Growth, detection, quantification, and inactivation of SARS-CoV-2

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Abstract

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 is the agent responsible for the coronavirus disease 2019 (COVID-19) global pandemic. SARS-CoV-2 is closely related to SARS-CoV, which caused the 2003 SARS outbreak. Although numerous reagents were developed to study SARS-CoV infections, few have been applicable to evaluating SARS-CoV-2 infection and immunity. Current limitations in studying SARS-CoV-2 include few validated assays with fully replication-competent wild-type virus. We have developed protocols to propagate, quantify, and work with infectious SARS-CoV-2. Here, we describe: (1) virus stock generation, (2) RT-qPCR quantification of SARS-CoV-2 RNA; (3) detection of SARS-CoV-2 antigen by flow cytometry, (4) quantification of infectious SARS-CoV-2 by focus-forming and plaque assays; and (5) validated protocols for virus inactivation. Collectively, these methods can be adapted to a variety of experimental designs, which should accelerate our understanding of SARS-CoV-2 biology and the development of effective countermeasures against COVID-19.

Keywords: Coronavirus, Titration, Plaque assay, Focus-forming assay, Flow cytometry, Virus inactivation, SARS-CoV-2

1. Introduction

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 is an enveloped virus with a single-stranded positive-sense RNA genome. Zoonotic transmission of SARS-CoV-2 from an as yet unidentified animal reservoir occurred in late 2019. Subsequent human-to-human transmission by respiratory droplets has resulted in the ongoing Coronavirus disease 2019 (COVID-19) pandemic that has infected millions of people worldwide (Wu et al., 2020b; Zhou et al., 2020; Zhu et al., 2020). The rapid spread and relatively high case fatality rate of COVID-19 has led to an urgent need to develop diagnostics, therapeutics, and vaccines.

The SARS-CoV-2 genome is comprised of approximately 30,000 nucleotides. The first two-thirds of the genome encodes for nonstructural proteins in open reading frames 1a and 1b that principally facilitate genome replication and viral RNA synthesis. The remaining one-third is comprised of genes encoding structural proteins such as spike (S), envelope (E), membrane (M), and nucleocapsid (N), which form the virion, and accessory proteins that regulate host cellular responses. Whole-genome phylogenetic analysis identified the SARS-like bat CoV (GenBank MG772933) as the closest known relative of SARS-CoV-2. Bats also are the reservoir host for SARS-CoV (Wu et al., 2020a). Alignment of SARS-CoV-2 to the consensus sequence of SARS-like CoV revealed 380 amino acid differences including 27 amino acid differences in the S protein and six substitutions in the receptor binding domain (RBD) (Wu et al., 2020a).

SARS-CoV entry is mediated by initial engagement of the RBD of the S protein with the human ACE2 receptor (Li et al., 2003, 2005), and recent studies have established that SARS-CoV-2 utilizes the same receptor for entry (Letko et al., 2020). The S protein also is a key target for neutralizing antibodies and vaccine strategies (Rockx et al., 2008; Sui et al., 2005; Zhu et al., 2007). Although the S protein of SARS-CoV and SARS-CoV-2 are structurally similar (Li et al., 2005; Walls et al., 2020; Wrapp et al., 2020), genetically similar (Walls et al., 2020), and use the same receptor (Lei et al., 2020; Li et al., 2003), neutralizing anti-SARS-CoV RBD antibodies (Abs) generally lack cross-reactivity to SARS-CoV-2 (Wrapp et al., 2020). However, polyclonal sera from mice immunized with recombinant SARS-CoV RBD protein inhibits SARS-CoV-2 infection (Walls et al., 2020). Recent studies have identified cross-reactive, non-neutralizing monoclonal Abs (mAbs) against SARS-CoV and SARS-CoV-2, which were isolated previously using phage display or hybridoma fusion screens (Joyce et al., 2020; ter Meulen et al., 2006; Tian et al., 2020; Tripp et al., 2005; Yuan et al., 2020). Competition binding studies show that two of these mAbs, CR3022 and 240CD, both recognize the SARS-CoV-2 RBD. A co-crystal structure revealed that CR3022 binds an epitope on the RBD distal to the binding site of ACE2 and SARS-CoV neutralizing antibodies (Yuan et al., 2020).

SARS-CoV-2 research must be performed in a biosafety level 3 laboratory by personnel equipped with a powered air-purifying respirator (PAPR). This limitation has compelled the development of many in vitro assays that utilize heterologous pseudotyped viruses expressing the SARS-CoV-2 S protein (Lei et al., 2020; Letko et al., 2020). However, this approach only can be used to study cellular and antibody interactions involving the S protein that principally affect attachment and entry. Here, we developed or adapted multiple methodologies to quantify SARS-CoV-2 infection in vitro using a patient isolate of SARS-CoV-2: 1) RT-qPCR quantification of viral RNA; 2) detection of viral antigen by flow cytometry; 3) focus-forming assay through immunostaining of the S protein and 4) plaque assay. We also have identified and validated chemical and heat treatment methods to inactivate replication-competent virions, which are compatible with downstream quantification assays. Together, the methodologies can be used to examine SARS-CoV-2 pathogenesis and antibody responses, and to screen for potential inhibitors of infection.

