Hepatitis C Virus Replication in Transfected and Serum-Infected Cultured Human Fetal Hepatocytes

Abstract

Understanding the pathogenesis of hepatitis C requires the availability of tissue culture models that sustain viral replication and produce infectious particles. We report on the establishment of a culture system of nontransformed human fetal hepatocytes that supports hepatitis C virus (HCV) replication after transfection with full-length in vitro-transcribed genotype 1a HCV RNA without adaptive mutations and infection with patient sera of diverse HCV genotypes. Transfected and infected hepatocytes expressed HCV core protein and HCV negative-strand RNA. For at least 2 months, transfected or infected cultures released HCV into the medium at high levels and usually with a cyclical pattern. Viral replication had some cytotoxic effects on the cells, which produced interferon (IFN)-␤ as a component of the antiviral response. Medium from transfected cells was able to infect naïve cultures in a Transwell system, and the infection was blocked by IFN-␣ and IFN-. Viral particles analyzed by sucrose density centrifugation had a density of 1.17 g/ml. Immunogold labeling with antibody against HCV envelope protein E2 decorated the surface of the viral particles, as visualized by electron microscopy. This culture system may be used to study the responses of nontransformed human hepatocytes to HCV infection, to analyze serum infectivity, and to clone novel HCVs from infected patients. (Am J

Modeling hepatitis C virus infection using human induced pluripotent stem cells

“…Thus, iHLCs support the complete HCV life cycle, including replication and release of infectious virions. Therefore, iHLCs sustain the entire HCV viral life cycle of at least genotype 2a, in accordance with prior reports showing that human fetal hepatocytes are capable of sustaining the HCV life cycle (22,23). Future work toward a fully personalized in vitro model of HCV infection would incorporate both personalized hepatocyte-like cells and isolates from HCV patients, including the most common HCV genotype in the United States, genotype 1a.…”

Entry and replication of recombinant hepatitis C viruses in cell culture

“…Fetal PHH Long-lived as compared to adult PHH [87][88][89][90]. Structures looking like bile canaliculi [87].…”

Cellular models for the screening and development of anti-hepatitis C virus agents

“…In addition, these models offer wide possibilities £ To the best of their knowledge, authors have no real or perceived conflicts of interests. to investigate antiviral drugs: i) the cells can be maintained in a differentiated phenotype, ii) they can be cultured in such a way as to maintain their drug metabolizing capacities (PichardGarcia et al, 2002), iii) they can be infected with HCVser of any genotype (including HCVpp and HCVcc particles), iv) the innate immune response is fully preserved, in contrast to what is observed in Huh-7 cells and derivatives, and other hepatoma cell lines (Keskinen et al, 1999;Lanford et al, 2003;Sumpter et al, 2005) ; indeed, fetal and adult human hepatocytes produce IFNβ and ISGs in response to IFN and HCV infection (Buck, 2008;Castet et al, 2002;Lazaro et al, 2007) a process that is likely to account for the low level of viral infectivity in these cells ; iv) finally, hepatocytes can be prepared from different patients so that the contribution of the interindividual variability of the host biology to antiviral drug response can be evaluated, in contrast to hepatoma cell lines of immortalized hepatocytes that are issued from a single individual; indeed previous analysis show that such diversity exists in terms of natural anti-viral response, response to IFN, receptor expression, etc. (Chevaliez & Pawlotsky, 2007a).…”

“…Virions were released from the infected cells into the medium and could be serially passaged three times into fresh liver cell cultures. More recently, Lazaro et al (Lazaro et al, 2007) characterized long-term, serum-free primary and passaged cultures of nontransformed hepatocytes from human fetal liver. After transfection of these hepatocytes with genotype 1a HCV (p90/HCVFLpU of genotype 1a) or infection with HCVser of genotypes 1, 2, and 3, medium analysis revealed up to 10 6 copies/ml of HCV RNA exhibiting a cyclic pattern; virus-like particles (density 1.17) were detected in the medium of transfected hepatocytes.…”

“…Several reports have shown that IFNα has a direct antiviral effect on HCV replication in different culture models including replicons (Huh-7), productive replicons (Huh-7.5), immortalized non-neoplastic hepatocytes (PH5CH8), fetal and adult primary human hepatocytes (Buck, 2008;Castet et al, 2002;Ikeda et al, 1998;Lanford et al, 2003;Lazaro et al, 2007;Lindenbach et al, 2005). On binding its receptor, IFN triggers the formation of a complex between a heterodimer of IFN receptors IFNAR1 and IFNRA2 and two members of the Janus kinase family, Jak1 and Tyk2.…”