A facile Q-RT-PCR assay for monitoring SARS-CoV-2 growth in cell culture

AbstractSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of the ongoing COVID-19 pandemic, has infected millions within just a few months and is continuing to spread around the globe causing immense respiratory disease and mortality. Assays to monitor SARS-CoV-2 growth depend on time-consuming and costly RNA extraction steps, hampering progress in basic research and drug development efforts. Here we developed a facile Q-RT-PCR assay that bypasses viral RNA extraction steps and can monitor SARS-CoV-2 replication kinetics from a small amount of cell culture supernatants. Using this assay, we screened the activities of a number of entry, SARS-CoV-2- and HIV-1-specific inhibitors in a proof of concept study. In line with previous studies which has shown that processing of the viral Spike protein by cellular proteases and endosomal fusion are required for entry, we found that E64D and apilimod potently decreased the amount of SARS-CoV-2 RNA in cell culture supernatants with minimal cytotoxicity. Surprisingly, we found that macropinocytosis inhibitor EIPA similarly decreased viral RNA in supernatants suggesting that entry may additionally be mediated by an alternative pathway. HIV-1-specific inhibitors nevirapine (an NNRTI), amprenavir (a protease inhibitor), and ALLINI-2 (an allosteric integrase inhibitor) modestly inhibited SARS-CoV-2 replication, albeit the IC50 values were much higher than that required for HIV-1. Taken together, this facile assay will undoubtedly expedite basic SARS-CoV-2 research, be amenable to mid-throughput screens to identify chemical inhibitors of SARS-CoV-2, and be applicable to a broad number of RNA and DNA viruses.ImportanceSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of the COVID-19 pandemic, has quickly become a major global health problem causing 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 facile Q-RT-PCR assay to monitor SARS-CoV-2 growth in cell culture supernatants that does not necessitate RNA extraction, and 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. This facile approach will undoubtedly expedite basic SARS-CoV-2 research, be amenable to screening platforms to identify therapeutics against SARS-CoV-2 and can be adapted to numerous other RNA and DNA viruses.

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

mSphere ◽

10.1128/msphere.00658-20 ◽

2020 ◽

Vol 5 (5) ◽

Author(s):

Christian Shema Mugisha ◽

Hung R. Vuong ◽

Maritza Puray-Chavez ◽

Adam L. Bailey ◽

Julie M. Fox ◽

...

Keyword(s):

Cell Culture ◽

Real Time ◽

Real Time Pcr ◽

Rna Extraction ◽

Quantitative Real Time Pcr ◽

Pcr Assay ◽

Dna Viruses ◽

Culture Supernatants ◽

Hiv 1 ◽

Rna And Dna

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.

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Nonpolio enteroviruses can also be recovered from non-reproducible cytopathology in L20B cell line

10.1101/158626 ◽

2017 ◽

Author(s):

J. A. Adeniji ◽

U.I. Ibok ◽

O.T. Ayinde ◽

A.O. Oragwa ◽

U.E. George ◽

...

Keyword(s):

Cell Culture ◽

Cell Line ◽

Rna Extraction ◽

Cdna Synthesis ◽

Pcr Assay ◽

Fecal Suspension ◽

Coxsackievirus A20 ◽

Culture Supernatants ◽

Seminested Pcr ◽

University Of Ibadan

ABSTRACTSamples showing cytopathology (CPE) on initial inoculation into L20B cell line but with no observed or reproducible CPE on passage in L20B or RD are considered negative for both poliovirus and nonpolio enteroviruses (NPEVs). The phenomenon is termed ‘non-reproducible CPE’. Its occurrence is usually ascribed to the likely presence of reoviruses, adenoviruses and other non-enteroviruses. This study aimed to investigate the likelihood that NPEVs are also present in cases with non-reproducible CPE.Twenty-six (26) cell culture suspensions were analyzed in this study. The suspensions were collected from the WHO National Polio Laboratory, Department of Virology, College of Medicine, University of Ibadan. The suspensions emanated from 13 L20B cell culture tubes that showed cytopathology within 5 days of inoculation with fecal suspension from AFP cases. However, on passage into one each of RD and L20B cell lines, the CPE was not reproducible. All samples were subjected to RNA extraction, cDNA synthesis, the WHO recommended VP1 RT-seminestedPCR assay, species resolution PCR assay, sequencing and phylogenetic analysis.Six (6) samples were positive for the VP1 RT-seminested PCR assay. Only four of which were positive by the species resolution PCR assay. The four amplicons were sequenced, however, only three (3) were successfully identified as Coxsackievirus A20 (2 isolates) and Echovirus 29 (1 isolate).The results of this study unambiguously showed the presence of NPEVs (particularly CVA20 and E29) in cell culture supernatants of samples with CPE on initial inoculation into L20B cell line but with no observed or reproducible CPE on passage in RD cell line. Therefore, like reoviruses, adenoviruses and other non-enteroviruses, NPEVs can also be recovered in cases with non-reproducible CPE.

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Effective optimization of SARS-CoV-2 laboratory testing variables in an era of supply chain constraints

Future Microbiology ◽

10.2217/fmb-2020-0094 ◽

2020 ◽

Vol 15 (15) ◽

pp. 1483-1487

Author(s):

Nikhil S Sahajpal ◽

Ashis K Mondal ◽

Allan Njau ◽

Sudha Ananth ◽

Kimya Jones ◽

...

