Infectious, Chimeric Hepatitis C Virus, Methods of Producing the Same and Methods of Use Thereof

Abstract

The present invention provides infectious recombinant Hepatitis C Viruses (HCV), and vectors, cells and animals comprising the same. The present invention provides methods of producing infectious recombinant HCV, and their use in identifying anti-HCV therapeutic agents, as well as sequences of HCV associated with HCV pathogenesis.

1 . A method of screening for anti-HCV therapeutics, said method comprising:
 a) contacting a cell with an isolated nucleic acid molecule encoding an infectious recombinant HCV genome, comprising a chimeric HCV genome;   b) contacting the cell in (a) with a candidate molecule;   c) independently contacting the cell in (a) with a placebo; and   d) determining the effects of the candidate molecule on HCV infection, replication, or cell-to-cell spread, versus the effects of said placebo;   wherein a decrease in the level of HCV infection, replication, or cell-to-cell spread indicates said candidate molecule is an anti-HCV therapeutic.   
     
     
         2 . A method of identifying HCV variants with improved growth in cell culture, said method comprising:
 a) contacting cells with an isolated nucleic acid molecule encoding an infectious recombinant HCV genome, comprising a chimeric HCV genome;   b) contacting cells with an isolated nucleic acid molecule comprising at least one mutation of the chimeric HCV genome in (a);   c) independently culturing said cells in (a) and said cells in (b); and   d) determining HCV infection or cell-to-cell spread, in said cells in (a) versus said cells in (b), whereby enhanced HCV infection or cell-to-cell spread in said cells in (b) indicates said HCV variant has improved growth in cell culture.   
     
     
         3 . A method of selecting for HCV variants with enhanced capacity to produce infectious virus, said method comprising:
 a) obtaining a culture of cells with HCV genomes that efficiently replicate intracellularly but produce moderate to undetectable levels of infectious virus particles;   b) passaging said culture from (a);   c) selecting a passaged cell culture from (b) where infectious virus production has occurred; and   d) selecting an HCV variant with enhanced capacity to produce infectious virus.   
     
     
         4 . The method of  claim 3 , wherein said HCV genome is a chimeric HCV genome. 
     
     
         5 . The method of  claim 4 , wherein said chimeric HCV genome comprises a structural core, E1 and E2 genes and nonstructural p7 and NS2 genes from a first HCV strain, and a 5′ non-coding region (NCR), nonstructural NS3, NS4A, NS4B, NS5A, NS5B genes and a 3′ non-coding region (NCR) from a second HCV strain, or wherein said genes or said non-coding regions are homologous to said genes or said non-coding regions from said first or from said second HCV strain. 
     
     
         6 . The method of  claim 5 , wherein said first HCV strain is strain J6 or H77. 
     
     
         7 . The method of  claim 5 , wherein said second HCV strain is strain JFH1. 
     
     
         8 . The method of  claim 3 , wherein said cell culture in step (c) is selected for a substantially higher proportion of NS5A-positive cells. 
     
     
         9 . The method of  claim 3 , further comprising the step (e) of isolating an HCV-specific nucleic acid from said HCV variant of step (d). 
     
     
         10 . The method of  claim 9 , further comprising a step (f) wherein said HCV-specific nucleic acid of step (e) is sequenced to reveal one or more mutations. 
     
     
         11 . The method of  claim 10 , further comprising the step (g) of reengineering one or more of said mutations revealed in step (f) into an HCV genome. 
     
     
         12 . An HCV variant virus produced by the method of  claim 3 . 
     
     
         13 . The HCV variant virus of  claim 12 , wherein said virus is infectious HCV. 
     
     
         14 . The infectious HCV of  claim 13 , wherein said infectious HCV is produced at a titer of 10 1 -10 6  TCID 50 /ml (50% end-point tissue-culture infectious dose per milliliter). 
     
     
         15 . The infectious HCV of  claim 13 , wherein said infectious HCV is produced at a titer of up to 10 5  TCID 50 /ml (50% end-point tissue-culture infectious dose per milliliter). 
     
     
         16 . The infectious HCV of  claim 13 , wherein said infectious HCV is produced at a titer of greater than 10 5  TCID 50 /ml (50% end-point tissue-culture infectious dose per milliliter). 
     
     
         17 . An isolated nucleic acid produced by the method of  claim 9 . 
     
     
         18 . A reengineered HCV genome produced by the method of  claim 11 . 
     
     
         19 . The reengineered HCV genome of  claim 18 , wherein said genome comprises two or three reengineered mutations. 
     
     
         20 . A method of identifying a JFH-1 like HCV isolate which can replicate and produce infectious virus in culture, said method comprising contacting an Huh-7.5 cell culture with an HCV isolate and determining if said isolate is capable of infection and spread within said cell culture. 
     
     
         21 . The method of  claim 12 , wherein an Huh-7.5 cell culture comprises an Huh-7.5 derivative that responds to productive HCV cell culture infection by expression of a reporter gene or dominant selectable marker. 
     
     
         22 . The method of  claim 12 , wherein said HCV isolate is obtained from a clinical sample.