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. 2010 May 20;6(5):e1000910.
doi: 10.1371/journal.ppat.1000910.

Persistent growth of a human plasma-derived hepatitis C virus genotype 1b isolate in cell culture

Erica Silberstein  1 , Kathleen Mihalik, Laura Ulitzky, Ewan P Plant, Montserrat Puig, Sara Gagneten, Mei-ying W Yu, Neerja Kaushik-Basu, Stephen M Feinstone, Deborah R Taylor
Affiliations

Persistent growth of a human plasma-derived hepatitis C virus genotype 1b isolate in cell culture

Erica Silberstein et al. PLoS Pathog. .

Abstract

HCV (hepatitis C virus) research, including therapeutics and vaccine development, has been hampered by the lack of suitable tissue culture models. Development of cell culture systems for the growth of the most drug-resistant HCV genotype (1b) as well as natural isolates has remained a challenge. Transfection of cultured cells with adenovirus-associated RNA(I) (VA RNA(I)), a known interferon (IFN) antagonist and inhibitor of dsRNA-mediated antiviral pathways, enhanced the growth of plasma-derived HCV genotype 1b. Furthermore, persistent viral growth was achieved after passaging through IFN-alpha/beta-deficient VeroE6 cells for 2 years. Persistently infected cells were maintained in culture for an additional 4 years, and the virus rescued from these cells induced strong cytopathic effect (CPE). Using a CPE-based assay, we measured inhibition of viral production by anti-HCV specific inhibitors, including 2'-C-Methyl-D-Adenosine, demonstrating its utility for the evaluation of HCV antivirals. This virus constitutes a novel tool for the study of one of the most relevant strains of HCV, genotype 1b, which will now be available for HCV life cycle research and useful for the development of new therapeutics.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Genotype 1b-persistently infected VeroE6 cells express HCV antigens.
Immunostaining of naïve VeroE6 (A–D, left panels) and LB-piVe cells (A–D, right panels). LB-piVe cells (A, C) and LB-piVe-enriched, panned cells (B, D). (E, F) Immunoblot of LB-piVe and J6/JFH-1-infected cell extracts, stained with anti-NS5A antibody (E) or anti-Core antibody (F). (G) HCV RNA extracted from filter-clarified supernatants from J6/JFH-1-infected Huh7.5 and VA RNAI-transfected LB-piVe cells, quantitated by real-time PCR. Error bars, ±SD.
Figure 2
Figure 2. HCV growth can be measured by observing cytopathic effects.
Light microscopy of (A) mock-infected VeroE6 (left panel) and LB-piVe cells (right panel) and (B) crystal violet-stained Huh7.5 cells 5 days after transfer of supernatant from A. (C) Mock-infected (left panel) and LB-piVe-infected Huh7.5 cells (from B, right panel) immunostained with anti-Core antibodies at 3 dpi. (D) Immunoblot of BB7 replicon containing Huh7.5 cells or LB-piVe-infected Huh7.5 cell extracts, stained with anti-NS5A antibody. (E) Histogram showing quantitative RNA titer (HCV RNA copies/ml, see Materials and Methods for details on HCV RNA quantification) and corresponding micrographs of crystal violet stained LB-piVe-infected Huh7.5 cells at 5 dpi (mag 200×).
Figure 3
Figure 3. Virus neutralization by anti-CD81 and anti-HCV antibodies.
(A) Huh7.5 cells were pre-incubated with anti-CD81 before infection with filter-clarified supernatants from LB-piVe cells or (B) J6/JFH-1-infected cells. M2, isotype-control antibody. J6/JFH-1 was titrated by an end-point dilution assay using indirect immunofluorescence. Wells were scored positive if at least 1 positive cell was detected. (C) LB-piVe was neutralized by incubation with human anti-HCIGIV or (D) anti-E2 monoclonal antibodies . LBpiVe virus titers in A, C and D were determined by a CPE-based TCID50 assay. HCV titers were calculated using the method of Reed and Muench . Error bars, ±SD.
