WO1999055366A1 - Chimeres du virus de l'hepatite c et virus de la diarrhee virale des bovins - Google Patents

Chimeres du virus de l'hepatite c et virus de la diarrhee virale des bovins Download PDF

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WO1999055366A1
WO1999055366A1 PCT/US1999/008850 US9908850W WO9955366A1 WO 1999055366 A1 WO1999055366 A1 WO 1999055366A1 US 9908850 W US9908850 W US 9908850W WO 9955366 A1 WO9955366 A1 WO 9955366A1
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hcv
bvdv
ntr
rna
virus
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PCT/US1999/008850
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WO1999055366A8 (fr
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Charles M. Rice
Ilya Frolov
M. Scott Mcbride
Young-Min Lee
Eugene V. Agapov
Tina M. Myers
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Washington University
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Priority to EP99919976A priority Critical patent/EP1071454A1/fr
Priority to CA002330086A priority patent/CA2330086A1/fr
Priority to AU37571/99A priority patent/AU3757199A/en
Publication of WO1999055366A1 publication Critical patent/WO1999055366A1/fr
Publication of WO1999055366A8 publication Critical patent/WO1999055366A8/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5256—Virus expressing foreign proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24311—Pestivirus, e.g. bovine viral diarrhea virus
    • C12N2770/24322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • This invention relates generally to the development of therapies for treating hepatitis C virus (HCV) and bovine viral diarrhea virus (BVDV) and more particularly to the identification of such therapies using chimeric viruses comprising a genomic sequence derived from HCV and bovine viral diarrhea virus (BVDV).
  • HCV hepatitis C virus
  • BVDV bovine viral diarrhea virus
  • Flavivirdae is an important family of human and animal RNA viral pathogens (Rice, CM. 1996. Flavivirdae: The viruses and their replication. In: Fields BN, Knipe DM, Howley PM., eds. Fields virology. Philadelphia: Lippincott-Raven Publishers, pp. 931-960.)
  • the three currently recognized genera of the Flavivirdae family exhibit distinct differences in transmission, host range, and pathogenesis.
  • members of the classical flavivirus genus such as yellow fever virus and dengue virus, are typically transmitted to vertebrate hosts via arthropod vectors and cause acute self-limiting disease (Monath TP, Heinz FX. 1996. Flaviviruses.
  • Flavivirdae genus The most recently characterized Flavivirdae genus is the hepacivirus genus, the sole member of which is the common and exclusively human pathogen, hepatitis C virus (HCV). HCV is transmitted by contaminated blood or blood products and is the most common agent of non- A, non-B hepatitis, affecting more that 1% of the population worldwide (Houghton, 1996. Hepatitis C viruses. In: Fields BN, Knipe DM, Howley PM., eds. Fields virology. Philadelphia: Lippincott-Raven Publishers, pp. 1035-1058.). Unlike flavivirus and pestivirus infections, which are usually eliminated by host immune response, chronic HCV infections are common and can cause mild to severe liver disease including cancer.
  • Virus particles consist of a lipid bilayer envelope with embedded transmembrane glycoproteins surrounding a protein-RNA nucleocapsid. Genome RNAs are single-stranded of positive polarity, and function as the sole mRNA species for translation of a single long open reading frame (ORF). This ORF is translated into a polyprotein which is processed by cellular and viral proteases into mature viral proteins. Structural proteins destined for incorporation into virus particles are encoded in the N-terminal portion of the polyprotein, while the nonstructural proteins which form components of the viral RNA replicase are encoded in the remainder.
  • Flavivirdae contain conserved cz ' s-elements important for translation, RNA replication, and packaging (Bukh et al., Proc. Natl. Acad. Sci. USA 59:4942-4946, 1992; Deng et al., Nucleic Acids Res. 27:1949-1957, 1993; Cahour et al., Virol. 207:68-76, 1995; Kolykhalov et al., J. Virol. 70:3363-3371, 1996; Men et al., J. Virol. 70:3930-3937, 1996; Tanaka et al., J. Virol. 70:3307-3312, 1996; Huang HV. 1997.
  • RNA viruses Evolution of the alphavirus promoter and the exacting sequences of RNA viruses. In: Saluzzo J-F, Dodet B. eds. Factors in the emergence of arbovirus disesases. Paris: Elsevier Press, pp. 65-79; Mandl et al., J. Virol. 72:2132-2140, 1998).
  • NTR nontranslated region
  • flavivirus genome RNAs are translated in a cap-dependent manner. These RNAs contain a 5' cap structure that is presumably added by virus-encoded RNA triphosphatases, guanylyl-, and methyl-transferases (Rice, 1996, supra).
  • RNAs appear to be uncapped and contain long 5' NTRs with cis RNA elements that function as internal ribosome entry sites (IRES) for translation initiation at the polyprotein AUG (Lemon et al., Semin. Virol. 5:274-288, 1997).
  • IRES internal ribosome entry sites
  • the 5' NTRs of HCV and BVDV have a similar structural and functional organization despite containing only short stretches of high sequence identity (Wang et al., Curr. Top. Microbiol fmmunol. 203:99-115, 1995; Lemon et al., 1997, supra).
  • the IRES within each NTR is located at the 3' end of the NTR at a position proximal to the AUG initiation codon of the ORF. Although the 5' terminal sequence of each of these viruses is apparently not required for IRES function (Rijnbrand et al., FEBSLett 365:115-119, 1995; Hyundai et al., Virology. 222:31-42, 1996; Rijnbrand et al., J.
  • sequences in the 5' NTR may be required for regulating translation versus initiation of negative-strand RNA synthesis. Such regulation could occur by direct interaction of 5' and 3' RNA elements or indirectly, via RNA-protein interactions. Sequences in the 5' NTR may also modulate packaging versus translation. Finally, sequences complementary to the 5' NTR, which are located at the 3' end of negative- strand RNA, are likely to function in the initiation of positive-strand RNA synthesis.
  • the HCV 3 ' NTR contains an internal polypyrimidine tract followed by a highly conserved sequence of 98 bases at the 3 ' terminus, which has been shown to be required for replication of HCV (U.S. Application Serial No. 08/811,566).
  • the present invention provides novel compositions and methods for studying HCV replication which are based on the discovery that chimeras of HCV and BVDV genomic sequences can be constructed that are able to replicate in cell culture.
  • the BVDV- specific sequence provides the chimeric viral nucleic acid with the ability to replicate in cell culture, while the HCV-specific sequence allows the chimeric viral nucleic acid to be used to screen possible compounds for anti-viral activity against HCV. It is believed that similar replication-competent chimeras can be constructed from HCV and other pestiviruses.
  • the present invention provides a novel, chimeric viral RNA in which at least one of the 5' NTR; ORF and 3' NTR regions is chimeric and comprises a nucleotide sequence from the corresponding region of a pestivirus in operable linkage with a nucleotide sequence from the corresponding region of an hepatitis C virus (HCV).
  • the chimeric viral RNA is replication-competent.
  • the pestivirus is BVDV.
  • the invention provides a polynucleotide comprising a DNA-dependent promoter operably linked to a cDNA of a chimeric viral RNA as described above and cells transiently transfected or stably transformed with the polynucleotide.
  • the cDNA may encode a dominant selectable marker or an assayable reporter.
  • the invention provides a method for identifying compounds having anti-HCV activity.
  • the method comprises providing a first cell containing a chimeric viral nucleic acid derived from HCV and a pestivirus as described above and a second cell containing the pestivirus, and then comparing the replication efficiency of the chimeric viral nucleic acid in the presence and absence of a test compound to the replication efficiency of the pestivirus in the presence and absence of the test compound, wherein a greater reduction in compound-induced replication efficiency of the chimeric viral nucleic acid than the pestivirus indicates the compound has anti-HCV activity.
  • the invention also provides a genetically-engineered virus which comprises a chimeric viral nucleic acid derived from HCV and a pestivirus as described above.
  • the genetically-engineered virus comprises virus particles containing at least one HCV structural protein and is useful in a vaccine against HCV.
  • the genetically-engineered virus is attenuated as compared to the pestivirus and is useful as a vaccine against the pestivirus.
  • the invention provides a replication-competent BVDV vector expressing a heterologous sequence.
  • the BVDV vector comprises the BVDV sequences encoding the BVDV replication machinery.
  • the replication- competent BVDV vector expresses an antigen and is useful as a vaccine.
  • Figure 1 is a schematic representation of the 5' NTRs of BVDV, HCV, and EMCV showing the position of the start codons of the ORF, and the boxes indicating the canonical IRES elements.
  • Figure 2 shows a schematic representation of BVDV and HCV chimeras, plaque phenotypes, reticulocyte translation efficiencies relative to parental BVDV, specific infectivities in MDBK cells, titers at 24 and 48 h post-transfection (or 72 h, as indicated), and an indication of whether pseudorevertants arose with results from BVDV, 5 'HCV, BVDV+HCV, and BVDV+HCVdelB3 chimeras shown in Fig.
  • Figure 3 illustrates the in vitro translation efficiency of BVDV RNA or chimeras showing bar graphs of the amount of N pro , the N-terminal protein in the BVDV ORF, expressed by the various constructs.
  • Figure 4 illustrates a schematic representation of EMCV chimeras, plaque phenotypes, reticulocyte translation efficiencies relative to parental BVDV, specific infectivities in MDBK cells, titers at 24 and 48 h post-transfection (or 72 h, as indicated), and an indication of whether pseudorevertants arose.
