Establishment of an infectious genotype 1b hepatitis C virus clone in human hepatocyte chimeric mice

Kimura, Takashi; Imamura, Michio; Hiraga, Nobuhiko; Hatakeyama, Tsuyoshi; Miki, Daiki; Noguchi, Chiemi; Mori, Nozomu; Tsuge, Masataka; Takahashi, Shoichi; Fujimoto, Yoshifumi; Iwao, Eiji; Ochi, Hidenori; Abe, Hiromi; Maekawa, Toshiro; Arataki, Keiko; Tateno, Chise; Yoshizato, Katsutoshi; Wakita, Takaji; Okamoto, Tomoyoshi; Matsuura, Yoshiharu; Chayama, Kazuaki

doi:10.1099/vir.0.83658-0

Cited by 34 publications

(31 citation statements)

References 24 publications

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“…This knowledge will further the development of intergenotypic recombinant cell culture systems containing yet undefined, minimal JFH1 elements that are critical for cell culture viability. Transfection of RNA transcripts of a genotype 1b cDNA clone into SCID-uPA mice engrafted with human hepatocytes was recently reported (34). In future studies, this model system might offer an alternative for studies of infectivity of cDNA clones in vivo.…”

Section: Discussionmentioning

confidence: 96%

Novel Infectious cDNA Clones of Hepatitis C Virus Genotype 3a (Strain S52) and 4a (Strain ED43): Genetic Analyses and In Vivo Pathogenesis Studies

Gottwein

Scheel

Callendret

et al. 2010

J Virol

126

156

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Previously, RNA transcripts of cDNA clones of hepatitis C virus (HCV) genotypes 1a (strains H77, HCV-1, and HC-TN), 1b (HC-J4, Con1, and HCV-N), and 2a (HC-J6 and JFH1) were found to be infectious in chimpanzees. However, only JFH1 was infectious in human hepatoma Huh7 cells. We performed genetic analysis of HCV genotype 3a (strain S52) and 4a (strain ED43) prototype strains and generated full-length consensus cDNA clones (pS52 and pED43). Transfection of Huh7.5 cells with RNA transcripts of these clones did not yield cells expressing HCV Core. However, intrahepatic transfection of chimpanzees resulted in robust infection with peak HCV RNA titers of ϳ5.5 log 10 international units (IU)/ml. Genomic consensus sequences recovered from serum at the times of peak viral titers were identical to the sequences of the parental plasmids. Both chimpanzees developed acute hepatitis with elevated liver enzymes and significant necroinflammatory liver changes coinciding with detection of gamma interferon-secreting, intrahepatic T cells. However, the onset and broadness of intrahepatic T-cell responses varied greatly in the two animals, with an early (week 4) multispecific response in the ED43-infected animal (3 weeks before the first evidence of viral control) and a late (week 11) response with limited breadth in the S52-infected animal (without evidence of viral control). Autologous serum neutralizing antibodies were not detected during the acute infection in either animal. Both animals became persistently infected. In conclusion, we generated fully functional infectious cDNA clones of HCV genotypes 3a and 4a. Proof of functionality of all genes might further the development of recombinant cell culture systems for these important genotypes.Hepatitis C virus (HCV) is a small, enveloped virus with a single-stranded RNA genome, approximately 9.6 kb in length. The genome consists of 5Ј and 3Ј untranslated regions (UTRs) and a single open reading frame (ORF), encoding structural proteins (Core, E1, and E2), p7, and nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) (22). Due to significant genetic heterogeneity, HCV was classified into 7 major genotypes and numerous subtypes, differing Ͼ30% and Ͼ20%, respectively, at the nucleotide level and at the amino acid level. Strains/isolates differ in 2 to 10% at the nucleotide/ amino acid level, and quasispecies typically differ in up to 2% at the nucleotide/amino acid level (70). As a main cause of liver cirrhosis and hepatocellular carcinoma, chronic HCV infection poses a major public health burden. There is no vaccine available, and combination therapy with alpha interferon and ribavirin is characterized by many side effects and contraindications, as well as low efficacy (22). Research on the HCV life cycle and new therapeutics requires well-characterized experimental models and reagents representing the different virus variants.Chimpanzees, the only animal model of HCV infection mirroring immunopathogenesis and viral persistence observed in human infections (4, 80), can be ...

