Diagnostic Method And Immunopathology Study Of Porcine Hemagglutinating Encephalomyelitis Virus

Porcine hemagglutinating encephalomyelitis (PHE), caused by hemagglutinating encephaomyelitis virus (HEV), is a hyperinfecting viral disease which infected swine acutely. HEV causes two distinct symptoms in sucking piglets: encephalomyelitis and the vomiting and wasting disease. The mortality rate of piglets under 3 week old is from 20% to 100%. PHE outbroke firstly in 1958, Ontario, in Canada, then PHE happened in Northern Ireland, England, Japan, Germany, the United States, Denmark, France, Australia, Belgium and other countries. When PHE outbroke in Argentina in August 2006, 1226 pigs died, the incidence rate is as high as 52.6%. As early as 1985, China reported PHE occurred in a Beijing's pig farm. Subsequently, PHE occurred in Jilin, Liaoning, Shandong and Taiwan and other regions. In 1962, HEV was firstly isolated from Canada's piglets suffering from encephalomyelitis by Andries K.. Pensaert MB, a Belgians, derived PHEV-VW572 from piglet's tonsils in 1972. The piglet clinical symptom showed not "encephalomyelitis" but "vomiting and failure". Afterwards, the virus strains isolated was the same pathogen as the previous isolated strain. In 2007, our laboratory isolated HEV from piglet's brain in a farm of Jilin Province, which is the first HEV isolation on the report in China. A large number of serological survey showed that, HEV infection was the worldwide and pose serious threat to development of pig farming industry.PHEV is a member of the genus Coronavirus, family Coronaviridae, order Nidovirales. Coronavirus is a large group of ribonucleic acid (RNA) viruses that infect a wide range of mammalian and avian species. They are pleomorphic positive-sense single-stranded RNA viruses measuring 60–220nm in diameter. The characteristic feature of the coronaviral morphology is the presence of long (12–24nm), widely spaced petal- or pear-shaped surface projections, which impart to the virus the appearance of a solar corona. Four structural proteins have been identified in the virus. They are the spike or peplomer (S) protein, membrane (M) protein, nucleocapsid (N) protein and the hemagglutinin-esterase (HE) protein.Pigs are the only species naturally susceptible to PHEV, mice and rats can be experimentally infected with PHEV. PHEV causes encephalomyelitis, or vomiting and wasting disease in suckling piglets. When piglets under 3 weeks old are infected with PHEV, their mortality rate is 100%, but pigs more than 3 weeks show no obvious clinical signs. Neonatal piglets receive usually maternal antibodies to PHEV from the colostrum of sows that protect against PHEV. However, once the piglets without maternal antibodies infect PHEV, they will die soon and few survive. The common diagnostic tests for porcine hemagglutinating encephalomyelitis (PHE) include epidemiological data, age susceptibility and disease course correlated with histopathological findings.The definitive diagnostic test for PHE include virus isolation, virus neutralization, immunohistochemistry (IHC). These procedures are labor intensive and time consuming. In order to establish specific, sensitive, rapid diagnosis method of HEV, the conservative region HEV was selected to design specific probes and primers; through optimizing reaction condition and identification of specificity, sensitivity, and reproducibility, a fluorescence quantitative RT-PCR method of HEV was established successfully; after detecting the different tissue samples by this established method, the result showed that the detection rate of brain is the highest. In order to establish specific, sensitive antibody detection method of pigs hemagglutinating encephalomyelitis, esterase protein (HE) gene of HEV was chose as a candidate gene. HE protein epitope-rich region of the gene was cloned, expressed in E. coli and purified. Western-blot test showed the purified protein well reacted with positive HEV serum. The indirect ELISA detection methods of HEV antibody was established through exploring and identifying a series of reaction conditions using purified HE protein. Serological survey in Jinzhou, Liaoning Province region using the established ELISA method showed the seropositive rate was differ in different farms from 0 to 100%.In order to study the HEV distribution and immune response in the host, mice were used as the experimental animal models. After intranasal inoculating HEV, mice were necropsied randomly at 1, 3, 5, 7, and 9d PI. Blood was collected and virus-specific antibody responses were determined by ELISA. Spleen cells were recovered, and tests were carried out to determine lymphocyte proliferation responses; and tests were carried out to detect cytokine; and carried out to detect the proportions of cluster of differentiationsubpopulations of T lymphocytes; through the above-mentioned test results, the rule of humoral and cellular immune at different infection time response was revealed. The results showed that the virus load differed in different organ in the same day PI; and the dynamic changes differed in the same organ in different days PI. Among this, the highest virus load was in the cerebrum. HEV located in pyramidal cells in the cerebrum, Purkinje cells in the cerebellum and epithelial cells of renal tubule. HEV infection caused immunosuppression in mice. Mice could not emerge the effective humoral immunity and cellular immunity.In order to study the immune response of HEV S protein, M protein and N protein, these protein were expressed in Pichia pastoris yeast and purified by using the Ni-NTA affinity columnin. Serums and spleens were collected from the mice on 7 days after the thrid immunization (interval two weeks each immunization) for detecting the anti-HEV antibodies and proliferative response of spleen cells. The results show that, S1 protein can induce significantly higher HEV antibody levels; M protein and N protein can induce the enhanced proliferative response of spleen cells, induced increased concentrations of INFγ.