Abstract
Multiple genotype 1a clones have been reported, including the very first hepatitis C virus (HCV) clone called H77. The replication ability of some of these clones has been confirmed in vitro and in vivo, although this ability is somehow compromised. We now report a newly isolated genotype 1a clone, designated HCV-RMT, which has the ability to replicate efficiently in patients, chimeric mice with humanized liver, and cultured cells. An authentic subgenomic replicon cell line was established from the HCV-RMT sequence with spontaneous introduction of three adaptive mutations, which were later confirmed to be responsible for efficient replication in HuH-7 cells as both subgenomic replicon RNA and viral genome RNA. Following transfection, the HCV-RMT RNA genome with three adaptive mutations was maintained for more than 2 months in HuH-7 cells. One clone selected from the transfected cells had a high copy number, and its supernatant could infect naïve HuH-7 cells. Direct injection of wild-type HCV-RMT RNA into the liver of chimeric mice with humanized liver resulted in vigorous replication, similar to inoculation with the parental patient’s serum. A study of virus replication using HCV-RMT derivatives with various combinations of adaptive mutations revealed a clear inversely proportional relationship between in vitro and in vivo replication abilities. Thus, we suggest that HCV-RMT and its derivatives are important tools for HCV genotype 1a research and for determining the mechanism of HCV replication in vitro and in vivo.
Highlights
Hepatitis C virus (HCV) is an enveloped positive-strand RNA virus that belongs to the Flaviviridae family [1]
Another breakthrough was made with the discovery of a genotype 2a Japan fulminant hepatitis (JFH)-1 strain that soon became well known for its vigorous replication as a replicon with no adaptive mutations [16]
The first PCR amplification was carried out with the generated cDNA and Phusion DNA polymerase (Finnzymes, Vantaa, Finland) using sense primers corresponding to nucleotides 9-28, 2952-2972, and 5963-5979 and antisense primers corresponding to nucleotides 4038-4054, 70427057, and 9549-9569
Summary
Hepatitis C virus (HCV) is an enveloped positive-strand RNA virus that belongs to the Flaviviridae family [1]. Some reports have been published about full-length replicons with structural proteins in addition to nonstructural proteins, little [13] or no [14,15] secretion of infectious virions was observed, which may have been partly due to adaptive mutations Another breakthrough was made with the discovery of a genotype 2a Japan fulminant hepatitis (JFH)-1 strain that soon became well known for its vigorous replication as a replicon with no adaptive mutations [16]. The new HCV clone, which was designated HCV-RMT (GenBank accession number, AB520610), was different from other genotype 1a clones because it did not require any artificially introduced adaptive mutations for the establishment of replicon cells With these features, our newly cloned HCV-RMT may be a useful tool for investigating the entire life cycle of genotype 1 HCV
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