an analyte (e.g. the amount of HIV-1 in a
patient's blood). Control and calibrator concepts for NAT assays
are well known and have been described in e.g. ISO standard
17511:2003 and Section III G. of FDA's (1999) "Guidance for
Industry In the Manufacture and Clinical Evaluation of In Vitro
Tests to Detect Nucleic Acid Sequences of Human Immunodeficiency
Viruses Types 1 and 2" and CLSI Guideline MM3-A2 Section 11.4.
[0058] Internal Quality Standards like Internal Controls (IC) and
internal Quantification Standards (QS) are used as follows
according to the Clinical and Laboratory Standards Institute, CLSI
Guideline MM3-A2, Section 7: "Inhibition of a nucleic acid test
results from the presence of substances that lead to a failure to
achieve a positive signal. Interference of a nucleic acid test
results in a reduction in assay performance leading to a
false-negative or false-positive result. Inhibitors of
amplification can be detected by the use of internal control
templates, also referred to as recovery templates, or simply,
internal controls. The control template can be added to the sample
either prior to, or after sample preparation. If it is added prior
to sample preparation, it can also serve as a control for the
nucleic acid extraction." Due to its resistance to RNase
degradation this invention can be added prior to sample preparation
and serve as a control for the nucleic acid extraction. It could be
even useful as a full process control including even earlier steps
than sample preparation like sample collection and sample
transport. The same guideline specifies a number of different
control types useful for "Detection of Inhibitors and Interfering
Substances" (Section 7.1, CLSI Guideline MM3-A2). A RNA virus
chimera described in this invention can be used to provide the
substances useful to serve as the internal controls described in
the guideline. These controls are known in the art also as
"unmodified target controls" (section 7.1.1., CLSI Guideline
MM3-A2), "modified target controls" (section 7.1.2) and
"Heterologous Internal Controls" (section 7.1.3, CLSI Guideline
MM3-A2).
[0059] Internal quality standards (IQS) like IC or QS, are assayed
simultaneously in the same reaction vessel as the target analyte. A
negative patient result (e.g. for HIV or HCV) is only reported as
"negative" or as "target not detected" if the IC or QS result falls
within an acceptable range. In case the IQS results falls outside
of the acceptance range the result is considered invalid and the
sample should be retested.
[0060] An internal quantification standard (QS) is a type of IQS
that allows precise quantification of the target analyte even in
the presence of inhibitors. Both QS and Internal control (IC)
generate signals distinguishable from the target signal. In the
case of PCR, the amplification product and the generated signal are
different and distinguishable from the target signal. IC and QS are
used to detect and compensate (QS) for inhibition. They help to
distinguish between a true negative and a false negative result. QS
and IC sequences are often synthetic materials since they are
usually designed to be different from the target, however, they
should still behave like the target analyte (e.g. a virus) and go
through the entire diagnostic process from sample preparation to
detection. Ideally an IQS would in addition also monitor sample
collection and transport.
[0061] U.S. Pat. No. 7,183,084 B2 and U.S. Pat. No. 7,192,745
describe a specialized type of internal quality control. The
parallel complementary control (PCC) has the same thermodynamic
properties as the target amplicon and therefore behaves in PCR like
the target nucleic acid. The parallel complementary control
provides one component to ensure an IQS behaves like the target
analyte, however as a piece of naked RNA it would not be resistant
to degradation by RNases when added into e.g. human plasma. To
fully achieve that the PCC behaves like the target analyte during
sample preparation, it needs to be encapsulated in a particle that
behaves like the target analyte and can be added to the patient
sample without being degraded. This invention is useful to provide
RNase resistant parallel complementary internal controls formulated
in a patient sample like matrix.
[0062] The chimeric RNA genome of the chimeric Pestivirus is useful
to provide the RNA based internal controls and quantification
standards described in U.S. Pat. No. 7,192,745.
[0063] This invention can further be used as a standard for the
quantification of RNA as described in U.S. Pat. No. 7,183,084 B2
and further provide an RNase resistant form of a parallel
complementary QS as described in U.S. Pat. No. 7,192,745.
[0064] Quantification Standards are commonly used in commercial NAT
assay kits. Two examples are the Roche COBAS AmpliPrep/COBAS TaqMan
HIV-1 Test kit or Roche COBAS AmpliPrep/COBAS TaqMan HCV Test kit.
The QS material is described in the Roche COBAS AmpliPrep/COBAS
TaqMan HIV-1 Test (P/N: 03542998 190) package insert (May 2007 Doc
Rev. 1.0) as "HIV-1 QS (HIV-1 Quantification Standard). An HCV QS
is used with the Roche COBAS AmpliPrep/COBAS TaqMan HCV Test and
described in the package insert.
[0065] Two different Pestivirus chimeras could be used as a RNase
resistant QS (Chimera Roche-1) and a positive control and a
calibrator material (Chimera Roche-2) for the Roche COBAS
AmpliPrep/COBAS TaqMan HIV-1 Test. Chimera Roche-2 would contain a
heterologous HIV sequence detectable by the assay. Two additional
chimeras would be required for the same purpose for the Roche COBAS
AmpliPrep/COBAS TaqMan HCV Test kit (Chimera Roche-3 as HCV PC and
Chimera Roche-4 as HCV QS).
[0066] Internal Controls are commonly used in commercial NAT assay
kits. The Pestivirus chimeras could serve as an internal control
(IC) for the detection of inhibition as used in the ABBOTT RealTime
HIV-1 test kit (69-6672/R1) or ABBOTT RealTime HCV test kit
(69-6675/R1). The Abbott Internal Controls are described in the
package insert under "Reagents" List No. 2G31Y for the HIV-1 test
and List No. 4J86Y for the HCV test. The IC target sequence for
both tests was derived from the hydroxypyruvate reductase gene from
Cucurbita pepo and delivered as an Armored RNA particle in negative
human plasma. A Pestivirus RNA chimera could be used to provide the
same sequence from the hydroxypyruvate reductase gene from
Cucurbita pepo. A Pestivirus RNA chimera based internal control
would mimic the target analyte better than Armored RNA, because it
resembles HIV and HCV more than the MS2 bacteriophage.
[0067] A Pestivirus chimera could be used as a RNase resistant IC
(Chimera Abbott-1) and a separate Pestivirus-HIV Chimera could be
provided to serve as low and high positive control in the Abbott
RealTime HIV-1 Control Kit (List No. 2G31-80) (Chimera Abbott-2).
Chimera Abbott-2 could also be used as calibrator material in the
Abbott realTime HIV-1 Calibrator Kit (List No. 2G31-70). One
additional Pestivirus-HCV chimera (Chimera Abbott-3) would be
required for controls and calibrators of the ABBOTT RealTime HCV
test. Unlike the Roche TaqMan assays, which use separate
Quantification Standards sequences for their HIV and HCV assay, the
Abbott realTime PCR assays, use a common IC sequence for their
HIV-1 and HCV assays.
[0068] The Pestivirus chimeras can be used as IC, QS, positive
control and calibrator in commercial RNA NAT assays. For example,
the chimeric RNA genome of the chimeric Pestivirus can be used as
Positive control and calibrator in commercial RNA NAT assays. A
second chimera would be required to serve as IC or QS. These
controls can be packaged together or separate from the other
required reagents. The materials can be lot specific or lot
independent.
[0069] FIG. 1 shows a schematic alignment of the genomes of the
Pestivirus BVDV virus and the Hepacivirus HCV virus. BVDV and HCV
display a similar genomic organization. Both viral RNAs contain a
long open reading frame (ORF; indicated as box) that is flanked by
nontranslated regions (NTRs) at the 5' and 3' ends (single lines).
The BVDV and HCV 5'NTRs contain internal ribosomal entry sites
(IRES) that mediate translation of a viral polyprotein. Inspection
of FIG. 1 shows that both viral RNAs encode a similar polyprotein
(C, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, NS5B-COOH) that is co-
and post-translationally processed by cellular and viral proteases
to give rise to structural (C, E1, E2, p7) and nonstructural
(NS2-NS5B) viral proteins (Lindenbach et al., 2007). The BVDV
genome contains an additional nonstructural protein, the
autoprotease N.sup.PRO and an additional envelope protein,
E.sup.RNS. The BVDV genome has a length of approximately 13 kb,
while the HCV genome has a genome size of ca. 10 kb.
[0070] FIG. 2 shows in diagram form the strategy for the insertion
of a heterologous sequence (HCV 5'NTR, amplicon) within the 3'NTR
of the Pestivirus BVDV. FIG. 2 shows the insertion within the 3'NTR
into a region between the two stem-loop structures SL.sub.STOP and
SLII. This region was rather tolerant to the insertion of a
variable sequence. However, the HCV 5'NTR should not be able to
re-initiate translation as this would interfere with the initiation
of replication at the immediate Tend. For that purpose, sequence
elements in the BVDV 3'NTR that ensure immediate translation
termination are left intact (Isken et al., 2004) The 5'NTR region
of the HCV is circled in FIG. 2, and an arrow indicates the
insertion of this sequence within the 3'NTR of Pestivirus BVDV. The
structure of the stem-loops found in the 5'NTR and the 3'NTR of the
HCV and the BVDV viruses is indicated. The known functions of the
different regions of the viral NTRs during translation and RNA
replication are indicated (Grassmann et al., 2005). The open
reading frame (ORF) is shown as a box.
[0071] FIG. 3 shows a diagrammatic model of the structure and
functions of the Pestivirus BVDV 3'NTR (Yu, et al., 1999; Isken et
al., 2004). A representative sequence of the 3'NTR from BVDV (BVDV
replicon D19c, which is highly homologous to the 3'NTR of BVDV
non-CP7; Behrens et al., J. Virol., 1998) is shown. The variable
region (3'V) is involved in the coordination of translation and RNA
replication by efficiently terminating translation. For this
activity, binding of NFAR proteins to the 3'V region involving the
UGA boxes is needed. The 3' variable region (3'V) and the 3'
constant region (3'C) are each shown boxed with dashed lines. The
3'V represents the sequence following the translational stop codon
(shown boxed; a stopping ribosome is schematized by a double
structure); the border between the 3'V and the following 3'C region
is indicated by juxtaposition of the dashed line boxes near the top
of SLII. The constant region (3'C) is shown as the remainder of the
3'NTR. The 3'C is the region where the replication complex
assembles, which is indicated by a large oval. The functional
interactions of the 3'NTR are indicated diagrammatically for: the
translating ribosome (double structure), the NFAR proteins
(collection of circles), and the replication complex (large oval).
SS indicates a highly conserved intervening region located between
SLII and SL I. The large arrow indicates an insertion site for the
heterologous RNA sequence for the formation of a stable and
functional Pestivirus RNA chimera.
[0072] FIG. 4 discloses a more detailed diagram of the 3'NTR
sequence of Pestivirus BVDV-D19c, which is highly homologous to the
3'NTR of BVDV non-CP7 showing the 3'V and the 3'C regions. As in
FIG. 3, the 3'V and the 3'C regions are indicated with dashed-line
boxes. The 3'V region contains the SL.sub.STOP (SLIII) stem-loop
and a portion of the SLII stem-loop, which ends after the end of
the 3'UGA-like box and is marked by a juxtaposition of the dashed
line boxes (at nucleotide 90; Deng and Brock, 1993). In FIG. 4, the
3'V region contained UGA box motifs, including: the 5'UGA box
(nucleotides 19-30), the UGA.sub.pos.cons. box (nucleotides 46-57)
and the 3'UGA-like box (nucleotides 79-90). As in FIG. 3, the large
arrow indicates a preferred insertion site immediately following
the SL.sub.STOP (SLIII) where a heterologous RNA sequence may be
inserted for the formation of a stable and functional Pestivirus
RNA chimera.
[0073] FIG. 5A shows an alignment of the 3'NTR for various strains
of Pestivirus. On the left is an alignment of the 3'NTRs of
different Pestivirus strains (Becher et al. J. Virol. 1998 72:
5165-5173). The sequences forming SL.sub.STOP (SLIII) and SLII are
indicated. With all of the different Pestivirus strains, the
predictions of the structural similarity of SLII and SLstop (SLIII)
are analogous. The boxed area indicates the position of the
UGA.sub.pos.cons. box, which is located within the 3'V region and
contains four nucleotides that are 100% conserved between all known
Pestivirus strains (marked by asterisks). A thick arrow indicates
the proposed border of the 3'V and 3'C regions of the 3'NTR of all
these Pestivirus strains (Deng and Brock, 1993). The
UGA.sub.pos.cons. box has been found to be present at nearly
identical positions in all pestiviral genomes, i.e. about 39-42
residues downstream of the ORF (Isken, 2003). The arrow indicates a
preferred site where the heterologous insert was introduced into
BVDV-non-CP7. FIG. 5B details the consensus sequence and conserved
positions of the UGA box sequences of different Pestivirus strains
(Isken et al., 2003). Given the analogous structural similarity of
the stem-loop structures within the 3'V and the consensus of
sequences within the UGA.sub.pos.cons. box, the 3'NTR, therefore,
may be commonly used for insertion of a heterologous sequence with
all Pestivirus. The site immediately following the
UGA.sub.pos.cons. box is an example of a preferred site for all
Pestivirus strains for insertion of a heterologous RNA sequence.
The procedure described for the construction of a 3'NTR viral
chimera can be used with all Pestivirus to create functional
chimeric viruses that may be, for example, used as standards and
controls for analytical assays.
Example 1
Construction of BVDV-Non-CP7 cDNA and Generation of Infectious
Non-Cytopathic Viral RNA
[0074] A modified cDNA of BVDV (type-1) strain CP7 was generated as
a starting material for all subsequent procedures (Becher et al.,
2000 J. Virol. 74: 7884-7894). The modification was performed such
that the CP7 insert (Tautz et al., J Virol. 1996 November;
70(11):7851-8) was removed to create a cDNA containing plasmid that
encoded a BVDV "non-CP7" RNA biotype (M. Behrens, unpublished
data). A similar construct was published earlier by Makoschey et
al., (Vaccine, 2004, Sep. 3; 22(25-26):3285-94.) The complete
sequence of the cDNA for the BVDV-non-CP7 is given as SEQ ID NO:
1.
