key: cord-1010807-83d0g8ix
authors: Mendoza, Emelissa J.; Manguiat, Kathy; Wood, Heidi; Drebot, Michael
title: Two Detailed Plaque Assay Protocols for the Quantification of Infectious SARS‐CoV‐2
date: 2020-05-31
journal: Curr Protoc Microbiol
DOI: 10.1002/cpmc.105
sha: c905479e3f7aa7c05e214ba1efad85a0b559c855
doc_id: 1010807
cord_uid: 83d0g8ix

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 (TCID(50)), 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

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), formerly known as 2019-nCoV, was identified as the causal agent of COronaVIrus Disease-19 (COVID-19), a pneumonia-like syndrome that emerged in Wuhan, Hubei, China in December 2019 (WHO, 2020c; Wu et al., 2020; Zhu et al., 2020) . Since then, SARS-CoV-2 has spread rapidly, and the pandemic was deemed a public health emergency of international concern in January 2020 (WHO, 2020d) . As of May 1, 2020, over 3 million cases and 216,000 fatalities have been reported worldwide (WHO, 2020a) . To date, there remains no therapeutic or vaccine effective for the treatment or prevention of SARS-CoV-2 infection (Sanders, Monogue, Jodlowski, & Cutrell, 2020) . Thus, clinical management of critically ill patients with COVID-19 relies heavily upon supportive Mendoza et al.

Current Protocols in Microbiology measures, such as mechanical ventilation and hemodynamic support (Poston, Patel, & Davis, 2020) . The enormous number of patients and limited resources have overwhelmed health care systems in their efforts to provide such support. Ongoing research involving infectious SARS-CoV-2 will be crucial in understanding viral-associated pathogenesis and conducting pre-clinical testing of medical countermeasures for the pathogen causing the COVID-19 pandemic.

Virus quantification assays are key tools for research involving SARS-CoV-2. Molecular tests detecting viral RNA can be used to quantify viral loads in clinical samples and virus titers of SARS-CoV-2 stocks prepared from cell culture supernatants Corman et al., 2020) . While this method is highly sensitive and specific, it is incapable of quantifying infectious SARS-CoV-2, particularly when a high proportion of non-infectious virions are produced during a lytic cycle. Tissue culture infectious dose−50 (TCID 50 ) measures infectious SARS-CoV-2 by detecting the presence or absence of cytopathic effect in cell culture upon infection with serial dilutions of a virus specimen (van Doremalen et al., 2020) . However, this only provides a qualitative measurement of infectious virus in TCID 50 units, which describe the amount of virus needed to induce 50% CPE in susceptible cells. 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 (Harcourt et al., 2020) . Infectious virus titers are measured in plaque-forming units (PFU) . As such, plaque assays remain the gold standard in quantifying concentrations of replication-competent lytic virions (Cooper, 1961; Juarez, Long, Aguilar, Kochel, & Halsey, 2013) .

In this article, we describe two detailed procedures to conduct SARS-CoV-2 plaque assays. In both plaque assays, a confluent monolayer of host cells is infected with serial dilutions of SARS-CoV-2 of unknown starting concentration. After adsorption, an immobilizing overlay is used to cover the infected monolayer, to prevent virus spread and restrict virus growth to foci of cells at the sites of initial infection. During incubation, zones of cell death develop as viral infection and replication are restricted to the surrounding monolayer, leading to plaque formation. After incubation, cells are stained to enhance the contrast between plaques and the uninfected monolayer. Plaques are then enumerated and used to calculate the titer of infectious virus in the specimen. The first plaque assay method that we describe (Basic Protocol) uses Noble agar as the matrix in a solid overlay and neutral red as the stain to enhance plaque visualization. The second plaque assay method that we describe (Alternate Protocol) uses carboxymethylcellulose (CMC) as the matrix in a liquid overlay and crystal violet as the stain to enhance plaque visualization. An overview of these protocols is shown in Figure 1 . We believe that both of these protocols would be useful for quantifying infectious SARS-CoV-2 in viral stocks produced from infected cell supernatant. We also believe that either method can be further optimized for other research purposes, such as quantifying infectious SARS-CoV-2 in specimens from animals experimentally infected with SARS-CoV-2.

This protocol outlines a plaque assay method that can be used for the quantification of SARS-CoV-2 plaque-forming units (PFUs) in virus specimens, including viral stocks prepared from infected cell culture supernatants, and with further optimization, bodily fluids from animals infected with SARS-CoV-2. In brief, 10-fold serial dilutions of specimens containing an unknown amount of SARS-CoV-2 are adsorbed onto a monolayer of susceptible cells. After adsorption, a solid overlay medium (S-OM) is applied to the cell monolayer to restrict virus growth to the originally infected foci of cells. These foci develop into plaques, which are visualized after the addition of a secondary S-OM containing neutral red, which is incorporated and bound by lysosomes in viable cells (Repetto, del Peso, & Zurita, 2008) . As a result, clear plaques can be distinguished from the brownish-red uninfected monolayer. Plaques are enumerated and used for the calculation of virus titers in PFU/ml. Vero CCL-81 cells are also susceptible to SARS-CoV-2 infection (Harcourt et al., 2020) . However, it may be necessary to optimize conditions of the protocol, such as infection medium, overlay diluent, and the number of days post-infection to enumerate plaques.