  • Figure 5 illustrates a pseudorevertant analyses showing in (Fig. 5 A) the relative positions of mutations detected within the plaque-purified variants of passaged BVDV+HCVdelBlB2B3, 5'EMCV, and 5 ⁇ CV, and in (Fig. 5B) the 5' terminal sequences of pseudorevertants of BVDV+HCVdelBlB2B3, 5'EMCV, and 5'HCV. Novel nucleotides or sequences are shown in bold upper case type. Pseudorevertants are numbered and designated by the suffix ".R". The upper case sequence in BVDV+HCVdelB 1B2B3 and
  • BVDV+HCVdelBlB2B3.Rl is a remnant of downstream BVDV 5' NTR sequences and was created during the cloning procedures.
  • Figure 6 illustrates the construction of derivatives of 5'HCV designed to contain 5' termini corresponding to the sequence detected within the three analyzed pseudorevertants.
  • Fig. 6A shows the 5' terminal sequence of the 5'HCV derivatives with the suffix (orig) designating a derivative containing the original 5' terminal sequence of the pseudorevertant; the suffix (cons) designating a derivative containing the consensus tetranucleotide sequence 5'-GUAU at the same position; and novel sequences shown in bold upper case type.
  • Fig. 6B shows plaque phenotypes, reticulocyte translation efficiencies relative to parental BVDV, specific infectivities in MDBK cells, and titers at 24 and 48 h post-transfection are indicated.
  • Figure 7 illustrates a single step growth curve for various chimeric constructs showing released virus titers measured by performing plaque assays on MDBK cells transfected with various constructs.
  • Figure 8 illustrates replication of BVDV RNA or chimeric derivatives in transfected MDBK cells.
  • Equal numbers of MDBK cells ( ⁇ 8 x 10 6 ) were electroporated with 5 Dg of each in vitro synthesized RNA.
  • MDBK cells were also transfected with infectious yellow fever 17D and Sindbis RNAs to provide molecular mass markers.
  • One fifth of the transfected cells were seeded on 35-mm dishes and incubated in D-MEM supplemented with 10% horse serum for 6 h at 37°C. The media were then replaced with 1 ml of fresh media containing 2 g/ml of actinomycin D and 40 Ci/ml of 3 H-uridine. Incubations were continued for 10 h at 37°C.
  • RNAs were isolated as described in Materials and Methods, and 1/4 of the samples was denatured in glyoxal and loaded on an agarose gel.
  • A Autoradiograph of the dried gel. Only the portion of the gel containing the genomic RNAs is shown.
  • B Amount of radioactivity contained within the displayed fragments as determined by scintillation counting. BVDV, lane 1; 5'HCV, lane 2; BVDV+HCVdelB2B3, lane 3;
  • Figure 10 illustrates the sequence of low copy number plasmid pACNR BVDV NADL (circular) harboring the functional cDNA of cytopathic BVDV NADL (positive sense cDNA 5' to 3'; nt 1-12578.
  • Figure 11 illustrates the sequence of infectious BVDV NADL (positive sense cDNA 5' to 3').
  • Figure 12 illustrates the sequence of infectious non-cytopathic BVDV NADL lacking clns (positive sense cDNA 5' to 3').
  • Figure 13 illustrates the sequence adapted HCV 5' NTR from 5'HCV/Rl.cons (positive sense cDNA 5' to 3'; only the sequence from the 5' base to the ATG initiating the polyprotein is shown).
  • Figure 14 illustrates the sequence of adapted HCV 5' NTR from 5'HCV/Rl.orig (positive sense cDNA 5' to 3'; only the sequence from the 5' base to the ATG initiating the polyprotein is shown).
  • Figure 15 illustrates the sequence of adapted HCV 5'NTR from 5'HCV/R2.cons (positive sense cDNA 5' to 3'; only the sequence from the 5' base to the ATG initiating the polyprotein is shown).
  • Figure 16 illustrates the sequence of adapted HCV 5' NTR from 5'HCV/R2.orig (positive sense cNDA 5' to 3'; only the sequence from the 5' base to the ATG initiating the polyprotein is shown).
  • Figure 17 illustrates the sequence of adapted HCV 5' NTR from 5'HCV/R3.cons (positive sense cDNA 5' to 3'; only the sequence from the 5'base to the ATG initiating the polyprotein is shown).
  • Figure 18 illustrates the sequence of adapted HCV 5'NTR from 5 ⁇ CV/R3.orig (positive sense cDNA 5' to 3'; only the sequence from the 5' base to the ATG initiating the polyprotein is shown).
  • Figure 19 illustrates the sequence of prototype HCV-BVDV chimera from pNADL/5 ⁇ R3.orig/3'H3'B with the adapted HCV 5'NTR from 5'HCV/R3.orig and tandem 3' NTR elements from HCV followed by BVDV (positive sense cDNA 5' to 3') as discussed in Example 5.
  • Figure 20 illustrates various deletions of the poly U track in the 3 'NTR HCV sequence of BVDV/HCV chimera p5H-3H33.
  • Figure 21 illustrates the schematic representation of functional HCV/-BVDV chimera from pCBV/p7.
  • Figure 22 illustrates the sequence of functional HCV-BVDV chimera from pCBV/p7
  • Figure 23 illustrates the schematic representation of a HCV/BVDV chimera with selectable marker.
  • Figure 24 illustrates the sequence of functional HCV-BVDV chimera from pCBV/p7/IRES-pac expressing a dominant selectable marker conferring resistance to puromycin (positive sense cDNA 5' to 3').
  • Figure 25 illustrates the schematic representation of a bicistronic HCV/BVDV chimera.
  • Figure 26 illustrates the sequence of functional bicistronic chimera expressing the entire HCV structural region derived from plasmid pNADL/BI#41/HCV str (positive sense cDNA 5' to 3')
  • HCV-BVDV chimeric RNAs which are replication competent. Such chimeras are useful in screening compounds in vitro for antiviral activity against HCV.
  • in vivo replication of HCV-BVDV chimeras according to the invention may be attenuated as compared to wild-type BVDV and thus may be useful in vaccinating animals against BVDV.
  • HCV chimeric structures described herein for BVDV are applicable to other pestiviruses. In the context of this disclosure, the following terms will be defined as follows unless otherwise indicated:
  • Cross-acting sequences means the nucleotide sequences from an RNA virus genome that are necessary for recognition of the genomic RNA by specific protein(s) of the RNA virus or host cell that carry out replication, transcription, translation or packaging of the genome.
  • Genetically-engineered virus means any virus whose genome is different than that of a wild-type virus due to a human-made deletion, insertion, or substitution of one or more nucleotides to the wild-type viral genome. "Infectious” when used to describe a virus means the virus is capable of entering cells and initiating a virus replication cycle, whether or not this leads to the production of new RNA virus particles.
  • Nucleotide sequence refers to DNA and the corresponding RNA sequence where relevant. It will be understood that sequences shown in the Figures are DNA versions of the RNA sequence and that chimeric molecules of the invention may comprises RNA molecules or cDNA copies of such RNA molecules.
  • Replication-competent as applied to a chimeric HCV-pestivirus RNA means the RNA is capable of RNA-dependent replication in at least one cell type that supports replication of the wild-type parental pestivirus.
  • the number of replicated RNA molecules produced by an HCV-pestivirus chimeric RNA of the invention is at least 10-fold higher than the limit of detection, which is typically 10 to 100 molecules. More preferably, chimeric RNA production by the HCV-pestivirus chimeric RNA is at least 10 2 to 10 3 -fold higher than the detection limit.
  • the replication-competent chimeric RNA replicates at an efficiency that is preferably, at least 0.001%, more preferably, at least 0.01%, more preferably, at least 0.1%, more preferably, at least 1%, more preferably at least 10% and most preferably at least 50% up to 90% that of the parental pestivirus in the same cell type.
  • Transfected cell means a cell containing an exogenously introduced nucleic acid molecule, and includes cells that are transiently transfected with the exogenous nucleic acid.
  • Transformed cell or “stably transformed cell” means a cell containing an exogenously introduced nucleic acid molecule which is present in the cytoplasm or nucleus of the cell and may be stably integrated into the chromosomal DNA of the cell.
  • Virus means a virion, virus particle or a viral genome.
  • a chimeric viral RNA according to the invention is designed to comprise a 5 ' NTR, an ORF, and a 3' NTR, at least one of which is a chimeric region containing two operably linked nucleotide sequences that are from the same region of a pestivirus and an HCV.
  • Pestivirus-specific sequences useful in the invention can be taken from the appropriate genomic region of any cytopathic or noncytopathic type I or type II BVDV isolate, classical swine fever virus (CSFV) isolate, or border disease viral isolate.
  • CSFV classical swine fever virus
  • HCV-specific sequences can be taken from any strain or isolate of HCV, including but not limited to HCV-1, HCV- la, HCV- lb, HCV-lc, HCV-2a, HCV-2b, HCV-2c, HCV-3a .
  • the parental pestivirus is a cytopathic strain of BVDV and the parental HCV strain is HCV-1.
  • the pestivirus- and HCV-specific sequences are operably linked in the chimeric region, meaning the sequences are arranged such that the resulting chimeric structure is functional in the context of replication of the pestivirus.
