Reviewer
Jean Kaoru Millet
  • INRA French National Institute for Agricultural Research
Research fields
  • Microbiology
Cytopathic Effect Assay and Plaque Assay to Evaluate in vitro Activity of Antiviral Compounds Against Human Coronaviruses 229E, OC43, and NL63
Authors:  Yanmei Hu, Chunlong Ma and Jun Wang, date: 02/05/2022, view: 5039, Q&A: 0

Coronaviruses are important human pathogens, among which the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent for the COVID-19 pandemic. To combat the SARS-CoV-2 pandemic, there is a pressing need for antivirals, especially broad-spectrum antivirals that are active against all seven human coronaviruses (HCoVs). For this reason, we are interested in developing antiviral assays to expedite the drug discovery process. Here, we provide the detailed protocol for the cytopathic effect (CPE) assay and the plaque assay for human coronaviruses 229E (HCoV-229E), HCoV-OC43, and HCoV-NL63, to identify novel antivirals against HCoVs. Neutral red was used in the CPE assay, as it is relatively inexpensive and more sensitive than other reagents. Multiple parameters including multiplicity of infection, incubation time and temperature, and staining conditions have been optimized for CPE and plaque assays for HCoV-229E in MRC-5, Huh-7, and RD cell lines; HCoV-OC43 in RD, MRC-5, and BSC-1 cell lines, and HCoV-NL63 in Vero E6, Huh-7, MRC-5, and RD cell lines. Both CPE and plaque assays have been calibrated with the positive control compounds remdesivir and GC-376. Both CPE and plaque assays have high sensitivity, excellent reproducibility, and are cost-effective. The protocols described herein can be used as surrogate assays in the biosafety level 2 facility to identify entry inhibitors and protease inhibitors for SARS-CoV-2, as HCoV-NL63 also uses ACE2 as the receptor for cell entry, and the main proteases of HCoV-OC43 and SARS-CoV-2 are highly conserved. In addition, these assays can also be used as secondary assays to profile the broad-spectrum antiviral activity of existing SARS-CoV-2 drug candidates.


Enterovirus Competition Assay to Assess Replication Fitness
Authors:  Valeria Lulla and Andrew E. Firth, date: 05/20/2019, view: 5113, Q&A: 0
In virology the difference between the fitness of two viruses can be determined by using various methods, such as virus titer, growth curve analysis, measurement of virus infectivity, analysis of produced RNA copies and viral protein production. However, for closely performing viruses, it is often very hard to distinguish the differences. In vitro competition assays are a sensitive tool for determining viral replication fitness for many viruses replicating in cell culture. Relative viral replication fitness is usually measured from multiple cycle growth competition assays. Competition assays provide a sensitive measurement of viral fitness since the viruses are competing for cellular targets under identical growth conditions. This protocol describes a competition assay for enteroviruses and contains two alternative formats for initial infections, which can be varied depending on specific goals for each particular experiment. The protocol involves infection of cells with competing viruses, passaging, RNA extraction from infected cells, RT-PCR and Sanger sequencing followed by comparative analysis of resulting chromatograms obtained under various initial infection conditions. The techniques are applicable to members of many virus families, such as alphaviruses, flaviviruses, pestiviruses, and other RNA viruses with an established reverse genetics system.
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