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.

2. Seed plates: Trypsinize (see Current Protocols article: Phelan & May, 2015) a confluent T-150 flask of Vero E6 cells and bring volume to 20 ml with cell maintenance medium. Use an automated cell counter to count cells and prepare a suspension at a density of 3 × 10 5 cells/ml in cell maintenance medium. For each specimen to be titrated, seed four 6-well plates with 6 × 10 5 cells per well by adding 2 ml of the suspension to each well. Incubate at 37°C in a 5% CO 2 incubator overnight to achieve 100% confluence the following day.

3. Specimen dilution: For each specimen to be titrated, label 2-ml microcentrifuge tubes as follows: Neg, 10 −1 , 10 −2 , 10 −3 , 10 −4 , 10 −5 , and 10 −6 . Add 900 μl of infection medium to each tube. Serially dilute specimens 10-fold by transferring 100 μl of each specimen to the appropriate tube labeled 10 −2 , vortexing, and transferring 100 μl to the subsequent tube, down to 10 −6 .

For high-titer specimens, it may be necessary to conduct further serial dilutions.

Label 6-well plates containing cells (see step 2) according to Figure 2 . Aspirate medium from cell monolayers under vacuum, leaving ∼100 μl of medium to prevent monolayer from drying. Using a Pipet-Aid and a serological pipette, wash cells with 1 ml of DPBS. Leave ∼100 μl DPBS to prevent monolayer from drying out. Dispense 100 μl of the infection medium from the microcentrifuge tube labeled "N" into designated wells, as depicted in Figure 1 . Dispense 100 μl of diluted specimens (10 −2 , 10 −3 , 10 −4 , 10 −5 , and 10 −6 ) into designated wells, changing pipette tips every time ( Figure 2 ). Incubate at 37°C in a 5% CO 2 incubator for 1 hr, rocking every 15 min.

Do not swirl the inoculum. Use a front-to-back and side-to-side movement to evenly distribute the inoculum across the monolayer.

Mendoza et al.

Current Protocols in Microbiology 5. Preparation of primary S-OM: Incubate overlay diluent at 44°C in a water bath. Microwave to dissolve 2% NA (solid matrix) and incubate at 44°C in a water bath. After rocking plates for the last time (∼10 min before incubation is over), prepare primary S-OM by combining equal amounts (1:1) of overlay diluent and 2% NA (solid matrix). Use Formula 1 to calculate the total volume of S-OM needed, followed by Formula 2 to calculate the overlay diluent and 2% NA (solid matrix) needed to prepare the primary S-OM.

Formula 1: Total volume of primary S-OM needed (in ml) = Number of specimens × 4 plates per specimen × 6 wells per plate × 3 ml primary S-OM per well × 1.2 (to account for pipetting error)

Formula 2: Volume of overlay diluent or 2% NA (solid matrix) needed for primary S-OM (in ml) = total volume of primary S-OM needed ÷ 2

The bottle of prepared primary S-OM can be placed on top of a Styrofoam mat to prevent it from cooling in the biological safety cabinet (BSC).

6. Primary solid overlay: Add 3 ml of primary S-OM to each well of the titration plates (step 4). Allow primary S-OM to solidify and incubate at 37°C in a 5% CO 2 incubator for 2 days.

7. Preparation: Incubate overlay diluent at 44°C in a water bath. Microwave to dissolve 2% NA (solid matrix) and incubate at 44°C in a water bath. Prepare secondary S-OM (composition: 1:1 overlay diluent:2% NA with 0.01% neutral red) by using Formula 3 to calculate the total volume of secondary S-OM needed. Use Formula 4 to calculate the volume of 0.33% NR needed, Formula 5 to calculate the volume of overlay diluent needed, and Formula 6 to calculate the volume of 2% NA (solid matrix) needed to prepare the secondary S-OM.

Formula 3: Total volume of secondary S-OM needed (in ml) = Number of specimens × 4 plates per specimen × 6 wells per plate × 2 ml secondary S-OM per well × 1.2 (to account for pipetting error)

Formula 4: Volume of 0.33% NR needed for secondary S-OM (in ml) = total volume of secondary S-OM needed × 0.03

Formula 5: Volume of overlay diluent needed for secondary S-OM (in ml) = (total volume of secondary solid overlay medium needed ÷ 2) − volume of 0.33% NR needed for secondary S-OM Formula 6: Volume of 2% NA (solid matrix) needed for secondary S-OM = total volume of secondary S-OM needed ÷ 2

The bottle of prepared secondary S-OM can be placed on top of a Styrofoam mat to minimize cooling in the BSC.