Keyword(s):

Supply Chain ◽

Rna Extraction ◽

Nasopharyngeal Swab ◽

Sample Collection ◽

Rt Pcr ◽

Pcr Assay ◽

Short Supply ◽

Viral Transport ◽

3D Printed ◽

Bridging Studies

RT-PCR-based assays for the detection of SARS-CoV-2 have played an essential role in the current COVID-19 pandemic. However, the sample collection and test reagents are in short supply, primarily due to supply chain issues. Thus, to eliminate testing constraints, we have optimized three key process variables: RNA extraction and RT-PCR reactions, different sample types and media to facilitate SARS-CoV-2 testing. By performing various validation and bridging studies, we have shown that various sample types such as nasopharyngeal swab, bronchioalveolar lavage and saliva, collected using conventional nasopharyngeal swabs, ESwab or 3D-printed swabs and, preserved in viral transport media, universal transport media, 0.9% sodium chloride or Amies media are compatible with RT-PCR assay for COVID-19. Besides, the reduction of PCR reagents by up to fourfold also produces reliable results.

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The Detection of Enteroviruses in Sewage Using Caco-2 Cells

Polish Journal of Microbiology ◽

10.33073/pjm-2013-014 ◽

2013 ◽

Vol 62 (1) ◽

pp. 97-100 ◽

Cited By ~ 3

Author(s):

MAGDALENA WIECZOREK ◽

ŁUKASZ KURYK ◽

AGNIESZKA WITEK ◽

ANNA DIUWE ◽

BOGUMIŁA LITWIŃSKA

Keyword(s):

Cell Culture ◽

Rt Pcr ◽

Pcr Assay ◽

Beef Extract

The work presented here demonstrates the utility of Caco-2 cells to detect enteroviruses in sewage. Viruses were concentrated by beef extract elution and organic flocculation prior to analysis by cell culture assays and RT-PCR. Enteroviruses were detected in all sewage samples, but only one sample was positive solely in RT-PCR assay. We proved that Caco-2 cells were more effective than RD and L20B cells in enterovirus isolation, depending on procedures used in the inoculation process.

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A rapid RT-PCR assay for the detection of HIV-1 in human plasma specimens

Experimental and Molecular Pathology ◽

10.1016/j.yexmp.2014.06.005 ◽

2014 ◽

Vol 97 (1) ◽

pp. 111-115 ◽

Cited By ~ 1

Author(s):

Paul R. Burchard ◽

Ahmad N. Abou Tayoun ◽

Axel Scherer ◽

Gregory J. Tsongalis

Keyword(s):

Human Plasma ◽

Rt Pcr ◽

Pcr Assay ◽

Hiv 1

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Development of an integrated cell culture—Real-time RT-PCR assay for detection of reovirus in biosolids

Journal of Virological Methods ◽

10.1016/j.jviromet.2006.10.001 ◽

2007 ◽

Vol 139 (2) ◽

pp. 195-202 ◽

Cited By ~ 19

Author(s):

Elizabeth M. Gallagher ◽

Aaron B. Margolin

Keyword(s):

Cell Culture ◽

Real Time ◽

Rt Pcr ◽

Pcr Assay

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Detecting HIV-1 Tat in Cell Culture Supernatants by ELISA or Western Blot

Methods in Molecular Biology - HIV Protocols ◽

10.1007/978-1-4939-3046-3_22 ◽

2016 ◽

pp. 329-342 ◽

Cited By ~ 3

Author(s):

Fabienne Rayne ◽

Solène Debaisieux ◽

Annie Tu ◽

Christophe Chopard ◽

Petra Tryoen-Toth ◽

...

Keyword(s):

Cell Culture ◽

Western Blot ◽

Culture Supernatants ◽

Hiv 1

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An Improved Multiplex IC-RT-PCR Assay Distinguishes Nine Strains of Potato virus Y

Plant Disease ◽

10.1094/pdis-02-13-0161-sr ◽

2013 ◽

Vol 97 (10) ◽

pp. 1370-1374 ◽

Cited By ~ 31

Author(s):

Mohamad Chikh-Ali ◽

Stewart M. Gray ◽

Alexander V. Karasev

Keyword(s):

Potato Virus ◽

Potato Virus Y ◽

Rna Extraction ◽

Leaf Tissue ◽

Rt Pcr ◽

Pcr Assay ◽

Potato Leaf ◽

Extraction Step ◽

First Time ◽

Virus Testing

A multiplex reverse-transcription polymerase chain reaction (RT-PCR) assay was previously developed to identify a group of Potato virus Y (PVY) isolates with unusual recombinant structures (e.g., PVYNTN-NW and SYR-III) and to differentiate them from other PVY strains. In the present study, the efficiency of this multiplex RT-PCR assay was validated and extended considerably to include five additional strains and strain groups not tested before. To make the multiplex RT-PCR assay more applicable and suitable for routine virus testing and typing, it was modified by replacing the conventional RNA extraction step with the immunocapture (IC) procedure. The results obtained using well-characterized reference isolates revealed, for the first time, that this multiplex RT-PCR assay is an accurate and robust method to identify and differentiate the nine PVY strains reported to date, including PVYO (both PVYO and PVYO-O5), PVYN, PVYNA-N, PVYNTN, PVYZ, PVYE, PVY-NE11, PVYN-Wi, and PVYN:O, which is not possible by any of the previously reported RT-PCR procedures. This would make the IC-RT-PCR procedure presented here a method of choice to identify PVY strains and assess the strain composition of PVY in a given area. The IC-RT-PCR protocol was successfully applied to typing PVY isolates in potato leaf tissue collected in the field.

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