PCR Detection Of Porcine Reproductive And Repriratory Syndrome Virus And Full-genome Sequencing And Analysis Of Some Positive Samples

Porcine Reproductive and Respiratory Syndrome(PRRS)is caused by Porcine Reproductive and Respiratory Syndrome Virus(PRRSV)which lead to reproductive failure in sows.A disease characterized by miscarriage,stillbirth,mummification and respiratory diseases in piglets.PRRSV is a non-segmented single-stranded positive-strand RNA virus.Due to the lack of proofreading function of RNA-dependent RNA polymerase,the gene is prone to deletion,insertion and mutation during replication,in turn,causes PRRSV to have high variability and strain diversity.In this study,molecular detection and identification techniques were used to investigate the prevalence of PRRSV in Henan and surrounding areas,and genetic evolution and recombinant analysis were performed to provide references for clinical diagnosis and effective prevention and control of PRRS.1.The molecular epidemiological survey of PRRSV in Henan and surrounding areas in 2017 and 2018 was carried out,and the NSP2 sequence was analyzed for genetic evolutionIn this experiment,241 clinical samples were tested by RT-PCR,and 121 of them were positive for PRRSV.Of the 121 positive samples,92 were NADC30-Like PRRSV,24 HP-PRRSV,3 classic PRRSV,1 European PRRSV and 1 HP-PRRSV with NADC30-Like Co-infection.Representative clinical samples were selected for NSP2 sequence determination,and 23 NSP2 sequences were obtained.Genetic and evolutionary analysis of 23 NSP2 sequences showed that there were 131 amino acid discontinuous deletions in the NSP2 region of 10 strains,which were highly consistent with the sequences of NADC30 strains.There were 10 strains with discontinuous deletion of 30 amino acids in the NSP2 region,which was highly consistent with the sequence of JXA1 strain.Two strains of NSP2 sequence have higher consistency with CH-la strain.Another strain of NSP2 has a high consistency with the Lelystad virus strain and has a continuous deletion of 54 amino acids in the NSP2 region.The NSP2 gene of PRRSV strain has a large degree of variation,and various genotypes co-exist in the pig population,which deepens the complexity of PRRSV epidemic in the field.Therefore,it is necessary to continuously monitor the prevalence and variation of PRRSV,so as to provide reference for the clinical diagnosis and effective prevention and control of PRRSV2.Whole gene sequencing and recombinant analysis of PRRSV NADC30 strainIn order to further understand the genetic variation of the NADC30 strain in Henan province,the whole gene sequences of some detected strains were determined,and a total of 10 NADC30 strains were obtained.The results of homology analysis showed that the homology of 10 strains with VR2332 was 84.8-86.8%,that with JXA1 was 83.4-88.5%,and that with NADC30 was 90.1-94.8%.Genetic and evolutionary analysis showed that 10 strains were located in the NADC30-like subgroup represented by NADC30,and the branches of 10 strains in the genetic and evolutionary tree were closer to HNjz15 and NADC30.Recombinant analysis showed that 4 of the 10 strains were not recombinant.Six strains were recombinant with HP-PRRSV,5 of which were recombined at nonstructural proteins and the other with recombination at both nonstructural and structural proteins.This study provides a reference for further analysis of variation and recombination of NADC30-Like strains in China.3.Isolation and identification of three NADC30-Like strainsIn this experiment,three clinical samples of NADC30-Like were inoculated with abrasive solution into PAM cells for virus isolation,and the cell cultures were identified by RT-PCR,and 3 strains of viruses were successfully isolated.