8. Secondary solid overlay: Add 2 ml of secondary S-OM to each well of the titration plates. Allow secondary S-OM to solidify and incubate at 37°C in a 5% CO 2 incubator overnight.

9. Enumerate plaques: Use a white-light transilluminator (light box) to aid in visualizing the plaques, which will appear as peach-colored circles on a brownish-red monolayer of cells ( Figure 3A ). The negative control should have a uniform monolayer, which can be used as a reference. Record the number of plaques observed per well at each virus dilution.

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 × 10 5 PFU/ml.

Titer calculation: Identify the virus dilution factor that yields 5-100 plaques per well (Baer & Kehn-Hall, 2014) . Use Formula 7 to calculate the average number of plaques at this dilution. Use Formula 8 to calculate the titer of SARS-CoV-2 in the specimen using the identified dilution factor and the inoculum volume of 0.1 ml. Refer to Figure 3B for an example plaque assay plate and interpretation. 11. Waste disposal: Storage of plates prior to enumeration is not recommended because the borders of the plaques become less distinct over time. Wells of each plate can be topped up with an appropriate disinfectant (such as a 5% Micro-Chem Plus solution) and incubated at room temperature overnight prior to disposal in biohazardous waste. All biohazardous waste should be autoclaved at 121°C for 1 hr.

This protocol can be used as an alternative to the Basic Protocol for the quantification of SARS-CoV-2 by plaque assay. The first steps in the protocol are identical to the Basic Protocol. However, after adsorption, a liquid overlay medium (L-OM) is applied to the cell monolayer to restrict virus growth to the originally infected foci of cells. In addition, instead of incorporating a vital stain into the secondary overlay (S-OM), the L-OM is removed from the monolayer, fixed, and stained with crystal violet, which binds to proteins and DNA within cells (Feoktistova, Geserick, & Leverkus, 2016 

1. Perform steps 1-2 of the Basic Protocol.

Incubate overlay diluent at 44°C in a water bath. After rocking plates for the last time (approximately 10 min before incubation is over), prepare L-OM by adding equal amounts (1:1) of overlay diluent and 3% CMC (liquid matrix). Use Formula 1 to calculate the total volume of L-OM needed, followed by Formula 2 to calculate the overlay diluent and 3% CMC (liquid matrix) needed to prepare the L-OM.

Formula 1: Total volume of L-OM needed (in ml) = Number of specimens × 4 plates per specimen × 6 wells per plate × 3 ml L-OM per well × 1.2 (to account for pipetting error)

Formula 2: Volume of overlay diluent or 3% CMC (liquid matrix) needed for L-OM (in ml) = total volume of L-OM needed ÷ 2 4. Liquid overlay: Add 3 ml of L-OM to each well of the titration plates (see Basic Protocol, step 4). Incubate at 37°C in a 5% CO 2 incubator for 3 days.

During this incubation, avoid movement of the plates, which can disrupt the liquid overlay and cause plaques to smear.

Mendoza et al.

Current Protocols in Microbiology 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 × 10 5 PFU/ml. 8. Enumerate plaques: Plaques will appear as clear circles on a purple monolayer of cells ( Figure 4A ). The negative control should have a uniform monolayer, which can be used as a reference. Record the number of plaques observed per well at each virus dilution.

9. Titer calculation: Identify the virus dilution factor demonstrating 5-100 plaques per well. Use Formula 7 (see Basic Protocol) to calculate the average number of plaques at this dilution. Use Formula 8 (see Basic Protocol) to calculate the titer of SARS-CoV-2 in the specimen using the identified dilution factor and the inoculum volume of 0.1 ml. Refer to Figure 4B for an example plaque assay plate and interpretation.

10. Waste disposal: Dispose of plates in biohazardous waste and autoclave at 121°C for 1 hr. Liquid waste containing disinfectant should be incubated at room temperature overnight prior to disposal. MEM (2×) , no phenol red (ThermoFisher Scientific #11935046) 40 ml FBS (ThermoFisher Scientific #16140071) 10 ml 200 mM l-glutamine (ThermoFisher Scientific #25030149) 10 ml 100× nonessential amino acids (NEAA; ThermoFisher Scientific #11140050) 7.5 ml 100× sodium bicarbonate (ThermoFisher Scientific #25080094) Store at 4°C up to 2 months

The accurate quantification of infectious virus specimens is crucial for a multitude of applications in virology. Plaque assays were established in 1952 as an adaptation of phage assays, which were used to calculate bacteriophage titers in plant biology (Cooper, 1961; Dulbecco & Vogt, 1953) . Although new techniques for viral titration continue to evolve, plaque assays continue to be the gold standard for the quantification of infectious virus (Baer & Kehn-Hall, 2014) .

