Genetic Variation Analysis Of PRRSV Epidemic Strains In Some Areas Of Henan And Preliminary Isolation And Identification Of Some Epidemic Strains

Porcine reproductive and respiratory syndrome(PRRS)mainly results in reproductive dysfunction in sows and respiratory symptoms in weaned piglets.Infection of PRRS can cause severe immunosuppression in pigs,extremely easy to mix with other pathogens will lead to a significant increase in the death rate of weaned piglets.The disease is caused by porcine reproductive and respiratoiy syndrome virus(PRRSV).PRRSV,as a RNA virus,has a very high probability of variation in its genome.These variations will directly lead to changes in virulence among strains and exacerbate the complexity of the epidemic in the field of PRRSV.,increasing the difficulty of prevention and control of PRRSV.In this study,in order to further understand the epidemic situation of epidemic PRRSV strains and the characteristics of the genetic variation of the epidemic PRRSV strains,provide an important reference basis for The formulation of reasonable prevention and control measures,we analyze the genetic variation and evolution of the ORF5,Nsp2 gene of epidemic PRRSV strains in Henan,and isolate and identify the epidemic strains.The GP5 protein encoded by ORF5 gene,is the most frequently mutated part of all the structural proteins of PRRSV,and GP5 protein can induce both high titer neutralizing antibody and non-neutralizing antibody.Therefore,there is a certain correlation between the variation of ORF5 gene and the pathogenicity of the strain.Many researchers often use it as an important target gene for PRRSV epidemiological investigation.In this study,PRRSV was performed on 152 tissue samples and 23 serum samples from all over Henan province,and 17 positive PRRSV samples were detected by RT-PCR.The ORF5 gene of the positive samples was amplified and sequenced.15 different ORF5 gene sequences were obtained.The full-length 603bp of 14 strains encoded 200 amino acids and one full-length 600bp encoded 199 amino acids.Nucleotide sequence alignment reveal that the homology of 15 strains was 81.6%-99.8%.The nucleotide homology is 84.7%-99.8%between the epidemic strains and the American strains represented by VR2332,only 61.4%-64%homology with European strains represented by LV strain.lt can be concluded that all of the 15 epidemic strains belong to the American type.The nucleotide homology was 84.7%-99.8%between the epidemic strains and classical VR2332 strains,the nucleotide homology with HP-PRRSV represented by JXA1 is 83.7%-99.3%,the nucleotide homology with NADC30-like represented by NADC30 is 83.3%-93.9%.The comparison of amino acid differential sites shows that there were significant variations in the core region of the decoy epitope 26aa-32aa and the core region of neutralizing antigen epitope in 15 strains of epidemic strains.After comparing the N-glycosylation sites of the epidemic strains,we found that the number of N-glycosylation sites of the epidemic strains has an increasing trend,and the increased N-glycosylation sites were mostly at 34aa and 35aa,which are closer to the core region of the neutralizing epitopes of GP5 protein.Nsp2 gene encodes the largest non-structural protein Nsp2,Nsp2 protein is the most variable part of all PRRSV proteins.Nsp2 protein plays an important role in virus proliferation and host immune response,the variation of these genes is closely related to the biological characteristics of the virus.In this study,the nsp2 gene of 17 PRRSV positive samples was amplified and sequenced.It was found that 10 different nsp2 gene sequences were obtained.The nucleotide homology of Nsp2 gene between epidemic strains was 59.5%-98.2%.The nucleotide homology between each epidemic strain with the classical strain represented by VR2332 was 61.6%-99.7%,60.7%-77.8%of the nucleotide homology with HP-PRRSV represented by JXA1 strain,the nucleotide holology with NADC30-like represented by NADC30 was 62.9%-90.8%.The comparison of amino acid differential sites shows that the characteristic deletion of NADC30 strain(111+1+19aa)was found in HeNan-AY1?HeNan-AY2?HeNan-BY3?HcNan-JZ23?HeNan-JZ29?HeNan-JZ28?HeNan-RN?HeNan-XX7?HeNan-ZMD epidemic strains.17 samples of PRRSV positive samples were inoculated into Marc-145 cells after aseptic grinding and 0.22nm filter filtration,and virus isolation was carried out by blind passage three generations.The third generation of HcNan-XX10,HeNan-JZ2,HcNan-AY1 showed cytopathic effect.RT-PCR and Q-PCR were used to detect and identify the virus,the results showed that the third generation cells of HeNan-XX10,HeNan-JZ2,HeNan-AY1 samples could detect positive PRRSV nucleic acid,and the viral load was more than 103copies/?L.The third generation cell cultures of the three epidemic strains were inoculated into monolayer PAM cells.CPE were observed caused by HeNan-XX10 epidemic strain after inoculation 12 hours,after inoculation 48 hours,however,CPE caused by HeNan-JZ2,HeNan-AY1 epidemic strain could be observed.