[0075] The plasmid encoding the BVDV-non-CP7 cDNA was linearized by
restriction digestion with the restriction endonuclease SmaI. In
vitro transcripts were generated by run-off transcription using SP6
RNA polymerase. The viral RNA was generated by in vitro
transcription using SP6 RNA polymerase. The in vitro generated
BVDV-non-CP7 RNAs were transfected into Marbin Darby Bovine Kidney
(MDBK) cells using standard protocols. Three (3) days post
transfection, the cell culture supernatant was collected and used
to either titrate the contained infectious virus particles or to
re-infect other MDBK cells. Three days after re-infection, virus
titers were determined and the cells examined for a cytopathic
effect using standard procedures (Tautz et al., J. Virology, 1996,
70 (11):7851-8). Thus, the non-cytopathic nature of the generated
virus was confirmed. The titrations revealed that 5.times.10.sup.6
to 5.times.10.sup.7 infectious virus particles were detectable per
ml of cell culture supernatant (see also Table 1 and Table 2, data
for BVDV-non-CP7).
Example 2
Generation of Chimeric BVDV-Non-CP7 cDNAs
[0076] The plasmid including the BVDV-non-CP7 cDNA (SEQ ID NO: 1)
was then used to introduce the HCV 5'NTR sequence (HCV Coni cDNA;
Lohmann et al., 1999) within the BVDV 3'NTR to obtain a functional
chimeric viral sequence (BVDV-non-CP7-HCV 5'NTR).
[0077] For that purpose, a synthetic DNA fragment was generated
commercially. This DNA fragment corresponded to the ClaI
(initiating at pos. 11047 of the BVDV-non-CP7 cDNA sequence)/SmaI
(initiating at pos. 12264 of BVDV-non-CP7 cDNA sequence) fragment
of the BVDV-non-CP7 cDNA (SEQ ID NO:1), but also included an HCV
5'NTR insert (Con 1 subtype 1 b isolate; Lohmann et al., 1999,
Science. Jul. 2: 285 (5424): 110-3) flanked by two restriction
sites (SnaBI and PacI) and an additional TAA trinucleotide. The
cDNA sequence of the HCV 5'NTR is given in SEQ ID NO: 2. This
heterologous insert was positioned such that in corresponding RNA
transcripts it was located between the UGA.sub.pos.cons. box and
the SLII stem-loop structure in the 3'V of the BVDV non-CP7 3'NTR
(see FIGS. 3 and 4 for diagram of a preferred insertion site).
[0078] Using standard recombinant cloning techniques, the
respective ClaI/SmaI fragment was cloned into the BVDV-non-CP7 cDNA
(SEQ ID NO:1) containing plasmid that was cut with the same
restriction sites. Performing several independent ligations and
screening of several hundred clones yielded recombinant plasmids
encoding the chimeric cDNA. The resultant chimeric cDNA clone was
termed BVDV-non-CP7-HCV 5'NTR cDNA (SEQ ID NO: 3). It thus encoded
a heterologous sequence at position 12134, i.e., essentially two
novel restriction sites and the HCV 5'NTR (SEQ ID NO: 2) placed
within the sequence encoding the BVDV 3'NTR to yield the
BVDV-non-CP7-HCV 5'NTR chimera (SEQ ID NO: 3, FIG. 6). Note that
the introduced HCV 5'NTR sequence was modified at two single
nucleotide positions to remove internal SmaI sites (c.fwdarw.t at
position 12269 and c.fwdarw.t at pos. 12456 of the BVDV-non-CP7-HCV
5' NTR cDNA) These nucleotide exchanges were necessary to allow
linearization of the BVDV-non-CP7-HCV 5' NTR cDNA encoding plasmid
by SmaI for run off transcription by SP6 RNA polymerase to generate
viral RNA molecules that ended with the correct 3' terminus. FIG. 6
shows the cDNA sequence of the BVDV-non-CP7-HCV 5'NTR chimera with
the restriction endonuclease sites indicated for ClaI, part of
SmaI, SnaBI and PacI. The nucleotide exchanges (c changed into t)
are also indicated.
[0079] Applying the same strategy, an insert encoding two
restriction sites, SnaBI and PacI) was introduced into the
BVDV-non-CP7 cDNA containing plasmid (SEQ ID NO: 1) at the same
position (insertion at position 12134). This construct was termed
BVDV-non-CP7+ cloning site, and the cDNA sequence is given in SEQ
ID No: 4. FIG. 7 shows the cDNA sequence of the BVDV-non-CP7+
cloning site chimera with the restriction endonuclease sites
indicated for ClaI, part of SmaI, for SnaBI and PacI.
[0080] The correctness of the respective inserts was verified by
restriction analysis of the cDNA encoding plasmids and sequencing
as shown in FIG. 8. FIG. 8 shows sequence details of the 3'NTR
region for the construction of the Pestivirus chimera sequences
(given as DNA sequence). FIG. 8 (A+B) shows a comparison of the
cDNA sequences of the 3'NTR of the BVDV-non-CP7+ cloning site
chimera and the sequence of the 3'NTR of BVDV-non-CP7.) FIG. 8
(A+C) shows a comparison of the sequence of the 3' NTR of the
BVDV-non-CP7-HCV 5'NTR chimera and the sequence of the 3'NTR of
BVDV-non-CP7.
[0081] In FIG. 8, the translational stop-codon (tga) at the end of
the open reading frame (ORF) is boxed, and pseudo-stop codons are
underlined. Additional and specifically changed sequences (taa) are
shown in italics. The SnaBI restriction site (tacgta) and the PacI
restriction site (ttaattaa) are shown in brackets. The 5'UGA box
(tattgtatataa) and the UGA.sub.pos.cons. box (tattatgtttaa) are
indicated by the small dashed-line boxes. FIG. 8A shows the 3' NTR
of BVDV-non-CP7 (SEQ ID NO: 5), which corresponds to that for the
wild type starting material. (As noted above, the complete sequence
of the BVDV-non-CP7 is given as SEQ ID NO: 1.) FIG. 8B shows the 3'
NTR of the BVDV-non-CP7+ cloning site chimera, which has the
SnaBI/PacI restriction endonuclease cloning site insert (SEQ ID NO:
6). (As noted above, the complete sequence of the BVDV-non-CP7+
cloning site is given in SEQ ID NO: 4, FIG. 7.) FIG. 8C shows the
sequence of the 3'NTR region for the BVDV-non-CP7-HCV 5'NTR chimera
(SEQ ID NO: 7). The sequence of the HCV 5'NTR insert (SEQ ID NO. 2)
is indicated by the large, dashed-line box. (As noted above, the
complete sequence of the BVDV-non-CP7-HCV-5'NTR chimera is given as
SEQ ID NO: 3, FIG. 6.)
Example 3
Stability of the cDNA Plasmid Constructs of the Newly-Generated
Pestivirus BVDV Chimeras
[0082] Several individually isolated cDNA plasmids encoding either
BVDV-non-CP7 (SEQ ID NO: 1), the BVDV-non-CP7+ cloning site chimera
(SEQ ID NO: 4), or the BVDV-non-CP7-HCV 5'NTR chimera (SEQ ID NO:
3) were grown in E. coli. The plasmids were prepared using standard
procedures and the authenticity of the inserts was verified by DNA
sequencing. The overall stabilities of the plasmids through several
passages in E. coli were verified by performing restriction
analysis using multiple restriction enzymes and sequencing.
Example 4
The Pestivirus BVDV Chimeric RNAs are Replication Competent and
Generate Infectious Virus Particles at Similar Titers as the
Original BVDV-Non-CP7 RNA
[0083] The plasmids encoding the chimeric cDNAs were linearized
with the restriction endonuclease SmaI. In vitro transcription was
performed with SP6 RNA polymerase using standard protocols. The
viral RNA then was transfected into Marbin Darby bovine kidney
cells (MDBK) using standard procedures. Three (3) days post
transfection, the virus titer contained in the cell culture
supernatant was measured by a standard titration protocol. The
transfection supernatant was then used to re-infect cells and to
re-harvest and re-titrate virus at 3 day intervals. As shown in
Table 1, the virus titers obtained after the initial transfection
and re-infection were found to be similar with experiments that
were performed with the RNAs of BVDV-non-CP7 and two BVDV-non-CP7+
cloning site chimeras (K17 and K25), respectively.
TABLE-US-00001 TABLE 1 Virus titers for BVDV-non-CP7 and
BVDV-non-CP7 + cloning site chimera after transfection and 1st
re-infection. BVDV- BVDV- non-CP7 + non-CP7 + BVDV- cloning site
cloning site Days post non-CP7 chimera, K17 chimera, K25 infection
Titer (/ml) 6.30E+05 9.40E+06 1.30E+06 3 (1st re-infection)
[0084] As shown in Table 2 (line 1) similar virus titers to those
found in Table 1 were also obtained with transfection and first
re-infection experiments when the BVDV-non-CP7 and the
BVDV-non-CP7-HCV 5'NTR chimera were tested, respectively. As also
shown in Table 2, similar virus titers were found throughout
subsequent re-infection experiments (up to 7 re-infection passages)
for the BVDV-non-CP7 and the BVDV-non-CP7-HCV 5'NTR chimera,
respectively.
TABLE-US-00002 TABLE 2 Virus titers at various times post-infection
for BVDV-non-CP7 and BVDV-non-CP7-HCV 5'NTR chimera Titer (/ml)
BVDV-nonCP7- HCV 5'NTR Days post BVDV-non-CP7 chimera trasfection
Days post infection 1.20E+06 5.10E+06 3 2.10E+07 1.30E+07 3 (1st
re-infection) 2.20E+05 4.90E+05 3 (2nd re-infection) 1.70E+06
6.10E+06 3 (3rd re-infection) 4.30E+06 3.20E+07 3 (4th
re-infection) 7.40E+07 9.30E+07 3 (7th re-infection).sup.1
.sup.1Titrations of 5th and 6th re-infections were not
performed
[0085] To test for viral protein synthesis, cells that were
infected with virus (see above) were investigated by
immunofluorescence (IF) to detect newly synthesized NS3 protein.
IF-detectable synthesis of NS3 protein unambiguously indicated RNA
replication. Protein synthesis that occurred by translation of the
viral RNA within the cell was not detectable. In other words, only
if the RNA amplified would sufficient protein be synthesized and
thus become detectable by IF testing (Behrens et al., 1998; J
Virol. 1998 March; 72(3):2364-72). We detected NS3 synthesis with
both chimeras (BVDV-nonCP7+ cloning site and BVDV-nonCP7-HCV
5'NTR), as well as with the BVDV-non-CP7 throughout all of the
performed transfection/infection experiments that are shown in
Tables 1 and 2. In sum, these results showed that following
transfection of the RNAs into host cells, the Pestivirus chimeras
BVDV-non-CP7+ cloning sites and BVDV-non-CP7-HCV 5'NTR showed
identical rates of viral protein synthesis and of infectious viral
particle formation.
Example 5
Growth of BVDV-Non-CP7 and the BVDV-Non-CP7-HCV 5'NTR Chimera
[0086] The growth characteristics of the BVDV-non-CP7-HCV 5'NTR
chimera was compared with that of the BVDV-non-CP7. As shown in
FIGS. 9 and 10, the growth behavior of the newly generated
BVDV-non-CP7-HCV 5'NTR chimera (measured by one-step growth curves
over a 72 hour time period) was indistinguishable from that
obtained for the BVDV-non-CP7. The data for FIGS. 9 and 10 is given
in Table 3.
TABLE-US-00003 TABLE 3 Growth curve data for BVDV-non-CP7 and
BVDV-non- CP7-HCV 5'NTR chimera (see FIGS. 9 and 10) Titer/ml TIME
(hr) BVDV-non-CP7 BVDV-non-CP7-HCV 5'NTR chimera 12 7.50E+02
4.50E+02 18 3.70E+03 2.50E+03 24 1.90E+04 1.90E+04 36 2.30E+06
2.20E+06 48 3.00E+08 4.00E+08 72 4.00E+08 5.00E+08
Example 6
Long Term Stability of the Pestivirus BVDV-Non-CP7-HCV 5'NTR
Chimera (7 Passages)
[0087] The Pestivirus BVDV-non-CP7-HCV 5' NTR chimera was passaged
(performing subsequent re-infection experiments) for up to seven
times (several weeks), without loss of titer (see Table 2; see
Example 4). As shown in Table 2, measuring the virus titers after
seven passages revealed no significant differences in the titer of
the BVDV-non-CP7-HCV 5'NTR chimera with respect to the titer of
BVDV-non-CP7 (commonly, average titers of 5.times.10.sup.7/ml cell
culture supernatant were measured). These findings allowed stable
manufacturing of larger amounts (>l liter) of virus in
culture.
[0088] To determine the stability of the genomic sequence of the
BVDV non-CP7-HCV 5'NTR chimera, after three passages as well as
after seven passages, total viral RNA was isolated from the
infected cells (using a standard procedure; Behrens et al., 1998; J
Virol. 1998 March; 72(3):2364-72.) and the BVDV 3'NTR amplified by
RT-PCR. After cloning of the PCR fragments (cloning site BamHI,
XbaI), five different clones were sequenced. The clones generated
from the 3.sup.rd passage showed no nucleotide exchanges (data not
shown). The clones generated from the 7.sup.th passage showed very
few changes: Two of five clones that were obtained after the
7.sup.th passage displayed one nucleotide exchange within the HCV
insert, while two clones showed mutations within the genomic BVDV
sequence, and one clone showed no changes. FIG. 11 shows the
sequence details for these five clones obtained after the 7.sup.th
passage: clone 1 (FIG. 11A) (SEQ ID NO: 8) had a g-t exchange in
the HCV insert; clone 2 (FIG. 11B) (SEQ ID NO: 9) had a c-t
exchange in the HCV insert; clone 3 (FIG. 11C) (SEQ ID NO: 10) had
a t-c exchange in the BVDV sequence; clone 4 (FIG. 11D) (SEQ ID NO:
11) had no sequence changes, and clone 5 (FIG. 11E) (SEQ ID NO: 12)
had g deleted in the BVDV sequence. The HCV insert in the BVDV
non-CP7-HCV 5'NTR chimera, therefore, had approximately the same
stability as the surrounding genomic BVDV sequence.
Example 7
Inactivation of the Pestivirus BVDV Chimera with
.beta.-propiolactone
[0089] Inactivation experiments of the BVDV-non-CP7-HCV 5'NTR virus
particles were performed. The culture supernatant of
BVDV-non-CP7-HCV 5'NTR infected MDBK cells (7th re-infection, virus
titer approximately 1.times.10.sup.8 per ml) was incubated with
various concentrations of .beta.-propiolactone for various times at
three different temperatures (4.degree. C., 25.degree. C. and
37.degree. C.) as indicated in Table 4. The .beta.-propiolactone is
a protein-modifying agent known to react with amides of the
NH.sub.3 group of lysine or arginine. Following incubation, the
culture supernatants were titrated and tested by immunofluorescence
(IF, see Example 4) to detect viral RNA replication, and thus
detection of infectious virus for the cultured cells. The data
obtained are shown in Table 4. When infectious virus was detectable
throughout several titrations (12.times.1:5 steps), it was scored
as (+)-IF. When no infectious virus was detectable throughout
several titrations (12.times.1:5 steps), it was scored (-)-IF. As
shown below, when the culture supernatant was incubated at 0.03%
.beta.-propiolactone at 37.degree. C. for 120 min, the Pestivirus
chimeric virus was inactivated, while there was no toxicity for the
cultured cells.