  • the chimeric viral RNA comprises a chimeric 5' NTR which comprises a BVDV-specific 5' terminal sequence of 5 '-(G/A)UAU and an IRES derived from HCV, with the ORF and the 3 ' NTR consisting of a sequence from the same regions of BVDV.
  • BVDV-specific sequences at the 5' terminus and in the ORF and 3' NTR are chosen such that they are functional in the context of BVDV, meaning the chimeric viral RNA expresses the replication machinery of BVDV and this replication machinery is capable of replicating the chimeric RNA.
  • translation of the BVDV ORF in the chimeric viral RNA is dependent upon a functional HCV IRES.
  • the presence of a functional HCV IRES in this chimera allows the chimera to be used to screen for compounds that target the HCV IRES and thereby inhibit translation of the BVDV ORF as well as replication of the chimeric virus.
  • Such compounds would be expected to also inhibit translation of the ORF in a wild-type HCV and consequently inhibit HCV replication.
  • BVDV 5 ' NTR chimeras include but are not limited to antisense RNAs, RNA decoys that bind proteins involved in recognition of the HCV-specific sequences, ribozymes, and small molecule inhibitors of critical RNA-protein interactions.
  • antisense RNAs include but are not limited to antisense RNAs, RNA decoys that bind proteins involved in recognition of the HCV-specific sequences, ribozymes, and small molecule inhibitors of critical RNA-protein interactions.
  • ribozymes small molecule inhibitors of critical RNA-protein interactions.
  • small molecule inhibitors of critical RNA-protein interactions small molecule inhibitors of critical RNA-protein interactions.
  • the use of such substances for therapeutic applications are known in the art. See, e.g., Amarzguioui M, et al., "Hammerhead ribozyme design and application.” Cell Mol Life Sci. 1998 Nov;54(l l):l 175-202; Welch PJ
  • the invention includes replication- competent HCV-pestivirus chimeras having a chimeric ORF.
  • a chimeric ORF is one comprising an HCV sequence encoding the structural proteins and a pestivirus sequence encoding the nonstructural proteins. It is believed that upon introduction into a cell, such a HCV-BVDV ORF chimera will produce HCV-like virus particles that will be released from the cell and capable of infecting cells normally infected by wild-type HCV, i.e., cells expressing an HCV receptor such as human CD81.
  • ORF chimeras would be useful to screen compounds for drugs that inhibit formation, release or entry of HCV particles.
  • ORF chimeras that produce virus particles containing at least one HCV structural protein would be useful as vaccines against HCV.
  • Other ORF chimeras contemplated by the invention include, for example, chimeras comprising a pestivirus sequence encoding structural proteins and an HCV sequence encoding one or more nonstructural proteins such as the NS3 protease, NS4A cofactor, NS5 A phosphoprotein/interferon resistance determinant and/or the NS5B polymerase.
  • ORF chimeras Replication of such ORF chimeras would be dependent upon the function of the HCV nonstructural protein(s) and these ORF chimeras could be used to screen for drugs that target the HCV nonstructural protein(s) as well as to screen for and map potential drug resistance mutations in HCV nonstructural proteins.
  • HCV- pestivirus ORF chimeras could be useful for developing alternative in vivo animal models for HCV replication and HCV-associated hepatocellular carcinoma to evaluate antivirals and anti-tumor agents.
  • the invention also provides replication-competent HCV-pestivirus chimeras having a chimeric 3 ' NTR which contains one or more conserved elements of the HCV 3 ' NTR.
  • Such 3 ' NTR chimeras would be useful for screening or evaluating compounds targeted against the HCV 3 ' NTR.
  • Compounds that could be screened include antisense RNA molecules, ribozymes and small molecule inhibitors of critical RNA-protein interactions.
  • One 3' NTR chimera according to the invention comprises a BVDV 5 ' NTR, BVDV ORF and a chimeric 3' NTR which consists of an HCV-specific sequence derived from the HCV 3' NTR immediately followed by a BVDV 3 ' NTR.
  • the HCV-specific 3 ' NTR that allows for replication in the context of BVDV has a deletion in the 3 ' NTR poly (U) tract but has all the other HCV 3 ' NTR elements, including the 98 bp 3 ' terminal conserved element.
  • HCV-pestivirus chimeras included within the scope of the invention include those comprising combinations of chimeric regions, i.e., 5' NTR and ORF chimeras; 5' NTR and 3' NTR chimeras; ORF and 3 ' NTR chimeras; and chimeric RNAs in which each of the 5 ' NTR, ORF and 3 ' NTR regions comprise an HCV sequence operably linked to a pestivirus sequence.
  • the invention also provides chimeric RNAs having two ORFs, or bicistronic HCV- pestivirus chimeras.
  • Bicistronic chimeras contemplated by the invention include structures in which the first ORF contains one or more HCV genes and is followed by a second IRES operably linked to a second ORF encoding the pestivirus replicase machinery. It is also contemplated the first ORF may encode a heterologous sequence such as an antigen.
  • HCV-pestivirus chimeras of the invention will be attenuated as compared to the parental wild-type pestivirus.
  • Such attenuated chimeric RNA genomes would be candidate vaccines in the form of live-attenuated virus particles or as RNA or cDNA "genetic" vaccines.
  • the invention also includes vaccines against HCV which comprise an immunogenically-effective amount of HCV-pestivirus particles or nucleic acid.
  • Anti-HCV vaccines comprising virus particles should preferably contain one or more HCV structural proteins.
  • compositions of the present invention can be administered by any suitable route known in the art including for example by injection such as intraperitoneal, intravenous, subcutaneous, intramuscular, transdermal, intrathecal or intracerebral injection. Administration can be either rapid as by injection or over a period of time as by slow infusion or administration of slow release formulation.
  • compositions according to the invention can be employed in the form of pharmaceutical or veterinary preparations. Such preparations are made in a manner well known in the pharmaceutical and veterinary arts.
  • One preferred preparation utilizes a vehicle of physiological saline solution, but it is contemplated that other pharmaceutically acceptable carriers such as physiological concentrations of other non-toxic salts, five percent aqueous glucose solution, sterile water or the like may also be used. It may also be desirable that a suitable buffer be present in the composition.
  • Such solutions can, if desired, be lyophilized and stored in a sterile ampoule ready for reconstitution by the addition of sterile water for ready injection.
  • the primary solvent can be aqueous or alternatively non-aqueous.
  • the carrier can also contain other pharmaceutically-acceptable excipients for modifying or maintaining the pH, osmolarity, viscosity, clarity, color, sterility, stability, rate of dissolution, or odor of the formulation.
  • the carrier may contain still other pharmaceutically-acceptable excipients for modifying or maintaining release or absorption or penetration across the blood-brain barrier.
  • excipients are those substances usually and customarily employed to formulate dosages for parenteral administration in either unit dosage or multi-dose form or for direct infusion into the cerebrospinal fluid by continuous or periodic infusion.
  • formulations containing a chimeric virus according to the invention are to be administered orally.
  • Such formulations are preferably encapsulated and formulated with suitable carriers in solid dosage forms.
  • suitable carriers, excipients, and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, gelatin, syrup, methyl cellulose, methyl- and propylhydroxybenzoates, talc, magnesium, stearate, water, mineral oil, and the like.
  • the formulations can additionally include lubricating agents, wetting agents, emulsifying and suspending agents, preserving agents, sweetening agents or flavoring agents.
  • the compositions may be formulated so as to provide rapid, sustained, or delayed release of the active ingredients after administration to the patient by employing procedures well known in the art.
  • the formulations can also contain substances that diminish proteolytic degradation and promote absorption such as, for example, surface active agents.
  • the specific dose is calculated according to the approximate body weight or body surface area of the patient or the volume of body space to be occupied. The dose will also be calculated dependent upon the particular route of administration selected. Such calculations can be made without undue experimentation by one skilled in the art. Exact dosages are determined in conjunction with standard dose-response studies. It will be understood that the amount of the composition actually administered will be determined by a practitioner, in the light of the relevant circumstances including the condition or conditions to be treated, the choice of composition to be administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the chosen route of administration. Dose administration can be repeated depending upon the pharmacokinetic parameters of the dosage formulation and the route of administration used.
  • Replication-competent HCV-pestiviruses are generated by choosing the HCV function or sequence element desired to be studied.
  • the HCV sequence can be obtained from a plasmid clone of a partial or full HCV genome using PCR to amplify a target region containing the desired sequence or by restriction enzyme digestion.
  • the HCV fragment is then inserted into the desired location of a clone of the pestivirus genome using standard techniques. Desired portions of the pestivirus genome may be deleted before or after addition of the HCV fragment.
  • the recombinant genome is then transfected into a cell that supports replication of the parental pestivirus genome and their ability to replicate using standard assays.
  • replication can be assessed by virus-induced cytopathic effect; plaque formation; detection of viral antigens and/or viral RNA accumulation; and by plaque assay measuring released infectious virus.
  • the inventors herein have found that the BVDV RNA replication machinery works in many cell types, including bovine, hamster, mouse and human cells. It has also been reported that BVDV RNAs can amplify in other cell types including human hepatoma lines and hepatocytes (Behrens SE, et al., J Virol. 1998 Mar;72(3):2364-72). The host cell range for a particular chimera will be dependent upon the properties of that chimera as empirically determined.
