Study On The Pathogenicity Of Porcine Encephalomyocarditis Virus Gxlc Strain

Encephalomyocarditis virus (EMCV) can cause an infectious disease characterized by acute encephalitis and myocarditis in piglets and severe reproductive failure in sows. EMCV can cause encephalitis, myocarditis and diabetes in mice. We infected mice and piglets with porcine EMCV GXLC strain respectively in order to analyze its pathogenicity in this study.Based on the conserved regions of the porcine EMCV 3D gene, cytokine gene IL-1β, TNF-α, IL-6, iNOS as well as housekeeping geneβ-actin of mouse and porcine, a pair of primers and a TaqMan probe were respectively designed, and related TaqMan real-time PCR assays were developed for detection of them with related recombinant plasmid as the standard samples. The correlation coefficient of the standard curve was over 0.998,10 copies/μL of standard plasmids could be detected, and there was no cross-reaction with FMDV, CSFV, PRRSV, PRV, PCV2 and normal BHK-21 cells; for detection of proinflammatory cytokines and the housekeeping gene, the fluorescent signals could only be detected by the reaction with standard plasmid or cDNA of each cytokine; the coefficient of variation was less than 2 percent for both intra- and inter-assay. The results indicated that the TaqMan real-time PCR assays we developed were highly specific, sensitive and reproducible which could be used as an effective tool for detection and quantification of EMC V.The porcine EMCV GXLC strain was used to infect healthy BALB/c mice and piglets of 4-week age to study the viral pathogenicity. After infection, not only the clinical manifestations, the gross pathological changes and histopathological changes were observed; but also the viral loads, the mRNA expression levels of IL-1β, TNF-α, IL-6 and iNOS in heart, brain and spleen of mice and piglets were determined; In addition, both the ratios of heart weight to body weight and the mice protein loads of IL-1β,TNF-αin mouse serum were detected; the antibody titers of piglet in serum were also detected. The results showed that:(1) the infected mice displayed significant neurological symptoms, and most of them died within 24 hours after showing obvious manifestations; the piglets manifested febrile responses and obvious symptoms such as listlessness。(2) for the autopsy, both gray necrosis lesions on the surface of heart and cerebral hemorrhage were found in mice; myocardial expansion and softness, cerebral hyperemia, swelling and hyperemia in lymph nodes were also abservated in piglets; (3) the ratio of heart weight to body weight and the protein loads of serum IL-1βand TNF-αin infected mice increased significantly, and high titers of anti-EMCV antibody was also detected in serum of piglest. (4) a large amount of viruses were detected in brain, heart, spleen and serum of infected mice and piglets, and the viral loads were closely related with clinical manifestations and pathological lesions.(5) for the histopathological observation of mice and piglet, we found degeneration and necrosis in neuron, microvascular hemorrhage and infiltration of inflammatory cells in cortex, large area of myocardial necrosis, degradation, atrophy and diffused inflammation, which result in the rates of histopathological lesions. (6) the mRNA expression levels of IL-1β, TNF-α, IL-6 and iNOS in heart, brain and spleen were significantly up-regulated, whose peak time was closely related to the time during which obvious clinical manifestations, histopathological lesions appeared. In conclusion, porcine EMCV GXLC strain is highly pathogenic to mice and piglets, and the cytokines such as IL-1β, TNF-α, IL-6 and iNOS play important roles in the pathogenic process of EMCV.