Etiology、detection Methods And Vaccine Development For Porcine And Encephalomyocarditis Virus Wild Isolate From Henan

Encephalomyocarditis(EMC) is a zoonosis disease caused by EMC virus(EMCV) was characterized by encephalitis, myocarditis and reproductive disorders. Rodents(rats and mice) are considered to be natural hosts of EMCV and are thought to be the primary reservoir and disseminators of the virus. Pig is infected widely and seriously, but a wide range of other species may also be affected, including humans. The first EMCV strain was isolated in China in 2005,however, EMCV infection in pigs is widespread and the positive rate of antibody is significant difference now. Although serious outbreak has not yet occurred, the potential threaten is on the rise. Henan province is located in the central plains, and location and resources advantages make her into a worthy breeding province. Moreover, complex geographical surroundings, rich biodiversity, and frequent activity of various rodents provide a objective condition for EMCV occurrence, spread and prevalence. However, there is no report on EMCV prevalence, pathogen characteristics and prevention and control researches in Henan province so far. Therefore, the present paper conducted a series of research works on six EMCV strains from different animals in Henan province, including virus isolation and detection, the whole genome sequencing and analysis, establishment of detection methods, pathogenicity test, gene expression and research and development of combined vaccine, to enrich the basic theory and clinical application in China,minimize the potential threaten of EMCV infection.1. Isolation, identification and whole genome sequence analysis of EMCV from different animal sourceIn or der to study the infection status of EMCV in the different animal hosts, cell inoculation and RT-PCR technology was used in the present paper, and the six strains from local aardvark,domesticated boar, mice and improved breed pig were isolated. This identified that EMCV infection is prevalent in pig farms in Henan province, and EMCV can infect different animal sources, which reminded us of taking fully consideration of the zoonosis ecology in the course of wild animal breeding activities.The clinical symptoms, biological characterizes and pathogenicity are various, after infectingdifferent hosts with different EMCV strains. The whole genome of six EMCV isolates generated a sequence of 7724-7735 bp in length, high identity with each other. The six strains belonged to the same group with other references from mice and pigs in China, suggesting cross-infection among them and same infection source. The phylogenetic tree based on the full length genome, EMCV was divided into G1,G2 and G3 groups. The six strains in this paper belonged to G1 group with other Chinese references, but the distribution is not complete. The results indicated that the isolated EMCV had a high pathogenicity to mice, and mice plays an important medium role in cross infection, transmission and prevalence of EMCV infection in different pig source.2. Research on EMCV diagnostic methodsEstablishment of rapid, specific and sensitive diagnosis methods is vital for disease diagnosis,epidemiological investigation, etiology research and development and research of vaccine. In the present paper, SYBR Green I Real-time PCR method was established with EMCV HNXX13 VP1 gene as the target sequence. The results identified the method had good amplification efficiency,high specificity and repeatability, moreover, higher sensitivity, and provided a technical platform for EMCV molecular epidemiological investigation, epidemic monitoring, etiology research and rapid diagnosis. Meanwhile, the prokaryotic expression and identification of VP1 gene were firstly conducted from domestic wild boar EMCV strain JZ1202, and the recombinant protein was used as envelope antigen to establish indirect ELISA method for detecting EMCV antibody in the serum. Applied to clinic, the survey found that the detection rate was 65.87%in Henan province, total serum positive rate was 45.3%. EMCV infection existed in different size and different stages pig farms, compared with large-scale pig farms, small and medium-sized had higher positive rate, moreover, mixed infection rates with PCV-2 as high as 75.14%, which filled the gap of EMCV molecular and serum epidemiological surveillance in Henan province.3. The study on influence of pathogenicity in piglets infection with EMCV after PCV-2The impact on growth and pathogenicity of piglets infection with EMCV after PCV-1 was conducted for the first time at home and abroad. The results found that PCV-2 early infection can cause piglets infection with EMCV more serious pathogenicity and lethality. The lymphocyte ratio and EMCV neutralizing antibody maintain at a low level after infection with EMCV and PCV-2 successively. The results confirmed that EMCV virus could be detected and isolated from anal swab after nine days EMCV alone infection, however, secondary infection could sustain to 12 days. This identified that the replication and detoxification of EMCV increased in pigs, but the coordination mechanism needs to be further replicated and verified.4. Research and development and immune efficacy detection of EMCV and PCV-2combined inactivated vaccineThe EMC inactivated vaccine has been successfully developed abroad, however, combined vaccines with other diseases are not still reported. The EMCV and PCV-2 combined inactivatedvaccine was firstly developed in the present paper. The immune effect of combined vaccine was higher than each single vaccine after vaccinating mice and piglets, in addition, could produce high level of antibodies. The effective resistence to EMCV and PCV-2 attack can offer joint immune for the two disease, and achieve the “one needle against the two disease” to satisfy the current potential demand for EMCV immune in pig production.5. Research on expression and immunogenicity of EMCV VP1 gene in baculovirus Insect baculovirus expression system applicated in EMCV VP1 gene fromThe VP1 gene of epidemic strains conducted by application of insect baculovirus expression has been rarely reported. Based on the baculovirus codon preference, EMCV VP1 gene of JZ1201 isolated from Henan was optimized and artificially synthesized, and stable expression of VP1 recombinant baculovirus was constructed. Specific and high level of neutralizing antibody was produced by immunizing mice, and division and proliferation of lymphocytes, and immunogenicity were significantly increased, which lay a foundation for further research on its function, subunit vaccine and development of diagnostic antigen.In conclusion, the study in this paper lay a solid foundation for epidemiological surveillance system, pathogenesis, and rapid diagnosis, immune mechanism and development of new vaccines,and provide a scientific theory basis for effective prevention and control of the disease.