2. Results and discussion

Propagation of SARS-CoV-2 in vitro. Isolates of SARS-CoV-2 from patients or animals often need to be propagated to generate high-titer virus stocks. We have tested several cell types and found African Green Monkey cell lines and derivatives thereof to be most permissive to SARS-CoV-2 infection. These include Vero-CCL81 (ATCC-CCL81), Vero-furin (Mukherjee et al., 2016), Vero E6 (ATCC-CRL1586), Vero-TMPRSS2 (Matsuyama et al., 2020), and MA104 (ATCC-CRL-2378.1) cells. Each cell type is sufficient to propagate SARS-CoV-2 using the protocol detailed below. All procedures should be completed only after appropriate safety training is obtained and using aseptic technique within a certified biosafety cabinet under BSL-3 containment.

2.1. Materials needed

Chosen cell type (Vero-CCL81, Vero-furin, Vero E6, Vero-TMPRSS2, and MA104 cells)

Standard media for chosen cell type (see Recipes)

Infection media (see Recipes)

SARS-CoV-2 seed stock

150 cm 2 (T150) tissue culture flasks

15 mL disposable polystyrene conical tubes with screw caps (e.g., Falcon)

50 mL disposable polystyrene conical tubes with screw caps (e.g., Falcon)

1.5 mL or 0.5-mL O-ring tubes

In a standard BSL2 laboratory, plate cells for infection one day prior into two T150 flasks in standard media for the chosen cell type. One flask serves as a mock-infected control and the other for infection. Plate cells so they will be ~80–90% confluent the following day. *For instance, plate 1 x 10 7 Vero CCL81 cells per T150 flask. Place flasks in a humidified 37 °C incubator with 5% CO₂ overnight.

Transfer flasks into BSL3 facility the following day. Rapidly thaw a SARS-CoV-2 stock at 37 °C. Calculate the volume of virus needed to infect at the desired multiplicity of infection (MOI) using the following formula:

( # of cells in a confluent T 150 ) x ( % confluency at present ) x ( desired MOI ) Virus titer in PFU / mL = Volume of virus needed ( mL )

Remove medium from T150 flasks. Replenish with 20 mL of fresh infection medium for mock-infected flask. Add 20 mL of infection medium containing virus from step #3 to flask for infection.

Incubate for 48–72 h at 37 °C monitoring daily for evidence of cytopathic effect (CPE) ( Fig. 1 ). Use the mock-infected flask as a control for subtle CPE.

SARS-CoV-2 causes cytopathic effect on Vero E6 cell monolayers. Vero E6 cells were inoculated with SARS-CoV-2 at an multiplicity of infection (MOI) of 0.01 plaque forming unit (PFU)/cell and monitored for cytopathic effect at the indicated timepoints. Images were collected using an EVOS XL Core Imaging System. Magnification is 10X for all images.

*Harvesting at 48–72 h post-inoculation has yielded the best titers in our hands; although, titers remain roughly the same when incubated for longer periods (4–5 days). CPE should be apparent by day 3 in Vero or MA104 cells.

To harvest virus, collect the cell culture supernatant by pipetting the media into two 15 mL conical tubes. Centrifuge at 450×g for 5 min at 4 °C to clarify supernatants and pellet cell debris. Combine the supernatant from all tubes into a single vessel and gently mix using a serological pipette to ensure homogeneity across aliquots of the stock. Pipette the supernatant into small aliquots (200–500 μL) in O-ring tubes. Store at −80 °C.

Real-time PCR assay for SARS-CoV-2 detection. Detection of viral RNA by reverse-transcription quantitative polymerase chain reaction (RT-qPCR) using a TaqMan probe is a highly-sensitive and specific method for measuring viral burden in a variety of specimens. Because CoVs generate subgenomic RNAs as a template for translation, the abundance of viral RNA varies for each gene and depends upon the gene position within the genome. Genes located closer to the 3′ end of the (+) sense genome will have a greater abundance of transcripts than those located at the 5′ end of the (+) sense genome. This should be considered when designing primer/probe combinations, as “N gene” transcripts will be more abundant than genomic RNA copies, which can be quantified by targeting sequences within the ORF1a gene. Many primer/probe combinations have been designed and validated, several of which are used in clinical diagnosis (CDC, 2020; Corman et al., 2020). In the clinical setting, precise copy-number quantitation of viral RNA is not necessary and instead sensitivity is paramount. However, quantitative assays are desirable for research applications, and may have utility in longitudinal studies of infected human subjects. RT-qPCR cycle threshold (Ct) values can be converted to transcript or genome copy number equivalents by generating an RNA standard curve, the design and production of which is described below.

2.2. Design of the primer/probe combination

The CoV replication strategy should be considered when designing a RT-qPCR assay. Primer/probe combinations targeting the N gene are most sensitive; those targeting the spike gene can also be used to titer spike-containing pseudoviruses; those targeting the ORF1a gene provide genome equivalents; and those targeting the leader sequence can give an estimation of the total number of viral transcripts ( Table 1 ). For a given viral gene target, a template (~500–1000 bp) for in vitro transcription can be generated by RT-PCR using primers that flank the intended target, with the forward (F) primer also including a 5′ T7 promoter sequence (Vogels et al., 2020). If multiple targets are desired, a single dsDNA fragment can be synthesized to include concatenated gene fragments, each of which spans the entirety of the target amplicons. This strategy also can be used to quantify host genes of interest (e.g., ACE2).

Table 1

Primer/probe combinations for detection of SARS-CoV-2 RNA.