Efficient culture of SARS-CoV-2 in human hepatoma cells enhances viability of the virus in human lung cancer cell lines permitting the screening of antiviral compounds

Abstract

Efforts to mitigate COVID-19 include screening of existing antiviral molecules that could be re-purposed to treat SARS-CoV-2 infections. Although SARS-CoV-2 propagates efficiently in African green monkey kidney (Vero) cells, antivirals such as nucleos(t)ide analogs (nucs) often exhibit decreased activity in these cells due to inefficient metabolization. Limited SARS-CoV-2 replication and propagation occurs in human cells, which are the most relevant testing platforms. By performing serial passages of a SARS-CoV-2 isolate in the human hepatoma cell line clone Huh7.5, we selected viral populations with improved viability in human cells. Culture adaptation led to the emergence of a significant number of high frequency changes (>90% of the viral population) in the region coding for the spike glycoprotein, including a deletion of nine amino acids in the N-terminal domain and 3 amino acid changes (E484D, P812R, and Q954H). We demonstrated that the Huh7.5-adapted virus exhibited a >3-Log10 increase in infectivity titers (TCID50) in Huh7.5 cells, with titers of ~8 Log10TCID50/mL, and >2-Log10 increase in the human lung cancer cell line Calu-1, with titers of ~6 Log10TCID50/mL. Culture adaptation in Huh7.5 cells further permitted efficient infection of the otherwise SARS-CoV-2 refractory human lung cancer cell line A549, with titers of ~6 Log10TCID50/mL. The enhanced ability of the virus to replicate and propagate in human cells permitted screening of a panel of nine nucs, including broad-spectrum compounds. Remdesivir, EIDD-2801 and to a limited extent galidesivir showed antiviral effect across these human cell lines, whereas sofosbuvir, uprifosbuvir, valopicitabine, mericitabine, ribavirin, and favipiravir had no apparent activity.ImportanceThe cell culture adapted variant of the SARS-CoV-2 virus obtained in the present study, showed significantly enhanced replication and propagation in various human cell lines, including lung derived cells otherwise refractory for infection with the original virus. This SARS-CoV-2 variant will be a valuable tool permitting investigations across human cell types, and studies of identified mutations could contribute to our understanding of viral pathogenesis. In particular, the adapted virus can be a good model for investigations of viral entry and cell tropism for SARS-CoV-2, in which the spike glycoprotein plays a central role. Further, as shown here with the use of remdesivir and EIDD-2801, two nucs with significant inhibitory effect against SARS-CoV-2, large differences in the antiviral activity are observed depending on the cell line. Thus, it is essential to select the most relevant target cells for pre-clinical screenings of antiviral compounds, facilitated by using a virus with broader tropism.

In vitro efficacy of Artemisinin-based treatments against SARS-CoV-2

“…Concentration-response experiments using the Danish SARS-CoV-2 isolate SARS-CoV-2/human/Denmark/DK-AHH1/2020 were performed employing a 96-well plate based high-throughput antiviral assay, allowing for multiple replicates per concentration, as described in Materials and Methods and Supplementary Information (Figures S3 and S4). 20 Seven replicates were measured at each concentration and a range of concentrations was evaluated to increase data accuracy when compared to the plaque-reduction assay, which was carried out in duplicates. Extracts or compounds were added to VeroE6 cells either 1.5 h prior to (pretreatment (pt)) or 1 h post infection (treatment (t)), respectively, followed by a two-day incubation of virus with extracts or compounds.…”

“…The SARS-CoV-2/human/Denmark/DK-AHH1/2020 virus for cell culture studies was obtained following inoculation of VeroE6 cells with patient swab sample, virus propagation in VeroE6 cells and generation of a sequence confirmed 2nd viral passage stock with an infectivity titer of 5.5 log TCID50/mL as described in Ramirez et al . 20…”

Hepatitis C Virus Protease Inhibitors Show Differential Efficacy and Interactions with Remdesivir for Treatment of SARS-CoV-2 in Vitro

“…EC50 against SARS-CoV-2 were in the micromolar range, with the lowest EC50 (15 μM for simeprevir) approaching EC50 of remdesivir, determined to be 2.5 μM in this study, which was in line with previously reported results. 34,35 However, EC50 of PI against SARS-CoV-2 were higher than EC50 against HCV: Initially developed HCV PI such as boceprevir and simeprevir were 10- to 1,000-fold less potent, while optimized HCV PI such as grazoprevir, glecaprevir and voxilaprevir were ̴ 1,000- to 100,000-fold less potent against SARS-CoV-2 than against different HCV isolates. 24,26–28,30,36–38 Boceprevir showed the highest SI of >27.6, while simeprevir showed one of the lowest SI of 3.9.…”

“…While remdesivir is the only antiviral directly targeting SARS-CoV-2 that is approved for treatment of patients with COVID-19, its clinical efficacy has recently been questioned. 9,69–73 Relatively limited clinical efficacy compared to high in vitro efficacy of remdesivir 34,35 might be due to its poor distribution to the lungs. 45 Improving drug formulation including development of inhalable formulations and combination with simeprevir might have the potential to increase clinical efficacy of remdesivir.…”

Reviews on Biological Activity, Clinical Trial and Synthesis Progress of Small Molecules for the Treatment of COVID-19

“…2,3,4,5,6,7. Some biological activities against COVID-19 are also listed based on literature reports [18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37].…”