Isolation And Identification Of NADC30-like Porcine Reproductive And Respiratory Syndrome Virus Strains And Sequencing And Analysis Of Whole Genomes

This study was conducted to isolate the NADC30-like virus strains and sequence the whole genome of them,so that we could understand the molecular characteristics and genetic evolution of the virus strains and conduct a further study on them.At the same time,detecting the genovariation of ORF5 and nsp2 genes in PRRSV strains which were the epidemic strains in Henan and surrounding areas,to reveal the latest development trend of PRRSV strains,and provide a scientific evidence for clinical diagnosis and the development of new vaccines and antiviral drugs.?.Establish the PCR methods of PRRSV typing virus strainsIn order to detect NADC30-like strains more quickly and accurately,this experiment was based on the molecular characteristics of the deletion of 131 amino acids and the no-deletion of 30 amino acids in the nsp2 region of the PRRSV NADC30 strain,the HP-PRRSV strain and the classical PRRSV strain,to design a pair of primers and establish a PCR method to classify these three types of strains.This experiment completed the establishment of the RT-PCR method,and verified the sensitivity and specificity of it,and tested the clinical samples.The results of the experiment showed that this method can distinguish the classical PRRSV strains,HP-PRRSV strains and NADC30 strains at the same time with good sensitivity and specificity.?.Molecular detection of PRRSV in Henan province and its surrounding areas during 2016 to 2017,and the analysis of mutations in ORF5 and nsp2 genesIn this study,756 clinical samples were detected by RT-PCR method while 139 positive samples were detected with a positive rate of 18.4%,and 42 nsp2 sequences and 50 GP5 sequences were amplified.Genetic evolution showed that the predominant strains in Henan were mainly American type strains,20 strains are highly homologous to the NADC30 strains and there is a discontinuous deletion of 131 amino acids in the nsp2 region.The sequences of the remaining 22 strains are highly homologous to HP-PRRSV strains,with a discontinuous deletion of 30 aminoacid in nsp2 region.Compared to 2016,the proportion of NADC30-like strains in Henan and surrounding areas had increased dramatically in 2017,and GP5 and nsp2 of the PRRSV epidemic strains had a significant variation,so it is necessary to continuously detect the prevalence and variation of PRRSV and provide a scientific evidence for clinical diagnosis and disease control and prevention.?.Isolation and identification of 20 samples of NADC30-like strainsAfter the virus isolation of the samples which were detected as NADC30-like strains inoculate with PAM cells,20 NADC30 strains were obtained.The RT-PCR results showed that the isolated strains we obtained could amplify the target bands,and the IPMA identification results showed that all the samples were positive for PRRSV.?.Sequencing and analysis of the whole gene of NADC30-like strains of PRRSVIn this study,the whole gene sequences of 20 NADC30-like strains were obtained,and their genetic evolution analysis and homology analysis were performed.The results showed that the homology of whole genome nucleotides between 20 strains was 86.8%-93.8%,the homology with VR2332 was 85.1%-88.2%,and the homology with CH-1a was 84.2%-90.8%.The homology with NADC30 is 88.3%-96.7%,and the homology with JXA1 is 83.5%-93.0%.The genetic evolution analysis showed that 19 strains belong to subtype 5 and 1 strain belongs to subtype 4.The results of recombination analysis of 20 strains showed that 19 strains were recombined,1 strain was not recombined while the recombination sites mostly occurred in non-structural proteins and the recombinant strain was mainly HP-PRRSV strains.This study provides a reference for further study on the mutation and genetic evolution of NADC30-like strains in China.