Robust hepatitis C genotype 3a cell culture releasing adapted intergenotypic 3a/2a (S52/JFH1) viruses.
OBJECTIVE
Recently, full viral life cycle hepatitis C virus (HCV) cell culture systems were developed for strain JFH1 (genotype 2a) and an intragenotypic 2a/2a genome (J6/JFH). We aimed at exploiting the unique JFH1 replication characteristics to develop culture systems for genotype 3a, which has a high prevalence worldwide.


METHODS
Huh7.5 cells were transfected with RNA transcripts of an intergenotypic 3a/JFH1 recombinant with core, E1, E2, p7, and NS2 of the 3a reference strain S52, and released viruses were passaged. Cultures were examined for HCV core and/or NS5A expression (immunostaining), HCV RNA titers (real-time PCR), and infectivity titers (50% tissue culture infectious dose). The role of mutations identified by sequencing of recovered S52/JFH1 viruses was analyzed by reverse genetics studies.


RESULTS
S52/JFH1 and J6/JFH viruses passaged in Huh7.5 cells showed comparable growth kinetics and similar peak HCV RNA and infectivity titers. However, analysis of S52/JFH1 viruses identified 9 putative adaptive mutations in core, E2, p7, NS3, and NS5A. All 7 S52/JFH1 recombinants with an amino acid change in p7 combined with a change in NS3 or NS5A, but only 2 of 9 recombinants with individual mutations (in p7 and NS3, respectively) were fully viable without the requirement for additional mutations. The biological relevance of our system was shown by studying dependence of 3a/JFH1 infection on CD81, and its impact on distribution of intracellular lipids.


CONCLUSIONS
We developed a robust intergenotypic recombinant cell culture system for HCV genotype 3a, providing a valuable tool for studies of 3a core-NS2 and related therapeutics.
Gottwein
Judith M
JM
Department of Infectious Diseases and Clinical Research Center, Hvidovre, Denmark.
Scheel
Troels K H
TK
Hoegh
Anne M
AM
Lademann
Jacob B
JB
Eugen-Olsen
Jesper
J
Lisby
Gorm
G
Bukh
Jens
J
D015703:Antigens, CD / Q000502:physiology; D002460:Cell Line; D005838:Genotype; D016174:Hepacivirus / Q000235:genetics / Q000472:pathogenicity / Q000502:physiology*; D006526:Hepatitis C / Q000235:genetics* / Q000378:metabolism / Q000473:pathology*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D012367:RNA, Viral / Q000378:metabolism; D060225:Tetraspanin 28; D014162:Transfection; D014758:Viral Core Proteins / Q000378:metabolism; D014779:Virus Replication / Q000502:physiology
D015703:Antigens, CD; C554798:CD81 protein, human; D012367:RNA, Viral; D060225:Tetraspanin 28; D014758:Viral Core Proteins