An experimental multiplex PCR/RT-PCR for detection of the main viruses associated with equine neurological disease

Viral neurological infections are important causes of morbidity and mortality in horses worldwide. Herein, we describe an end-point multiplex PCR/RT-PCR for simultaneous and differential detection of major equine encephalitis-related agents: rabies virus (RABV), equine herpesvirus type 1 (EHV-1), alphaviruses [Venezuelan equine encephalitis virus (VEEV), Eastern equine encephalitis virus (EEEV), Western equine encephalitis virus (WEEV) and Madariaga virus (MADV)] and flaviviruses [West Nile virus (WNV), Saint Louis encephalitis virus (SLEV), Rocio virus (ROCV) and Japanese encephalitis virus (JEV)]. The multiplex assay successfully amplified all targets either individually or simultaneously: alphavirus (381 bp-experimentally evaluated for MADV and potentially applicable to EEEV and WEEV based on primer design and sequence conservation), EHV-1 (255 bp), RABV (173 bp), VEEV (119 bp), and flavivirus (97-101 bp-experimentally evaluated using ROCV, with the potential to also detect WNV, SLEV and JEV, based on primer design and sequence conservation). Furthermore, the assay demonstrated analytical sensitivity and specificity that support its use for the comprehensive etiological diagnosis of equine neurological diseases. Overall, the multiplex PCR/RT-PCR described here may contribute to the diagnosis of viral neurological infections in horses, reducing costs and turnaround time, while contributing to prevention and control measures.