Isolation And Identification Of PRRSV Shaanxi-1 Strain And Genomic Sequecing And Phylogenetic Analysis Of The Virus

Porcine reproductive and respiratory syndrome virus (PRRSV), a member of the Arteriviridae, is widespread among the swine population, causing reproductive failure in sows and respiratory disorders in pigs of all ages, and has all economic impact on the pig industry worldwide. An emerged highly pathogenic porcine reproductive and respiratory syndrome virus(HP-PRRSV), occurred in the pigs in main swine industry provinces of mainland China in 2006, which made a big loss in the raise of pigs. The sequence and antigenic analysis of the PRRSV isolated in recent years, and indicates that there have many disparities by analyzing the virulence, gene sequence and antigenicity between isolated strains.In this study, clinical samples were collected from pigs suspected of having PRRS from Huaxian farm of Shaanxi Province. Isolation and identification of the virus prove to Isolate PRRSV successfully. In order to research the genetic variation and molecular characteristics of the virus, a whole gene was sequenced and blasted with the other reported strains. The main research are as follows:l.Isolation and identication of PRRSVThe samples that were collected from pigs suspected of having PRRS from Huaxian farm of Shaanxi Province. The virus isolates were propagated and plaque-purified in Marc-145 cells, and the virus propagation in Marc-145 cell were determined by IFA and RT-PCR. The results show that Marc-145 cells inoculated virus can see specific green fluorescent, and amplified fragments with the expected size of Nsp2 Mutant fragments consistent with the positive control amplification of JXA1. The results suggested that the isolate was obtained, which was designated as PRRSV Shaanxi-1.2.Cloning and sequencing of PRRSV genomeRNA was extracted from the PRRSV obtained by subculturing the virus on Marc-145 cells (3th-passage). RT-PCR was amplified using primers based on VR-2332 and HB-1(sh)/2002, and sub-sequences of whole PRRSV Shaanxi-1 genome was cloned respectively. The products of RT-PCR were inserted into pMD18-T vector, then transformed into DH5αidentified by bacteria PCR and double digestion finally sent to Nanjing Jinsite biotechnology Incorporated Company for sequencing, which using Blast and DNAStar 5.0 software for analysis. The results show that the Shaanxi-1 gene is 15323bp (not including the poly (A) tail), Shaanxi-1, JXA1, HUN4, jiangxi-3 and other HP-PRRSV strains have the identical fragment length, but have some differences from the former Chinese strains( CH-la, HB-l (sh) / 2002 ), mainly in the 5 UTR, ORF1a and 3 UTR regions.3.Genetic characteristics analysis of PRRSV genomeAccording to the overlaps of the whole genome segments, the sequence was assembled by DNAStar. The whole sequence was compared with the strains published in GenBank, meanwhile, the genetic variation and molecular characteristics of Shaanxi-1 were analyzed. The results showed as follows: Shaanxi-1 was related to the North American PRRSV genotype. Shaanxi-1 shared above 98.0% identity with the structural protein genes of strains isolated in China after 2006. American-type strains based on ORF5 sequence can be initially divided into two subgroups. Shaanxi-1 and Shaanxi-2 (wild boar isolate) have close genetic distance with JXA1, Henan-1, HuN4, HUB2, and CH-la, belonging to the same subgroup.The molecular features of Shaanxi-1 were consistent with the HP-PRRSV: loss of base "A" at position 120 in 5'UTR, missed base "G" at position 19 in 3'UTR. In Nsp2, the deletion of"L"were observed at position 481 and sequencial 29 amino acids were lost at position 532⁵60. Shaanxi-1 shared a high identity in virulence site which contained 9 amino acids with HP-PRRSV, which obviously different from the vaccine strains and traditional attenuated strains, however, the positions of Shaanxi-1 ORF2 S²³,ORF3 L¹⁴³S²⁴⁸,ORF4 I⁶⁶P⁶⁷ and Shaanxi-2 Nsp2 G¹⁹¹,ORF2 F⁵⁰,ORF3 A²²⁵,ORF4 G⁴³ I66,ORF7 R₄₈S₄₉ were same as vaccine strains and traditional attenuated strains.The genome of one isolate of Porcine respiratory and reproduc-tive syndrome virus (PRRSV) from Huaxian farm, designated Shaanxi-1, was sequenced and analyzed. Comparative analysis with the genomic sequences of Shaanxi-2 (wild boar isolate) and other typical strains. Combine with the pathogenicity tests on piglets of Shaanxi-1 and Shaanxi-2, Meanwhile, preliminary understanding of the relationship between the pathogenic features of Shaanxi-1 and Shaanxi-2 mutants and the occurrence of high swine fever. which made us further understanding the source, genetic background and evolutionary direction of PRRSV in Shaanxi Province, and enriched PRRSV gene bank and layed a good foundation for depth research of PRRSV gene function, reveal molecular mechanism of PRRSV pathogenesis, develop PRRSV DNA vaccine, and ultimately, effective prevent and control of PRRS.