TABLE-US-00004 TABLE 4 Determination of inactivation of Pestivirus
BVDV-non-CP7-HCV 5'NTR chimera. .beta.-propiolactone Concentration
TIME .degree. C. 0.01% 0.03% 0.05% 0.1% 0.3% 60 min 4.degree. C.
IF+ IF+ IF+.sup. nd nd 120 min 4.degree. C. IF+ IF+ IF-.sup.2 nd nd
240 min 4.degree. C. IF+ .sup. IF-.sup.1 IF-.sup.2 nd nd 15 min
25.degree. C. IF+ .sup. IF-.sup.1 IF-.sup.1 nd nd 30 min 25.degree.
C. IF+ .sup. IF-.sup.1 IF-.sup.1 nd nd 60 min 25.degree. C. nd nd
nd IF-.sup.2 IF-.sup.3 120 min 25.degree. C. IF+ .sup. IF-.sup.1
IF-.sup.1 nd nd 180 min 25.degree. C. IF+ .sup. IF-.sup.1 IF-.sup.1
nd nd 15 min 37.degree. C. IF+ .sup. IF-.sup.1 IF-.sup.1 nd nd 30
min 37.degree. C. IF+ .sup. IF-.sup.1 IF-.sup.1 nd nd 120 min
37.degree. C. IF+ ##STR00001## IF-.sup.1 nd nd Legend:
.sup.1Initial dilution toxic for cells; .sup.2Initial dilution and
1:5 dilution toxic for cells; .sup.3Initial dilution, 1:5 dilution,
and 1:25 dilution toxic for cells. In bold, optimal inactivation
condition.
Example 8
Testing of HCV-BVDV Chimera for Positive HCV Signal in Real-Time
PCR and Estimation of Titer
[0090] Two DNA plasmid BVDV chimera clones (K4 and K8) expected to
contain the HCV 5'NTR sequence were transcribed into full length
viral RNA. MDBK cells were transfected with viral RNA and cultured.
The cells produced infectious viral particles, which were
subsequently harvested and tested for containing the expected HCV
5'NTR RNA sequence (see Example 3).
[0091] Viral RNA was extracted from clone K4 and K8 supernatant, as
well as a HCV control, using the QIAamp Virus MinElute Kit
(QIAGEN). Reverse transcription real time PCR was performed using
the Roche TaqMan HCV ASR. K4 and K8 BVDV-HCV chimeras showed high
titer HCV signals indicating successful integration of the HCV
5'NTR into BVDV (Table 5). Based on the observed Ct values it was
estimated both chimeras produced a signal equivalent to about 300
million HCV IU/mL. One IU has previously been estimated to be
roughly equivalent to 2.63 viral particles. The estimated titer of
7.8E+08 virus particles/mL is higher than the observed 9.30E+07
infectious particles/mL (Table 2) because only about one in 10
viral particles is infectious. HCV patient samples rarely exceed 10
million IU/mL.
[0092] It should be noted that International Units (IU) are a
different unit of measure than the previously mentioned "infectious
particles" in Table 2. The HCV control showed the expected
value.
TABLE-US-00005 TABLE 5 Estimation of BVDV-non-CP-7-HCV 5'NTR
chimera titer using the Roche HCV ASR Dilution Final Conc Samples
Ct QS Ct Factor log IU/ml IU/ml HCV-BVDV K8 20.3 31.4 10 7.46
2.9.E+08 HCV-BVDV K4 20.2 31.4 10 7.49 3.1.E+08 HCV control 26.6
31.3 1 5.30 2.0.E+05
Example 9
The HCV Signal for BVDV-NonCP7-HCV 5'NTR Chimera was from RNA
[0093] We tested whether the positive HCV signal in Example 6 was
generated from RNA or an unlikely DNA contamination from the cDNA
plasmid encoding the BVDV-non-CP7-HCV 5'NTR chimera RNA. The
BVDV-nonCP7-HCV 5'NTR K4 and K8 chimera samples were extracted
using a QIAamp Virus MinElute Kit (QIAGEN). PCR amplifications were
performed using two separate preparations of the TaqMan One-Step
RT-PCR Master Mix Reagents (ABI) and a reverse transcription PCR
was performed using an HCV PCR assay based on real time detection.
One master mix was prepared with no reverse transcriptase (RT),
while the other master mix contained the reverse transcriptase
enzyme. The RT enzyme is required for converting RNA into cDNA,
which can be used during PCR by a thermostable DNA polymerase as
substrate for amplification. RNA is no substrate for the DNA
polymerase. Without the RT, it was expected if RNA were the source
of the HCV signal, no DNA template would be made, and thus no
amplification by DNA polymerase could occur. However without RT, if
DNA were the source of the HCV signal, it would serve as the
template for DNA polymerase and a HCV signal would be
generated.
[0094] BVDV-non-CP7-HCV 5 NTR chimera samples were prepared with
both master mixes, and run on the same 96-well plate on the ABI
7300. As shown in Table 6, the BVDV-non-CP7-HCV 5 NTR chimera
samples and HCV samples only amplified when the reverse
transcriptase (RT) was present. This allowed the conclusion that
the BVDV-non-CP7-HCV 5 NTR chimera samples contain RNA, not DNA.
Also, the HCV control signal originated as expected from RNA.
TABLE-US-00006 TABLE 6 BVDV-non-CP7-HCV 5'NTR chimera signal is
derived from RNA sample Ct with RT Ct without RT
BVDV-non-CP7-HCV-5'NTR chimera, 26.5 no signal K8
BVDV-non-CP7-HCV-5'NTR chimera, 26.7 no signal K4 HCV control 33.1
no signal
Example 10
The BVDV-Non-CP7-HCV 5'NTR Chimera is RNase Resistant
[0095] We tested to determine whether the BVDV-non-CP7-HCV 5'NTR
chimera RNA was resistant to RNase degradation because its RNA was
protected within an intact virion, or if it existed as
non-encapsulated, free RNA vulnerable to enzymatic digestion with
RNase.
[0096] The two BVDV-non-CP7-HCV 5'NTR chimeras K4 and K8 and an HCV
control were tested under two conditions. In one experiment, an
RNase digest was done prior to extraction (QIAamp Virus MinElute
Kit (QIAGEN)). Loss of signal in PCR amplification would indicate
free, unprotected RNA. In a second control condition, no RNase was
added to the HCV-BVDV chimera samples before extraction. If the
chimeric RNA was non-encapsulated, the RNase digest would degrade
the free RNA. This degradation would either eliminate the HCV
signal completely, or shift the Ct to a significantly higher
value.
[0097] As shown in Table 7, the Ct values for the BVDV-non-CP7-HCV
5 NTR chimeras K4 and K8 did not significantly change after RNase
treatment, indicating the RNA is encapsulated, and therefore
protected from RNA degradation. To control RNase digestion was
working, we extracted the HCV RNA and treated it with RNase prior
to amplification. The HCV signal was eliminated completely,
confirming the RNase digest was working as expected.
TABLE-US-00007 TABLE 7 Resistance of BVDV-non-CP7-HCV 5'NTR chimera
RNA to RNase Ct: No RNase Ct: RNase Digest Ct: RNase Digest Sample
Treatment Before Extraction After Extraction BVDV-non-CP7-HCV 34.9
34.8 Not tested 5'NTR chimera K8 BVDV-non-CP7-HCV 34.0 35.2 Not
tested 5'NTR chimera K4 HCV 38.1 37.7 Negative
Example 11
Use of BVDV-Non-CP7-HCV 5'NTR Chimera as an HCV Calibrator for HCV
NAT Amplification Assays
[0098] The performance of a known HCV calibrator (AcroMetrix
OptiQuant HCV) consisting of intact naturally occurring HCV virions
in plasma was compared with the performance of a BVDV-non-CP7-HCV 5
NTR chimeric HCV calibrator, which also consisted of intact virions
in plasma. First, a value assignment of the 0.05%
.beta.-propiolactone inactivated BVDV-non-CP7-HCV 5 NTR stock
material in plasma was conducted using an HCV Acrometrix Primary
Standard (APS). The HCV APS is metrologically traceable to the
Second HCV WHO Standard (NIBSC code: 96/798). The BVDV-non-CP7-HCV
5 NTR chimera sample was diluted to the same levels as the
OptiQuant HCV panel members: 5E6, 5E5, 5E4, 5E3, 5E2, 5E1 IU/ml.
The OptiQuant HCV panel had also been value assigned using the HCV
APS. RNA extraction for the calibrators was performed using the
QIAamp Virus MinElute Kit (QIAGEN) on the automated QIAcube
instrument (QIAGEN). Real-time PCR was performed using a reverse
transcription PCR HCV assay based on real time detection.
[0099] As shown in FIG. 12, the measured Ct values were plotted
against the log of HCV IU/ml. The linear regression lines were
equivalent for HCV and BVDV-non-CP7-HCV 5 NTR chimera panels within
the measurement error: y=-3.80x+44.0 for BVDV-non-CP7-HCV 5 NTR
chimera, and y=-3.82x+44.3 for OptiQuant HCV. The slopes of the two
calibrators showed approximately equivalent PCR efficiencies: 87%
for the BVDV-non-CP7-HCV 5 NTR chimera calibrator and 86% for the
OptiQuant HCV calibrator. The coefficients of determination
indicated a high degree of linearity: 0.98 for HCV-BVDV, and 0.99
for the OptiQuant HCV calibrator. The use of BVDV-non-CP7-HCV 5 NTR
chimeric calibrator gave equivalent data compared to a known HCV
calibrator that closely resembles patient samples, such as the
AcroMetrix OptiQuant HCV panel.
Example 12
Use of BVDV-Non-CP7-HCV 5'NTR Chimera as an Internal Quantification
Standard (QS)
[0100] In this experiment the HCV signal generated with the
BVDV-non-CP7-HCV 5 NTR chimera functioned as QS for a quantitative
West Nile Virus (WNV) assay. Four replicates of a WNV panel at 1E6,
1E5, 1E4, and 1E3 copies/ml were extracted using the Qiagen QIAcube
with the Qiagen QIAamp MinElute Virus Spin Kit. 19.4 .mu.l of BVDV
at a concentration of 1E4 IU/ml was added as an internal
quantification standard (QS) to the carrier RNA solution following
the MinElute sample extraction protocol. 12.34 .mu.l of the RNA
eluate was amplified using WNV assay reagents, which also contained
HCV-5'NTR primers and probes. The PCR reaction was run on an
Applied Biosystems, ABI PRISM 7300. The data were analyzed using
the Applied Biosystems Sequence Detection Software version 1.4.
[0101] The results of the linear regression analysis are shown in
FIG. 13. A slope of 2.891 and a coefficient of determination of
0.996 was obtained from the equation of the line.
[0102] This experiment demonstrated usefulness of a BVDV chimera as
an internal Quantification Standard (QS) in a quantitative NAT
assay. The calibration curve for a quantitative WNV assay was
generated by subtracting the WNV Ct value from the QS
(BVDV-non-CP7-HCV 5 NTR) Ct and plotting the difference in Ct
against the known WNV concentration in copies/mL (cp/mL).
Example 12
Generation of a cDNA or Plasmid from Purified Chimeric Pestivirus
RNA
[0103] Donis and Vassilev described in U.S. Pat. No. 6,001,613 the
generation of plasmid containing the cDNA of BVDV and producing
infectious BVDV. The same methods and more recent methods known in
the art can be used to purify viral RNA from the pestivirus-chimera
and use the purified RNA to generate cDNA of this chimera. The cDNA
can be cloned into a plasmid. The cDNA or plasmid would be useful
as the starting point for generating new virus as described in
Examples 3 and 4.
