Duplex Real-Time RT-PCR Assays for the Detection and Typing of Epizootic Haemorrhagic Disease Virus.

Cyril Viarouge,Stephan Zientara,Corinne Sailleau,Emmanuel Breard,Damien Vitour,Houssam Attoui

doi:10.1371/journal.pone.0132540

Abstract

Epizootic haemorrhagic disease virus (EHDV) may cause severe clinical episodes in some species of deer and sometimes in cattle. Laboratory diagnosis provides a basis for the design and timely implementation of disease control measures. There are seven distinct EHDV serotypes, VP2 coding segment 2 being the target for serotype specificity. This paper reports the development and validation of eight duplex real-time RT-PCR assays to simultaneously amplify the EHDV target (S9 for the pan-EHDV real-time RT-PCR assay and S2 for the serotyping assays) and endogenous control gene Beta-actin. Analytical and diagnostic sensitivity and specificity, inter- and intra-assay variation and efficiency were evaluated for each assay. All were shown to be highly specific and sensitive.

Highlights

Epizootic haemorrhagic disease virus (EHDV) is a double-stranded RNA non-enveloped virus belonging to the Reoviridae family, genus Orbivirus
This paper describes the development and validation of eight duplex realtime RT-PCR assays for the detection and serotyping of each EHDV serotype (EHDV-1/-3, -2, -4, -5, -6, -7 and -8): a duplex pan-EHDV real-time RT-PCR assay that amplifies a portion of segment 9 of all EHDV strains coupled with a real-time RT-PCR that amplifies the beta-actin RNA of ruminant cells and a set of seven duplex serotyping real-time RT-PCR assays that amplify both the EHD segment 2 portion of each serotype and the beta-actin gene
All the infected biological samples used in this study were taken either from naturally infected domestic cattle or buffaloes, or from experimentally infected animals [13,14,17,22]: 40 blood samples infected by EHDV-1 (Reunion Island and French Guiana); 32 blood samples infected by EHDV-6 (Reunion Island and French Guiana); 24 blood samples infected by EHDV-6

Summary

Introduction

Epizootic haemorrhagic disease virus (EHDV) is a double-stranded RNA non-enveloped virus belonging to the Reoviridae family, genus Orbivirus. It is transmitted by Culicoides biting midges. EHDV has a 10-segmented RNA genome that encodes seven structural proteins (VP1-VP7) divided into two capsids and five nonstructural proteins (NS1-NS4, NS3a) [1,2,3,4,5]. In 2006, an exotic strain of EHDV-6 was isolated from moribund and dead white-tailed deer (Odocoileus virginianus) in Indiana and Illinois. This EHDV-6 strain originates from a reassortment between co-circulating strains of serotypes 2 and 6 [9]. EHDV-6 and -2 were reported in the Caribbean basin (Martinique, Guadeloupe Islands and French Guiana), while EHDV-1 was detected in French Guiana

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