A Real-Time PCR Assay for Bat SARS-Like Coronavirus Detection and Its Application to Italian Greater Horseshoe Bat Faecal Sample Surveys

Andrea Balboni,Santino Prosperi,Laura Gallina,Mara Battilani,Alessandra Palladini

doi:10.1100/2012/989514

Andrea Balboni, Santino Prosperi + Show 3 more

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https://doi.org/10.1100/2012/989514

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Abstract

Bats are source of coronaviruses closely related to the severe acute respiratory syndrome (SARS) virus. Numerous studies have been carried out to identify new bat viruses related to SARS-coronavirus (bat-SARS-like CoVs) using a reverse-transcribed-polymerase chain reaction assay. However, a qualitative PCR could underestimate the prevalence of infection, affecting the epidemiological evaluation of bats in viral ecology. In this work an SYBR Green-real time PCR assay was developed for diagnosing infection with SARS-related coronaviruses from bat guano and was applied as screening tool in a survey carried out on 45 greater horseshoe bats (Rhinolophus ferrumequinum) sampled in Italy in 2009. The assay showed high sensitivity and reproducibility. Its application on bats screening resulted in a prevalence of 42%. This method could be suitable as screening tool in epidemiological surveys about the presence of bat-SARS-like CoVs, consequently to obtain a more realistic scenario of the viral prevalence in the population.

Highlights

Among the human viral epidemics which have emerged in the past ten years, one of the most important is represented by the outbreak of severe acute respiratory syndrome (SARS) which appeared in China in 2002-2003, rapidly causing a human epidemic [1]
The primers used for the PCR reaction generally amplified a fragment of the RNA-dependent RNA polymerase gene (RdRp, the 12th nonstructural protein encoded by ORF1a, b), a conserved feature of the coronavirus genome which was frequently used for subsequent phylogenetic analysis [4, 8, 10,11,12,13,14,15,16]
The standard curve was generated by plotting the real-time PCR threshold cycle numbers (Ct) of each dilution against the known copy numbers of recombinant plasmid

Summary

Introduction

Among the human viral epidemics which have emerged in the past ten years, one of the most important is represented by the outbreak of severe acute respiratory syndrome (SARS) which appeared in China in 2002-2003, rapidly causing a human epidemic [1].Several studies showed, firstly, that a new coronavirus was the aetiological agent of SARS, the SARS-CoV [2], and subsequently bats were the natural reservoir for several viruses closely related genetically to the SARS-CoV, the SARS-like coronaviruses (SARS-like CoVs) [3]. The primers used for the PCR reaction generally amplified a fragment of the RNA-dependent RNA polymerase gene (RdRp, the 12th nonstructural protein encoded by ORF1a, b), a conserved feature of the coronavirus genome which was frequently used for subsequent phylogenetic analysis [4, 8, 10,11,12,13,14,15,16] This technique may not have an optimal sensitivity, causing an underestimation of the true prevalence of infection, which may be due to a low amount of viral clearance in the faeces during the infection, too low to be detected by traditional PCR methods

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