Novel cell culture-adapted genotype 2a hepatitis C virus infectious clone.
Although the recently developed infectious hepatitis C virus system that uses the JFH-1 clone enables the study of whole HCV viral life cycles, limited particular HCV strains have been available with the system. In this study, we isolated another genotype 2a HCV cDNA, the JFH-2 strain, from a patient with fulminant hepatitis. JFH-2 subgenomic replicons were constructed. HuH-7 cells transfected with in vitro transcribed replicon RNAs were cultured with G418, and selected colonies were isolated and expanded. From sequencing analysis of the replicon genome, several mutations were found. Some of the mutations enhanced JFH-2 replication; the 2217AS mutation in the NS5A interferon sensitivity-determining region exhibited the strongest adaptive effect. Interestingly, a full-length chimeric or wild-type JFH-2 genome with the adaptive mutation could replicate in Huh-7.5.1 cells and produce infectious virus after extensive passages of the virus genome-replicating cells. Virus infection efficiency was sufficient for autonomous virus propagation in cultured cells. Additional mutations were identified in the infectious virus genome. Interestingly, full-length viral RNA synthesized from the cDNA clone with these adaptive mutations was infectious for cultured cells. This approach may be applicable for the establishment of new infectious HCV clones.
Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.
Tomoko
T
Date
Takanobu
T
Kato
Junko
J
Kato
Hitoshi
H
Takahashi
Kenichi
K
Morikawa
Daisuke
D
Akazawa
Asako
A
Murayama
Keiko
K
Tanaka-Kaneko
Tetsutaro
T
Sata
Yasuhito
Y
Tanaka
Masashi
M
Mizokami
Takaji
T
Wakita
D000818:Animals; D018929:Cell Culture Techniques; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D005838:Genotype; D016174:Hepacivirus / Q000235:genetics*; D006526:Hepatitis C / Q000235:genetics / Q000821:virology*; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism; D006801:Humans; D008297:Male; D051379:Mice; D016513:Mice, SCID; D008875:Middle Aged; D009154:Mutation; D010802:Phylogeny; D017422:Sequence Analysis, DNA; D013997:Time Factors; D014162:Transfection
D018076:DNA, Complementary