A multiplex real-time PCR assay for detection of equid herpesvirus 1 and 4

Equid herpesvirus (EHV) 1 and -4 are common viral pathogens of horses that can cause upper respiratory disease, neurological disease, abortion, and death. As characteristic alphaherpesviruses, both EHV-1 and EHV-4 can establish latency, resulting in a lifelong carrier state in infected animals. Here we describe the development and validation of a rapid and sensitive multiplex real-time PCR assay (EHV1-4MP) that simultaneously detects EHV-1 and EHV-4 and includes an endogenous internal control - melanocortin 1 receptor (MC1R) - targeting the equid genome. The EHV1-4MP assay analytical sensitivity was determined to be approximately two copies for EHV-1, four copies for EHV-4, and 10 copies for the equid MC1R gene per reaction. Analytical specificity was determined using a panel of 28 equine respiratory pathogens and commensal equine microorganisms. The EHV1-4MP assay detected reference and clinical isolates of EHV-1 and EHV-4, and did not detect other equid herpesviruses such as EHV-2, EHV-3, EHV-5, or several other viral and bacterial pathogens of horses. Importantly, the EHV1-4MP assay developed here has improved specificity compared to existing assays and is able to exclude the closely related EHV-3, EHV-8, and EHV-9 viruses. Diagnostic performance was evaluated using 60 clinical samples including upper respiratory swabs and washes, blood, placenta, lung, and brain. The EHV1-4MP assay results were in 100% concordance with singleplex EHV-1 and EHV-4 assays. Our results demonstrate that the EHV1-4MP real-time assay developed here offers rapid, sensitive, and simultaneous detection of EHV-1 and EHV-4.