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

Equine herpesvirus (EHV) 1 and EHV-4 are common viral pathogens of horses that can cause upper respiratory disease, neurological disease, abortion, or 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 targeting the equid genome. The EHV1-4MP assay analytical sensitivity was determined to be 15 genome copies for EHV-1, EHV-4, and equid MC1R 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 equine 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 -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. ImportanceEquine herpesvirus (EHV) 1 and -4 are highly contagious and ubiquitous pathogens that commonly cause fever and respiratory disease, and can lead to outbreaks, abortion, neurological disease, or death. 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 as well as an endogenous equid control and an exogenous DNA control, with improved specificity compared to existing assays.