  • pseudorevertants can frequently be isolated that are capable of stable replication. Such pseudorevertants will have one or more deletions or base substitutions in the HCV and/or pestivirus sequences. Information derived from these gain-of- function mutations can be used to define the elements necessary for generating stable, replication-competent chimeras of HCV and a pestivirus.
  • the invention provides a method for screening compounds for antiviral activity against HCV.
  • the method involves comparing a test compound's effect on replication of a chimeric HCV-pestivirus RNA molecule as described above with the compound's effect on replication of the parental pestivirus.
  • Compounds which have a greater effect on replication of the chimeric virus than the pestivirus are likely directed against the HCV portion of the chimera.
  • the method is performed by providing duplicate cell cultures containing a chimeric viral RNA which is replication-competent in that cell, treating one of the culture with the test compound, and then measuring the replication efficiency of the chimeric RNA in both cultures. Any effect induced by the compound is compared against the compound's effect on replication of the parental pestivirus in cells of the same type. This control assay is preferably performed at the same time using the same culture conditions.
  • the cells used in the screening assay can be prepared by transiently transfecting the cells with the desired chimeric RNA molecule as described below.
  • the chimeric RNA molecule can be constitutively expressed in the cell by transfecting the cell with a polynucleotide comprising a cDNA of the chimeric RNA operably linked to a DNA-dependent promoter.
  • the chimeric cDNA may include a selectable marker, which would allow for selection of cells expressing the chimeric RNA. It is also envisioned the selectable marker could be a dominant marker that allows selection of cells expressing chimeras having adaptive mutations or selection of cells permissive for virus replication (Frolov et al., J. Virol. 73:3854-3865, 1999). It is also contemplated the cDNA could express a reporter gene that could be assayed to measure RNA replication.
  • chimeric virus particles are incubated with a cell permissive for infection by the pestivirus in the presence or absence of the test compound and then replication of the chimeric virus is measured and compared to the replication of the parental pestivirus incubated with the same cell type in the presence or absence of the test compound.
  • Inhibition of replication can be measured in many ways, including assaying for the reduction of virus-induced cytopathic effect; inhibition of plaque formation, reduced production of viral antigens as detected by immunofluoresence assay; reduced viral RNA accumulation; reduction in released infectious virus from treated and untreated control and chimera samples using a plaque assay.
  • a cell line that is designed for pestivirus-specific transactivation of a reporter gene could be used directly or in lieu of a plaque assay.
  • the reporter gene is operably linked to a promoter that is activated upon infection by the chimeric virus and production of the viral transactivator protein.
  • Example 2 This example illustrates the construction of chimeras for expressing additional functional portions of the HCV genome by addition of further HCV sequence downstream from the functional or adapted HCV 5'NTR chimeras fused in-frame to the BVDV ORF.
  • Figure 21 involves fusion of HCV sequences to BVDV sequences in the p7 protein coding region (at a convenient BseRI restriction site).
  • Both HCV and BVDV encode a p7 protein that is located immediately downstream of the E2 protein.
  • the p7 protein is a small hydrophobic protein of unknown function.
  • pCBV/p7 consists of the first 79 bases of the BVDV 5'NTR encoding stem loop structure Bl' and Bl, followed by the entire HCV 5'NTR, the entire HCV structural protein coding region and the first 36 amino acids of HCV p7 fused to the C-terminal 31 amino acids of BVDV p7.
  • the fused p7 gene is followed by the remainder of the BVDV ORF including the entire nonstructural region and the BVDV 3' NTR.
  • Transfection of MDBK cells with the RNA corresponding to this sequence leads to replication of the chimeric RNA and production of the expected HCV and BVDV polyprotein cleavage products. Variations on this strategy are envisioned in which all or part of the HCV polyprotein and cis elements important for RNA packaging can be expressed in viable chimeras.
  • the BVDV replicase regions for either cytopathic or non-cytopathic pestiviruses can be used.
  • Transfection of cells permissive for HCV particle, assembly, release and reinfection with this chimeric RNA can be used to make HCV-like particles.
  • These particles and this infection system can be used (i) to screen for specific inhibitors of HCV particle, assembly, release and reinfection, (ii) for identifying antibodies capable of neutralizing HCV infectivity and (iii) as live or inactivated vaccines.
  • this embodiment of the invention demonstrates that the BVDV RNA replication machinery can be used for expression of heterologous RNA and polypeptide sequences and can be used as a vehicle for RNA or DNA "genetic" vaccination in which the BVDV replicase amplifies the level of antigen expression by cytoplasmic RNA-dependent replication.
  • Example 3 This example illustrates chimeric RNA's that are modified to express dominant selectable markers, assayable markers or FACS sortable markers.
  • Such variants can be used to select for chimeras capable of replication in particular cell types, or to screen for cell types that are permissive for replication of the chimeric RNA.
  • Selectable markers include, but are not limited to, the genes encoding puromycin resistance (puromycin N-acetyl transferase; PAC), neomycin resistance, blasticidin resistance, hygromycin resistance, etc.
  • Assayable markers include, but are not limited to, the genes encoding B-galactosidase, luciferase, B-glucuronidase, etc.
  • Easily sortable molecules include single chain antibodies, cell surface markers, and non-toxic protein markers like green fluorescent protein.
  • the RNA encoded by pCBV/p7 was modified to include a cassette at the beginning of the BVDV 3 'NTR that is comprised of the EMCV IRES driving the gene encoding PAC.
  • This chimeric RNA can replicate, expresses PAC and confers resistance to puromycin resistance.
  • This property can be used to select for variants of the chimera that are capable of noncytopathic replication in desired cells type and also provides a means of showing that cells harbor a functional chimeric RNA. Desired variants can be identified, cloned and further characterized as described in Example 1.
  • this location in the BVDV genome and this strategy for expressing heterologous genes may also be applied to using infectious attenuated pestiviruses as gene expression vectors and as chimeric live vaccines against other animal pathogens.
  • FIG. 25 A specific example is shown in Figure 25 and its sequence as Figure 26.
  • the 5' sequences are identical to that of pCBV/p7 except that the HCV ORF continues to include the first 246 amino acids of NS4B.
  • the HCV sequence is followed by the EMCV IRES fused to BVDV Npro, the N-terminal 10 aa of BVDV C, the C-terminal 19 aa of C, 9 N-terminal amino acids of Erns, 48 C-terminal amino acids of E2 and the remainder of the BVDV NADL ORF and 3' NTR.
  • the constructed BVDV ORF encodes a functional BVDV RNA replicase. The deletions in the N-terminal portion of this ORF were designed to preserve proper membrane topology and processing of the replicase.
  • the bicistronic chimeric RNA can replicate upon transfection of permissive BVDV host cells.
  • This example illustrates 3'NTR chimeras.
  • initial attempts to recover viable chimeric viruses in which the BVDV 3'NTR was completely replaced by that of HCV were unsuccessful, a strategy similar to that detailed in Example 1 has produced chimeras that harbor the conserved elements of the HCV 3'NTR.
  • An initial tandem 3'NTR construct was made in which the HCV 3'NTR was engineered to follow the BVDV ORF.
  • the complete BVDV 3'NTR was position 3' to the HCV 3' NTR after a short heterologous sequence. This sequence of this parental construct, which replicated poorly, is shown in Figure 19 RNAs transcribed from this plasmid were of low specific infectivity suggesting that revertants or pseudorevertants might have arisen.
  • pBVDV is a derivative of pACNR/BVDV NADL which contains a G ⁇ T transversion at nt 14994 that creates an Xba I site upstream of the T7 promoter (T. Myers & CM. Rice, unpubl.). To facilitate construction of the chimeras, subclones were created.
  • This fragment was digested with Sacl and ligated into pGEM3Zf(-) which had been digested with Sma I and Sac I to produce the subclone pGEM498-Sacl.
  • a fragment containing the BVDV 5' NTR was synthesized by PCR amplification of pBVDV with primers #183 (5'- TTTTCTAGATAATACGACTCACTATAGTATACGAGAATTAGAAAAGGCACTCG) and #480 (5'-GGGGGCTGGCACGTGCCATGTACA).
  • pGemXbal-Sacl contains a tandem fusion of the BVDV 5' NTR, the HCV 5' NTR, and the 5' portion of the BVDV N pr0 gene.
  • pBVDV + HCV was created by digesting pGEMXbal-Sacl with Xba I and Sac I and ligating the fragment into pBVDV digested with the same two enzymes, and as such pBVDV + HCV contains the T7 promoter, followed by the entire 385-nt 5' NTR of BVDV, a GT dinucleotide (nt 386-387), the entire 341-nt 5' NTR of HCV (nt 388-728), and the sequence of the BVDV NADL strain including the ORF and 3' NTR.
  • pBVDV + HCV containing deletions within the BVDV 5' NTR and/or the HCV 5' NTR were created in the subclone pGEMXbal-Sacl, as described below, prior to ligation into Sba I- and Sac I-digested pBVDV.
  • pGEMXbal-Sacl was digested with 4/7 II and BsrG I.
  • pGEMXbal-Sacl was digested with Avr II and BsrG I.
  • pGEMXbal- Sacl was digested with SnaB I and BsrG I.
  • pGEMXbal-Sacl was digested with Avr II and Xcm I.