The plaque assay using the double overlay method (Basic Protocol) described in this article has been adapted for SARS-CoV-2 from previously characterized procedures (Berry et al., 2004; Harcourt et al., 2020; Ströher et al., 2004) . Noble agar, the solid matrix used in the overlay in the Basic Protocol, is considered a traditional overlay matrix for plaque assays. However, it comes with disadvantages. Heating is required to liquefy the matrix immediately prior to application, which warrants the need to work quickly when applying this type of overlay since it can re-solidify as it cools. The secondary overlay in this protocol uses neutral red to stain the monolayer and enhance the visualization of plaques. As a vital stain, neutral red has the advantage of being applied during incubation, enabling live monitoring of plaque formation. However, the contrast between plaques and viable cells stained with neutral red is not as distinct as that produced by crystal violet and fixation in the Alternate Protocol, and requires a light box to further enhance visualization.

The plaque assay using the fixation and staining method (Alternate Protocol) described in this article has been adapted for SARS-CoV-2 from previously characterized procedures (Schneider et al., 2012; Vicenzi et al., 2004; Wang, Sakhatskyy, Chou, & Lu, 2005) . In contrast to the Basic Protocol, this method uses a liquid overlay matrix, which confers benefits over solid/semi-solid overlay matrices, including the ease of removal prior to fixation. In addition, liquid overlays can be applied to cell monolayers at room temperature, eliminating issues of re-solidification during the application process. However, the disadvantage of the liquid overlay matrix is that disruption of the liquid overlays can result in smeared plaques, and thus great care must be taken to prevent movement of plates during the incubation period. The fixation and subsequent staining with crystal violet used to enhance plaque visualization in the protocol has several advantages, including increased contrast between plaques from the purple monolayers, as well as the inactivation of the virus. In addition, fixation enables plates to be stored and plaques to be enumerated at a later time. However, some disadvantages of this method includes the generation of formaldehyde waste and the additional need to properly inactivate that chemical.

As summarized in Table 1 , each protocol has advantages and disadvantages that can be assessed to select the method best suited to each laboratory's resources and preferences. A comparison conducted in our laboratory demonstrated that there were no significant differences in viral titers calculated using either method.

Since SARS-CoV-2 is a BSL-3 pathogen, procedures using SARS-CoV-2 specimens require BSL-3 facilities, equipment, and operational practices. Additional personal protective equipment (PPE), including 

See Table 2 for commonly encountered problems, causes, and solutions. A plaque assay plate exhibiting commonly encountered problems is depicted in Figure 5 . Figure 3 for an example interpretation of a schematic 6-well SARS-CoV-2 plaque assay plate. In our laboratory, we have found that the titers of most of our SARS-CoV-2 stocks passaged in Vero E6 and titrated by plaque assays using Vero E6 range from 10 5 to 10 6 PFU/ml.

Current Protocols in Microbiology 

Minimize movement of plates during incubation by leaving plates untouched throughout the incubation and reducing the number of times the incubator door is opened and closed.

"Crescent moon" along edge of well Drying of cell monolayer (a) Leave ∼50-100 μl of liquid in each well when aspirating medium and DPBS; (b) rock plates every 10-15 min during adsorption incubation; (c) work in small batches of plates to prevent the monolayer from drying out between each aspiration and medium addition Large plaque-like spot Scratching of the monolayer by pipette tip during medium addition or removal Avoid making touching the monolayer with pipette tip by hovering above the well and aiming along the edges when adding medium. Minimize the force used when using a serological pipette tip to remove medium.

Medium added directly to monolayer at high speed Aim medium along the edges at low speed

The Basic Protocol and Alternate Protocol can both be completed in 5-7 days, including the time needed to seed plates for the plaque assay. However, if starting Vero E6 cells from cryostocks, it would be necessary to passage the cell line at least three times prior to use in either assay to remove excess cryopreservative and allow cells to return to normal growth, extending the duration by a few days; this is not taken into account in the protocols. If plates are seeded as described in the protocols on a Monday, the plaque assay can begin on Tuesday and end with plaque enumeration on Friday. Using this schedule, the secondary overlay would be applied on Thursday for the Basic Protocol.

Mendoza et al.

Current Protocols in Microbiology 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.

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Development of this protocol was supported by the Public Health Agency of Canada. We thank Dr. Darwyn Kobasa, Dr. Amrit Boese, and Anders Leung for providing the SARS-CoV-2 stock, and for their constructive commentary (Public Health Agency of Canada). We would also like to thank Kristina Dimitrova (Public Health Agency of Canada) and Dr. Yohannes Berhane (Canadian Food Inspection Agency) for their constructive commentary regarding the optimization of the protocols.