Efficient cell culture system for hepatitis C virus genotype 6A
The present invention relates to nucleic acid sequences that encode hepatitis C viruses (HCV) of genotype 6a that are useful in the fundamental research of HCV as well as in the search of antivirals and vaccines against HCV. In particular, the present invention relates to nucleic acid sequences that comprises HCVs, which are capable of expressing said virus when transfected into cells and are capable of replication or infectivity in cultured cells.
1. An isolated nucleic acid molecule, which encodes human hepatitis C virus of genotype 6a,
strain HK2, wherein said molecule encodes an amino acid sequence with a sequence identity of at least 95% to that of SEQ ID NO: 3 and wherein the said molecule comprises the following adaptive mutations I774M, R945G, Q1024R, P1117L, V1279A, T1291N, T1370I, L1387R, S1470G, A1673S, V1825A, D2424G, N2805D, V2854M, A2927T, D2987G and Y2989F with reference to SEQ ID NO: 62, or
strain HK6a, wherein said molecule encodes an amino acid sequence with a sequence identity of at least 95% to that of SEQ ID NO: 4 and wherein said molecule comprises the following adaptive mutations F350S, N417T, V775M, P1118L, N1283T, T1292N, S1312P, A1674S, D2425G, N2806D, E2869G, A2928T, D2988G and Y2990F with reference to SEQ ID NO: 63.
2. The nucleic acid molecule according to claim 1 , wherein said molecule encoding strain HK2 further comprises the following adaptive mutations F349S and N416T with reference to SEQ ID NO: 62.
3. The nucleic acid molecule according to claim 1 , wherein said molecule encoding strain HK6a comprises a further adaptive mutation being T1371I with reference to SEQ ID NO: 63.
4. The nucleic acid molecule according to claim 1 , wherein said molecule encoding strain HK2 comprises at least 96% sequence identity to SEQ ID NO: 3.
5. The nucleic acid molecule according to claim 1 , wherein said molecule encoding strain HK6a comprises at least 96% sequence identity to SEQ ID NO: 4.
6. The nucleic acid molecule according to claim 1 , which is strain HK2cc SEQ ID NO: 19.
7. The nucleic acid molecule according to claim 1 , which is strain HK6acc SEQ ID NO: 20.
8. The nucleic acid molecule according to claim 1 , wherein said molecule is capable of generating a HCV infectivity titer of 102 FFU/ml (focus forming units)/ml or above following transfection and/or subsequent viral passage.
9. A method for producing a hepatitis C virus particle, comprising culturing an isolated cell comprising the nucleic acid molecule of claim 1 and producing hepatitis C virus particles therefrom.
10. The method according to claim 9 , wherein the isolated cell is Huh7.5.
11. An immunogenic composition comprising a hepatitis C virus particle produced by the method of claim 9 .
12. A method for producing an immunogenic response in a subject comprising administering a therapeutically effective amount of the hepatitis C virus particle produced by the method of claim 9 to the subject.
13. A method for producing an isolated cell, which replicates human hepatitis C virus and produces a virus particle comprising introducing the nucleic acid molecule of claim 1 into an isolate cell and culturing the isolated cell to produce the human hepatitis C virus particle.
14. The method according to claim 13 , further comprising infecting other cells with the produced human hepatitis C virus particle.