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Cloned genome of infectious hepatitis C virus of genotype 2A and uses thereof

a technology cloned genome, which is applied in the field of cloned genome of infectious hepatitis c virus of genotype 2a, can solve the problems of hcv remaining a serious public health problem, progress has been hindered, and there is no useful cell culture system

Inactive Publication Date: 2007-06-21
YANAGI MASAYUKI +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As a result of the inability to develop a universally effective therapy against HCV infection, it is estimated that there are still more than 25,000 new infections yearly in the U.S. (Alter, M. J. 1997 Hepatology 26:62S-65S) Moreover, since there is no vaccine for HCV, HCV remains a serious public health problem.
Despite the intense interest in the development of vaccines and therapies for HCV, progress has been hindered by the absence of a useful cell culture system and the lack of any small animal model for laboratory study.
For example, while replication of HCV in several cell lines has been reported, such observations have turned out not to be highly reproducible.
Consequently, HCV has been studied only by using clinical materials obtained from patients or experimentally infected chimpanzees, an animal model whose availability is very limited.
Unfortunately, the viability of these clones was not tested in vivo and concerns were raised about the infectivity of these cDNA clones in vitro (Fausto, N.

Method used

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  • Cloned genome of infectious hepatitis C virus of genotype 2A and uses thereof
  • Cloned genome of infectious hepatitis C virus of genotype 2A and uses thereof
  • Cloned genome of infectious hepatitis C virus of genotype 2A and uses thereof

Examples

Experimental program
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example 1

Sequence Analysis of HCV Strain HC-J6CH

[0120] As minor deviations from the consensus amino acid sequence were found previously to render full-length HCV cDNA clones noninfectious (Yanagi, M. et al. 1997 PNAS USA 94:8738-8743; Yanagi, M. et al. 1998 Virology 244:161-172), the consensus sequence of the cloning source of genotype 2a (strain HC-J6CH) was determined prior to constructing any full-length clones. In brief, a plasma pool containing strain HC-J6CH was prepared from acute phase plasmapheresis units collected from a chimpanzee experimentally infected with HC-J6 (Okamoto, H. et al. 1991 J. Gen. Virol. 72:2697-2704). The HCV genome titer of this pool was 105.4 genome equivalents (GE) / ml (Quantiplex HCV RNA bDNA 2.0, Chiron) and the infectivity titer was 104 chimpanzee infectious doses / ml.

[0121] The consensus sequence of the 5′ UTR of HC-J6CH (nts. 17-340) was deduced from 5 clones containing nts. 17-297 and 8 clones containing nts. 86-340. The 5′ UTR of the various clones was ...

example 3

A Consensus Molecular Clone of Genotype 2a is Infectious in vivo

[0135] In order to prove that the genotype 2a portion used in the 4 intertypic chimeric cDNA clones indeed represented the infectious sequence, a consensus full-length cDNA clone of HC-J6CH (pJ6CF) was constructed. The core sequence of the T7 promoter, a 5′ guanosine residue and the full-length sequence of HC-J6CH (9711 nts) were cloned into pGEM-9Zf vector using NotI / XbaI sites. Within the HCV sequence there were no deduced amino acid differences and only 4 nucleotide differences (at nucleotide positions 1822, 5494, 9247 and 9289) from the consensus sequence of HC-J6CH as determined in the present study. The silent mutation at position 1822 was within the structural region and so was al. so-present in the four intertypic chimeras. The 5′ terminal 16 nts and the 3′ terminal 82 nts were deduced from previously published HCV genotype 2a sequences (Okamoto, H. et al. 1991 J. Gen. Virol. 72:2697-2704, Tanaka, T. et al. 199...

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Abstract

The present invention discloses nucleic acid sequence which encodes infectious hepatitis C virus of strain HC-J6CH, genotype 2a, and the use of the sequence, and polypeptides encoded by all or part of the sequence, in the development of vaccines and diagnostics for HCV and in the development of screening assays for the identification of antiviral agents for HCV.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation of U.S. application Ser. No.: 09 / 980,559, filed May 14, 2002, which is the U.S. National Phase of International Application No.: PCT / US00 / 15446, filed Jun. 2, 2000, designating the United States of America and published in English on Dec. 14, 2000 as WO 00 / 75338, which claims the benefit of U.S. Provisional Application No. 60 / 137,693 filed Jun. 4, 1999, all of which are hereby expressly incorporated by reference in their entireties.FIELD OF THE INVENTION [0002] The present invention relates to molecular approaches to the production of nucleic acid sequence which comprises the genome of infectious hepatitis C virus. In particular, the invention provides a nucleic acid sequence which comprises the genome of an infectious hepatitis C virus of genotype 2a. The invention therefore relates to the use of the nucleic acid sequence and polypeptides encoded by all or part of the sequence in the development of va...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68C12P21/06
CPCA61K2039/525A61K2039/53C07K14/005C12N2770/24222
Inventor YANAGI, MASAYUKIBUKH, JENSEMERSON, SUZANNE U.PURCELL, ROBERT H.
Owner YANAGI MASAYUKI