Sequence CWU 1
1
12112267DNApestivirus type 1misc_featureBVDV-non-CP7 1gtatacgagg
ttaggcaagt tctcgtatac atattggaca ctctaaaaat aattaggcct 60aggggacaaa
aatcctcctt agcgaaggcc gaaaagaggc taaccatgcc cttagtagga
120ctagcaaaat aaggggggta gcaacagtgg cgagttcgtt ggatggctga
agccctgagt 180acagggtagt cgtcagtggt tcgacgcttt ggaggacaag
cctcgagatg ccacgtggac 240gagggcatgc ccacagcaca tcttaacctg
gacaggggtc gttcaggtga aaacggttta 300accaaccgct acgaatacag
tctgatagga tgctgcagag gcccactgta ttgctactga 360aaatctctgc
tgtacatggc acatggagtt gatcacaaat gaacttttat acaaaacata
420caaacaaaaa cccgctggag tggaggaacc agtatacgac caagctggta
accctttgtt 480tggagaaaga ggagtgattc atccgcagtc aacgctaaaa
cttccacata aaagagggga 540gcgtgaagtc cccaccaatc tggcttcttt
accaaaaaga ggtgactgca ggtcgggtaa 600cagcaagggg cctgtgagtg
gaatctactt aaaaccaggg ccgttattct accaagatta 660taaaggacct
gtctatcata gagccccatt ggagtttttt gaggaggcgt ctatgtgtga
720gacaactaaa agaataggga gagtaactgg tagtgacagc agattatacc
acatttacgt 780gtgtattgat gggtgcataa tagtcaagag tgctacaaaa
gaccgccaga aagtactcaa 840gtgggtccac aacaagctaa actgccccct
atgggtttca agctgctccg acacaaaaga 900tgaaggggtg gtgaggaaga
agcaacaaaa gccagatagg ttggaaaagg ggagaatgaa 960gataacacct
aaggagtcag agaaagacag taagaccaag ccgccagatg ctacgatagt
1020ggtagatgga gtcaagtatc aggtaaagaa aaaaggaaaa gtcaagagca
agaacaccca 1080ggacggctta taccacaaca aaaataaacc tcaagagtcg
cgcaagaaac tagagaaagc 1140cctattggcc tgggcaataa tagccctggt
tttctttcaa gtcacaatgg gagagaacat 1200aacgcaatgg aacttacaag
ataatggaac ggaaggcata caacgagcca tgtttcaaag 1260aggagtgaat
agaagtttac atgggatctg gccagagaaa atctgtacag gtgttccttc
1320ccacctggcc actgacacag aattgaaggc aattcatggt atgatggatg
caagtgagaa 1380gacaaattat acgtgctgca gactccaacg ccatgaatgg
aacaaacatg gttggtgcaa 1440ctggtacaac attgaacctt ggatcctcct
tatgaataaa actcaggcca accttactga 1500gggtcagcca ctaagggagt
gtgccgtcac atgccggtat gatcgagata gtgacctgaa 1560tgtagtaaca
caagccaggg atagccccac accattaaca ggttgcaaga aaggcaaaaa
1620cttttccttt gcaggcatat tggtacaagg gccttgcaac tttgaaatag
ccgtaagtga 1680tgtgctgttc aaagagcatg attgcactag tgtgattcaa
gacacagctc actacctcgt 1740agacgggatg accaactccc tagagagtgc
caggcaaggg accgcgaaac taacaacttg 1800gctgggcagg cagcttggga
tactaggaaa gaaactggaa aacaagagta agacatggtt 1860tggggcatat
gcagcctctc cctattgtga ggtagaacgg aagcttggtt acatctggta
1920tacaaagaat tgcactccag cctgtttgcc taggaataca aagatcatcg
gccccggtag 1980gtttgacacc aatgccgagg atggtaaaat actgcatgag
atggggggtc acttgtcgga 2040ggtgctacta ctctcagtgg tagtgctgtc
cgatttcgct ccagagacag ccagtgtgat 2100atacttgatt cttcatttct
ccatcccaca aggacacact gatatacaag attgtgacaa 2160aaaccaacta
aacctcaccg tagaactcac aacagcagaa gtaataccag gctcagtttg
2220gaacttgggt aaatatgttt gtgtaagacc agattggtgg ccttatgaga
cagccacagt 2280cctggtgatt gaagaggtgg gtcaagtaat taaggttgtc
ttaagggcgt taaaagatct 2340gacgcgcatt tggaccgctg ctacaaccac
tgcattcttg gtttgtctgg tgaaggtagt 2400gagaggccaa gtgttacaag
gtatactgtg gctgatgctc ataacagggg cgcaagggta 2460cccagactgc
aaacccggct tttcatacgc catagccaaa aatgatgaga ttggcccact
2520tggagctaca ggcctcacca ctcagtggta cgaatactcg gatgggatgc
ggctgcagga 2580ctcagtagtt gaagtttggt gtaaaaatgg agagatcaaa
tatctaatca gatgcgggag 2640ggaagccagg tatctggctg ttctacacac
gagagccttg ccgacatctg tagtatttga 2700aaaaattttt gatgggaaag
aacaagagga catagtagaa atggatgaca actttgaatt 2760cggtctttgc
ccgtgtgatg ctagaccctt gataagggga aaatttaata caacacttct
2820aaatgggcca gccttccaga tggtttgccc tataggatgg acagggactg
taagctgtac 2880actggccaat aaggatacgt tagccacaat cgttgtgaga
acgtataaga gggtcaggcc 2940ttttccatat aggcaggact gtgtcaccca
gaaaaccatc ggggaagacc tctacgactg 3000tgccttagga gggaattgga
cttgtgtgcc gggggatgca ctacgatatg tagctgggcc 3060cgttgagtct
tgtgagtggt gtggttacaa gtttttaaaa agtgagggtc tgccgcattt
3120cccaatcggc aaatgcaggc tgaagaatga gagtggctat agacaagtgg
atgagacttc 3180ttgcaacaga aacggcgtgg ctatagtgcc atctggcacg
gtcaaatgca agatagggga 3240cacggtggtg caagtcattg caatggatga
taaactaggg cctatgcctt gcaaaccaca 3300tgaaatcata tccagtgagg
ggccagtgga aaagacggca tgcaccttca actacacaag 3360aacattaaaa
aacaagtact ttgagcccag ggataactat tttcaacaat acatgttaaa
3420gggggagtac caatattggt ttgacctaga gatcactgac caccaccgag
attacttcgc 3480tgagtccctg ttggtgatag tagttgcact cctgggtggc
aggtacgtgc tttggctgct 3540ggtcacatac atgatcttat cagaacagat
ggcctcgggt gtccagtatg gggcaggtga 3600aatagtgatg atgggcaact
tgttaacaca tgacagtgtt gaagtggtga catatttctt 3660actactatac
ctactactaa gagaggaaaa caccaaaaaa tgggtcatac ttatatacca
3720catcatagta atgcatcctc taaaatcggt gacggtgata ttgctaatgg
ttggggggat 3780ggcaaaggct gaaccaggtg cccaggggta cctagagcag
gtagacctta gttttacgat 3840gattacgatc atcgtaatag gtctggttat
agctaggcgt gatcccactg tggtgccact 3900agtcactata gtcgcggcac
tgaagatcac aggactaggc tttgggcccg gagtggatgc 3960agctatggca
gttctcacct taaccctact gatgactagt tatgtgacag actacttcag
4020gtataaaagg tggatacaat gtatcctcag cttagtagcc ggggtgttcc
ttatccggac 4080cctcaaacat ctaggtgaac tcaaaacccc tgagctgacc
ataccaaatt ggaggccact 4140aaccttcata ctattatacc tgacttcagc
aacagttgtt acaagatgga aaattgatat 4200agctggcata ttcctgcaag
gggcccctat ccttttgatg atcgccaccc tatgggctga 4260cttcttgact
cttgttctga tcctacccac ctacgaatta gccaagctgt actacctaaa
4320gaacgtcaag actgacgtgg agaagagttg gctggggggg ttagactaca
ggacaattga 4380ctctgtctat gatgtggatg aaagtggaga aggcgtgtac
ctcttcccgt ccagacagaa 4440gaaaaataag aatatcagca tactcttgcc
cctcatcaga gctacgctaa taagttgtat 4500tagcagcaaa tggcagatgg
tgtatatggc ttacttaacc ctggacttta tgtactacat 4560gcacagaaag
gttattgaag agatatcagg gagtaccaat gtgatgtcta gagtgatagc
4620agcacttata gaattaaact ggtccatgga agaagaagag agcaagggct
taaagaagtt 4680ttttatacta tctggaaggt tgaggaacct tataataaag
cataaggtta ggaaccagac 4740tgtggcaagc tggtatgggg aggaagaagt
ctacggcatg ccaaaagtcg taaccataat 4800aagggcctgc acgctaaaca
agaacaaaca ttgcataata tgcacagtat gtgaggctag 4860aaagtggaag
ggaggcaact gccctaaatg cggccgccac gggaagccca tcatttgtgg
4920gatgactcta gcggattttg aagaaaggca ctacaagaga atttttataa
gggaaggtaa 4980ctttgaagga cccttcaggc aggaatacaa tgggtttgta
caatacaccg ctagggggca 5040attgttcctg agaaatttac ccatattggc
aaccaaagta aaaatgatca tggtaggcaa 5100cctaggagag gaaatcggtg
atctagaaca cctaggatgg atcctaaggg gacctgccgt 5160gtgcaagaaa
ataactgagc acgaaaaatg ccatgtcaac atactggaca agctgactgc
5220gttttttgga gttatgccaa gagggactac accaagggct ccggtgagat
tcccaacagc 5280actactaaag gtaaggaggg gattggaaac cggttgggct
tacacgcatc aaggtggcat 5340aagctcagta gaccatgtga ccgctggcaa
ggatctattg gtttgtgaca gtatgggtag 5400aactagagtg gtttgccaaa
gcaacaacaa gttaactgat gagacagaat atggtgtcaa 5460gacggactcc
ggatgtccag atggtgccag atgctatgta ttaaacccag aggcagtaaa
5520tatatcaggg tccaagggag ctgtcgtaca cctccaaaaa acgggtgggg
aatttacatg 5580tgttactgca tcaggtacac cggccttctt cgacctgaaa
aatttgaaag gatggtcggg 5640tctacccata tttgaagcct ccagcggcag
agtggttggc agagtcaaag tgggaaagaa 5700tgaggaatcc aaacccacaa
aattaatgag tggtatccaa actgtttcta aaaatacggc 5760cgatttaaca
gaaatggtca agaagataac cagcatgaac aggggggact ttaggcagat
5820aacccttgca acaggggcag ggaagaccac tgagctccca aaagcagtga
tagaggagat 5880aggacgacac aaacgggtac tagtgctcat accattaaga
gcagcagctg agtcagtcta 5940tcaatacatg agattgaaac acccaagtat
ctcctttaac ctgagaatag gggacatgaa 6000agaaggggat atggcaaccg
ggatcaccta cgcctcatat ggatattttt gccaaatgcc 6060acaaccaaag
ctcagagcag caatgataga gtattcatac atatttctgg atgagtatca
6120ctgcgctact cctgagcagt tggctgttat aggaaaaatt cacagatttt
ctgagagcat 6180aagagtggtt gccatgactg ccaccccagc agggtcagta
accacaacag ggcaaaaaca 6240cccaatagaa gaattcatag cccctgaggt
gatgaaaggg gaggaccttg gaagccagtt 6300ccttgacata gcggggttaa
agatccctgt agaggagatg aagggtaaca tgttggtttt 6360cgtgcccacg
aggaacatgg cagttgaagt agccaagaaa ctaaaagcca agggctacaa
6420ctcagggtat tactacagtg gggaagaccc agctaacttg agagtggtaa
catcacagtc 6480cccatacgtc gtggtagcca ctaatgccat cgagtcaggg
gtaacgctgc cagatttaga 6540tacagttgtt gacacaggtc tgaaatgtga
gaagagggtg agggtgtctt ccaaaatacc 6600ctttatagta acaggcctta
agagaatggc tgtcactgtg ggcgaacagg ctcagcggag 6660aggcagggta
ggtagagtga aacccggtag gtattataga agtcaggaaa cagcaaccgg
6720gtcaaaggac taccactatg acttgttaca ggcacagagg tacgggatcg
aagatgggat 6780caacgtaaca aagtccttta gggagatgaa ttatgactgg
agcctgtatg aggaagacag 6840cttgctgata acccagctgg agatactgaa
caatctactc atctctgaag atttaccagc 6900agctgttaaa aacatcatgg
caagaactga tcacccagag cctatccagc ttgcatataa 6960cagttatgag
gtccaagtcc ctgtgctgtt cccaaaaata aggaatgggg aggtcacaga
7020cacttacgag aactactcat tcctaaatgc aaggaaacta ggggaagacg
tgcccgtgta 7080cgtttatgcc accgaagatg aagatctggc tgtggacctt
ctaggcttgg actggccaga 7140cccagggaat cagcaagtag tggagactgg
gaaggcactg aagcaagtgg taggactgtc 7200ctctgccgaa aatgccttgc
tcatagccct atttgggtat gtaggatacc aagccttgtc 7260aaaaagacac
gtcccaatga tcacagacat atacactata gaagatcaaa gactagagga
7320cacaacccac cttcaatatg cgcccaatgc cataagaact gaggggaagg
agactgaact 7380aaaggaatta gcagtgggtg acttggacaa aatcatgggt
tccatctcgg actatgcatc 7440agagggattg aatttcgtaa ggtcccaagc
agaaaagatg agatctgccc ccgctttcaa 7500agaaaacgtg gaagctgcta
aagggtacgt ccaaaagttt attgattctc tcatagaaaa 7560taaagaaacc
ataatcagat atggcctgtg gggaacacac acggcactct acaagagtat
7620tgccgcgaga ttgggtcatg aaactgcatt cgctacacta gtgataaagt
ggctggcctt 7680cgggggtgag tcggtgtcag accacatgag acaagcagct
gtcgacctgg ttgtttatta 7740tgtgatcaat aagccctcct tcccagggga
ttctgaaacc caacaggaag gaaggcgatt 7800cgtcgccagc ctgttcatct
ccgctttggc aacctacaca tacaaaactt ggaattacaa 7860caacctctcc
aaggtagtag aaccagcctt agcatacctc ccctatgcta ccaatgcact
7920aaaaatgttt accccgacca gactggagag cgtagttata cttagtacca
caatatacaa 7980aacttacctc tcaataagga agggaaagag tgatggactg
ttgggtacag ggatcagtgc 8040agcaatggag attctatcac agaacccagt
gtcggtaggt atatctgtca tgctgggggt 8100gggggcgatt gccgcgcaca
atgccattga gtctagtgaa caaaaaagga ccctgttgat 8160gaaagtgttt
gtaaaaaact tcctggacca ggcggcaaca gatgagctgg taaaggaaaa
8220cccagagaaa ataataatgg ccctatttga agcagtccag acaattggca
accccttgag 8280gctcatatat cacctgtatg gggtttacta caaaggctgg
gaagcaaaag aactatcaga 8340gagaacagca ggcaggaacc tgttcacctt
gataatgttc gaagccttcg aactactagg 8400gatggactct gaagggaaga
