Two Detailed Plaque Assay Protocols for the Quantification of Infectious SARS-CoV-2

Abstract

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has been identified as the causal agent of COronaVIrus Disease-19 (COVID-19), an atypical pneumonia-like syndrome that emerged in December 2019. While SARS-CoV-2 titers can be measured by detection of viral nucleic acid, this method is unable to quantitate infectious virions. Measurement of infectious SARS-CoV-2 can be achieved by tissue culture infectious dose-50 (TCID₅₀ ), which detects the presence or absence of cytopathic effect in cells infected with serial dilutions of a virus specimen. However, this method only provides a qualitative infectious virus titer. Plaque assays are a quantitative method of measuring infectious SARS-CoV-2 by quantifying the plaques formed in cell culture upon infection with serial dilutions of a virus specimen. As such, plaque assays remain the gold standard in quantifying concentrations of replication-competent lytic virions. Here, we describe two detailed plaque assay protocols to quantify infectious SARS-CoV-2 using different overlay and staining methods. Both methods have several advantages and disadvantages, which can be considered when choosing the procedure best suited for each laboratory. These assays can be used for several research purposes, including titration of virus stocks produced from infected cell supernatant and, with further optimization, quantification of SARS-CoV-2 in specimens collected from infected animals. © 2019 The Authors. Basic Protocol: SARS-CoV-2 plaque assay using a solid double overlay method Alternate Protocol: SARS-CoV-2 plaque assay using a liquid overlay and fixation-staining method.

Keywords: COVID-19; COVID19; SARS-CoV-2; plaque assay; virus quantification.

Figure 1

Graphical protocol overview of two plaque assay methods for the quantification of infectious SARS‐CoV‐2.

Figure 2

Plate layout for plaque assays conducted in 6‐well plates. Vero E6 cells grown in 6‐well plates are inoculated with 100 μl of 10‐fold serial dilutions of SARS‐CoV‐2 specimen from 10⁻² to 10⁻⁶, as well as infection medium as a negative control. Four replicates are prepared for each titrated specimen.

Figure 3

(A) Representative plaque assay plate processed by the Basic Protocol, which uses a solid double overlay method. (B) Schematic example of a 6‐well SARS‐CoV‐2 plaque assay plate processed by the Basic Protocol after 3 dpi. The negative control shows an intact monolayer stained brownish red. The 10⁻⁶ dilution appears similar to the negative control, indicating the absence of SARS‐CoV‐2 plaque‐forming units. The 10⁻⁵ and 10⁻³ dilutions show 2 and >100 peach‐colored plaques, but since these values are less than 5 and greater than 100, they will not be used in the calculation of the virus titer. The 10⁻⁴ dilution shows 21 plaques, and thus these values will be used in the calculation of the virus titer. The titer from this plate is 2.1 × 10⁵ PFU/ml.

Figure 4

(A) Representative plaque assay plate processed by the Alternate Protocol, which uses a liquid overlay and fixation‐staining method. (B) Schematic example of a 6‐well SARS‐CoV‐2 plaque assay plate processed by the Alternate Protocol after 3 dpi. The negative control shows an intact monolayer stained purple. The 10⁻⁶ dilution appears similar to the negative control, indicating the absence of SARS‐CoV‐2 plaque‐forming units. The 10⁻⁵ and 10⁻³ dilutions show 2 and >100 clear‐colored plaques, but since these values are less than 5 and greater than 100, they will not be used in the calculation of the virus titer. The 10⁻⁴ dilution shows 21 plaques, and thus these values will be used in the calculation of the virus titer. The titer from this plate is 2.1 × 10⁵ PFU/ml.

Figure 5

A representative plaque assay plate exhibiting commonly encountered problems. (A) “Shooting star” appearance of plaques in the first two wells is encountered when the liquid overlay is disrupted during the incubation. (B) “Crescent moon” along edge of wells can occur if the cell monolayer is allowed to dry upon aspirating cell culture medium/DPBS or if inoculum is not evenly distributed across the monolayer by rocking during adsorption. (C) A large plaque‐like spot that differs from the other plaques in size, morphology, and border distinction can occur when the monolayer is scratched by a serological pipette tip or medium is added directly to monolayer at high speed.