Establishment of persistent hepatitis C virus infection and replication in vitro.

Abstract

Hepatitis C virus (HCV) is a major cause of chronic viral hepatitis. Development of anti-viral strategies has been hampered by the lack of efficient cell systems to propagate HCV in vitro. To establish a long-term culture system, we tested human hepatoma (HuH7, HepG2) and porcine non-hepatoma (PK15, STE) cell lines, as well as several culture and infection conditions. As a marker for virus replication, minus-strand HCV RNA in infected cells was detected by an enhanced detection system using nested RT-PCR followed by hybridization analysis. Short-term efficiency of HCV infection (10 days) was slightly increased by addition of polyethylene glycol (PEG) and/or dimethyl sulfoxide (DMSO) to culture media during inoculation of HuH7, PK15 and STE cells, but no augmentation in long-term culture was achieved, suggesting enhanced attachment of HCV to cells rather than more efficient infection. A

Schistosoma mansoni soluble egg antigens enhance HCV replication in mammalian cells

“…The susceptibility of HepG2 cells to HCV infection and its support for viral replication agree with previous reports [19,21,25]. The sensitivity of human anti-HCV-antibody positive sera to detect HCV antigens in infected HepG2 agrees with the observations of El-Awady et al [21].…”

“…Infection of HepG2 cells was performed according to protocols described previously with some modifications [19,25]. In 12-well plates, cells were grown for 24 hours to semi-confluence in complete medium, washed three times with FBS-free medium then inoculated with sterile filtered HCVinfected serum through a 0.22 µm syringe filter to a final concentration of 10%.…”

“…Due to restricted availability of primary hepatocytes, the immortalized human hepatoma cell line HepG2 was later successfully used to host HCV replication in vitro [19][20][21]. In addition, several reports demonstrated replication and assembly of HCV-genotype 4 in peripheral blood mononuclear cells (PBMCs) from Egyptian infected patients [22][23].…”

Association of low-density lipoprotein receptor polymorphisms and outcome of hepatitis C infection

“…38 Furthermore, continued stimulation of LDLR expression enhanced virus replication in vitro. 39 The serum concentration of beta-lipoproteins is thought to influence HCV concentration, as the former could inhibit the binding of the virus to its proposed receptor, the LDLR. Indeed, it was demonstrated that beta-lipoproteins negatively affected the level of HCV-Ag.…”

Hepatitis C virus population dynamics in human lymphocytes and hepatocytes infected in vitro.

“…To date, HCV has been shown to replicate in vitro in human lymphocytes (Cribier et al, 1995 ;Kato et al, 1995 ;Mizutani et al, 1996 b ;Shimizu et al, 1992Shimizu et al, , 1993Shimizu & Yoshikura, 1994), human fibroblasts (Zibert et al, 1995), chimpanzee hepatocytes (Lanford et al, 1994) and human hepatocytes (Ikeda et al, 1997(Ikeda et al, , 1998Ito et al, 1996 ;Seipp et al, 1997). Evidence that HCV possesses cell tropism come from the findings that an HCV population with a limited hypervariable region 1 (HVR1) sequence, which is located in the N-terminal region of the second envelope glycoprotein (E2) (Hijikata et al, 1991 b ;Weiner et al, 1991), became predominant in cultured cells, despite the complicated quasispecies of the HVR1 in the primary inoculum (Hijikata et al, 1995 ;Ikeda et al, 1997 ;Kato et al, 1995 ;Nakajima et al, 1996 ;Sugiyama et al, 1997 b).…”