Isolation And Identification Of Porcine Reproductive And Respiratory Syndrome Virus Strains And Sequencing And Analysis In Some Areas Of China From 2020 To 2022

Porcine reproductive and respiratory syndrome(PRRS)is a viral infectious disease caused by Porcine reproductive and respiratory syndrome virus(PRRSV),which has caused significant economic losses to the world’s pig industry.and is one of the most important infectious diseases in pig diseases.At present,the clinical prevalence of PRRSV in China is complex and diverse,with multiple types of virus strains coexisting.Different types of viruses are prone to recombination,and under the influence of infection pressure and external environment,their gene sequences are prone to variation,resulting in high variability and strain diversity of PRRSV.Therefore,analyzing the sequence variation characteristics and genetic evolution of clinical epidemic strains through sequencing and virus isolation can help detect the emergence of new epidemic strains in a timely manner,providing reference basis for clinical diagnosis and the development of new vaccines.The purpose of this study was to conduct molecular detection on disease samples suspected of PRRS infection in some regions of China from 2020 to 2022,and to sequence and analyze the genetic evolution of ORF5,NSP2 genes and the entire genome of representative samples.At the same time,some samples were selected for virus strain isolation and identification.To understand the genetic variation and evolutionary trends of current clinical epidemic strain.1.Molecular detection of PRRSV and sequencing analysis of ORF5 and NSP2 genes in clinical samplesIn this study,a total of 186 PRRSV positive samples were detected by RT-PCR in 1143 samples,with a positive rate of 16.3%.18 representative samples were selected according to the disease situation of the pig farm from which the samples came and the collection site.PRRSV typing primers were used for typing.At the same time,gene sequences of NSP2 and ORF5 were amplified.18 gene sequences of NSP2 and ORF5 were obtained by sequencing.Typing results showed that 15 of the 18 positive samples were NADC30-like and 3 samples were HP-PRRSV.The results of amino acid analysis showed that the NSP2 gene of 15 NADC30 samples had 131 discontinuous deletion of amino acids,which had the same deletion pattern as NADC30 samples,and 4 samples had 1-5 additional deletion of amino acids.The NSP2 gene of 3 HP-PRRSV samples showed 30 discontinuous deletion patterns,which was the same as that of HP-PRRSV without insertion or deletion of additional amino acids.The results of genetic evolution based on ORF5 gene showed that 5NADC30 samples were located in Sublineage1.8,8 NADC30 samples were located in Sublineage1.5,and 2 NADC30 samples were located in Lineage3.3 HP-PRRSV samples were located in the Lineage8 branch.In conclusion,the current clinical circulating strains of PRRSV showed diversification,and the sequence difference among different strains was large.Therefore,continuous monitoring of PRRSV prevalence and variation were conducive to timely discovery the situation of new PRRSV.2.Complete gene sequencing and recombination analysis of PRRSVDue to the differences in the genetic evolution of ORF5 and NSP2 genes in 18 positive samples,In order to further explore the genetic evolutionary relationship between these sequences,the whole gene sequence amplification sequencing was performed on 18 samples.Finally,13 whole gene sequences were amplified from 15NADC30 samples and 2 whole gene sequences were amplified from 3 HP-PRRSV samples.The results of 2 HP-PRRSV gene sequences were consistent with those of NSP2 and ORF5 gene sequences.Among the 13 NADC30 gene sequences,the results of 4 NADC30 gene sequences were consistent with the results of NSP2 gene,and the results of the other 9 NADC30 gene sequences were consistent with the results of NSP2 gene,but not consistent with the results of ORF5 gene.The results of recombination analysis showed that 12 of the 15 whole gene sequences were recombined,and all the 12 recombinant samples were NADC30-like.The recombination regions mostly occurred in the non-structural proteins and structural proteins ORF2a~ORF6.The 12 recombinant sequences all took NADC30-like strains as the main parent.This study provides reliable data support for the subsequent studies on the pathogenicity and molecular characteristics of the recombinant strains.3.Isolation and identification of PRRSVSix clinical samples were selected from 18 positive samples and inoculated with PAM cells for virus isolation.Among them,5 samples showed cytopathic changes such as shrinkage,aggregation,and rupture after 3 days of inoculation with PAM cells.Cell cultures were harvested for continuous passage.RT-PCR was performed on P3 cell culture using 436-F/436-R identification primers.The results showed that 5samples could be amplified with specific target bands.Then,specific green fluorescence was observed by IFA identification using N protein monoclonal antibody.