Development of immunohistochemistry and in situ hybridisation for the detection of SARS-CoV and SARS-CoV-2 in formalin-fixed paraffin-embedded specimens

Fabian Z X Lean,Kevin R Bewley,Ian Brown,Samuel P Smith,Alejandro Nuñez,Miles W Carroll,Mart M Lamers,Rebecca Shipley,Debby Schipper,Nigel Temperton,Ashley C Banyard,Bart L Haagmans,Sharon M Brookes

doi:10.1038/s41598-020-78949-0

Abstract

The rapid emergence of SARS-CoV-2, the causative agent of COVID-19, and its dissemination globally has caused an unprecedented strain on public health. Animal models are urgently being developed for SARS-CoV-2 to aid rational design of vaccines and therapeutics. Immunohistochemistry and in situ hybridisation techniques that facilitate reliable and reproducible detection of SARS-CoV and SARS-CoV-2 viral products in formalin-fixed paraffin-embedded (FFPE) specimens would be of great utility. A selection of commercial antibodies generated against SARS-CoV spike protein and nucleoprotein, double stranded RNA, and RNA probe for spike genes were evaluated for the ability to detect FFPE infected cells. We also tested both heat- and enzymatic-mediated virus antigen retrieval methods to determine the optimal virus antigen recovery as well as identifying alternative retrieval methods to enable flexibility of IHC methods. In addition to using native virus infected cells as positive control material, the evaluation of non-infected cells expressing coronavirus (SARS, MERS) spike as a biosecure alternative to assays involving live virus was undertaken. Optimized protocols were successfully applied to experimental animal-derived tissues. The diverse techniques for virus detection and control material generation demonstrated in this study can be applied to investigations of coronavirus pathogenesis and therapeutic research in animal models.

Highlights

A novel coronavirus, SARS-CoV-2, emerged in Central China late 2019, primarily causing a respiratory disease termed COVID-191
SARS-CoV-2 is a Betacoronavirus classified under the order Nidovirales and family Coronaviridae, with a RNA genome that is closely related to SARS-CoV3
A rabbit monoclonal spike antibody and rabbit polyclonal nucleoprotein antibody (Sino Biological, 40143-T62) were identified to be suitable for IHC on formalin-fixed paraffin-embedded (FFPE) cell pellets in which specific cytoplasmic chromogen granules were observed in SARS-CoV and SARS-CoV-2 infected cells but not on uninfected cells (Fig. 1a–f)

Summary

Introduction

A novel coronavirus, SARS-CoV-2, emerged in Central China late 2019, primarily causing a respiratory disease termed COVID-191. This virus spread rapidly across the globe and was declared a pandemic by the WHO on the 11th of March 2020. SARS-CoV-2 is a Betacoronavirus classified under the order Nidovirales and family Coronaviridae, with a RNA genome that is closely related to SARS-CoV3. SARS-CoV-2 and SARS-CoV cellular entry is dependent on the receptor angiotensin-converting enzyme 2 (ACE2). Following the activation of spike protein by the cellular priming proteases TMPRSS2 and cathepsins, fusion of viral envelope and cellular membranes occur, allowing the viral genome to enter the host cell[4]. The spike protein, consisting of S1 and S2 subunits, is a major target

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