Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of human coronavirus disease 2019 (COVID-19), emerged in Wuhan, China, in December 2019. The virus rapidly spread globally, resulting in a public health crisis including almost 5 million cases and 323,256 deaths as of May 21, 2020. Here, we describe the identification and evaluation of commercially available reagents and assays for the molecular detection of SARS-CoV-2 in infected FFPE cell pellets. We identified a suitable rabbit polyclonal anti-SARS-CoV spike protein antibody and a mouse monoclonal anti-SARS-CoV nucleocapsid protein (NP) antibody for cross-detection of the respective SARS-CoV-2 proteins by IHC and immunofluorescence assay (IFA). Next, we established RNAscope in situ hybridization (ISH) to detect SARS-CoV-2 RNA. Furthermore, we established a multiplex FISH (mFISH) to detect positive-sense SARS-CoV-2 RNA and negative-sense SARS-CoV-2 RNA (a replicative intermediate indicating viral replication). Finally, we developed a dual staining assay using IHC and ISH to detect SARS-CoV-2 antigen and RNA in the same FFPE section. It is hoped that these reagents and assays will accelerate COVID-19 pathogenesis studies in humans and in COVID-19 animal models.
Highlights
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiologic agent of human coronavirus disease 2019 (COVID-19), initially emerged in Wuhan, Hubei Province, China, in December 2019 [1,2,3]
We identified 1 rabbit polyclonal antibody against SARS-CoV S (Sino Biological, 40150-T62-COV2) and a mouse monoclonal antibody against SARS-CoV nucleocapsid protein (NP) (Sino Biological, 40143-MM05) that did not stain uninfected, but stained SARS-CoV-2–infected, FFPE cell pellets (Figure 1, A–D)
We have previously reported the development of RNAscope in situ hybridization (ISH) assays to detect various high-consequence viruses including Ebola virus (EBOV; Filoviridae: Ebolavirus), Marburg virus (MARV; Filoviridae: Marburgvirus), Lassa virus (LASV; Arenaviridae: Mammarenavirus), and Nipah virus (NiV; Paramyxoviridae: Henipavirus) in FFPE animal tissues [26,27,28,29]
Summary
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiologic agent of human coronavirus disease 2019 (COVID-19), initially emerged in Wuhan, Hubei Province, China, in December 2019 [1,2,3]. The virus is classified as a betacoronavirus (Nidovirales: Coronaviridae) together with the other 2 highly virulent human pathogens severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) [6]. The SARS-CoV-2 genomes shares 79.6% and 50.0% nucleotide sequence identity with the genomes of SARS-CoV and MERS-CoV, respectively [5]. Similar to SARS-CoV, SARS-CoV-2 virions use their spike (S) glycoproteins to engage host-cell angiotensin I–converting enzyme 2 (ACE2) to gain entry into host cells and host-cell transmembrane serine protease 2 (TMPRSS2) for S priming [7]
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