Isolation And Identification Of Xinxiang2015 Strain PRRSV And Cloning And Analysis Of Nsp2 And GP5 Genes

Porcine reproductive and respiratory syndrome(PRRS)is a reproductive disorder and respiratory infectious disease caused by porcine reproductive and respiratory syndrome virus(PRRSV).It is characterized by anorexia,fever,miscarriages in the later stages of pregnancy,stillbirths,mummy fetuses,respiratory illness or death in young pigs.The disease was first detected in the Midwestern United States in 1987,and later found in North American,German,French,Dutch and other European countries and Asian countries.From the beginning,because of not understanding the cause,some countries in Europe named the disease as "pig mysterious disease",and due to the purpleness of the ears of some sick pigs,it was also called "swine blue ear disease",and it also called as "Pig infertility and respiratory syndrome" and "epidemic abortion and respiratory syndrome".PRRSV was first isolated by Guo Baoqing and others in China in 1996.PRRSV now exists widely in China with a severe epidemic situation.In order to understand the prevalence and evolution of PRRSV in Xinxiang city,this laboratory collected the suspected PRRSV materials sent from a pig farm in the area.The PRRSV nucleic acid identification was performed on the submitted disease materials,and the positive samples were selected for virus isolation and identification.The results showed that there was a nucleic acid positive rate of 80% in the suspected samples,and one PRRSV strain was successfully isolated from one of the positive samples.The virus was found to bind to PRRSV specific N protein antibodies by indirect immunoassay(IFA).The isolated PRRSV representative strain provides molecular evidence for the further study of the molecular evolution of porcine reproductive and respiratory syndrome virus in Xinxiang.In order to further study the sequence characteristics of Xinxiang2015 strain of PRRSV,this study uses molecular biology technology to extract the total RNA after virus infected cells,and then reverse transcription,PCR amplification,target gene recovery,T vector ligation,transformation E.coli,picking the positive clone,sequence sequencing and sequence analysis have cloned the full-length sequences of the non-structural protein NSP2 and structural protein GP5 protein of PRRSV of Xinxiang2015 strain,respectively.The experimental results of NSP2 proteinshowed that the Nsp2 gene of PRRSV of Xinxiang2015 strain was cloned in this experiment.The gene was 3483 bp in length.Nucleotide homology with Nsp2 gene in VR-2332 strain,Ch-1a strain,HuN4 strain,and NHNAN-XINX strain were 80.5%,96.0%,96.2%,and 67.8%,respectively.Compared with the classic PRRSV and highly pathogenic PRRSV,there are 3 consecutive discontinuous deletions of amino acids in the sequence,that is,in addition to 2 discontinuous deletions with high pathogenicity,there are also 4 amino acid deletions at positions 485 to 488;The experimental results of GP5 protein show that the Gp5 protein gene is 603 bp in length.Compared with the GP5 gene in VR-2332 strain,Lelystad virus strain,Ch-1a strain,HuN4 strain and HENAN-XINX strain,the homology was 89.4%,49.6%,94.9%,99.3%,and 85.7%,respectively.The phylogenetic tree analysis results of NSP2 protein and Gp5 protein showed that Xinxiang2015 strain PRRSV belonged to highly pathogenic PRRSV(HP-PRRSV).The results of this experiment further confirmed that the PRRSV prevalent in recent years around Henan Province was highly pathogenic PRRSV and provided evidence for understanding the evolutionary study of porcine reproductive and respiratory syndrome virus nationwide.