taaggaacct gtctgggaat tatgtcctgg atttgatcta 8460cagcctacat
aaacagataa atagaggctt gaaaaaaata gtcttggggt gggctcccgc
8520accatttagt tgcgactgga ctcctagtga tgagagaatt aggttaccca
caaacaacta 8580tctaagagta gaaactaagt gtccatgtgg ctatgagatg
aaagcactaa ggaacgttgg 8640tggcagtctt accaaagtgg aggagaaagg
accttttctc tgcaggaaca ggcttggtag 8700agggccggtc aactatagag
tcacaaagta ctatgatgac aacctcaaag agataaaacc 8760agttgctaaa
ctagaaggat ttgtggatca ctattacaaa ggtgttacag caaggataga
8820ttatggcaga gggaaaatgc tattagctac tgataaatgg gaggtggagc
acggtgttgt 8880cactaggttg gcaaagagat ataccggagt tggattcaag
ggagcatacc tgggtgatga 8940acccaaccac cgcgacctag tagaaagaga
ctgtgcaact ataacaaaaa atacagtgca 9000gtttttaaaa atgaagaaag
gctgtgcatt tacctatgac ttaaccctgt ccaatttaac 9060caggttaatt
gaattggtac acaaaaataa cctagaagag aaagacatac cagcagccac
9120agtaacgaca tggctggctt atacttttgt aaatgaagat attgggacta
taaaaccagt 9180actaggagag agagtggtca ccgacccagt ggtggatgtt
aacttacaac cagaagtaca 9240agtggataca tcagaggttg ggatcacttt
agttggtagg gcagccttaa tgacgacagg 9300tactacaccc gtagtcgaaa
aaacagagcc caatgctgat ggtggtccaa gctccataaa 9360gattgggttg
gatgaaggaa gatacccagg acctggactg caagaccgca ccttgaccga
9420tgaaatacat tctagggatg aaaggccctt tgttctagtc ctgggctcaa
aaaattctat 9480gtcaaataga gctaaaactg ctagaaacat caacttatac
aaggggaata accccaggga 9540gattagagat ctgatggcac aggggcgtat
gctagttgtg gccttaaagg attttaaccc 9600tgagttgtct gaactagttg
atttcaaggg gactttctta gacagggaag ccttggaagc 9660tctcagcctg
gggcggccaa agtccaagca ggtgaccaca gccacagtta gggagttatt
9720agagcaggag gtacaagttg agatccccag ttggtttgga gcaggtgatc
cagtcttctt 9780ggaagtgact ttgaagggtg acagatatca cttagtagga
gatgtagata gagtgaaaga 9840tcaagcgaag gagcttgggg ccacggacca
gacaagaata gtgaaggaag tgggtgcaag 9900aacctatacc atgaagctgt
ctagttggtt tcttcaggca acaaataaac agatgagctt 9960gaccccttta
tttgaggagc tattgctacg ttgcccccct aaaataaaga gcaataaagg
10020gcacatggca tcagcttacc aactagcaca gggaaactgg gagccccttg
actgtggagt 10080tcacctgggc accatacctg ccaggagggt aaaaatccac
ccatatgaag cttacctgaa 10140actgaaggat ttattggaag aagaagaaaa
gaaaccaaag tgtagagaca cagtaataag 10200agaacacaac aagtggatcc
tcaaaaaagt gaggcaccag ggtaatctca atacaaagaa 10260aatcctcaac
cctggaaagc tatcagaaca gctagataga gaagggcata aaagaaacat
10320ttataacaat cagattggca ccataatgac ggaagcagga agtaggttgg
aaaaattacc 10380agtcgtcaga gcccaaactg acactaaaag cttccatgag
gcaatcagag ataagataga 10440caagaatgaa aatcagcaga gcccaggact
gcatgataaa ttgttagaga tctttcatac 10500aatagcccaa cccagcctaa
gacacaccta cagtgacgtg acgtgggagc aacttgaggc 10560aggggttaat
agaaaggggg ctgctggctt tctagagaag aagaatgttg gagaagtact
10620ggactcagag aagcacctgg tggaacaact gatcagagat ttgaaaacag
gaaggaagat 10680aagatattat gagacagcaa taccaaaaaa tgagaagaga
gatgtcagtg atgattggca 10740atcaggggac ttagtagatg agaagaaacc
aagggtgatt caataccctg aagctaaaac 10800aagactagcc atcactaaag
taatgtacaa ctgggtgaaa cagcagcccg tcgtgatccc 10860agggtatgaa
gggaagaccc cattatttaa cattttcaac aaggtgagga aggaatggga
10920tttgttcaat gaaccagtag ctgtgagttt cgacactaag gcttgggaca
cccaagtaac 10980tagtagagat ctacggctta ttggtgaaat tcaaaaatat
tactacagga aagagtggca 11040caaattcatc gataccatta ctgaccatat
ggtggaggtg cccgtcataa cggcagatgg 11100tgaggtatac ataagaaatg
gacaaagggg tagtggccag ccagacacaa gtgcaggcaa 11160tagcatgcta
aacgtgttaa caatgatgta tgccttctgt gaaagtacgg gggttccata
11220caagagtttc aatagggttg caaggatcca tgtctgtggg gatgacggct
tcctaataac 11280agagaagggg ctgggattaa agtttgccaa caatgggatg
caaattctgc acgaagcagg 11340caagcctcaa aagataactg agggggaaag
aatgaaagtt gcctataggt tcgaggacat 11400agaattctgc tctcatacac
cagtccccgt taggtggtct gataacacca gcagttacat 11460ggccggcaga
gacactgccg ttatattatc aaagatggca acaagattgg attcaagtgg
11520agaaaggggt actatagcat atgaaaaagc agtggccttt agttttttgc
tgatgtactc 11580ctggaatcct cttgtgagga ggatctgtct actggtcctt
tcacagcagc cagagacaac 11640tccatcaacc cagaccactt actattataa
aggagaccca ataggagcct acaaagatgt 11700aataggtaag aatttgtgtg
aattaaaaag gacgggtttt gaaaaattgg ccaatttaaa 11760cctaagcctg
tccacgttag gaatctggtc caaacataca agtaaaagaa tcatccaaga
11820ctgtgtaacc atcgggaaag aggaaggcaa ttggctggtc aatgccgaca
ggttgatatc 11880tagcaaaact ggccatttgt acatacctga caaaggttat
acattacaag ggaaacatta 11940tgaacaactt caactgcagg caagaactag
cccagtcacg ggagtaggga cggagagata 12000taaactaggc cctatagtaa
acctgctgct gaggaggttg agagttctgc ttatggcagc 12060tgtcggtgcc
agcagttgaa ataatgtatg tatatattgt atataaatct gtatttgtat
12120atattatgtt taaatttagt tgagattagt agtgatatat agttatctac
ctcaagctaa 12180cactacactc aatgcacaca gcactttagc tgtatgaggg
tacacccgac gtccacggtt 12240ggactaggga aaacccttaa cagcccc
122672341DNAHepatitis C virusmisc_featureHCV 5' NTR 2gccagccccc
gattgggggc gacactccac catagatcac tcccctgtga ggaactactg 60tcttcacgca
gaaagcgtct agccatggcg ttagtatgag tgtcgtgcag cctccaggac
120cccccctctc gggagagcca tagtggtctg cggaaccggt gagtacaccg
gaattgccag 180gacgaccggg tcctttcttg gatcaacccg ctcaatgcct
ggagatttgg gcgtgccccc 240gcgagactgc tagccgagta gtgttgggtc
gcgaaaggcc ttgtggtact gcctgatagg 300gtgcttgcga gtgcctcggg
aggtctcgta gaccgtgcac c 341312625DNApestivirus type
1misc_featureBVDV non-CP7-HCV 5'NTR chimera 3gtatacgagg ttaggcaagt
tctcgtatac atattggaca ctctaaaaat aattaggcct 60aggggacaaa aatcctcctt
agcgaaggcc gaaaagaggc taaccatgcc cttagtagga 120ctagcaaaat
aaggggggta gcaacagtgg cgagttcgtt ggatggctga agccctgagt
180acagggtagt cgtcagtggt tcgacgcttt ggaggacaag cctcgagatg
ccacgtggac 240gagggcatgc ccacagcaca tcttaacctg gacaggggtc
gttcaggtga aaacggttta 300accaaccgct acgaatacag tctgatagga
tgctgcagag gcccactgta ttgctactga 360aaatctctgc tgtacatggc
acatggagtt gatcacaaat gaacttttat acaaaacata 420caaacaaaaa
cccgctggag tggaggaacc agtatacgac caagctggta accctttgtt
480tggagaaaga ggagtgattc atccgcagtc aacgctaaaa cttccacata
aaagagggga 540gcgtgaagtc cccaccaatc tggcttcttt accaaaaaga
ggtgactgca ggtcgggtaa 600cagcaagggg cctgtgagtg gaatctactt
aaaaccaggg ccgttattct accaagatta 660taaaggacct gtctatcata
gagccccatt ggagtttttt gaggaggcgt ctatgtgtga 720gacaactaaa
agaataggga gagtaactgg tagtgacagc agattatacc acatttacgt
780gtgtattgat gggtgcataa tagtcaagag tgctacaaaa gaccgccaga
aagtactcaa 840gtgggtccac aacaagctaa actgccccct atgggtttca
agctgctccg acacaaaaga 900tgaaggggtg gtgaggaaga agcaacaaaa
gccagatagg ttggaaaagg ggagaatgaa 960gataacacct aaggagtcag
agaaagacag taagaccaag ccgccagatg ctacgatagt 1020ggtagatgga
gtcaagtatc aggtaaagaa aaaaggaaaa gtcaagagca agaacaccca
1080ggacggctta taccacaaca aaaataaacc tcaagagtcg cgcaagaaac
tagagaaagc 1140cctattggcc tgggcaataa tagccctggt tttctttcaa
gtcacaatgg gagagaacat 1200aacgcaatgg aacttacaag ataatggaac
ggaaggcata caacgagcca tgtttcaaag 1260aggagtgaat agaagtttac
atgggatctg gccagagaaa atctgtacag gtgttccttc 1320ccacctggcc
actgacacag aattgaaggc aattcatggt atgatggatg caagtgagaa
1380gacaaattat acgtgctgca gactccaacg ccatgaatgg aacaaacatg
gttggtgcaa 1440ctggtacaac attgaacctt ggatcctcct tatgaataaa
actcaggcca accttactga 1500gggtcagcca ctaagggagt gtgccgtcac
atgccggtat gatcgagata gtgacctgaa 1560tgtagtaaca caagccaggg
atagccccac accattaaca ggttgcaaga aaggcaaaaa 1620cttttccttt
gcaggcatat tggtacaagg gccttgcaac tttgaaatag ccgtaagtga
1680tgtgctgttc aaagagcatg attgcactag tgtgattcaa gacacagctc
actacctcgt 1740agacgggatg accaactccc tagagagtgc caggcaaggg
accgcgaaac taacaacttg 1800gctgggcagg cagcttggga tactaggaaa
gaaactggaa aacaagagta agacatggtt 1860tggggcatat gcagcctctc
cctattgtga ggtagaacgg aagcttggtt acatctggta 1920tacaaagaat
tgcactccag cctgtttgcc taggaataca aagatcatcg gccccggtag
1980gtttgacacc aatgccgagg atggtaaaat actgcatgag atggggggtc
acttgtcgga 2040ggtgctacta ctctcagtgg tagtgctgtc cgatttcgct
ccagagacag ccagtgtgat 2100atacttgatt cttcatttct ccatcccaca
aggacacact gatatacaag attgtgacaa 2160aaaccaacta aacctcaccg
tagaactcac aacagcagaa gtaataccag gctcagtttg
2220gaacttgggt aaatatgttt gtgtaagacc agattggtgg ccttatgaga
cagccacagt 2280cctggtgatt gaagaggtgg gtcaagtaat taaggttgtc
ttaagggcgt taaaagatct 2340gacgcgcatt tggaccgctg ctacaaccac
tgcattcttg gtttgtctgg tgaaggtagt 2400gagaggccaa gtgttacaag
gtatactgtg gctgatgctc ataacagggg cgcaagggta 2460cccagactgc
aaacccggct tttcatacgc catagccaaa aatgatgaga ttggcccact
2520tggagctaca ggcctcacca ctcagtggta cgaatactcg gatgggatgc
ggctgcagga 2580ctcagtagtt gaagtttggt gtaaaaatgg agagatcaaa
tatctaatca gatgcgggag 2640ggaagccagg tatctggctg ttctacacac
gagagccttg ccgacatctg tagtatttga 2700aaaaattttt gatgggaaag
aacaagagga catagtagaa atggatgaca actttgaatt 2760cggtctttgc
ccgtgtgatg ctagaccctt gataagggga aaatttaata caacacttct
2820aaatgggcca gccttccaga tggtttgccc tataggatgg acagggactg
taagctgtac 2880actggccaat aaggatacgt tagccacaat cgttgtgaga
acgtataaga gggtcaggcc 2940ttttccatat aggcaggact gtgtcaccca
gaaaaccatc ggggaagacc tctacgactg 3000tgccttagga gggaattgga
cttgtgtgcc gggggatgca ctacgatatg tagctgggcc 3060cgttgagtct
tgtgagtggt gtggttacaa gtttttaaaa agtgagggtc tgccgcattt
3120cccaatcggc aaatgcaggc tgaagaatga gagtggctat agacaagtgg
atgagacttc 3180ttgcaacaga aacggcgtgg ctatagtgcc atctggcacg
gtcaaatgca agatagggga 3240cacggtggtg caagtcattg caatggatga
taaactaggg cctatgcctt gcaaaccaca 3300tgaaatcata tccagtgagg
ggccagtgga aaagacggca tgcaccttca actacacaag 3360aacattaaaa
aacaagtact ttgagcccag ggataactat tttcaacaat acatgttaaa
3420gggggagtac caatattggt ttgacctaga gatcactgac caccaccgag
attacttcgc 3480tgagtccctg ttggtgatag tagttgcact cctgggtggc
aggtacgtgc tttggctgct 3540ggtcacatac atgatcttat cagaacagat
ggcctcgggt gtccagtatg gggcaggtga 3600aatagtgatg atgggcaact
tgttaacaca tgacagtgtt gaagtggtga catatttctt 3660actactatac
ctactactaa gagaggaaaa caccaaaaaa tgggtcatac ttatatacca
3720catcatagta atgcatcctc taaaatcggt gacggtgata ttgctaatgg
ttggggggat 3780ggcaaaggct gaaccaggtg cccaggggta cctagagcag
gtagacctta gttttacgat 3840gattacgatc atcgtaatag gtctggttat
agctaggcgt gatcccactg tggtgccact 3900agtcactata gtcgcggcac
tgaagatcac aggactaggc tttgggcccg gagtggatgc 3960agctatggca
gttctcacct taaccctact gatgactagt tatgtgacag actacttcag
4020gtataaaagg tggatacaat gtatcctcag cttagtagcc ggggtgttcc
ttatccggac 4080cctcaaacat ctaggtgaac tcaaaacccc tgagctgacc
ataccaaatt ggaggccact 4140aaccttcata ctattatacc tgacttcagc