  • pGEMXbal-Sacl was digested with AVR II and Bsg I.
  • pBVDV + HCVdelB2B3H3 dideletion of nt 67-374, 518-704
  • subclone pGEMXbal-SacidelB2B3 was digested with Sma I.
  • p5'HCV was created by digesting p90/HCVliongpU with Xba I and Nru I and ligating the fragment into pBVDV + HCV digested with the same two enzymes.
  • EMCV plasmid pEC g
  • p5'EMCV contains the entire 710 nt of the 5' NTR of EMCV, followed by the open reading frame of BVDV and the 3' NTR.
  • One extra G residue was added between the T7 promoter and the first nucleotide of the EMCV 5' NTR to facilitate efficient in vitro transcription.
  • Convenient restriction sites within the BVDV 5' NTR or the EMCV 5' NTR were used to create additional chimeras.
  • the plasmid pBVDV + EMCVdelA contains nt 1-378 of BVDV 5' NTR fused with nt 45-710 of EMCV (the BsrG I site of BVDV ligated to the EcoR V site of EMCV), pBVDV + EMCVdelB3A contains nt 1-173 of BVDV fused with nt 45-710 of EMCV (the Afl II site of BVDV ligated to the EcoR V site of ⁇ MCV).
  • pBVDV + ⁇ MCVdelB2B3 A contains nt 1 -66 of BVDV fused with nt 45 -710 of EMCV (the Avr II site of BVDV ligated to the EcoR V site of ⁇ MCV).
  • pBVDV + ⁇ MCVdelB3ABC contains nt 1- 173 of BVDV fused with nt 161-710 of EMCV (the Afl II site of BVDV ligated to the / > spl405 site of EMCV).
  • pBVDV + EMCVdelB2B3ABC nt 1-66 of BVDV fused with nt 161-710 of EMCV (the Avr II site of BVDV ligated to the Ps/?1406 site of EMCV).
  • pBVDV + EMCVdelB3A-H contains nt 1-101 of BVDV fused with nt 289-710 of EMCV (the Nhe I site of BVDV ligated to the Avr II site of EMCV).
  • pBVDV + EMCVdelB2B3A-H contains nt 1-62 of BVDV fused with nt 289-710 of EMCV (the Avr II site of BVDV ligated to the Avr II site of EMCV).
  • the schematics of the chimeric 5' NTRs are presented in Figures 2 and 4. All other heterologous 5' NTRs used in the study were generated by PCR using an oligonucleotide complementary to nt256-272 of the HCV 5' NTR and primers containing the sequence of the Xba I restriction site followed by the T7 promoter, the heterologous sequences found in sequenced pseudorevertants, or sequences corresponding to different regions of the HCV 5' NTR.
  • MDBK cells were obtained from M. Collett (ViroPharma, Inc.) and BT cells were obtained from the American Type Culture Collection (Rockville, Maryland). Cells were grown in Dulbecco's modified Eagle medium (D-MEM) supplemented with 10% horse serum and sodium pyruvate. Transcriptions and transfections
  • All the designed plasmids were digested to completion with Sda I (&e83871), purified by phenol extraction, precipitated by ethanol, and dissolved in water.
  • the transcription reactions were performed sin the T7 Megascript kit (AMBION) using the conditions recommended by the manufacturer. Reactions were incubated at 37°C for 1 h, and 3 H-UTP was added to the reaction to quantify the RNA synthesis. The quality of the synthesized RNAs was checked by agarose gel electrophoresis, and samples containing 50-60% of full-length RNA were used for electroporations and in vitro translations. The reaction mixtures were aliquoted and stored at -70°C prior to electroporation or in vitro translations.
  • Transfection was performed by electroporation of MDBK cells using previously described conditions (Mendez et al., 1998, supra). Two micrograms of in vitro synthesized RNA, corresponding to approximately 1 ⁇ g of the full-length transcript, were used per electroporation. In standard experiments, ten- fold dilutions of electroporated cells were seeded in 6-well tissue culture plates containing 5 x 10 5 naive MDBK cells per well. After 1 h of incubation at 37°C in an 5% C0 2 incubator, cells were overlaid with 3 ml of 0.6% LE Sea Kem agarose (FMC Bioproducts) containing minimal essential medium supplemented with 5% horse serum.
  • FMC Bioproducts 0.6% LE Sea Kem agarose
  • Plaques were stained with crystal violet after 3 days incubation at 37°C. The rest of the transfected cells was seeded into 100-mm dishes and incubated for approximately 48 h or until cytopathic effect was observed in virtually all cells. Samples of the media were taken at 24 and 48 h, and virus titers were determined as described above and previously (Mendez et al., 1998, supra). Analysis of the 5' ends of viral genomes
  • Plaques were typically isolated from the agarose overlay without staining with neutral red.
  • Virus was eluted in 1 ml of D-MEM/10% horse serum for several hours and was used to infect 5 x 10 5 MDBK cells in 35-mm dishes. After 1 h of virus adsorption of 37 °C, an additional 1 ml of D-MEM/10% horse serum was added to the dishes, and incubation was continued for 36-48 h until cytopathic effect was observed in virtually all cells.
  • RNAs Fifty microliters of harvested viral stocks were clarified by low speed centrifugation, and viral RNAs were isolated by TRIzol reagent (Gibco-BRL) using the protocol recommended by the manufacturer. Sequencing of the 5' termini was performed using an oligonucleotide/cDNA-ligation strategy described elsewhere (Troutt et al., Proc. Natl. Acad. Sci. USA 59:9823-9825, 1992). The primer SI (5'-GTCGTTTCACACATGGATCC), complementary to nt 710-729 of the BVDV genome, was used for cDNA synthesis.
  • a phosphorylated oligonucleotide tag (5'-GACTGTTGTGGCCTGCAGGGCCGAATT) with an amino group on the 3' terminus was ligated to the first strand cDNA (Troutt et al., 1992, supra).
  • the primers for PCR amplification were as follows: primer A (5'-GCCCTGCAGGCCACAACAGTC), complementary to the tag; primer B (5'-TCAGGCAGTACCACAA) complementary to nt 281-296 of the HCV 5' NTR; and primer C (5'-GGAATGCTCGTCAAGAAGACAG), complementary to nt 268-289 of the EMCV 5' NTR.
  • the primer pairs of A + B or A + C were used for analysis of the pseudorevertants of 5'HCV and BVDV + HCVdelBlB2B3 or 5'EMCV, respectively.
  • 5'HCV pseudorevertants one tenth of the ligation mixture was used for an additional PCR reaction. This fragment was synthesized using primer S 1 , describe above, and a primer corresponding to nt 147-175 of the HCV genome. Fragments were purified by agarose gel elecfrophoresis and cloned into the plasmid pRS2. Multiple independent clones were sequenced by the standard dideoxy-mediated chain termination methods using the Sequenase version 2.0 DNA Sequencing Kit (USB). Cell-free translation
  • RNA-free translation reactions were performed in reticulocyte extracts (Promega) using conditions recommended by the manufacture. Usually 0.1-1 ⁇ g of the same in vitro synthesized RNAs used in transfection experiments were used in 25 ⁇ l translation reactions. After 45 min of incubation at 30 °C, 2 ⁇ l were dissolved in 10 ⁇ l of sample buffer, and those samples were analyzed by sodium dodecyl sulfate PAGE. Labeled proteins were visualized by autoradiography of the dried gel. The efficiency of translation was measured using phosphorimager analysis (Molecular Dynamics) by comparing the radioactivity in the band corresponding to the N pro protein.
  • phosphorimager analysis Molecular Dynamics
  • RNAs were isolated from the cells by using TRIzol reagent as recommended by the manufacturer (Gibco-BRL). After denaturation with glyoxal in dimethylsulfoxide, cellular RNAs were analyzed by elecfrophoresis in a 1% agarose gel containing a 10 mM phosphate buffer. Pieces of the dried gel containing the approp ⁇ ate RNA bands were excised, and their radioactivity measured by liquid scintillation counting.
  • EMCV is a member of the cardiovirus genus withm the family Ptcornaviridae. While not a member of the Flaviviridae, EMCV is similar to HCV and BVDV m that it is a positive- strand RNA virus shown to contain an IRES withm its 5' NTR (Jang et al., J virol 62:2636- 2643, 1988).
  • the HCV IRES and the BVDV IRES have been classified as type 3 IRESs, while the EMCV IRES is classified as a type 2 IRES (Lemon & Honda, Siemin Virol 5:274-288, 1997).
  • these three IRESs as well as IRESs from other members of the Flaviviridae and the Ptcornaviridae have been proposed to contain a common structural core (Le et al., Virus Genes 72: 135-147, 1996).
  • the secondary structure of the 385-nt BVDV 5' NTR has not been as extensively studied, but is proposed to be similar to that of HCV (Brown et al., 1992) with four discrete hairpins (referred to here as Bl', Bl, B2, and B3) and a pseudoknot at the base of B3 (Rijnbrand et al., 1997).
  • the secondary structure of the longer (>700 nt) EMCV 5' NTR consists of a se ⁇ es of hairpms A-M (Duke et al., 1992; Hoffman & Palmenberg, 1996).
  • a revised model of the EMCV 5' NTR suggests moderately different secondary structures for the C and G subregions, and significantly different secondary structures for the I-M subregion (Palmenberg & Sgro, 1997).
