Molecular Detection of SARS-CoV-2 in Formalin Fixed Paraffin Embedded Specimens

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 more than one million cases and tens of thousands of deaths. Here, we describe the identification and evaluation of commercially available reagents and assays for the molecular detection of SARS-CoV-2 in infected formalin fixed paraffin embedded (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 immunohistochemistry (IHC) and immunofluorescence assay (IFA). Next, we established RNAscope in situ hybridization (ISH) to detect SARS-CoV-2 RNA. Furthermore, we established a multiplex fluorescence ISH (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. These reagents and assays will accelerate COVID-19 pathogenesis studies in humans and in COVID-19 animal models.


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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiologic agents of human for S priming (7). 43 Bats are speculated to be the natural reservoir of SARS-CoV-2 because numerous other 44 betacoronaviruses are of chiropteran origin (8,9). However, although the COVID-19 pandemic 45 may have begun with a bat-to-human transmission event, it appears that close to all human 46 infections trace back to respiratory droplets produced by infected people and fomites (respiratory 47 droplet landing sites) (10, 11). Human infections lead to various degrees of disease severity, 48 ranging from asymptomatic infection or mild symptoms to fatal pneumonia. Older patients or 49 patients with chronic medical conditions are more vulnerable to becoming critically ill with poor 50 prognosis (12). The most common symptoms and clinical signs of COVID-19 are fever, cough, 51 Liu et al.
SARS-CoV-2 detection in FFPE specimens 4 dyspnea, and myalgia with medium incubation period of 4 days (13-15). Ground-glass opacity is 52 the most common radiologic finding on chest CT upon admission (13-15). Bilateral diffuse 53 alveolar damage, alveolar hemorrhage and edema, interstitial fibrosis and inflammation, and type 54 II pneumocyte hyperplasia are observed in post-mortem human lungs (16)(17)(18). 55 At the time of writing, there are no animal models that truly mimic the disease spectrum and 56 pathogenesis of COVID-19. However, small animals (e.g., human ACE2 transgenic laboratory 57 mice (19), cats (20), domestic ferrets (20, 21), golden hamsters (22)), and nonhuman primates 58 (e.g., rhesus monkeys (23, 24), crab-eating macaques (25)), are used to study SARS-CoV-2 59 infection as alveolar damage, interstitial inflammation, and viral shedding occur in these animal 60 models to various degree. It is hoped that further development of these and other animal models 61 will help overcome the current roadblock to evaluating the efficacy of candidate medical 62 countermeasures (MCMs) against and the pathogenesis of COVID-19.

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Detection of viral antigen using IHC or IFA techniques and detection of viral nucleic acids using 64 ISH within infected, but inactivated, human or animal model tissues greatly facilitates detection 65 of viral infection and thereby pathogenesis and MCM efficacy studies. These techniques become 66 paramount in particular for studies of a potential pathogen that does not cause overt, or causes 67 only mild, disease, such as SARS-CoV-2 in the currently available animal models. Viral antigen-68 based immunostaining has been used to detect SARS-CoV-2 antigen in both post-mortem human 69 and animal tissues (1, 16, 22, 25). However, the antibodies used in these studies were produced 70 in-house and therefore are not commonly available. Identification and characterization of 71 commercially available anti-SARS-CoV-2 antibodies and ISH assays that can be used to detect 72 SARS-CoV-2 in FFPE tissues are therefore critically needed. 73 Liu et al.
SARS-CoV-2 detection in FFPE specimens 5 Here, we describe the evaluation of a rabbit polyclonal anti-SARS-CoV S antibody and a mouse 74 monoclonal anti-SARS-CoV NP antibody that are commercially available and, in IHC and IFA, 75 recognized respective SARS-CoV-2 proteins in FFPE specimens. We also identify two 76 commercially available ISH assays that can be used to efficiently detect SARS-CoV-2 RNA in 77 such specimens and develop a dual staining assay using IHC and ISH to detect SARS-CoV-2 S 78 and RNA in the same FFPE section. CoV NP that may cross react with SARS-CoV-2 (Supplemental Table 1). Additionally, we also 87 identified a rabbit monoclonal antibody against SARS-CoV-2 S (Supplemental Table 1). To   Table 2). 105 As expected, the forty ZZ positive-sense RNA probe 2 binding to SARS-CoV-2 positive-sense 106 RNA resulted in a stronger signal than the twenty ZZ positive-sense RNA probe 1 (Figure 2A (26,28,29). Here, we tested mFISH to 115 detect SARS-CoV-2 replication in FFPE specimens using positive-sense RNA probe 2 and 116 negative-sense RNA probe 2 (Supplemental Table 2). Consistent with the RNAscope ISH  to identify antibodies that binds their targets in FFPE tissues compared to frozen section tissues.

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The FFPE specimen-compatible rabbit and mouse anti-SARS-CoV-2 antibodies we 145 characterized here can be used to map the cellular targets of SARS-CoV-2 in various organs 146 using multiplex IFA in addition to detecting viral infection. RNAscope ISH is a relatively novel 147 ISH platform with high-sensitivity and low-background due to its unique "ZZ" probe design   Table 1) and incubated at room temperature for 45 min. Subsequently, sections 187 Liu et al.

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were rinsed, and the peroxidase-labeled polymer (secondary antibody) was applied for 30 min.  Table 2). Tissue sections were deparaffinized with xylene, underwent a series of 214 ethanol washes and peroxidase blocking, and were then heated in kit-provided antigen retrieval 215 buffer and digested by kit-provided proteinase. Sections were exposed to ISH target probe pairs   Table 1) overnight at 4°C, 237 following the Fast Red substrate ISH procedure described above using positive-sense RNA probe 238 2 (Supplemental Table 2). One day later, sections were rinsed, and the peroxidase-labeled