aacagttgtt acaagatgga aaattgatat 4200agctggcata ttcctgcaag
gggcccctat ccttttgatg atcgccaccc tatgggctga 4260cttcttgact
cttgttctga tcctacccac ctacgaatta gccaagctgt actacctaaa
4320gaacgtcaag actgacgtgg agaagagttg gctggggggg ttagactaca
ggacaattga 4380ctctgtctat gatgtggatg aaagtggaga aggcgtgtac
ctcttcccgt ccagacagaa 4440gaaaaataag aatatcagca tactcttgcc
cctcatcaga gctacgctaa taagttgtat 4500tagcagcaaa tggcagatgg
tgtatatggc ttacttaacc ctggacttta tgtactacat 4560gcacagaaag
gttattgaag agatatcagg gagtaccaat gtgatgtcta gagtgatagc
4620agcacttata gaattaaact ggtccatgga agaagaagag agcaagggct
taaagaagtt 4680ttttatacta tctggaaggt tgaggaacct tataataaag
cataaggtta ggaaccagac 4740tgtggcaagc tggtatgggg aggaagaagt
ctacggcatg ccaaaagtcg taaccataat 4800aagggcctgc acgctaaaca
agaacaaaca ttgcataata tgcacagtat gtgaggctag 4860aaagtggaag
ggaggcaact gccctaaatg cggccgccac gggaagccca tcatttgtgg
4920gatgactcta gcggattttg aagaaaggca ctacaagaga atttttataa
gggaaggtaa 4980ctttgaagga cccttcaggc aggaatacaa tgggtttgta
caatacaccg ctagggggca 5040attgttcctg agaaatttac ccatattggc
aaccaaagta aaaatgatca tggtaggcaa 5100cctaggagag gaaatcggtg
atctagaaca cctaggatgg atcctaaggg gacctgccgt 5160gtgcaagaaa
ataactgagc acgaaaaatg ccatgtcaac atactggaca agctgactgc
5220gttttttgga gttatgccaa gagggactac accaagggct ccggtgagat
tcccaacagc 5280actactaaag gtaaggaggg gattggaaac cggttgggct
tacacgcatc aaggtggcat 5340aagctcagta gaccatgtga ccgctggcaa
ggatctattg gtttgtgaca gtatgggtag 5400aactagagtg gtttgccaaa
gcaacaacaa gttaactgat gagacagaat atggtgtcaa 5460gacggactcc
ggatgtccag atggtgccag atgctatgta ttaaacccag aggcagtaaa
5520tatatcaggg tccaagggag ctgtcgtaca cctccaaaaa acgggtgggg
aatttacatg 5580tgttactgca tcaggtacac cggccttctt cgacctgaaa
aatttgaaag gatggtcggg 5640tctacccata tttgaagcct ccagcggcag
agtggttggc agagtcaaag tgggaaagaa 5700tgaggaatcc aaacccacaa
aattaatgag tggtatccaa actgtttcta aaaatacggc 5760cgatttaaca
gaaatggtca agaagataac cagcatgaac aggggggact ttaggcagat
5820aacccttgca acaggggcag ggaagaccac tgagctccca aaagcagtga
tagaggagat 5880aggacgacac aaacgggtac tagtgctcat accattaaga
gcagcagctg agtcagtcta 5940tcaatacatg agattgaaac acccaagtat
ctcctttaac ctgagaatag gggacatgaa 6000agaaggggat atggcaaccg
ggatcaccta cgcctcatat ggatattttt gccaaatgcc 6060acaaccaaag
ctcagagcag caatgataga gtattcatac atatttctgg atgagtatca
6120ctgcgctact cctgagcagt tggctgttat aggaaaaatt cacagatttt
ctgagagcat 6180aagagtggtt gccatgactg ccaccccagc agggtcagta
accacaacag ggcaaaaaca 6240cccaatagaa gaattcatag cccctgaggt
gatgaaaggg gaggaccttg gaagccagtt 6300ccttgacata gcggggttaa
agatccctgt agaggagatg aagggtaaca tgttggtttt 6360cgtgcccacg
aggaacatgg cagttgaagt agccaagaaa ctaaaagcca agggctacaa
6420ctcagggtat tactacagtg gggaagaccc agctaacttg agagtggtaa
catcacagtc 6480cccatacgtc gtggtagcca ctaatgccat cgagtcaggg
gtaacgctgc cagatttaga 6540tacagttgtt gacacaggtc tgaaatgtga
gaagagggtg agggtgtctt ccaaaatacc 6600ctttatagta acaggcctta
agagaatggc tgtcactgtg ggcgaacagg ctcagcggag 6660aggcagggta
ggtagagtga aacccggtag gtattataga agtcaggaaa cagcaaccgg
6720gtcaaaggac taccactatg acttgttaca ggcacagagg tacgggatcg
aagatgggat 6780caacgtaaca aagtccttta gggagatgaa ttatgactgg
agcctgtatg aggaagacag 6840cttgctgata acccagctgg agatactgaa
caatctactc atctctgaag atttaccagc 6900agctgttaaa aacatcatgg
caagaactga tcacccagag cctatccagc ttgcatataa 6960cagttatgag
gtccaagtcc ctgtgctgtt cccaaaaata aggaatgggg aggtcacaga
7020cacttacgag aactactcat tcctaaatgc aaggaaacta ggggaagacg
tgcccgtgta 7080cgtttatgcc accgaagatg aagatctggc tgtggacctt
ctaggcttgg actggccaga 7140cccagggaat cagcaagtag tggagactgg
gaaggcactg aagcaagtgg taggactgtc 7200ctctgccgaa aatgccttgc
tcatagccct atttgggtat gtaggatacc aagccttgtc 7260aaaaagacac
gtcccaatga tcacagacat atacactata gaagatcaaa gactagagga
7320cacaacccac cttcaatatg cgcccaatgc cataagaact gaggggaagg
agactgaact 7380aaaggaatta gcagtgggtg acttggacaa aatcatgggt
tccatctcgg actatgcatc 7440agagggattg aatttcgtaa ggtcccaagc
agaaaagatg agatctgccc ccgctttcaa 7500agaaaacgtg gaagctgcta
aagggtacgt ccaaaagttt attgattctc tcatagaaaa 7560taaagaaacc
ataatcagat atggcctgtg gggaacacac acggcactct acaagagtat
7620tgccgcgaga ttgggtcatg aaactgcatt cgctacacta gtgataaagt
ggctggcctt 7680cgggggtgag tcggtgtcag accacatgag acaagcagct
gtcgacctgg ttgtttatta 7740tgtgatcaat aagccctcct tcccagggga
ttctgaaacc caacaggaag gaaggcgatt 7800cgtcgccagc ctgttcatct
ccgctttggc aacctacaca tacaaaactt ggaattacaa 7860caacctctcc
aaggtagtag aaccagcctt agcatacctc ccctatgcta ccaatgcact
7920aaaaatgttt accccgacca gactggagag cgtagttata cttagtacca
caatatacaa 7980aacttacctc tcaataagga agggaaagag tgatggactg
ttgggtacag ggatcagtgc 8040agcaatggag attctatcac agaacccagt
gtcggtaggt atatctgtca tgctgggggt 8100gggggcgatt gccgcgcaca
atgccattga gtctagtgaa caaaaaagga ccctgttgat 8160gaaagtgttt
gtaaaaaact tcctggacca ggcggcaaca gatgagctgg taaaggaaaa
8220cccagagaaa ataataatgg ccctatttga agcagtccag acaattggca
accccttgag 8280gctcatatat cacctgtatg gggtttacta caaaggctgg
gaagcaaaag aactatcaga 8340gagaacagca ggcaggaacc tgttcacctt
gataatgttc gaagccttcg aactactagg 8400gatggactct gaagggaaga
taaggaacct gtctgggaat tatgtcctgg atttgatcta 8460cagcctacat
aaacagataa atagaggctt gaaaaaaata gtcttggggt gggctcccgc
8520accatttagt tgcgactgga ctcctagtga tgagagaatt aggttaccca
caaacaacta 8580tctaagagta gaaactaagt gtccatgtgg ctatgagatg
aaagcactaa ggaacgttgg 8640tggcagtctt accaaagtgg aggagaaagg
accttttctc tgcaggaaca ggcttggtag 8700agggccggtc aactatagag
tcacaaagta ctatgatgac aacctcaaag agataaaacc 8760agttgctaaa
ctagaaggat ttgtggatca ctattacaaa ggtgttacag caaggataga
8820ttatggcaga gggaaaatgc tattagctac tgataaatgg gaggtggagc
acggtgttgt 8880cactaggttg gcaaagagat ataccggagt tggattcaag
ggagcatacc tgggtgatga 8940acccaaccac cgcgacctag tagaaagaga
ctgtgcaact ataacaaaaa atacagtgca 9000gtttttaaaa atgaagaaag
gctgtgcatt tacctatgac ttaaccctgt ccaatttaac 9060caggttaatt
gaattggtac acaaaaataa cctagaagag aaagacatac cagcagccac
9120agtaacgaca tggctggctt atacttttgt aaatgaagat attgggacta
taaaaccagt 9180actaggagag agagtggtca ccgacccagt ggtggatgtt
aacttacaac cagaagtaca 9240agtggataca tcagaggttg ggatcacttt
agttggtagg gcagccttaa tgacgacagg 9300tactacaccc gtagtcgaaa
aaacagagcc caatgctgat ggtggtccaa gctccataaa 9360gattgggttg
gatgaaggaa gatacccagg acctggactg caagaccgca ccttgaccga
9420tgaaatacat tctagggatg aaaggccctt tgttctagtc ctgggctcaa
aaaattctat 9480gtcaaataga gctaaaactg ctagaaacat caacttatac
aaggggaata accccaggga 9540gattagagat ctgatggcac aggggcgtat
gctagttgtg gccttaaagg attttaaccc 9600tgagttgtct gaactagttg
atttcaaggg gactttctta gacagggaag ccttggaagc 9660tctcagcctg
gggcggccaa agtccaagca ggtgaccaca gccacagtta gggagttatt
9720agagcaggag gtacaagttg agatccccag ttggtttgga gcaggtgatc
cagtcttctt 9780ggaagtgact ttgaagggtg acagatatca cttagtagga
gatgtagata gagtgaaaga 9840tcaagcgaag gagcttgggg ccacggacca
gacaagaata gtgaaggaag tgggtgcaag 9900aacctatacc atgaagctgt
ctagttggtt tcttcaggca acaaataaac agatgagctt 9960gaccccttta
tttgaggagc tattgctacg ttgcccccct aaaataaaga gcaataaagg
10020gcacatggca tcagcttacc aactagcaca gggaaactgg gagccccttg
actgtggagt 10080tcacctgggc accatacctg ccaggagggt aaaaatccac
ccatatgaag cttacctgaa 10140actgaaggat ttattggaag aagaagaaaa
gaaaccaaag tgtagagaca cagtaataag 10200agaacacaac aagtggatcc
tcaaaaaagt gaggcaccag ggtaatctca atacaaagaa 10260aatcctcaac
cctggaaagc tatcagaaca gctagataga gaagggcata aaagaaacat
10320ttataacaat cagattggca ccataatgac ggaagcagga agtaggttgg
aaaaattacc 10380agtcgtcaga gcccaaactg acactaaaag cttccatgag
gcaatcagag ataagataga 10440caagaatgaa aatcagcaga gcccaggact
gcatgataaa ttgttagaga tctttcatac 10500aatagcccaa cccagcctaa
gacacaccta cagtgacgtg acgtgggagc aacttgaggc 10560aggggttaat
agaaaggggg ctgctggctt tctagagaag aagaatgttg gagaagtact
10620ggactcagag aagcacctgg tggaacaact gatcagagat ttgaaaacag
gaaggaagat 10680aagatattat gagacagcaa taccaaaaaa tgagaagaga
gatgtcagtg atgattggca 10740atcaggggac ttagtagatg agaagaaacc
aagggtgatt caataccctg aagctaaaac 10800aagactagcc atcactaaag
taatgtacaa ctgggtgaaa cagcagcccg tcgtgatccc 10860agggtatgaa
gggaagaccc cattatttaa cattttcaac aaggtgagga aggaatggga
10920tttgttcaat gaaccagtag ctgtgagttt cgacactaag gcttgggaca
cccaagtaac 10980tagtagagat ctacggctta ttggtgaaat tcaaaaatat
tactacagga aagagtggca 11040caaattcatc gataccatta ctgaccatat
ggtggaggtg cccgtcataa cggcagatgg 11100tgaggtatac ataagaaatg
gacaaagggg tagtggccag ccagacacaa gtgcaggcaa 11160tagcatgcta
aacgtgttaa caatgatgta tgccttctgt gaaagtacgg gggttccata
11220caagagtttc aatagggttg caaggatcca tgtctgtggg gatgacggct
tcctaataac 11280agagaagggg ctgggattaa agtttgccaa caatgggatg
caaattctgc acgaagcagg 11340caagcctcaa aagataactg agggggaaag
aatgaaagtt gcctataggt tcgaggacat 11400agaattctgc tctcatacac
cagtccccgt taggtggtct gataacacca gcagttacat 11460ggccggcaga
gacactgccg ttatattatc aaagatggca acaagattgg attcaagtgg
11520agaaaggggt actatagcat atgaaaaagc agtggccttt agttttttgc
tgatgtactc 11580ctggaatcct cttgtgagga ggatctgtct actggtcctt
tcacagcagc cagagacaac 11640tccatcaacc cagaccactt actattataa
aggagaccca ataggagcct acaaagatgt 11700aataggtaag aatttgtgtg
aattaaaaag gacgggtttt gaaaaattgg ccaatttaaa 11760cctaagcctg
tccacgttag gaatctggtc caaacataca agtaaaagaa tcatccaaga
11820ctgtgtaacc atcgggaaag aggaaggcaa ttggctggtc aatgccgaca
ggttgatatc 11880tagcaaaact ggccatttgt acatacctga caaaggttat
acattacaag ggaaacatta 11940tgaacaactt caactgcagg caagaactag
cccagtcacg ggagtaggga cggagagata 12000taaactaggc cctatagtaa
acctgctgct gaggaggttg agagttctgc ttatggcagc 12060tgtcggtgcc
agcagttgaa ataatgtatg tatatattgt atataaatct gtatttgtat
12120atattatgtt taaatacgta gccagccccc gattgggggc gacactccac
catagatcac 12180tcccctgtga ggaactactg tcttcacgca gaaagcgtct
agccatggcg ttagtatgag 12240tgtcgtgcag cctccaggac cccccctctc
gggagagcca tagtggtctg cggaaccggt 12300gagtacaccg gaattgccag
gacgaccggg tcctttcttg gatcaacccg ctcaatgcct 12360ggagatttgg
gcgtgccccc gcgagactgc tagccgagta gtgttgggtc gcgaaaggcc
12420ttgtggtact gcctgatagg gtgcttgcga gtgcctcggg aggtctcgta
gaccgtgcac 12480cttaattaat aatttagttg agattagtag tgatatatag
ttatctacct caagctaaca 12540ctacactcaa tgcacacagc actttagctg