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Section: Discussionmentioning

confidence: 96%

Novel Infectious cDNA Clones of Hepatitis C Virus Genotype 3a (Strain S52) and 4a (Strain ED43): Genetic Analyses and In Vivo Pathogenesis Studies

Gottwein

Scheel

Callendret

et al. 2010

J Virol

126

156

View full text Add to dashboard Cite

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“…Human hepatocyte-chimeric mice were intravenously injected with 50 μl of the human serum samples positive for HCV genotype 1b. The serum HCV RNA titer in human hepatocyte-chimeric mice was detected by nested PCR, as previously described (38,39). All animal protocols described in this study were performed in accordance with the guidelines and with approval of the Ethics Review Committee of Animal Experimentation of the Graduate School of Biomedical Sciences, Hiroshima University.…”

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confidence: 99%

Adoptive immunotherapy with liver allograft–derived lymphocytes induces anti-HCV activity after liver transplantation in humans and humanized mice

Ohira¹,

Ishiyama²,

Tanaka³

et al. 2009

J. Clin. Invest.

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After liver transplantation in HCV-infected patients, the virus load inevitably exceeds pre-transplantation levels. This phenomenon reflects suppression of the host-effector immune responses that control HCV replication by the immunosuppressive drugs used to prevent rejection of the transplanted liver. Here, we describe an adoptive immunotherapy approach, using lymphocytes extracted from liver allograft perfusate (termed herein liver allograft-derived lymphocytes), which includes an abundance of NK/NKT cells that mounted an anti-HCV response in HCV-infected liver transplantation recipients, despite the immunosuppressive environment. This therapy involved intravenously injecting patients 3 days after liver transplantation with liver allograft-derived lymphocytes treated with IL-2 and the CD3-specific mAb OKT3. During the first month after liver transplantation, the HCV RNA titers in the sera of recipients who received immunotherapy were markedly lower than those in the sera of recipients who did not receive immunotherapy. We further explored these observations in human hepatocyte-chimeric mice, in which mouse hepatocytes were replaced by human hepatocytes. These mice unfailingly developed HCV infections after inoculation with HCV-infected human serum. However, injection of human liver-derived lymphocytes treated with IL-2/OKT3 completely prevented HCV infection. Furthermore, an in vitro study using genomic HCV replicon-containing hepatic cells revealed that IFN-γ-secreting cells played a pivotal role in such anti-HCV responses. Thus, our study presents what we believe to be a novel paradigm for the inhibition of HCV replication in HCV-infected liver transplantation recipients. IntroductionLiver failure and hepatocellular carcinoma (HCC) due to chronic hepatitis C infection are the most common indications for liver transplantation (LT), and the incidences of both have been projected to increase further in the future. Recurrent HCV infection of the allograft is universal, occurs immediately after LT, and is associated with accelerated progression to cirrhosis, graft loss, and death (1, 2). This reflects the suppression of those host-effector immune responses that usually control HCV replication, suggesting that the immunosuppressive environment may play a major role in the rapid progression of recurrent HCV infection after LT (3, 4). Further, the immunosuppressive condition described above is considered to increase the incidence of cancer recurrence after LT in HCC patients. We recently proposed the novel strategy of adjuvant immunotherapy for preventing the recurrence of HCC after LT; this immunotherapy involves intravenously injecting LT recipients with activated liver allograft-derived NK cells (5, 6). Since the immunosuppressive regimen currently used after LT reduces the adaptive immune components but effectively maintains the innate components of cellular immunity (7-9), the augmenta-

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“…RNA extraction and quantitation of HCV by real-time polymerase chain reaction (PCR) were performed as described previously [19]. Briefly, RNA was extracted from mice serum, livers, or cellular lysate using SepaGene RVR (Sankojunyaku, Tokyo, Japan) and reverse transcribed with a random hexamer and a reverse transcriptase (ReverTraAce; TOYOBO, Osaka, Japan) according to the instructions provided by the manufacturer.…”

Section: Quantitation Of Hcv Rna and Isg Mrnasmentioning

confidence: 99%

“…We and other groups had reported that this mouse model is useful for evaluating anti-HCV drugs such as IFN-a and anti-NS3 protease in vivo [17][18][19].…”

Section: Introductionmentioning

confidence: 98%

ME3738 enhances the effect of interferon and inhibits hepatitis C virus replication both in vitro and in vivo

Abe

Imamura

Hiraga

et al. 2011

Journal of Hepatology

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Establishment of an infectious genotype 1b hepatitis C virus clone in human hepatocyte chimeric mice

Cited by 34 publications

References 24 publications

Novel Infectious cDNA Clones of Hepatitis C Virus Genotype 3a (Strain S52) and 4a (Strain ED43): Genetic Analyses and In Vivo Pathogenesis Studies

Novel Infectious cDNA Clones of Hepatitis C Virus Genotype 3a (Strain S52) and 4a (Strain ED43): Genetic Analyses and In Vivo Pathogenesis Studies

Adoptive immunotherapy with liver allograft–derived lymphocytes induces anti-HCV activity after liver transplantation in humans and humanized mice

ME3738 enhances the effect of interferon and inhibits hepatitis C virus replication both in vitro and in vivo

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