Isolation And Identification Of The Porcine Parvovirus And Cloning And Sequencing Analysis Of NS1 Gene,vp2 Gene

porcine parvovirus is one of the major pathogens that caused Reproductive failure. Progravid phase swine, which is infected with PPV, will suffer embryonic resorption, fetal mummification, abortion or stillbirths, that means great loss to pig industry. In this study, we survey randomly scattered pig farm in Taian area, to know the PPV infection and epidemic status; then the clinic swine sample is used to isolate and identify PPV in order to analysis the reason of PPV and study the etiology feature of PPV; the PPV isolated is Cloned and sequenced and the molecular biology character of PPV is also studied. This study provide useful knowledge for the study of PPV.The latex agglutination test to 100 scattered pig serum in Taian area show 34 positive samples (seropositivity rate 34%), of which sows, gilts and boars are 18(36%) , 9(30%) and 7(35%) respectively. The result indicated the infection rate of PPV is high in this area.20 clinic swine samples taken from clinic swine (heart, liver, kindey, lung and brain) are isolated and identified use PK-15 cell. There are 4 that produce pathological change in PK-15 cell. Take physico-chemical property identify, akaryocyte HI spectrum experiment, specificity experiment and PCR identify. The isolated virus is insensitivity to ether, chloroform, acid and trypsogen, good at heat resistance. Good at agglutinating erythrocyte of cavia cobaya, cat, rat, mouse and chicken, bad at agglutinating erythrocyte of cow and swine. 8 unit anti-PPV antibody can lower the HA titer of the virus to be tested above 8 times. It indicated the antibody had specific depressant effect to HA of the isolated virus. PCR amplificated a 1740bp fragment, the above results altogether indicated that the virus isloated is PPV.The PPV isolated from clinic swine samples is named as PPV-TA. The NS1gene and VP2 gene of PPV-TA are amplified by PCR using special primers for NS1 and VP2 designed according to PPV sequences in Gene Bank. As a result, the NS1 gene of PPV-TA include 1989 bp and encodes 662 amino acids, the nucleotide sequence identity is from 98.3% to 99.9% compared with PPV NS1 genes of the reference strains. PPV-TA NS1 protein has three glycosylation sites, which are 356NIS,446NFS,513NLT, the phosphorylation sites of NS1 protein of PPV-TA are Thr435 and Ser473, which are identical to the reference PPV strains. The VP2 gene of PPV-TA includes 1740 bp and encodes 579 amino acids, and the VP2 genes identity is from 98. 3 % to 99.8% between PPV-TA and the reference PPV strains. The 378D, 383H, 436S of VP2 protein of PPV-TA play a role on the tissue tropism, which are similar to PPV NADL-2 and PPV-china strains. PPV-TA VP2 protein has 9 linearity antigen sites, which are 21-GNESGGGGGGGGG-33,168-DLTASLMVALDTNNT-182,239-HSDIMFYTIENA VPIHLLRTGD-260,310-ANTRKGYHQTINNSYT-325,326-EATAIRPAQVGYNTPY M-342,367-DEPNGAIRFTMDYQHGH-383,376-TMDYQHGHLTTSS-388,385-TTS SQELERYTFNPQ-399 , 437-NMHFMNTL NTYGPLTAL-453 , same to the PPV NADL-2 and PPV china strains. The NS1 gene and VP2 gene of PPV-TA are comparatively conservative with some changes. That indicated bionomics of different strain are different. The study enrich the molecular epidemiology and etiology data of PPV.