A Simplified Quantitative Real-Time PCR Assay for Monitoring SARS-CoV-2 Growth in Cell Culture - PubMed (original) (raw)

. 2020 Sep 2;5(5):e00658-20.

doi: 10.1128/mSphere.00658-20.

Hung R Vuong # 1, Maritza Puray-Chavez 1, Adam L Bailey 2, Julie M Fox 3, Rita E Chen 2 3, Alex W Wessel 2 3, Jason M Scott 3, Houda H Harastani 3, Adrianus C M Boon 1 2 3, Haina Shin 3, Sebla B Kutluay 4

Affiliations

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

Christian Shema Mugisha et al. mSphere. 2020.

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.

Keywords: SARS-CoV-2; antivirals; assay development; viral entry; virus replication.

Copyright © 2020 Shema Mugisha et al.

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Figures

FIG 1

FIG 1

Development of a simplified qRT-PCR assay for SARS-CoV-2 viral RNA detection in cell culture supernatants. (A) Serially diluted RNA standards were either directly subjected to qRT-PCR or processed as described in the modified protocol detailed in the text prior to qRT-PCR. Log2 copy numbers are plotted against the cycle threshold (CT) values. Linear regression analysis was done to obtain the equations. Data show averages of results from three independent biological replicates. Error bars show standard errors of the means (SEM). (B) Comparison of the efficiency and detection ranges for quantifying SARS-CoV-2 RNA using purified RNA or lysed supernatants from virus stocks. Data are derived from three independent replicates. Error bars show the SEM. (C) Vero E6 cells were infected at a multiplicity of infection (MOI) of 0.01, and cell culture supernatants were analyzed for SARS-CoV-2 RNA following the conventional RNA extraction protocol versus the modified protocol developed here at various times postinfection. Cell-associated viral RNA was analyzed in parallel following RNA extraction for reference. Data are from three independent biological replicates. Error bars show the SEM. (D) Illustration of the efficiency and detection ranges of TaqMan-based and SYBR green-based qRT-PCR assays quantifying known amounts of SARS-CoV-2 RNA. Data are from 2 or 3 independent replicates. Error bars show the SEM. (E to L) The indicated viruses were subjected to RNA or DNA extraction (extracted) and diluted 10-fold or used directly following dilution (nonextracted) in the SYBR green-based (E to I) or TaqMan-based (J and L) qRT-PCR assay as described above. Samples were normalized such that equivalent amounts of the original virus stock were added to PCRs for extracted and nonextracted samples. Plots show the corresponding cycle threshold values (Ct, y axis) per virus dilution (x axis). Data are from two independent replicates, with error bars showing the SEM.

FIG 2

FIG 2

A compound screen to validate SARS-CoV-2-specific inhibitors and entry pathways. Vero E6 cells were infected with SARS-CoV-2 at an MOI of 0.01, and inhibitors were added concomitantly at the concentrations shown in the figures following virus adsorption. Supernatants from infected cells were lysed and used in a SYBR green-based qRT-PCR to quantify the viral RNA in cell culture supernatants. Compound cytotoxicity was monitored by the use of a RealTime-Glo MT cell viability assay kit (Promega) in parallel plates. Data show the cumulative results from 2 to 5 independent biological replicates. Error bars show the SEM. (A) Bafilomycin A. (B) Dynasore. (C) Latrunculin B. (D) EIPA. (E) E64D. (F) Apilimod. (G) Camostat mesylate. (H) Remdesivir.

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