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Hepatitis C virus: propagation, quantification, and storage.

ABSTRACT: Hepatitis C virus (HCV) is a leading cause of chronic liver diseases, including chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV is well known for its restricted tropism and it does not replicate well in animal species other than humans and chimpanzees. Since classical in vitro propagation of natural HCV isolates is not possible, a protocol for the rescue of infectious virus from cDNA clones (genotype 1a pH77S and genotype 2a pJFH-1) transfected as RNA into permissive cells is described here. Because these two molecular clones behave differently in their ability to propagate and produce infectious virus, different methods for propagation of these two viral strains are described. Methods for infectious virus titration, which can be accomplished by counting foci of infected cells following immunostaining for viral antigen expression in cells infected with serial dilutions of a virus harvest (focus forming unit, or FFU, assay), are also provided.

SUBMITTER: Yi M

PROVIDER: S-EPMC3039288 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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Hepatitis C virus: propagation, quantification, and storage.

Yi MinKyung M

Current protocols in microbiology 20101101

Hepatitis C virus (HCV) is a leading cause of chronic liver diseases, including chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV is well known for its restricted tropism and it does not replicate well in animal species other than humans and chimpanzees. Since classical in vitro propagation of natural HCV isolates is not possible, a protocol for the rescue of infectious virus from cDNA clones (genotype 1a pH77S and genotype 2a pJFH-1) transfected as RNA into permissive cells is des ...[more]

PMID: 21053255

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Project description:BackgroundScrub typhus is a leading cause of serious febrile illness in rural Southeast Asia. The causative agent, Orientia tsutsugamushi, is an obligate intracellular bacterium that is transmitted to humans by the bite of a Leptotrombidium mite. Research into the basic mechanisms of cell biology and pathogenicity of O. tsutsugamushi has lagged behind that of other important human pathogens. One reason for this is that O. tsutsugamushi is an obligate intracellular bacterium that can only be cultured in mammalian cells and that requires specific methodologies for propagation and analysis. Here, we have performed a body of work designed to improve methods for quantification, propagation, purification and long-term storage of this important but neglected human pathogen. These results will be useful to other researchers working on O. tsutsugamushi and also other obligate intracellular pathogens such as those in the Rickettsiales and Chlamydiales families.MethodologyA clinical isolate of O. tsutsugamushi was grown in cultured mouse embryonic fibroblast (L929) cells. Bacterial growth was measured using an O. tsutsugamushi-specific qPCR assay. Conditions leading to improvements in viability and growth were monitored in terms of the effect on bacterial cell number after growth in cultured mammalian cells.Key resultsDevelopment of a standardised growth assay to quantify bacterial replication and viability in vitro. Quantitative comparison of different DNA extraction methods. Quantification of the effect on growth of FBS concentration, daunorubicin supplementation, media composition, host cell confluence at infection and frequency of media replacement. Optimisation of bacterial purification including a comparison of host cell lysis methods, purification temperature, bacterial yield calculations and bacterial pelleting at different centrifugation speeds. Quantification of bacterial viability loss after long term storage and freezing under a range of conditions including different freezing buffers and different rates of freezing.ConclusionsHere we present a standardised method for comparing the viability of O. tsutsugamushi after purification, treatment and propagation under various conditions. Taken together, we present a body of data to support improved techniques for propagation, purification and storage of this organism. This data will be useful both for improving clinical isolation rates as well as performing in vitro cell biology experiments.

Project description:Hepatitis C virus (HCV) can cause acute and chronic infection that is associated with considerable liver-related morbidity and mortality. In recent years, there has been a shift in the treatment paradigm with the discovery and approval of agents that target specific proteins vital for viral replication. We employed a cell culture-adapted strain of HCV and human hepatoma-derived cells lines to test the effects of our novel small-molecule compound (AO13) on HCV. Virus inhibition was tested by analyzing RNA replication, protein expression, and virus production in virus-infected cells treated with AO13. Treatment with AO13 inhibited virus spread in cell culture and showed a 100-fold reduction in the levels of infectious virus production. AO13 significantly reduced the level of viral RNA contained within cell culture fluids and reduced the cellular levels of HCV core protein, suggesting that the compound might act on a late step in the viral life cycle. Finally, we observed that AO13 did not affect the release of infectious virus from infected cells. Docking studies and molecular dynamics analyses suggested that AO13 might target the NS5B RNA polymerase, however, real-time RT-PCR analyses of cellular levels of HCV RNA showed only an ∼2-fold reduction in viral RNA levels in the presence of AO13. Taken together, this study revealed that AO13 showed consistent, but low-level antiviral effect against HCV, although the mechanism of action remains unclear. IMPORTANCE The discovery of curative antiviral drugs for a chronic disease such as HCV infection has encouraged drug discovery in the context of other viruses for which no curative drugs currently exist. Since we currently face a novel virus that has caused a pandemic, the need for new antiviral agents is more apparent than ever. We describe here a novel compound that shows a modest antiviral effect against HCV that could serve as a lead compound for future drug development against other important viruses such as SARS-CoV-2.

Dataset Information

Hepatitis C virus: propagation, quantification, and storage.

Publications

Hepatitis C virus: propagation, quantification, and storage.

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