Inhibition of novel β coronavirus replication by a combination of interferon-α2b and ribavirin

Abstract

The identification of a novel β coronavirus, nCoV, as the causative agent of severe respiratory illness in humans originating in Saudi Arabia, Qatar and Jordan has raised concerns about the possibility of a coronavirus pandemic similar to that of SARS-CoV. As a definitive treatment regimen has never been thoroughly evaluated for coronavirus infections, there is an urgent need to rapidly identify potential therapeutics to address future cases of nCoV. To determine an intervention strategy, the effect of interferon-α2b and ribavirin on nCoV isolate hCoV-EMC/2012 replication in Vero and LLC-MK2 cells was evaluated. hCoV-EMC/2012 was sensitive to both interferon-α2b and ribavirin alone in Vero and LLC-MK2 cells, but only at relatively high concentrations; however, when combined, lower concentrations of interferon-α2b and ribavirin achieved comparable endpoints. Thus, a combination of interferon-α2b and ribavirin, which are already commonly used in the clinic, may be useful for patient management in the event of future nCoV infections.

Figure 1. Interferon-α2b and/or ribavirin treatment of hCoV-EMC/2012-infected Vero cells.

Vero cells were infected with hCoV-EMC/2012 at an MOI of 0.001 for 1 h and subsequently treated with interferon-α2b (IFN-α2b) and/or ribavirin at the indicated concentration. On day 5 post-infection cells were photographed and cytopathic effect was assessed (A). Cell lysates were collected and subjected to western blotting with serum from a rabbit immunized with whole inactivated hCoV-EMC/2012 (B). β-actin was used as loading control (actin).

Figure 2. Replication of novel human coronavirus hCoV-EMC/2012 in response to interferon-α2b or ribavirin treatment in Vero cells.

Vero cells were infected with hCoV-EMC/2012 at an MOI of 0.001 for 1 h and subsequently treated with interferon-α2b (IFN-α2b) or ribavirin at the indicated concentration. At 1 and 3 days post-infection, supernatants were removed and subsequently analyzed for viral load by real time quantitative RT-PCR (A,B) and infectious virus titers by 50% tissue culture infectious dose (TCID₅₀) assay (C, D). Viral loads are shown as TCID₅₀ equivalents/ml ±SD, in response to increasing concentrations of IFN-α2b (A) or ribavirin (B). Viral titers are TCID₅₀/ml ±SD in response to increasing concentrations of IFN-α2b (C) or ribavirin (D).

Figure 3. Replication of novel human coronavirus hCoV-EMC/2012 in response to interferon-α2b or ribavirin treatment in LLC-MK2 cells.

LLC-MK2 cells were infected with hCoV-EMC/2012 at an MOI of 0.001 for 1 h and subsequently treated with interferon-α2b (IFN-α2b) or ribavirin at the indicated concentration. At 1 and 3 days post-infection, supernatants were removed and subsequently analyzed for viral load by real time quantitative RT-PCR (A,B) and infectious virus titers by 50% tissue culture infectious dose (TCID₅₀) assay (C, D). Viral loads are shown as TCID₅₀ equivalents/ml ±SD, in response to increasing concentrations of IFN-α2b (A) or ribavirin (B). Viral titers are TCID₅₀/ml ±SD in response to increasing concentrations of IFN-α2b (C) or ribavirin (D).

Figure 4. Replication of novel human coronavirus hCoV-EMC/2012 in response to combined treatment with interferon-α2b and ribavirin in Vero cells.

Vero cells were infected with hCoV-EMC/2012 at an MOI of 0.001 for 1 h and subsequently treated with interferon-α2b (IFN-α2b) and/or ribavirin at the indicated concentration. At 3 days post-infection, supernatants were removed and subsequently analyzed for infectious virus titers by 50% tissue culture infectious dose (TCID₅₀) assay. Viral titers are shown as TCID₅₀/ml ±SE in response to increasing concentrations of IFN-α2b, ribavirin or the combination of both.