tatgagggta cacccgacgt ccacggttgg 12600actagggaaa acccttaaca gcccc
12625412281DNApestivirus type 1misc_featureBVDV-non-CP7 + cloning
site; BVDV-non-CP7 with SnaBI, PacI restriction endonuclease insert
4gtatacgagg ttaggcaagt tctcgtatac atattggaca ctctaaaaat aattaggcct
60aggggacaaa aatcctcctt agcgaaggcc gaaaagaggc taaccatgcc cttagtagga
120ctagcaaaat aaggggggta gcaacagtgg cgagttcgtt ggatggctga
agccctgagt 180acagggtagt cgtcagtggt tcgacgcttt ggaggacaag
cctcgagatg ccacgtggac 240gagggcatgc ccacagcaca tcttaacctg
gacaggggtc gttcaggtga aaacggttta 300accaaccgct acgaatacag
tctgatagga tgctgcagag gcccactgta ttgctactga 360aaatctctgc
tgtacatggc acatggagtt gatcacaaat gaacttttat acaaaacata
420caaacaaaaa cccgctggag tggaggaacc agtatacgac caagctggta
accctttgtt 480tggagaaaga ggagtgattc atccgcagtc aacgctaaaa
cttccacata aaagagggga 540gcgtgaagtc cccaccaatc tggcttcttt
accaaaaaga ggtgactgca ggtcgggtaa 600cagcaagggg cctgtgagtg
gaatctactt aaaaccaggg ccgttattct accaagatta 660taaaggacct
gtctatcata gagccccatt ggagtttttt gaggaggcgt ctatgtgtga
720gacaactaaa agaataggga gagtaactgg tagtgacagc agattatacc
acatttacgt 780gtgtattgat gggtgcataa tagtcaagag tgctacaaaa
gaccgccaga aagtactcaa 840gtgggtccac aacaagctaa actgccccct
atgggtttca agctgctccg acacaaaaga 900tgaaggggtg gtgaggaaga
agcaacaaaa gccagatagg ttggaaaagg ggagaatgaa 960gataacacct
aaggagtcag agaaagacag taagaccaag ccgccagatg ctacgatagt
1020ggtagatgga gtcaagtatc aggtaaagaa aaaaggaaaa gtcaagagca
agaacaccca 1080ggacggctta taccacaaca aaaataaacc tcaagagtcg
cgcaagaaac tagagaaagc 1140cctattggcc tgggcaataa tagccctggt
tttctttcaa gtcacaatgg gagagaacat 1200aacgcaatgg aacttacaag
ataatggaac ggaaggcata caacgagcca tgtttcaaag 1260aggagtgaat
agaagtttac atgggatctg gccagagaaa atctgtacag gtgttccttc
1320ccacctggcc actgacacag aattgaaggc aattcatggt atgatggatg
caagtgagaa 1380gacaaattat acgtgctgca gactccaacg ccatgaatgg
aacaaacatg gttggtgcaa 1440ctggtacaac attgaacctt ggatcctcct
tatgaataaa actcaggcca accttactga 1500gggtcagcca ctaagggagt
gtgccgtcac atgccggtat gatcgagata gtgacctgaa 1560tgtagtaaca
caagccaggg atagccccac accattaaca ggttgcaaga aaggcaaaaa
1620cttttccttt gcaggcatat tggtacaagg gccttgcaac tttgaaatag
ccgtaagtga 1680tgtgctgttc aaagagcatg attgcactag tgtgattcaa
gacacagctc actacctcgt 1740agacgggatg accaactccc tagagagtgc
caggcaaggg accgcgaaac taacaacttg 1800gctgggcagg cagcttggga
tactaggaaa gaaactggaa aacaagagta agacatggtt 1860tggggcatat
gcagcctctc cctattgtga ggtagaacgg aagcttggtt acatctggta
1920tacaaagaat tgcactccag cctgtttgcc taggaataca aagatcatcg
gccccggtag 1980gtttgacacc aatgccgagg atggtaaaat actgcatgag
atggggggtc acttgtcgga 2040ggtgctacta ctctcagtgg tagtgctgtc
cgatttcgct ccagagacag ccagtgtgat 2100atacttgatt cttcatttct
ccatcccaca aggacacact gatatacaag attgtgacaa 2160aaaccaacta
aacctcaccg tagaactcac aacagcagaa gtaataccag gctcagtttg
2220gaacttgggt aaatatgttt gtgtaagacc agattggtgg ccttatgaga
cagccacagt 2280cctggtgatt gaagaggtgg gtcaagtaat taaggttgtc
ttaagggcgt taaaagatct 2340gacgcgcatt tggaccgctg ctacaaccac
tgcattcttg gtttgtctgg tgaaggtagt 2400gagaggccaa gtgttacaag
gtatactgtg gctgatgctc ataacagggg cgcaagggta 2460cccagactgc
aaacccggct tttcatacgc catagccaaa aatgatgaga ttggcccact
2520tggagctaca ggcctcacca ctcagtggta cgaatactcg gatgggatgc
ggctgcagga 2580ctcagtagtt gaagtttggt gtaaaaatgg agagatcaaa
tatctaatca gatgcgggag 2640ggaagccagg tatctggctg ttctacacac
gagagccttg ccgacatctg tagtatttga 2700aaaaattttt gatgggaaag
aacaagagga catagtagaa atggatgaca actttgaatt 2760cggtctttgc
ccgtgtgatg ctagaccctt gataagggga aaatttaata caacacttct
2820aaatgggcca gccttccaga tggtttgccc tataggatgg acagggactg
taagctgtac 2880actggccaat aaggatacgt tagccacaat cgttgtgaga
acgtataaga gggtcaggcc 2940ttttccatat aggcaggact gtgtcaccca
gaaaaccatc ggggaagacc tctacgactg 3000tgccttagga gggaattgga
cttgtgtgcc gggggatgca ctacgatatg tagctgggcc 3060cgttgagtct
tgtgagtggt gtggttacaa gtttttaaaa agtgagggtc tgccgcattt
3120cccaatcggc aaatgcaggc tgaagaatga gagtggctat agacaagtgg
atgagacttc 3180ttgcaacaga aacggcgtgg ctatagtgcc atctggcacg
gtcaaatgca agatagggga 3240cacggtggtg caagtcattg caatggatga
taaactaggg cctatgcctt gcaaaccaca 3300tgaaatcata tccagtgagg
ggccagtgga aaagacggca tgcaccttca actacacaag 3360aacattaaaa
aacaagtact ttgagcccag ggataactat tttcaacaat acatgttaaa
3420gggggagtac caatattggt ttgacctaga gatcactgac caccaccgag
attacttcgc 3480tgagtccctg ttggtgatag tagttgcact cctgggtggc
aggtacgtgc tttggctgct 3540ggtcacatac atgatcttat cagaacagat
ggcctcgggt gtccagtatg gggcaggtga 3600aatagtgatg atgggcaact
tgttaacaca tgacagtgtt gaagtggtga catatttctt 3660actactatac
ctactactaa gagaggaaaa caccaaaaaa tgggtcatac ttatatacca
3720catcatagta atgcatcctc taaaatcggt gacggtgata ttgctaatgg
ttggggggat 3780ggcaaaggct gaaccaggtg cccaggggta cctagagcag
gtagacctta gttttacgat 3840gattacgatc atcgtaatag gtctggttat
agctaggcgt gatcccactg tggtgccact 3900agtcactata gtcgcggcac
tgaagatcac aggactaggc tttgggcccg gagtggatgc 3960agctatggca
gttctcacct taaccctact gatgactagt tatgtgacag actacttcag
4020gtataaaagg tggatacaat gtatcctcag cttagtagcc ggggtgttcc
ttatccggac 4080cctcaaacat ctaggtgaac tcaaaacccc tgagctgacc
ataccaaatt ggaggccact 4140aaccttcata ctattatacc tgacttcagc
aacagttgtt acaagatgga aaattgatat 4200agctggcata ttcctgcaag
gggcccctat ccttttgatg atcgccaccc tatgggctga 4260cttcttgact
cttgttctga tcctacccac ctacgaatta gccaagctgt actacctaaa
4320gaacgtcaag actgacgtgg agaagagttg gctggggggg ttagactaca
ggacaattga 4380ctctgtctat gatgtggatg aaagtggaga aggcgtgtac
ctcttcccgt ccagacagaa 4440gaaaaataag aatatcagca tactcttgcc
cctcatcaga gctacgctaa taagttgtat
4500tagcagcaaa tggcagatgg tgtatatggc ttacttaacc ctggacttta
tgtactacat 4560gcacagaaag gttattgaag agatatcagg gagtaccaat
gtgatgtcta gagtgatagc 4620agcacttata gaattaaact ggtccatgga
agaagaagag agcaagggct taaagaagtt 4680ttttatacta tctggaaggt
tgaggaacct tataataaag cataaggtta ggaaccagac 4740tgtggcaagc
tggtatgggg aggaagaagt ctacggcatg ccaaaagtcg taaccataat
4800aagggcctgc acgctaaaca agaacaaaca ttgcataata tgcacagtat
gtgaggctag 4860aaagtggaag ggaggcaact gccctaaatg cggccgccac
gggaagccca tcatttgtgg 4920gatgactcta gcggattttg aagaaaggca
ctacaagaga atttttataa gggaaggtaa 4980ctttgaagga cccttcaggc
aggaatacaa tgggtttgta caatacaccg ctagggggca 5040attgttcctg
agaaatttac ccatattggc aaccaaagta aaaatgatca tggtaggcaa
5100cctaggagag gaaatcggtg atctagaaca cctaggatgg atcctaaggg
gacctgccgt 5160gtgcaagaaa ataactgagc acgaaaaatg ccatgtcaac
atactggaca agctgactgc 5220gttttttgga gttatgccaa gagggactac
accaagggct ccggtgagat tcccaacagc 5280actactaaag gtaaggaggg
gattggaaac cggttgggct tacacgcatc aaggtggcat 5340aagctcagta
gaccatgtga ccgctggcaa ggatctattg gtttgtgaca gtatgggtag
5400aactagagtg gtttgccaaa gcaacaacaa gttaactgat gagacagaat
atggtgtcaa 5460gacggactcc ggatgtccag atggtgccag atgctatgta
ttaaacccag aggcagtaaa 5520tatatcaggg tccaagggag ctgtcgtaca
cctccaaaaa acgggtgggg aatttacatg 5580tgttactgca tcaggtacac
cggccttctt cgacctgaaa aatttgaaag gatggtcggg 5640tctacccata
tttgaagcct ccagcggcag agtggttggc agagtcaaag tgggaaagaa
5700tgaggaatcc aaacccacaa aattaatgag tggtatccaa actgtttcta
aaaatacggc 5760cgatttaaca gaaatggtca agaagataac cagcatgaac
aggggggact ttaggcagat 5820aacccttgca acaggggcag ggaagaccac
tgagctccca aaagcagtga tagaggagat 5880aggacgacac aaacgggtac
tagtgctcat accattaaga gcagcagctg agtcagtcta 5940tcaatacatg
agattgaaac acccaagtat ctcctttaac ctgagaatag gggacatgaa
6000agaaggggat atggcaaccg ggatcaccta cgcctcatat ggatattttt
gccaaatgcc 6060acaaccaaag ctcagagcag caatgataga gtattcatac
atatttctgg atgagtatca 6120ctgcgctact cctgagcagt tggctgttat
aggaaaaatt cacagatttt ctgagagcat 6180aagagtggtt gccatgactg
ccaccccagc agggtcagta accacaacag ggcaaaaaca 6240cccaatagaa
gaattcatag cccctgaggt gatgaaaggg gaggaccttg gaagccagtt
6300ccttgacata gcggggttaa agatccctgt agaggagatg aagggtaaca
tgttggtttt 6360cgtgcccacg aggaacatgg cagttgaagt agccaagaaa
ctaaaagcca agggctacaa 6420ctcagggtat tactacagtg gggaagaccc
agctaacttg agagtggtaa catcacagtc 6480cccatacgtc gtggtagcca
ctaatgccat cgagtcaggg gtaacgctgc cagatttaga 6540tacagttgtt
gacacaggtc tgaaatgtga gaagagggtg agggtgtctt ccaaaatacc
6600ctttatagta acaggcctta agagaatggc tgtcactgtg ggcgaacagg
ctcagcggag 6660aggcagggta ggtagagtga aacccggtag gtattataga
agtcaggaaa cagcaaccgg 6720gtcaaaggac taccactatg acttgttaca
ggcacagagg tacgggatcg aagatgggat 6780caacgtaaca aagtccttta
gggagatgaa ttatgactgg agcctgtatg aggaagacag 6840cttgctgata
acccagctgg agatactgaa caatctactc atctctgaag atttaccagc
6900agctgttaaa aacatcatgg caagaactga tcacccagag cctatccagc
ttgcatataa 6960cagttatgag gtccaagtcc ctgtgctgtt cccaaaaata
aggaatgggg aggtcacaga 7020cacttacgag aactactcat tcctaaatgc
aaggaaacta ggggaagacg tgcccgtgta 7080cgtttatgcc accgaagatg
aagatctggc tgtggacctt ctaggcttgg actggccaga 7140cccagggaat
cagcaagtag tggagactgg gaaggcactg aagcaagtgg taggactgtc
7200ctctgccgaa aatgccttgc tcatagccct atttgggtat gtaggatacc
aagccttgtc 7260aaaaagacac gtcccaatga tcacagacat atacactata
gaagatcaaa gactagagga 7320cacaacccac cttcaatatg cgcccaatgc
cataagaact gaggggaagg agactgaact 7380aaaggaatta gcagtgggtg
acttggacaa aatcatgggt tccatctcgg actatgcatc 7440agagggattg
aatttcgtaa ggtcccaagc agaaaagatg agatctgccc ccgctttcaa
7500agaaaacgtg gaagctgcta aagggtacgt ccaaaagttt attgattctc
tcatagaaaa 7560taaagaaacc ataatcagat atggcctgtg gggaacacac
acggcactct acaagagtat 7620tgccgcgaga ttgggtcatg aaactgcatt
cgctacacta gtgataaagt ggctggcctt 7680cgggggtgag tcggtgtcag
accacatgag acaagcagct gtcgacctgg ttgtttatta 7740tgtgatcaat
aagccctcct tcccagggga ttctgaaacc caacaggaag gaaggcgatt
7800cgtcgccagc ctgttcatct ccgctttggc aacctacaca tacaaaactt
ggaattacaa 7860caacctctcc aaggtagtag aaccagcctt agcatacctc
ccctatgcta ccaatgcact 7920aaaaatgttt accccgacca gactggagag
cgtagttata cttagtacca caatatacaa 7980aacttacctc tcaataagga
agggaaagag tgatggactg ttgggtacag ggatcagtgc 8040agcaatggag
attctatcac agaacccagt gtcggtaggt atatctgtca tgctgggggt
8100gggggcgatt gccgcgcaca atgccattga gtctagtgaa caaaaaagga
ccctgttgat 8160gaaagtgttt gtaaaaaact tcctggacca ggcggcaaca
gatgagctgg taaaggaaaa 8220cccagagaaa ataataatgg ccctatttga
agcagtccag acaattggca accccttgag 8280gctcatatat cacctgtatg
