Study On The Etiology And Detection Method For Porcine Encephalomyocarditis Virus

This study were focused on the establishment and application of etiological and serological diagnostic methods for detection of encephalomyocarditis virus (EMCV), the isolation and identification of EMCV Guangxi isolate (GXLC strain) and analysis of the molecular characteristics of GXLC strain genome, and the pathogenic experiment of porcine EMCV GXLC strain to mice and piglets. The study made a solid foundation on further research of EMCV in pathogenesis, immune mechanism and integrated prevention and control technology.The main conclusions of this study could be summarized as follows:First, the one-step RT-PCR, SYBR Green I real-time RT-PCR and TaqMan real-time RT-PCR assays for detection of EMCV were established. These methods provided an effective technology platform for rapid diagnosis of EMCV with high specificity, sensitivity and reproducibility.Second, EMCV structural protein VP1was expressed through prokaryotic expression vector (E. coli). An indirect ELISA using VP1as coated antigen was established for detection of EMCV antibody. A serological survey of part of the pig farms in Guangxi showed that80.79%serum samples and100%pig farms were positive for EMCV antibody, indicating that EMCV infection was common in Guangxi.Third, the Guangxi isolate of EMCV (GXLC strain) was isolated and identificated and its complete genome was sequenced and analyzed, including homology analysis of nucleotide and deduced amino acid sequence, comparison of amino acid sequence and phylogenetic analysis based on nucleotide sequence. The molecular characteristics of structural protein VP1gene and non-structural protein3D gene were analyzed in detail. The results showed that the genome sequence of GXLC strain was highly homologous to other domestic and foreign EMCV isolates, and all EMCV isolates can be devided into group Ⅰ and group Ⅱ, of which GXLC strain and other Chinese strains were distributed in la subgroup.Fourth, the TaqMan real-time RT-PCR assays for detection of mouse cytikines IL-1β, IL-6, TNF-α and iNOS were established. And also, the multiplex TaqMan real-time RT-PCR assays for detection of porcine cytokines IL-1β, IL-6, TNF-α and iNOS were established. These detection methods provided effective technology platforms for qualitative and quantitative detection of these cytokines.Fifth, the pathogenic experiments of porcine EMCV GXLC strain to mice and piglets were carried out. The results showed that the mice and piglets infected with EMCV displayed obvious viremia, clinical symptoms and pathological changes. The IL-1β, IL-6and TNF-α mRNA expression in tissues from the infected animals were significant upregulation, and the IL-1β and TNF-a protein levels in serum were significant increase (only mice measure). The peak period of cytokine expression was closely related to the time of appearing clinical symptoms, pathological damages and death peak (only mice death occur), and the cytokine expression levels were closely related the severity of pathological damages. The results indicated that the EMCV GXLC strain was highly pathogenic to mice and piglets and the proinflammatory cytokines IL-1β, IL-6and TNF-α played an important role in the pathogenic process of EMCV.