  • HI is nonessential for IRES function (Reynolds et al., 1995; Rijnbrand et al., 1995; Nissan et al., 1996b; Reynolds et al., 1996; Kamoshita et al., 1997) and its deletion has actually increased translation efficiency in some analyses (Rijnbrand et al., 1995; Hyundai et al., 1996b).
  • Most studies have found that hairpin H2 and H3 and the pseudoknot are essential for IRES function (Wang et al., 1993; Rijnbrand et al., 1995; Nissan et al, 1996b).
  • H2 may not be essential (Tsukiyama-Kohara et al., 1992; Urabe et al., 1997).
  • the 3' boundary of the HCV IRES is more controversial. The IRES clearly extends to the AUG initiation codon.
  • sequences affecting the efficiency of translation initiation extend into the ORF (Reynolds et al., 1995; Nissan et al., 1996a; Nissan et al., 1996b; Lu & Wimmer, 1996).
  • the BVDV IRES probably requires hairpins B2 and B3 and the pseudoknot for function, with B 1 ' and B 1 probably not required for IRES activity (Poole et al., 1995; Rijnbrand et al., 1997).
  • hairpins H-L have been shown to be required for IRES function in mono- or dicistronic constructs (Jang & Wimmer, 1990; Duke et al., 1992).
  • the remaining portion of the EMCV 5' NTR is thought to be required for RNA replication or unknown steps in viral replication that are important for pathogenesis (Duke et al., 1990; Martin & Palmenberg, 1996).
  • BVDV 5' NTR and the HCV 5' NTR are proposed to have similar RNA secondary structure and functional organization, an experiment was performed to test whether the BVDV 5' NTR could be replaced by the HCV 5' NTR.
  • p5' HCV has an exact replacement of the BVDV 5' NTR with that of HCV (Fig. 2A) while the coding sequence and 3' NTR of p5'HCV are identical to pBVDV. Positioning of the HCV 5' NTR in such a manner was necessary since translation initiation from the HCV IRES begins at or near the AUG start codon (Honda et al., 1996a; Reynolds et al., 1995; Reynolds et al., 1996; Rijnbrand et al.,
  • BVDV 5' NTR contains signals that are required for a step in replication other than translation which are not present in the 5' HCV chimera.
  • 5' HCV RNA Given the low specific infectivity of 5' HCV RNA, an experiment was performed to test the effect of placing the BVDV 5' NTR sequence upstream of the HCV 5' NTR, resulting in tandem BVDV and HCV 5' NTRs (called BVDV + HCV).
  • This arrangement actually decreased translation efficiency (Fig. 3, lane 14 vs. lane 1) yet restored infectivity (Fig. 2A).
  • the plaques produced by BVDV + HCV were also heterogeneous in size, indicating that this virus was unstable.
  • RT-PCR analysis indicated that pseudorevertants had indeed arisen in which portions of the BVDV and/or HCV 5' NTRs had been deleted (data not shown). These data show that sequences in the BVDV 5' NTR required for virus replication can function when placed upstream of a functional HCV IRES driving translation of the BVDV polyprotein.
  • Hairpins Bl' and Bl in conjunction with the HCV IRES are sufficient for stable and efficient BVDV replication
  • the sequences withm the BVDV 5' NTR that restored replication m the context of the HCV 5' NTR were mapped using three deletion variants.
  • the deletion BVDV + HCVdelB3 removed a large portion of hairpm B3; the deletion withm BVDV + HCVdelB2B3 removed hairpms B2 and B3, and the deletion within BVDV + HCVdelBlB2B3 removed hairpms Bl, B2 and B3.
  • the specific infectivities of RNAs from these deletion mutants were near that of BVDV RNA (Fig. 2).
  • RT-PCR analyses and sequencing indicated that BVDV + HCV delB3 and BVDV + HCVdelB2B3 were stably propagated and produced homogeneous plaques slightly smaller than those of wild-type BVDV (data not shown). In contrast, BVDV + HCVdelBlB2B3 produced smaller heterogeneous plaques.
  • BVDV + HCV delB2B3Hl virus produced smaller plaques than BVDV + HCV delB2B3, indicating that hairpm HI may augment replication of the chimera.
  • BVDV + HCV delB2B3HlH2 RNA was not infectious (Fig. 2B) and was translated poorly (Fig. 3, lane 11). Diminished HCV IRES activity might be due to deletion of hairpm H2 or juxtaposition of BVDV hairp s Bl' and Bl with H3.
  • the following experiment was performed to determine whether the BVDV 5' NTR could be replaced by the 5' NTR of a more phylogenetically distant virus, EMCV.
  • a derivative of BVDV was created, called 5' EMCV, that contains an exact replacement of the BVDV 5' NTR with the EMCV 5' NTR plus an additional guanosine residue at the 5' terminus for more efficient transc ⁇ ption initiation of T7 polymerase (Fig. 4A).
  • the specific infectivity of 5' EMCV RNA was more than three orders of magnitude lower than BVDV RNA, indicating that it was defective for replication, although its specific infectivity was higher than that of 5' HCV RNA (compare Figs. 4A and 2A).
  • 5' EMCV Similar to 5' HCV, 5' EMCV produced heterogeneous plaques, and sequence analysis indicated that pseudorevertants had a ⁇ sen The lower specific infectivity of 5' EMCV RNA was not likely because of a defect in translation, since the translation efficiency of 5' EMCV RNA was about threefold higher in vitro than that of BVDV RNA (Fig. 3, lane 20 vs. lane 19).
  • BVDV + HCV Similar to BVDV + HCV, it was also determined whether the BVDV 5' NTR at the 5' end of the 5' EMCV RNA would increase its specific infectivity BVDV + EMCVdelA (Fig. 4A) contained the entire BVDV 5' NTR m tandem with the EMCV 5' NTR lacking a portion of hairpm A. BVDV + EMCVdelA RNA had a specific infectivity near that of BDVD RNA (compare Figs. 4A and 2A) despite having a lower translation efficiency than 5' EMCV (Fig 3, lane 21 vs.
  • BVDV + EMCVdelB3A-H and BVDV + EMCVdelB2B3A-H in which a portion of hairpm H is deleted, were not infectious (Fig. 4B) and were inefficiently translated m vitro (Fig. 3, lanes 22 and 23). It should be noted that all of the BVDV + EMCV chimeras produced plaques of heterogeneous size, indicating some instability.
  • BVDV + HCVdelBlB2B3 did not replicate stably as indicated by the heterogeneity m the size of plaques produced by this virus.
  • 5' RACE analysis and sequencing indicated that nt 1-26 had been deleted in the pseudorevertants, removing a large portion of Bl' which was apparently deleterious m the absence of Bl. This deletion results m the 5' terminal sequence 5'GUAUCG which is identical to the first six bases of BVDV genome RNA (Fig. 5) and is repeated at positions 27-32.
  • the small plaque variant sequencing of multiple clones revealed heterogeneity oat the extreme 5' end, with either G of A identified as the 5' base.
  • the fused heterologous sequence began with the tetranucelotide sequence 5'-(G/A) UAU (Fig. 5B).
  • sequencing of the entire 5' NTR and a portion of the N pro coding region revealed only these changes at the 5' termini.
  • 5' NTR sequence changes are sufficient for the pseudorevertant phenotypes
  • derivatives of 5' HCV were created with the changes determined by 5' RACE (Fig. 6A) and analyzed the specific infectivities of these RNAs (Fig. 6B).
  • a derivative called 5' HCV.R1 orig was engineered which contained a 5' NTR consisting of the dinucleotide 5' -GU at the 5' terminus of HCV nt 35-341. This results in a 5' terminus consisting of 5'-GUAA.
  • 5'HCV.Rl orig RNA had a specific infectivity at least four orders of magnitude higher than 5' HCV RNA (Figs. 6B and 2A). This demonstrates that this 5' NTR structure is sufficient for phenotypic reversion to high specific infectivity. However, small plaques and considerable heterogeneity were observed for 5'HCV.Rl orig suggesting that additional mutations may be present in the original small plaque variant.
  • the engineered derivative 5'HCV.R2orig had a 5' NTR consisting of 22 nt of Tsp509I-homologous sequence followed by HCV nt 22-341.
  • 5'HCV.R3orig Another construct, called 5'HCV.R3orig was made, which has the 12 nt of the other heterologous sequence fused to the intact HCV 5' NTR. Specific infectivities for both these derivatives were essentially the same as observed for wild type BVDV RNA (2-4 x 10 6 PFU/ ⁇ g; Fig. 6B). Transfection with these transcripts produced medium plaques, as observed for the original variants, and this phenotype was stable upon passaging. These results show that the altered 5'NTR sequences were responsible for the pseudorevertant phenotypes rather than changes elsewhere in their genomes.
  • 5' HCV.Rlcons had the tetranucleotide sequence 5'-GUAU fused to HCV nt 35-341.
  • 5'HCV.R2cons had the 5'-GUAU tetranucleotide sequence fused to HCV nt 22-341.
  • 5'HCV.R3cons contained the tetranucleotide sequence 5'-Guau fused to the intact 5' terminus of the HCV NTR.
  • RNAs from all three of these derivatives had specific infectivities more than five orders of magnitude higher than 5'HCV and comparable to parental BVDV (Fig. 6B).