gggtttacta caaaggctgg gaagcaaaag aactatcaga 8340gagaacagca
ggcaggaacc tgttcacctt gataatgttc gaagccttcg aactactagg
8400gatggactct gaagggaaga taaggaacct gtctgggaat tatgtcctgg
atttgatcta 8460cagcctacat aaacagataa atagaggctt gaaaaaaata
gtcttggggt gggctcccgc 8520accatttagt tgcgactgga ctcctagtga
tgagagaatt aggttaccca caaacaacta 8580tctaagagta gaaactaagt
gtccatgtgg ctatgagatg aaagcactaa ggaacgttgg 8640tggcagtctt
accaaagtgg aggagaaagg accttttctc tgcaggaaca ggcttggtag
8700agggccggtc aactatagag tcacaaagta ctatgatgac aacctcaaag
agataaaacc 8760agttgctaaa ctagaaggat ttgtggatca ctattacaaa
ggtgttacag caaggataga 8820ttatggcaga gggaaaatgc tattagctac
tgataaatgg gaggtggagc acggtgttgt 8880cactaggttg gcaaagagat
ataccggagt tggattcaag ggagcatacc tgggtgatga 8940acccaaccac
cgcgacctag tagaaagaga ctgtgcaact ataacaaaaa atacagtgca
9000gtttttaaaa atgaagaaag gctgtgcatt tacctatgac ttaaccctgt
ccaatttaac 9060caggttaatt gaattggtac acaaaaataa cctagaagag
aaagacatac cagcagccac 9120agtaacgaca tggctggctt atacttttgt
aaatgaagat attgggacta taaaaccagt 9180actaggagag agagtggtca
ccgacccagt ggtggatgtt aacttacaac cagaagtaca 9240agtggataca
tcagaggttg ggatcacttt agttggtagg gcagccttaa tgacgacagg
9300tactacaccc gtagtcgaaa aaacagagcc caatgctgat ggtggtccaa
gctccataaa 9360gattgggttg gatgaaggaa gatacccagg acctggactg
caagaccgca ccttgaccga 9420tgaaatacat tctagggatg aaaggccctt
tgttctagtc ctgggctcaa aaaattctat 9480gtcaaataga gctaaaactg
ctagaaacat caacttatac aaggggaata accccaggga 9540gattagagat
ctgatggcac aggggcgtat gctagttgtg gccttaaagg attttaaccc
9600tgagttgtct gaactagttg atttcaaggg gactttctta gacagggaag
ccttggaagc 9660tctcagcctg gggcggccaa agtccaagca ggtgaccaca
gccacagtta gggagttatt 9720agagcaggag gtacaagttg agatccccag
ttggtttgga gcaggtgatc cagtcttctt 9780ggaagtgact ttgaagggtg
acagatatca cttagtagga gatgtagata gagtgaaaga 9840tcaagcgaag
gagcttgggg ccacggacca gacaagaata gtgaaggaag tgggtgcaag
9900aacctatacc atgaagctgt ctagttggtt tcttcaggca acaaataaac
agatgagctt 9960gaccccttta tttgaggagc tattgctacg ttgcccccct
aaaataaaga gcaataaagg 10020gcacatggca tcagcttacc aactagcaca
gggaaactgg gagccccttg actgtggagt 10080tcacctgggc accatacctg
ccaggagggt aaaaatccac ccatatgaag cttacctgaa 10140actgaaggat
ttattggaag aagaagaaaa gaaaccaaag tgtagagaca cagtaataag
10200agaacacaac aagtggatcc tcaaaaaagt gaggcaccag ggtaatctca
atacaaagaa 10260aatcctcaac cctggaaagc tatcagaaca gctagataga
gaagggcata aaagaaacat 10320ttataacaat cagattggca ccataatgac
ggaagcagga agtaggttgg aaaaattacc 10380agtcgtcaga gcccaaactg
acactaaaag cttccatgag gcaatcagag ataagataga 10440caagaatgaa
aatcagcaga gcccaggact gcatgataaa ttgttagaga tctttcatac
10500aatagcccaa cccagcctaa gacacaccta cagtgacgtg acgtgggagc
aacttgaggc 10560aggggttaat agaaaggggg ctgctggctt tctagagaag
aagaatgttg gagaagtact 10620ggactcagag aagcacctgg tggaacaact
gatcagagat ttgaaaacag gaaggaagat 10680aagatattat gagacagcaa
taccaaaaaa tgagaagaga gatgtcagtg atgattggca 10740atcaggggac
ttagtagatg agaagaaacc aagggtgatt caataccctg aagctaaaac
10800aagactagcc atcactaaag taatgtacaa ctgggtgaaa cagcagcccg
tcgtgatccc 10860agggtatgaa gggaagaccc cattatttaa cattttcaac
aaggtgagga aggaatggga 10920tttgttcaat gaaccagtag ctgtgagttt
cgacactaag gcttgggaca cccaagtaac 10980tagtagagat ctacggctta
ttggtgaaat tcaaaaatat tactacagga aagagtggca 11040caaattcatc
gataccatta ctgaccatat ggtggaggtg cccgtcataa cggcagatgg
11100tgaggtatac ataagaaatg gacaaagggg tagtggccag ccagacacaa
gtgcaggcaa 11160tagcatgcta aacgtgttaa caatgatgta tgccttctgt
gaaagtacgg gggttccata 11220caagagtttc aatagggttg caaggatcca
tgtctgtggg gatgacggct tcctaataac 11280agagaagggg ctgggattaa
agtttgccaa caatgggatg caaattctgc acgaagcagg 11340caagcctcaa
aagataactg agggggaaag aatgaaagtt gcctataggt tcgaggacat
11400agaattctgc tctcatacac cagtccccgt taggtggtct gataacacca
gcagttacat 11460ggccggcaga gacactgccg ttatattatc aaagatggca
acaagattgg attcaagtgg 11520agaaaggggt actatagcat atgaaaaagc
agtggccttt agttttttgc tgatgtactc 11580ctggaatcct cttgtgagga
ggatctgtct actggtcctt tcacagcagc cagagacaac 11640tccatcaacc
cagaccactt actattataa aggagaccca ataggagcct acaaagatgt
11700aataggtaag aatttgtgtg aattaaaaag gacgggtttt gaaaaattgg
ccaatttaaa 11760cctaagcctg tccacgttag gaatctggtc caaacataca
agtaaaagaa tcatccaaga 11820ctgtgtaacc atcgggaaag aggaaggcaa
ttggctggtc aatgccgaca ggttgatatc 11880tagcaaaact ggccatttgt
acatacctga caaaggttat acattacaag ggaaacatta 11940tgaacaactt
caactgcagg caagaactag cccagtcacg ggagtaggga cggagagata
12000taaactaggc cctatagtaa acctgctgct gaggaggttg agagttctgc
ttatggcagc 12060tgtcggtgcc agcagttgaa ataatgtatg tatatattgt
atataaatct gtatttgtat 12120atattatgtt taaatacgta ttaattaatt
tagttgagat tagtagtgat atatagttat 12180ctacctcaag ctaacactac
actcaatgca cacagcactt tagctgtatg agggtacacc 12240cgacgtccac
ggttggacta gggaaaaccc ttaacagccc c 122815355DNApestivirus type
1misc_featureBVDV-non-CP7 3' NTR 5tacgtagcca gcccccgatt gggggcgaca
ctccaccata gatcactccc ctgtgaggaa 60ctactgtctt cacgcagaaa gcgtctagcc
atggcgttag tatgagtgtc gtgcagcctc 120caggaccccc cctctcggga
gagccatagt ggtctgcgga accggtgagt acaccggaat 180tgccaggacg
accgggtcct ttcttggatc aacccgctca atgcctggag atttgggcgt
240gcccccgcga gactgctagc cgagtagtgt tgggtcgcga aaggccttgt
ggtactgcct 300gatagggtgc ttgcgagtgc ctcgggaggt ctcgtagacc
gtgcacctta attaa 3556205DNApestivirus type
1misc_featureBVDV-non-CP7 + cloning site chimera, 3'NTR, SnaBI and
PacI restriction endonuclease insert 6tgaaataatg tatgtatata
ttgtatataa atctgtattt gtatatatta tgtttaaata 60cgtattaatt aatttagttg
agattagtag tgatatatag ttatctacct caagctaaca 120ctacactcaa
tgcacacagc actttagctg tatgagggta cacccgacgt ccacggttgg
180actagggaaa acccttaaca gcccc 2057549DNApestivirus type
1misc_featureBVDV-non-CP7- HCV 5' NTR chimera, 3'NTR 7tgaaataatg
tatgtatata ttgtatataa atctgtattt gtatatatta tgtttaaata 60cgtagccagc
ccccgattgg gggcgacact ccaccataga tcactcccct gtgaggaact
120actgtcttca cgcagaaagc gtctagccat ggcgttagta tgagtgtcgt
gcagcctcca 180ggaccccccc tctcgggaga gccatagtgg tctgcggaac
cggtgagtac accggaattg 240ccaggacgac cgggtccttt cttggatcaa
cccgctcaat gcctggagat ttgggcgtgc 300ccccgcgaga ctgctagccg
agtagtgttg ggtcgcgaaa ggccttgtgg tactgcctga 360tagggtgctt
gcgagtgcct cgggaggtct cgtagaccgt gcaccttaat taataattta
420gttgagatta gtagtgatat atagttatct acctcaagct aacactacac
tcaatgcaca 480cagcacttta gctgtatgag ggtacacccg acgtccacgg
ttggactagg gaaaaccctt 540aacagcccc 5498524DNApestivirus type
1misc_featureBVDV-non-CP7 - HCV 5' NTR chimera, BamHI / XbaI cloned
PCR fragment, clone 1 8ggatccgctg tcggtgccag cagttgaaat aatgtatgta
tatattgtat ataaatctgt 60atttgtatat attatgttta aatacgtagc cagcccccta
ttgggggcga cactccacca 120tagatcactc ccctgtgagg aactactgtc
ttcacgcaga aagcgtctag ccatggcgtt 180agtatgagtg tcgtgcagcc
tccaggaccc cccctctcgg gagagccata gtggtctgcg 240gaaccggtga
gtacaccgga attgccagga cgaccgggtc ctttcttgga tcaacccgct
300caatgcctgg agatttgggc gtgcccccgc gagactgcta gccgagtagt
gttgggtcgc 360gaaaggcctt gtggtactgc ctgatagggt gcttgcgagt
gcctcgggag gtctcgtaga 420ccgtgcacct taattaataa tttagttgag
attagtagtg atatatagtt atctacctca 480agctaacact acactcaatg
cacacagcac tttagctgtc taga 5249524DNApestivirus type
1misc_featureBVDV non-CP7 - HCV 5'NTR chimera, BamHI / XbaI cloned
PCR fragment, clone 2 9ggatccgctg tcggtgccag cagttgaaat aatgtatgta
tatattgtat ataaatctgt 60atttgtatat attatgttta aatacgtagc cagcccccga
ttgggggcga cactccacca 120tagatcactc ccctgtgagg aactactgtc
ttcacgcaga aagcgtctag ccatggcgtt 180agtatgagtg tcgtgcagcc
tccaggaccc cccctctcgg gagagccata gtggtctgcg 240gaaccggtga
gtacaccgga attgccagga cgaccgggtc ctttcttgga tcaacccgct
300caatgcctgg agatttgggc gtgcccccgc gagactgcta gccgagtagt
gttgggtcgc 360gaaaggcctt gtggtactgt ctgatagggt gcttgcgagt
gcctcgggag gtctcgtaga 420ccgtgcacct taattaataa tttagttgag
attagtagtg atatatagtt atctacctca 480agctaacact acactcaatg
cacacagcac tttagctgtc taga 52410524DNApestivirus type
1misc_featureBVDV-non-CP7- HCV 5' NTR chimera, BamHI / Xba I cloned
PCR fragment, clone 3 10ggatccgctg tcggtgccag cagttgaaat aatgtatgta
tatattgtat ataaatctgt 60atttgtatat attatgttta aatacgtagc cagcccccga
ttgggggcga cactccacca 120tagatcactc ccctgtgagg aactactgtc
ttcacgcaga aagcgtctag ccatggcgtt 180agtatgagtg tcgtgcagcc
tccaggaccc cccctctcgg gagagccata gtggtctgcg 240gaaccggtga
gtacaccgga attgccagga cgaccgggtc ctttcttgga tcaacccgct
300caatgcctgg agatttgggc gtgcccccgc gagactgcta gccgagtagt
gttgggtcgc 360gaaaggcctt gtggtactgc ctgatagggt gcttgcgagt
gcctcgggag gtctcgtaga 420ccgtgcacct taattaataa tttagttgag
attagtagtg atatatagtt atctacctca 480agccaacact acactcaatg
cacacagcac tttagctgtc taga 52411524DNApestivirus type
1misc_featureBVDV-non-CP7 - HCV 5'NTR chimera, BamHI / XbaI cloned
PCR fragment, clone 4 11ggatccgctg tcggtgccag cagttgaaat aatgtatgta
tatattgtat ataaatctgt 60atttgtatat attatgttta aatacgtagc cagcccccga
ttgggggcga cactccacca 120tagatcactc ccctgtgagg aactactgtc
ttcacgcaga aagcgtctag ccatggcgtt 180agtatgagtg tcgtgcagcc
tccaggaccc cccctctcgg gagagccata gtggtctgcg 240gaaccggtga
gtacaccgga attgccagga cgaccgggtc ctttcttgga tcaacccgct
300caatgcctgg agatttgggc gtgcccccgc gagactgcta gccgagtagt
gttgggtcgc 360gaaaggcctt gtggtactgc ctgatagggt gcttgcgagt
gcctcgggag gtctcgtaga 420ccgtgcacct taattaataa tttagttgag
attagtagtg atatatagtt atctacctca 480agctaacact acactcaatg
cacacagcac tttagctgtc taga 52412523DNApestivirus type
1misc_featureBVDV-non-CP7 - HCV 5'NTR chimera, BamHI / XbaI cloned
PCR fragment, clone 5 12ggatccgctg tcgtgccagc agttgaaata atgtatgtat
atattgtata taaatctgta 60tttgtatata ttatgtttaa atacgtagcc agcccccgat
tgggggcgac actccaccat 120agatcactcc cctgtgagga actactgtct
tcacgcagaa agcgtctagc catggcgtta 180gtatgagtgt cgtgcagcct
ccaggacccc ccctctcggg agagccatag tggtctgcgg 240aaccggtgag
tacaccggaa ttgccaggac gaccgggtcc tttcttggat caacccgctc
300aatgcctgga gatttgggcg tgcccccgcg agactgctag ccgagtagtg
ttgggtcgcg 360aaaggccttg tggtactgcc tgatagggtg cttgcgagtg
cctcgggagg tctcgtagac 420cgtgcacctt aattaataat ttagttgaga
ttagtagtga tatatagtta tctacctcaa 480gctaacacta cactcaatgc
acacagcact ttagctgtct aga 523
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References