Um multiplex PCR/RT-PCR para a detecção de vírus associados a doenças neurológicas em equinos

Abstract

Viral neurological infections are important causes of morbidity and mortality in horses, affecting animals of all ages and breeds. The Rabies virus (RABV), Equine herpesvirus type 1 (EHV-1), alphaviruses [Eastern equine encephalitis virus (EEEV), Western equine encephalitis virus (WEEV), Venezuelan equine encephalitis virus (VEEV) and Madariaga virus (MADV)], and flaviviruses [West Nile virus (WNV), Saint Louis encephalitis virus (SLEV), Rocio virus (ROCV) and Japanese encephalitis virus (JEV)] are the main viral agents of equine encephalitis or encephalomyelitis. Infections by these viruses result in similar clinical manifestations, emphasizing the need for laboratory diagnosis for the correct identification of the agent. In order to contribute to a faster diagnosis of these infections, we developed a multiplex PCR/RT-PCR (end-point) assay for differential and simultaneous detection of these agents. Initially, specific and high-coverage primers were designed for the detection of RABV, EHV-1, and flaviviruses. For alphavirus detection, a primer set was designed for EEEV, WEEV and MADV, and another for VEEV. Subsequently, the concentration of primers, enzymes, and the annealing/extension temperature of the multiplex assay were optimized, along with the evaluation of agarose gel concentration for the correct differentiation of targets. After reaction optimization, the analytical sensitivity for RABV, EHV-1 and MADV was determined. The analytical specificity of the multiplex was evaluated against other potential agents of neurological diseases in horses: Streptococcus equi subsp. equi, Trypanosoma evansi, Sarcocystis neurona, Neospora spp. and Equine herpesvirus type 4 (EHV-4). The multiplex PCR/RT-PCR amplified all targets individually and simultaneously: alphaviruses (EEEV-WEEV-MADV, 381bp), EHV-1 (255bp), RABV (173bp), VEEV (119bp), and flaviviruses (WNV-SLEV-ROCV-JEV, 96-100bp). The detection limit of the multiplex PCR/RT-PCR was 1.07, 3.4, and 53.7 TCID50 for EHV-1, RABV and MADV, respectively. The analytical sensitivity for flaviviruses (WNV, SLEV, ROCV, and JEV) and other alphaviruses (EEEV, VEEV, WEEV, and VEEV) was not evaluated due to the lack of access to biosafety level 3 containment. No unspecific amplifications of other potential agents of neurological diseases in horses were observed. In conclusion, the simultaneous and differential detection of the main agents of equine neurological diseases, combined with high analytical specificity, makes the multiplex assay described here a useful tool for the differential diagnosis and surveillance of these neurological infections in horses.