A Simplified Quantitative Real-Time PCR Assay for Monitoring SARS-CoV-2 Growth in Cell Culture

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected millions within just a few months, causing severe respiratory disease and mortality. Assays to monitor SARS-CoV-2 growth in vitro depend on time-consuming and costly RNA extraction steps, hampering progress in basic research and drug development efforts. Here, we developed a simplified quantitative real-time PCR assay that bypasses viral RNA extraction steps and can monitor SARS-CoV-2 growth from a small amount of cell culture supernatants. In addition, we show that this approach is easily adaptable to numerous other RNA and DNA viruses. Using this assay, we screened the activities of a number of compounds that were predicted to alter SARS-CoV-2 entry and replication as well as HIV-1-specific drugs in a proof-of-concept study. We found that E64D (inhibitor of endosomal proteases cathepsin B and L) and apilimod (endosomal trafficking inhibitor) potently decreased the amount of SARS-CoV-2 RNA in cell culture supernatants with minimal cytotoxicity. Surprisingly, we found that the macropinocytosis inhibitor ethylisopropylamiloride (EIPA) similarly decreased SARS-CoV-2 RNA levels in supernatants, suggesting that entry may additionally be mediated by an alternative pathway. HIV-1-specific inhibitors nevirapine (a nonnucleoside reverse transcriptase inhibitor [NNRTI]), amprenavir (a protease inhibitor), and allosteric integrase inhibitor 2 (ALLINI-2) modestly inhibited SARS-CoV-2 replication, albeit the 50% inhibitory concentration (IC50) values were much higher than that required for HIV-1. Taking the data together, this simplified assay will expedite basic SARS-CoV-2 research, be amenable to mid-throughput screening assays (i.e., drug, CRISPR, small interfering RNA [siRNA], etc.), and be applicable to a broad number of RNA and DNA viruses. IMPORTANCE Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of the coronavirus disease 2019 (COVID-19) pandemic, is continuing to cause immense respiratory disease and social and economic disruptions. Conventional assays that monitor SARS-CoV-2 growth in cell culture rely on costly and time-consuming RNA extraction procedures, hampering progress in basic SARS-CoV-2 research and development of effective therapeutics. Here, we developed a simple quantitative real-time PCR assay to monitor SARS-CoV-2 growth in cell culture supernatants that does not necessitate RNA extraction and that is as accurate and sensitive as existing methods. In a proof-of-concept screen, we found that E64D, apilimod, EIPA, and remdesivir can substantially impede SARS-Cov-2 replication, providing novel insight into viral entry and replication mechanisms. In addition, we show that this approach is easily adaptable to numerous other RNA and DNA viruses. This simplified assay will undoubtedly expedite basic SARS-CoV-2 and virology research and be amenable to use in drug screening platforms to identify therapeutics against SARS-CoV-2.

Systematic analysis of SARS-CoV-2 infection of an ACE2-negative human airway cell

“…To begin to decipher the mechanism(s) of SARS-CoV-2 entry into H522 cells, we performed infections in the presence of compounds that interfere with SARS-CoV-2 entry, including camostat mesylate (TMPRSS2 inhibitor) (Hoffmann et al, 2020; Shang et al, 2020; Shema Mugisha et al, 2020a), E64D (broad spectrum inhibitor of proteases, including endosomal cathepsins) (Ou et al, 2020; Shema Mugisha et al, 2020a), bafilomycin A (inhibitor of vATPase) (Ou et al, 2020; Shema Mugisha et al, 2020a) and apilimod (inhibitor of PIKfyve) (Kang et al, 2020; Ou et al, 2020; Shema Mugisha et al, 2020a). We additionally included a specific inhibitor of AAK1 kinase (SGC-AAK1-1); AAK1 promotes CME through phosphorylation of the AP2M1 subunit of the AP2 complex (Agajanian et al, 2019; Conner and Schmid, 2002, 2003).…”

Amilorides inhibit SARS-CoV-2 replication in vitro by targeting RNA structures

“…To determine the antiviral activity and potency of the lead small molecules against SARS-CoV-2 we utilized a simplified Q-RT-PCR assay to monitor SARS-CoV-2 viral RNA levels in supernatants of infected Vero E6 cells. 32 Similar to Remdesivir, DMA-135 and 155 led to a dose dependent 10-30-fold decrease in cell-free viral RNA levels within 24 hours of infection with an approximate IC 50 of 10 and 16 μM, respectively ( Figure 5 ).…”

“…Analysis of SARS-CoV-2 growth in cell culture supernatants was performed using a simplified RT-qPCR assay as explained before. 32 In brief, Vero E6 cells were infected with SARS-CoV-2 at an MOI of 0.1 i.u./cell in 96-well plates, virus inoculum was removed 1 hour post-adsorption and replaced with media containing serial dilutions of the DMA compounds. 5 μL of cell culture media containing released virions were collected at 24 hpi and processed as detailed in previous studies.…”

“…5 μL of cell culture media containing released virions were collected at 24 hpi and processed as detailed in previous studies. 32 Viral RNA levels were quantitated by RT-qPCR using primers specific to SARS-CoV-2 N gene and a standard curve derived from in vitro synthesized RNA encoding N.…”

Repurposing Approved Drugs for Guiding COVID-19 Prophylaxis: A Systematic Review

“…Furthermore, a large-scale drug repurposing study apilimod was responsible for blocking viral replication in human pneumocyte-like cells derived from induced pluripotent stem cells with IC 50 raging from 0.088 to 0.012 μM, as well as exhibited antiviral activity in a primary human lung explant model (Huang et al, 2020). Other study with Vero E6 cell viral replication monitored by QRT-PCR assays, indicated that apilimod potentially decreased the amount of SARS-CoV-2 RNA in cell culture supernatants, with an IC 50 of 12 mM and low cell toxicity (Shema Mugisha et al, 2020). Baloxavir marboxyl is an influenza A and B antiviral, which inhibits the cap-dependent endonuclease necessary for viral replication, and this is the first representative of influenza-type PB2 inhibitors.…”