Figure 4
Figure 4. Inhibition of HCV by antivirals.
(A) LB-piVe cells (B) and J6/JFH1-infected Huh7.5 cells were treated with increasing concentrations of 2′-C-Me-A. The EC50 values were evaluated from dose response curves employing GraphPad Prism 3.0 software. (C) Non-specific siRNA (IRR) or HCV-specific siRNA (313) transfected Huh7.5 cells were inoculated with LB-piVe and stained with crystal violet (left). Filter-clarified culture supernatants were titrated by a CPE-based end-point dilution assay (right). (D) Non-specific siRNA (IRR) or HCV-specific siRNA (313) transfected Huh7.5 cells were inoculated with J6/JFH-1 and stained with anti-Core antibodies at 3 dpi (left). Fluorescent foci were counted in triplicate wells, and titers were calculated as the mean number of foci per ml (FFU/ml, right). (E) LB-piVe cells were transfected with pVA and then treated with 0, 10, 100 and 1000 IU/ml of Universal Type 1 IFN for 24, 48 and 72 hr. Viral titers were determined as in B. (F) Huh7.5 cells were inoculated with J6/JFH-1 and treated with 0, 10, and 1000 IU/ml of Universal Type 1 IFN for 72 hr. Viral titers were determined by infecting Huh7.5 cells in the absence of pVA. (G) LB-piVe titers in the absence of pVA, determined in naïve Huh7.5 cells with (+IFN) or without (−IFN) the addition of 0, 10, and 1000 IU/ml of Universal Type I IFN to the culture media. (H) Infection of naïve, non-transfected VeroE6 cells with genotype 1b-infectious human plasma (LB; [28], [29]) and treated with 1000 IU/mL IFN. HCV RNA copies were determined per µg of GAPDH RNA. HCV titers were calculated using the method of Reed and Muench . Error bars, ±SD.
Figure 5
Figure 5. VA RNAI stimulates replication of J6/JFH-1.
(A) J6/JFH-1-infected VeroE6 cells and (B) J6/JFH-1-infected Huh 7.5 cells immunostained at 4 dpi with anti-NS5A antibody. Nuclei were visualized using DAPI staining. (C) Huh 7.5 cells (left) transfected with a defective (dl1, center) , or wild-type VA RNAI (+VA RNAI, right) , , infected with J6/JFH-1 (center, right) and immunostained with anti-Core antibodies. (D) Titration of J6/JFH-1 from culture supernatants in C by an end-point dilution assay using indirect immunofluorescence. Wells were scored positive if at least 1 positive cell was detected. The TCID50 was calculated using the method of Reed and Muench . (E) J6/JFH-1 RNA (relative to GAPDH RNA) in cell lysates from infected Huh7.5 or VeroE6 cells after transfection with a defective (dl1) or wild-type (WT) VA RNAI , and quantitated by real-time PCR. Error bars, ±SD.
Figure 6
Figure 6. VA RNAI stimulates replication of HCV and increases RNA stability.
(A) LB-piVe cells transfected with pVA (+VA RNAI ) or dl1 (−VA RNAI). Filter-clarified culture supernatants were collected on days 0, 2, 4 and 6 (post-transfection) for virus titer determination by CPE-based TCID50 assay. (B) VeroE6 or Huh7.5 cells transfected with pVA or dl1 and inoculated with genotype 1b-infectious human plasma (LB; [28], [29]). HCV RNA was extracted from cell lysates at the indicated time points and the copy number was determined by quantitative RT-PCR using an HCV standard. Values represent the mean relative increase in HCV RNA relative to GAPDH RNA (+VA RNAI versus −VA RNAI). (C) Values in B expressed as log10 HCV RNA copies/ml. The double-ended arrow indicates the 60 fold difference in B reflected in the HCV RNA copies. (D) Infection of naïve, transfected VeroE6 cells with genotype 1b-infectious human plasma (LB; [28], [29]) treated with 2′-C-Me-A. HCV RNA copies were determined per µg of total cellular RNA. Error bars, ±SD.
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