  • the tetranucleotide sequence 5'-GUAU is important for efficient BVDV RNA accumulation
  • RNA replication of the 5' HCV chimera was severely impaired to a level below detection (Fig. 8, lane 2).
  • every 5' NTR alteration of 5' HCV that increased RNA specific infectivity and allowed efficient virus growth led to readily detectable viral RNA accumulation.
  • BVDV + HCVdelB2B3Hl displayed reduced RNA synthesis compared to BVDV + HCVdelB2B3 (Fig. 8, lane 4 vs. lane 3) perhaps explaining its small plaque phenotype and suggesting a possible positive role for HI in replication of this chimera.
  • 5'HCV.Rlorig which had exhibited plaque heterogeneity and slow growth, accumulated less RNA when compared to 5 ⁇ CV.Rlcons (Fig.
  • 5'HCV.R2orig and 5'HCV.R2cons showed similar RNA accumulation (Fig. 8, lane 9 vs. lane 10) consistent with their medium plaque phenotypes; and 5'HCV.R3cons exhibited reduced RNA synthesis compared to 5 ⁇ CV.R3orig (Fig. 8, lane 8 vs. lane 7), consistent with their small-versus medium-plaque phenotypes.
  • RNA phenotypes are complex, the most striking result is that addition of the Bl' Bl hairpins, addition of heterologous 5' sequences terminating with 5'-GUAU or simply fusion of this tetranucleotide sequence with the HCV 5' NTR or short 5' truncations of the HCV 5' NTR all dramatically upregulated RNA accumulation. This occurred without increasing translation efficiency, at least as measured in a cell-free assay (Fig. 3, compare lanes 3-8 to lane 1), suggesting that these sequences function at the level of RNA replication or stability. Discussion
  • the work presented here helps to define the requirements for a functional BVDV 5'NTR.
  • the BVDV-specific 5' NTR sequences required for efficient replication in cell culture are minimal and consist of the 5' terminal sequence, 5'-GUAU.
  • the sequence 5'- AUAU, detected for some pseudorevertants, may also be functional but this was not tested for technical reasons.
  • This simple 5'-terminal tetranucleotide sequence which is conserved among pestivirses (Ruggli et al., 1996; Becher et al., 1998), was shown to function in the context of functional IRES elements derived from the hepacivirus HCV or the picomavirus EMCV. As discussed below, this may indicate that the 5' signals required for BVDV RNA replication are rather simple or that elements in these heterologous IRESs can functionally replace deleted BVDV sequences.
  • BVDV genome RNA Sequences at the extreme 5' end of BVDV genome RNA could modulate the efficiency of RNA accumulation by affecting RNA stability, translation, promoter efficiency, or some combination of these processes. At this time, we can not distinguish among these possibilities but favor an effect on RNA replication.
  • the complement of the BVDV 5' sequence at the 3' end of the negative-strand RNA presumably functions in the initiation of positive-strand RNA synthesis.
  • AUAC-3' at the 3'terminus fo minus-strand RNA may be important for positive-strand RNA synthesis.
  • RNA replication signals within the 5' NTR appear to be rather simple, it is possible that the signals important for RNA replication actually extend into the IRES and are more complicated. For instance, the 5'HCV pseudorevertants were more stable and grew to higher titers than the 5'EMCV counterparts, despite the fact that the 5'EMCV RNAs were translated more efficiently in vitro. This may indicate that the BVDV and HCV IRESs contain signals important for RNA synthesis that are absent in the EMCV IRES.
  • BVDV and cellular RNAs is a rare event compared to the processes which lead to deletion of terminal viral sequences.
  • Poliovirus chimeras dependent upon a functional HCV IRES have been reported (Lu & Wimmer, 1996). Interestingly, viable poliovirus chimeras were produced only when HCV sequences included both the IRES and the N-terminal portion of the HCV ORF. Nucleotide sequences or structures in the downstream ORF can modulate HCV IRES translational efficiency (see Reynolds et al., 1995; Honda et al., 1996a) but it was also suggested that the N-terminal portion of the HCV core polypeptide might be involved. In the case of our 5' HCV pseudorevertants, there is no requirement for HCV C protein sequences.
  • HCV nt 359-394 and BVDV nt 405-440 are identical at 21 of 36 positions, although identity within this sequence may be attributed to a high adenosine content.
  • luciferase (LUC) and chloramphenicol acetyl transferase (CAT) reporter genes previously used to detect HCV IRES activity also have adenosine- or purine-rich regions in relatively the same position as the HCV ORF and BVDV ORF. It this region is indeed important for IRES activity, this may explain why some have observed that the HCV IRES does not require a portion of the HCV ORF for translation of CAT or LUC (Tsukiyama-Kohara et al., 1992; Wang et al., 1993). Point mutations and insertions within this region of HCV have been shown to reduce HCV IRES activity in vitro (Honda et al., 1996a,b).
  • Bl' and Bl are conserved among different strains of BVDV and similar hairpins are present in border disease virus and CSFV (Deng & Brock, 1993; Becher et al., 1998), Bl' and Bl were dispensable for BVDV replication, provided that the 5' tetranucleotide sequence 5'-(G/A)UAU remained. This may indicate a role for Bl' and Bl in viral replication in vivo that we do not observe in cell culture. It will be interesting to test the phenotype of chimeras that lack Bl' and Bl in vivo to determine if they are attenuated and might serve as useful BVDV vaccines.
  • BVDV-HCV chimeras that are dependent on a functional HCV IRES may have another practical application. It may be possible to use these chimeras to screen for anti-HCV therapeutics that target the HCV IRES.
  • Other researchers have shown antisense oligonucleotide-mediated inhibition of HCV gene expression in hepatocytes by targeting the oligonucleotides to the HCV IRES (Hanecak et al., 1996). It will be of interest to measure the efficacy of antisense oligonucleotides or ribozymes (Lieber et al., 1996) against replicating virus, and these chimeras are more useful than HCV for this purpose since they are able to replicate efficiently in cell culture.
  • BVDV is believed to be a reasonable model of HCV replication not only because of homology and conserved motifs within the 5' NTR but also because of similarities in overall genetic organization (Rice, 1996) and polyprotein processing strategy (Tautz et al., 1997; Xu et al., 1997).
  • a polynucleotide comprising a chimeric viral RNA which comprises:
  • an open reading frame (ORF) region (b) an open reading frame (ORF) region; and (c) a 3 ' nontranslated region (3 ' NTR); wherein at least one of said regions is chimeric and comprises a first nucleotide sequence from a pestivirus in operable linkage with a first nucleotide sequence from an hepatitis C virus (HCV), and wherein said chimeric viral RNA is replication-competent.
  • ORF open reading frame
  • HCV hepatitis C virus
  • the polynucleotide of claim 1 wherein the chimeric region is the 5 ' NTR and the first pestivirus nucleotide sequence is from a bovine viral diarrhea virus (BVDV).
  • BVDV bovine viral diarrhea virus
  • BVDV nucleotide sequence is located at the 5' terminus of the chimeric 5' NTR and comprises 5' RUAU.
  • HCV hepatitis C vims
  • chimeric region is the 5 ' NTR and the first pestivims nucleotide sequence is from a bovine viral diarrhea vims (BVDV).
  • BVDV bovine viral diarrhea vims
  • BVDV nucleotide sequence is located at the 5 ' terminus of the chimeric 5 ' NTR and comprises 5 ' RUAU.
  • a genetically-engineered vims comprising a chimeric RNA genome which comprises:
  • an open reading frame (ORF) region (b) an open reading frame (ORF) region; and (c) a 3 ' nontranslated region (3 ' NTR); wherein at least one of said regions is chimeric and comprises a first nucleotide sequence from a pestivims in operable linkage with a first nucleotide sequence from an hepatitis C virus (HCV), and wherein said chimeric RNA genome is replication-competent.
  • ORF open reading frame
  • HCV hepatitis C virus
  • BVDV bovine viral diarrhea vims
  • BVDV nucleotide sequence is located at the 5 ' terminus of the chimeric 5 ' NTR and comprises 5 ' RUAU and the first HCV nucleotide sequence in the chimeric 5 ' NTR comprises an internal ribosome entry site (IRES).
  • IRS internal ribosome entry site
  • a vaccine against bovine viral diarrhea virus comprising an immunogenically-effective amount of a genetically-engineered vims comprising a chimeric RNA genome having:
  • a 3 ' nontranslated region (3 ' NTR); wherein at least one of said regions is chimeric and comprises a first nucleotide sequence from BVDV in operable linkage with a first nucleotide sequence from an hepatitis C virus (HCV), and wherein the genetically-engineered vims is attenuated as compared to BVDV.
  • HCV hepatitis C virus
  • the chimeric region is the 5 ' NTR and the BVDV nucleotide sequence is located at the 5 ' terminus of the chimeric 5 ' NTR and comprises 5 ' RUAU and the first HCV nucleotide sequence in the chimeric 5 ' NTR comprises an internal ribosome entry site (IRES).
  • IRS internal ribosome entry site
  • a polynucleotide comprising a chimeric viral RNA which comprises: (a) a 5 ' nontranslated region (5 ' NTR);
  • a 3 ' nontranslated region (3 ' NTR); wherein at least one of said regions is chimeric and comprises a first nucleotide sequence from a pestivims in operable linkage with a heterologous nucleotide sequence and wherein said chimeric viral RNA is replication-competent.

