Variation Analysis Of NSP2 And ORF5 Genes Of PRRSV Strain In XinJiang

we collected some viscera and blood serum from clinical syndrome piglets in Xinjiang hogpens, and made pathological section; and molecular clinical diagnosis of RT-PCR test kit was used for detecting variation of Porcine Reproductive and Respiratory Syndrome Virus(Nsp2,1594～1680) from viscera of piglets, and used ELISA test kit to check valence antibody of PRRSV with serum, the result were both positive.The positive sample inoculated into Marc-145 cell, after three blind passages, the results showed that the virus could induce typical Cytopathogenic Effect (CPE) on Marc-145 line, PRRSV was positive in the cells and conformed by using RT-PCR test kit, RT-PCR method showed that 3 strains of PRRSV had been successfully isolated; and named XJ-a, XJ-b, XJ-c respectively; and on fourth passage, its infection titer was 10-5.33-10-5.88TCID50/0.1ml, and the detection of IFA was positive.According to the two sequences of NSP2(1062bp)and ORF5(603bp) of ATCC-VR2332 North American type PRRSV published in GeneBank, designed and synthesized special primers of P1/P2 and P5/P6 to build RT-PCR method for amplifing gene of NSP2 and ORF5, about 1062bp and 603bp cDNA fragment of 3 isolates were amplified by RT-PCR and sequenced after being directly cloned into the pUCm-T vector; and transformed into DH5a, and cultured again, choosed the positive strain for identification by PCR method and digested by EcoR I and Xho I.Finally, the sequences of NSP2and ORF5 genes of 3 isolates were compared with the sequences of other domestic and foreign PRRSV strains has published in Genbank by using DNAStar software, and the length of Nsp2 and ORF5 sequence was 1062bp and 603 bp respectively, the results indicated 3 isolates relationship are near with American type of VR2332 PRRSV, deducted amino acid sequence of NSP2 homology was above 77.6%～78.7% with VR2332, it had only 42.5% and 43.8% homologies with those of Europen LV isolates, sequence homology between 3 isolates and domestic American type was 77.2%～98.6%; and deducted amino acid sequence of ORF5 homology was above 86.6%～87.2% with VR2332, it had only 56.1% and 56.4% homologies with those of Europen LV isolates, sequence homology between 3 isolates and domestic American type was 85.7%～99.2%, especially with Jiangxi, Shandong, Henan, Hunan strains, their relationship are quite near, deducted amino acid of NSP2 and ORF5 sequence homology are both above 96%, and the antigenicity of their is extremely similar. It is concluded that strains of Xinjiang is genetically closer to strains isolated from North American than those from Europen, and there had being happened some variance between Xinjiang strain and other domestic American type.