Pathological Study Of HP-PRRS And The Sequence Analysis Of HP-PRRSV

The Porcine Reproductive and Respiratory Syndrome (PRRS) is one of the economically most important swine diseases worldwide, is one of the most economically influential infectious diseases in the industry of swine cultivation. Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent, a member of the family Arteriviridae in the order Nidovirales, PRRS is characterised by reproductive failure of sows and respiratory problems of piglets and growing pigs. Genomic analysis of PRRSV has shown that the virus genome varies from 15kb to 15.5 kb and comprises at least 9 open reading frames (ORFs) that encode nearly 20 mature proteins. PRRSV with different geograpHic origins and antigenic can be classified into 2 major genotypes, the European type (type I, EU-type) and North American type (type II, NA-type), Lelystad virus and isolation of VR2332 is the European prototype and the North American prototype PRRSV, respectively. The unprecedented large-scale porcine high fever disease (PHFD) outbreaks in 2006 swept over nearly half of the People's Republic of China and infected >2,000,000 pigs, dead >40,000 pigs, which posed great concern to the global swine industry and to public health.This study is for analying the true cause of porcine high fever disease, through to investigate 55 cases of observing the morbidity situation, the epidemiology and the clinical symptoms in the suburban country of Beijing and to15 pig farms in the surrounding areas, and through anatomical examine, the histopathological observe, immunohistochemical stains and observing under electron microscope to the disease pig and death pig, meanwhile making a nucleicacid and antibody test to the frozen samples and serum, respectively. The study result indicates that the positive rate of PRRSV nucleicacid is 60%, PCV-2 nucleicacid is 32.7%, and the rate of PRRSV and PCV-2 mixed infection is 18.2%. In this porcine high fever disease, the main pathogen is PRRSV. The invasion characteristics are different with the typical PRRS which formerly appeared, meanwhile the PCV-2 has mixed infection. The PRRS histopathological groups mostly are interstitial pneumonia, nonsuppurative encepHalitis, hemorrhage necrotizing lympHadenitis and acute inflammatory splenectasis, degenerate myocarditis, acute interstitial nepHritis accompanying to bleed, necrotic amygdalitis, endometritis of sow, alterative hepatitis, eosinopHilic inclusion in cytoplasm of hepatocytes. The positive organ of PRRSV are lung, spleen, lympHaden, amygdala, brain, liver, kidney, suprarene, stomach, intestines and heart. The positive cells of PRRSV are monocaryon-macropHage, epithelial cells, mucosa cellula epithelialis, lympH-cells, vascular endothelial cells, glandular epithelium cells (pulmonary epithelial cells, hepatocyte, enal tubular epithelial cells, and so on), caliciform cell, fibrocyte, fibroblast, cadiocyte, neurocyte, gliocyte and chondrocyte. In order to analyze the outbreak of high morbidity and mortality, In this study 4 tissue samples which without inoculated PRRS vaccine and were positive by RT-PCR identifying inoculated to MARC-145 cells for virus isolation. By RT-PCR, IFA and electron microscopy analysis proved that all the 4 strains of virus were PRRSV, the TCID50 were 10-6.5/0.1mL, named BJSY-1, BJPG, BJSD and BJBLZ strains. Further designed 16 overlapping primers, RT-PCR products were directly purified, sequenced, and spliced, we had got 4 complete genome sequence of PRRSV. Using molecular biology software gene comparison and evolution analysis, the results showed: The separation BJSY-1, BJPG, BJSD and BJBLZ strain complete genome sequences were 15319nt, 15315nt, 15345nt and 15316nt, respectively, through the audit by National Center for Biotechnology Information (NCBI), GenBank accession number were FJ950744, FJ950746, FJ950747 and FJ950745. The nucleotide homology of this Studiy 4 and the North American type strain were 83.3% to 99.4%, and the European-type strains is about only 58.3%. All the strains belong to the North America-based subgroupâ…¡, also belong to HP-PRRSV. ORF6 gene homology with the North American and European strains were the highest, followed by the ORF2 gene, is again ORF7, ORF4 and ORF5 gene, the lowest was ORF3 gene.In this syudy, we chose the lowest variability ORF6 gene sequences of HP-PRRSV BJSY-1 strain, designed and synthesized a pair of specific primers. The amplified ORF6 gene fragment was then cloned into the prokaryotic expression vector pET-32a(+),the recombinant plasmid pET-ORF6 was transformed into host cell BL21, after the successful construction, we grope appropriate conditions to induce M protein, and gained the solublefusion protein with immunocompetence in the HIS-Taq?,which was about 39kDa by purification and recycle. We gained an antigen which is stable and have better immunogenicity in this study which lay on foundation for preparation monoclonal antibody or as detect antigen.We considered that it is difficult to find the swine which antibody and antigen were all negative, so, tried to select the experimental animal which susceptive for PRRSV instead of porcine on research PRRSV, simultaneously another reason was verify if HP-PRRSV strain which have increased pathogenicity could infected other animals. In this study, we did the artificial infection experiment on BALB/c mice and Japanese big ear rabbits with different doses, then observed the variation of temperature and weight, According to the experimental design regular necroscopy and drawn materials for detecting nucleic acid and pathological examination on HP-PRRSV BJSY-1 strain. The results showed that: the change of the temperature and weight of the mice and the rabbits was no abnormalities, it was negative to detecting nucleicacid and not show distinctive histopathological changes on PRRSV. This study indicated that the HP-PRRSV strain did not infected BALB/c mice and Japanese big ear rabbits.