Abstract
BackgroundBy using the hepatitis C virus (HCV) genotype 2a JFH-1 or its chimeric strains, a HCV infection system has been previously developed through several methods– such as in vitro-transcribed JFH1-RNA transfection or stable transfection of the JFH1 cDNA into human hepatoma Huh-7 cell line or its derivatives. However, other reliable methods for delivery of the HCV genome into cells are still worth trying. The helper-dependent adenovirus (HDAd) is devoid of all viral coding sequences and has a package capacity of 37 kb, which is suitably large for the delivery of the HCV genome. Here we report a new method for delivery of the HCV genome into Huh-7 and HepG2 cells by using the HDAd vector.ResultsOur results demonstrated that the infection of Huh-7 cells with the HDAdJFH1 virus led to efficient HCV replication and virion production. We found that the HCV viral RNA levels could reach 107 copies per milliliter (ml) in the culture medium. HDAdJFH1-infected Huh-7 cells could be cultured for 8 passages with the culture medium remaining infectious for naïve Huh-7 cells throughout this period. This infection system proved effective for evaluating the anti-HCV effects of IFN-α in Huh-7 cells. Co-infection of HepG2 cells with the HDAdJFH1 and HDAdmiR-122 virus also resulted in HCV expression and replication.ConclusionThis is the first report of an HDAd-based strategy for HCV replication and production in vitro.
Highlights
By using the hepatitis C virus (HCV) genotype 2a JFH-1 or its chimeric strains, a Hepatitis C virus (HCV) infection system has been previously developed through several methods– such as in vitro-transcribed JFH1-Rinonucleic acid (RNA) transfection or stable transfection of the JFH1 Complementary DNA (cDNA) into human hepatoma Huh-7 cell line or its derivatives
Construction of a helper-dependent adenoviral vector expressing the HCV RNA genome An Ad5 vector has been previously used for the introduction of the hepatitis B viral genome into cultured cells and mice, and it was found that high-titer hepatitis B virions were secreted into the culture medium of infected hepatoma cells and the sera of infected mice [16]
We aimed to develop a simple and reliable method for packaging and delivering the HCV genome into cells to establish a novel HCV infection system
Summary
By using the hepatitis C virus (HCV) genotype 2a JFH-1 or its chimeric strains, a HCV infection system has been previously developed through several methods– such as in vitro-transcribed JFH1-RNA transfection or stable transfection of the JFH1 cDNA into human hepatoma Huh-7 cell line or its derivatives. In 2005, researchers discovered a genotype 2a isolate JFH1 from a Japanese patient with fulminant hepatitis that could exhibit complete the virus life cycle after transfection of in vitro transcribed full-length JFH1 RNA into Huh-7 or Huh7.5 cells This system is able to produce infectious viral particles in cell culture (HCVcc) [6,7]. It was found that stable human hepatoma cell lines containing a chromosomally integrated cDNA copy of the JFH1 genome with a hepatitis delta virus ribozyme at the 3′ end can constitutively produce infectious viral particles [8] These methods have proven to be effective in generating infectious HCV cell culture models in Huh-7 cell line and its derivatives
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