Genetic Analysis Of Structural Protein Gene Sequences Of Porcine Reproductive And Respiratory Syndrome Virus In Some Provinces In China

Porcine Reproductive and Respiratory Syndrome (PRRS) is a infectious swine disease caused by Porcine Reproductive and Respiratory Syndrome Virus(PRRSV),it was characterized by breeding failure of sows and respiratory disease of piglets.Highly Pathogenic Porcine Reproductive and Respiratory Syndrome(HP-PRRS)is a high deadly infectious swine disease which was triggered by mutative PRRSV. HPRRSV has a strong pathogenic ability, it can spread quickly in pig farms and lead to high mortality and mortality of pigs.This study isolated some PRRSVs and established a detective method for PRRSV detection.We also analyzed the homology and evolutionary characterizations of PRRSV structural protein genes.A pair of primers was designed to detect PRRSV based on the ORF2-7 genes of JXA1 strian which is available on GenBank.The length of the expected fragment was 690bp. An accurate RT-PCR method proper for large scale PRRSV detection was established after many times improvement according to the specificity assay, sensibility assay, repeat assay and criterion assay. 200 samples from 10 provinces of Shandong, Fujian, Guangxi, Yunnan, Hennan, Hubei, Hunan, Liaoning, Shanxi and Zhejiang were detected by the established RT-PCR method.And 89 samples were positive.20 PRRSV-suspected samples detected positive by the established RT-PCR method in Chapter 2 from Shandong, Fujian, Guangxi, Yunnan, Hennan, Hubei, Hunan, Liaoning, Shanxi and Zhejiang were incubated on Marc-145 cells after proper treatment.The PRRSV specific CPE appeared in the 2nd passage on Marc-145 cells. TCID50 of PRRSV strains of 08SDCXA and 09HUB5 were 106.50 and 107.714 respectively.The viron particles were observed by TSE (transmission electron microscope) after being separated according nonlinear sucrose density gradient centrifugation and negative staining. 50-60nm PRRSV-like round viron particles were seen with envelope between the sucrose range in the density of 35% to 45% , Which proved it were PRRSV particles.20 PRRSV strains were isolated from positive samples of 6 provinces obtained during 2007-2009 and proved by etiology identification in chapter 3. The ORF2-7 genes sequences were determined with new designed primers. The nucleotide and amino acid sequences were analyzed by DNAStar software. The analysis result showed the nucleotide identity of each ORF among the 20 PRRSV strains followed as below:ORF2(96.2%-99.7%),ORF3(97.0%-99.7%), ORF4(96.3%- 99.6%), ORF5(91.9%-99.5%), ORF6(96.4%-100%), ORF7(99.6%-100%). The aa identity of each ORF among 20 strains followed as: ORF2(94.9%-99.6%), ORF3(95.7%-99.2%), ORF4(94.4%-99.4%), ORF5(89.6%-99.0%), ORF6(96.0%-99.4%), ORF7(97.6%-99.2%). The 20 obtained ORF2-7 genes were compared with 10 domestic PRRSV strains of CH-1a, BJ-4, JXA1, HB-1, HB-2, HUN4, CC-1, GD2007, YN2008, SX2009 and 10 foreign PRRSV strains of VR-2332,LV, MLV, NVSL97-7895,PA8,LMY,EDRD-1,SP,07BJ,01CB1.The nucleotide identity followed as: ORF2(56.4%-99.9%), ORF3(55.3% -99.9%), ORF4(56.1%-100%), ORF5(52.7%-99.8%), ORF6(62.5%-100%),ORF7(57.3%-100%). The aa identity followed as:ORF2(57.2%-99.6%),ORF3(52.9%-99.6%), ORF4(64.2%-99.4%),ORF5(54.2%-99.5%), ORF6(75.9%-99.4%), ORF7 (56.5%-99.2%). The analysis of 20 structural protein genes suggested the PRRSV strains isolated from April 2007 to December 2009 had high homology with no relationship with time and space.The 20 PRRSV strains also had extreme high homology with JXA1 strain and remarkable mutations extent compared with VR-2332 strain.This suggested HP-PRRSVs were the dominant PRRSV strains in China recently.