Updated and Validated Pan-Coronavirus PCR Assay to Detect All Coronavirus Genera.

Myndi G Holbrook,Theo Bestebroer,Simon J Anthony,Neeltje Van Doremalen,Vincent J Munster,Isamara Navarrete-Macias

doi:10.3390/v13040599

Abstract

Coronavirus (CoV) spillover events from wildlife reservoirs can result in mild to severe human respiratory illness. These spillover events underlie the importance of detecting known and novel CoVs circulating in reservoir host species and determining CoV prevalence and distribution, allowing improved prediction of spillover events or where a human–reservoir interface should be closely monitored. To increase the likelihood of detecting all circulating genera and strains, we have modified primers published by Watanabe et al. in 2010 to generate a semi-nested pan-CoV PCR assay. Representatives from the four coronavirus genera (α-CoVs, β-CoVs, γ-CoVs and δ-CoVs) were tested and all of the in-house CoVs were detected using this assay. After comparing both assays, we found that the updated assay reliably detected viruses in all genera of CoVs with high sensitivity, whereas the sensitivity of the original assay was lower. Our updated PCR assay is an important tool to detect, monitor and track CoVs to enhance viral surveillance in reservoir hosts.

Highlights

Coronaviruses (CoVs) are a large virus family comprising four genera—Alphacoronavirus (α-CoV), Betacoronavirus (β-CoV), Gammacoronavirus (γ-CoV) and Deltacoronavirus (δ-CoV)
Specific spillover events resulting in new CoV infections in humans have occurred in recent history
MERS-CoV is closely related to bat CoVs such as PREDICT/PDF-2180 [12,13], suggesting that bats could be an ancestral reservoir

Summary

Introduction

Coronaviruses (CoVs) are a large virus family comprising four genera—Alphacoronavirus (α-CoV), Betacoronavirus (β-CoV), Gammacoronavirus (γ-CoV) and Deltacoronavirus (δ-CoV). Malayan pangolins (Manis javanica) have been investigated as an intermediate host, but the most likely natural animal reservoir are bats from the Rhinolophus genus [16,17,18,19,20,21] This is the third CoV spillover into the human population in the twenty-first century—highlighting the importance of tracking CoVs in their natural and intermediate reservoirs. Increased human-to-wildlife interface has raised the risk of CoV spillover events into the human population, and consensus PCR assays have been widely implemented in programs like PREDICT, they have not been systematically evaluated for sensitivity or updated to account for all the new diversity that has been discovered in recent years. The described novel PCR is suitable for detecting and monitoring CoV circulation in reservoir species

Primer Design and Optimization

Viruses Selected for PCR Establishment

RNA Extractions and cDNA Synthesis for Viral Templates

Pan-CoV PCR

Phylogenetic Tree Analysis

Primer Design