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Abstract

L'invention concerne un polynucléotide comprenant un ARN viral chimérique qui contient : une région non traduite 5' (5' NTR), une région à cadre de lecture ouvert (ORF) et une région non traduite 3' (3' NTR), au moins une des régions étant chimérique. La région chimérique comprend une première séquence nucléotidique provenant d'un pestivirus en liaison fonctionnelle avec une séquence nucléotidique hétérologue. L'ARN viral chimérique est compétente pour une réplication. De préférence, la séquence de pestivirus provient d'un virus de diarrhée virale des bovins et la séquence nucléotidique hétérologue provient d'un virus de l'hépatite C. L'invention concerne également une méthode d'identification de composés possédant une activité antivirale contre le virus de l'hépatite C, un virus d'ARN chimérique mis au point par génie génétique et un vaccin contre le virus de la diarrhée virale des bovins.
PCT/US1999/008850 1998-04-24 1999-04-23 Chimeres du virus de l'hepatite c et virus de la diarrhee virale des bovins WO1999055366A1 (fr)

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EP99919976A EP1071454A1 (fr) 1998-04-24 1999-04-23 Chimeres du virus de l'hepatite c et virus de la diarrhee virale des bovins
CA002330086A CA2330086A1 (fr) 1998-04-24 1999-04-23 Chimeres du virus de l'hepatite c et virus de la diarrhee virale des bovins
AU37571/99A AU3757199A (en) 1998-04-24 1999-04-23 Chimeras of hepatitis c virus and bovine viral diarrhea virus

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000075352A2 (fr) * 1999-06-04 2000-12-14 The Government Of The United States Of America As Represented By The Secretary, Department Of Health And Human Services Genomes recombinants vhc/bvdv et utilisation de ceux-ci
WO2001038538A1 (fr) * 1999-11-23 2001-05-31 Viropharma Incorporated Compositions de polymerase et procedes d'utilisation correspondants
EP1149901A1 (fr) * 2000-04-21 2001-10-31 Akzo Nobel N.V. Mutants de pestvirus et vaccins les contenants
WO2004092386A2 (fr) * 2003-04-11 2004-10-28 The Government Of The United States Of America As Represented By The Secretary Of Health And Human Services Induction d'une reponse de lymphocytes t avec des replicons de pestivirus recombinant ou des cellules dendritiques a transfection de replicon de pestivirus recombinant
EP1558276A2 (fr) * 2002-11-08 2005-08-03 The Administrators of the Tulane Educational Fund Inhibiteurs de fusion du flavivirus
US7009044B1 (en) 1999-06-04 2006-03-07 The United States Of America As Represented By The Department Of Health And Human Services HCV/BVDV chimeric genomes and uses thereof
EP1743652A1 (fr) 2002-08-26 2007-01-17 Pfizer Products Inc. Vaccin contre les infections de l'appareil génital et du système respiratoire chez les bovins
WO2014151401A1 (fr) 2013-03-15 2014-09-25 Zoetis Llc Protection croisée de bovins contre une infection par b. trehalosi par un vaccin multivalent
US8932606B2 (en) 2008-11-24 2015-01-13 Life Technologies Corporation Chimeric pestivirus with insertion in 3′ nontranslated region (3′NTR) with stable replication and rnase resistance

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
FROLOV I. MCBRIDE M S, RICE C M: "CIS-ACTING RNA ELEMENTS REQUIRED FOR REPLICATION OF BOVINE VIRAL DIARRHEA VIRUS-HEPATITIS C VIRUS 5' NONTRANSLATED REGION CHIMERAS", RNA, COLD SPRING HARBOR LABORATORY PRESS, US, vol. 04, 1 November 1998 (1998-11-01), US, pages 1418 - 1435, XP002919368, ISSN: 1355-8382, DOI: 10.1017/S1355838298981031 *
LU H-H, WIMMER E: "POLIOVIRUS CHIMERAS REPLICATING UNDER THE TRANSLATIONAL CONTROL OF GENETIC ELEMENTS OF HEPATITIS C VIRUS REVEAL UNUSUAL PROPERTIES OF THE INTERNAL RIBOSOMAL ENTRY SITE OF HEPATITIS C VIRUS", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, US, vol. 93, 20 February 1996 (1996-02-20), US, pages 1412 - 1417, XP002919370, ISSN: 0027-8424, DOI: 10.1073/pnas.93.4.1412 *
MALET I, ET AL.: "YELLOW FEVER 5' NONCODING REGION AS A POTENTIAL ELEMENT TO IMPROVE HEPATITIS C VIRUS PRODUCTION THROUGH MODIFICATION OF TRANSLATIONAL CONTROL", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, ACADEMIC PRESS INC. ORLANDO, FL, US, vol. 02, no. 02, 18 December 1998 (1998-12-18), US, pages 257 - 264, XP002919369, ISSN: 0006-291X, DOI: 10.1006/bbrc.1998.9740 *
VASSILEV V B, COLLETT M S, DONIS R O: "AUTHENTIC AND CHIMERIC FULL-LENGTH GENOMIC CDNA CLONES OF BOVINE VIRAL DIARRHEA VIRUS THAT YIELD INFECTIOUS TRANSCRIPTS", JOURNAL OF VIROLOGY., THE AMERICAN SOCIETY FOR MICROBIOLOGY., US, vol. 71, no. 01, 1 January 1997 (1997-01-01), US, pages 471 - 478, XP002919371, ISSN: 0022-538X *
VENUGOPAL K, GOULD E A: "TOWARDS A NEW GENERATION OF FLAVIVIRUS VACCINES", VACCINE, ELSEVIER LTD, GB, vol. 12, no. 11, 1 January 1994 (1994-01-01), GB, pages 966 - 975, XP002919372, ISSN: 0264-410X, DOI: 10.1016/0264-410X(94)90329-8 *

Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7009044B1 (en) 1999-06-04 2006-03-07 The United States Of America As Represented By The Department Of Health And Human Services HCV/BVDV chimeric genomes and uses thereof
WO2000075352A3 (fr) * 1999-06-04 2001-11-15 Us Health Genomes recombinants vhc/bvdv et utilisation de ceux-ci
WO2000075352A2 (fr) * 1999-06-04 2000-12-14 The Government Of The United States Of America As Represented By The Secretary, Department Of Health And Human Services Genomes recombinants vhc/bvdv et utilisation de ceux-ci
WO2001038538A1 (fr) * 1999-11-23 2001-05-31 Viropharma Incorporated Compositions de polymerase et procedes d'utilisation correspondants
EP1149901A1 (fr) * 2000-04-21 2001-10-31 Akzo Nobel N.V. Mutants de pestvirus et vaccins les contenants
JP2002325575A (ja) * 2000-04-21 2002-11-12 Akzo Nobel Nv ペスチウイルス変異体及びそれを含有するワクチン
US7037508B2 (en) 2000-04-21 2006-05-02 Akzo Nobel Nv Pestivirus mutants and vaccines containing the same
EP1743652A1 (fr) 2002-08-26 2007-01-17 Pfizer Products Inc. Vaccin contre les infections de l'appareil génital et du système respiratoire chez les bovins
EP2360168A1 (fr) * 2002-11-08 2011-08-24 The Administrators of the Tulane Educational Fund Inhibiteurs de fusion du flavivirus
EP1558276A2 (fr) * 2002-11-08 2005-08-03 The Administrators of the Tulane Educational Fund Inhibiteurs de fusion du flavivirus
EP1558276A4 (fr) * 2002-11-08 2006-09-13 Univ Tulane Inhibiteurs de fusion du flavivirus
US7416733B2 (en) 2002-11-08 2008-08-26 The Administrators Of The Tulane Educational Fund Flavivirus fusion inhibitors
US7854937B2 (en) 2002-11-08 2010-12-21 The Administrators Of The Tulane Educational Fund Flavivirus fusion inhibitors
EP2338894A1 (fr) * 2002-11-08 2011-06-29 The Administrators of the Tulane Educational Fund Inhibiteurs de fusion du flavivirus
US8153360B2 (en) 2002-11-08 2012-04-10 The Administrators Of The Tulane Educational Fund Flavivirus fusion inhibitors
WO2004092386A3 (fr) * 2003-04-11 2005-05-12 Us Gov Health & Human Serv Induction d'une reponse de lymphocytes t avec des replicons de pestivirus recombinant ou des cellules dendritiques a transfection de replicon de pestivirus recombinant
WO2004092386A2 (fr) * 2003-04-11 2004-10-28 The Government Of The United States Of America As Represented By The Secretary Of Health And Human Services Induction d'une reponse de lymphocytes t avec des replicons de pestivirus recombinant ou des cellules dendritiques a transfection de replicon de pestivirus recombinant
US8932606B2 (en) 2008-11-24 2015-01-13 Life Technologies Corporation Chimeric pestivirus with insertion in 3′ nontranslated region (3′NTR) with stable replication and rnase resistance
US8986673B2 (en) 2008-11-24 2015-03-24 Life Technologies Corporation Replication stable and RNase resistant chimeras of pestivirus with insertion in 3′ nontranslated region (3′NTR)
WO2014151401A1 (fr) 2013-03-15 2014-09-25 Zoetis Llc Protection croisée de bovins contre une infection par b. trehalosi par un vaccin multivalent

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