Isolation And Identification Of PRRSV Shaanxi-2 Strain Isolated From Wild Boar And Genomic Sequecing And Phylogenetic Analysis Of The Virus

Porcine reproductive and respiratory syndrome (PRRS), caused by porcine reproductive and respiratory syndrome virus (PRRSV), is characterized by reproductive failure in breeding animals and respiratory distress in all ages of pigs. PRRSV is a member of the family Arteriviridae in the order Nidovirales, the genome is approximately 15 kb in length and contains nine open reading frames (ORF).PRRSV is widely spreading in China since isolation in 1996. In April 2006, a highly pathogenic disease, caused by unknown agents and characterized by high fever and a high proportion of deaths in pigs of all ages, first emerged in some farms in Jiangxi Province of China. In the next several months, the disease spread rapidly to most provinces of China and resulted in death of millions of pigs and a significant economic loss of the swine industry of China. A highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) was found to be the main pathogen caused the disease named"swine highly fever disease". In the early winter of 2006, an unknown disease, of which the symptoms and epidemiological characteristics were similar to"swine highly fever disease"and induced a great large number of pigs death in Shaanxi Province. The PRRSV in this study was isolated from an infected wild boar of the Xi'an Wildlife Zoo when a epidemiological investigation of"swine highly fever disease"was raised in Shaanxi. 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:1. Samples collected of the lungs and lymph nodes were treated by grind and inoculated to Marc-145 cells. The CPE was observed in passage 2. The PRRSV was identified by antibody neutralization assay, RT-PCR, indirect immunofluorescence assay (IFA) and then named Shaanxi-2.2. RNA was extracted from the PRRSV obtained by passaging the virus on Marc-145 cells (5th-passage).RT-PCR was performed using primers based on VR-2332 and HB-1(sh)/2002 and sub-sequences of whole PRRSV Shanxi-2 genome was cloned respectively.The products of RT-PCR were inserted into pMD18-T vector then transformed into DH5αand identified by bacteria PCR and double digestion finally sent to Beijing AuGCT biotechnology co. for sequencing. According to the overlaps of the segments whole genome was assembly, multi-sequence alignment was carried out using DNAStar. The sequence was compared with the strains published in GenBank, meanwhile, the genetic variation and molecular characteristics of Shaanxi-2 were analyzed. The results showed as follows: Shaanxi-2 had an identity of 60.7% with the Europe strain LV4.2.1, 89.2% to 94.9% with the American-type strains BJ-4, CH-la, CH-1R isolated in China before 2006, and above 98.8% with the HP-PRRSV strains isolated in China after 2006. In all of non-structural proteins, Nsp2 had the largest variation, which had a identity of 76.5% with BJ-4 and 98.3% with JXA1 at the amino acid level. Compared with the structural protein genes of strains isolated before 2006, ORF3 and ORF5 got the largest variation comparatively, ORF6 and ORF7 showed more conservative. But compared with HP-PRRSV strains isolated after 2006, ORF7 had the largest variation comparatively, ORF3~ORF6 are more conservative. The molecular features of Shaanxi-2 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 posiion 481 and sequencial 29 amino acids were lost at position 533~561. Shaanxi-2 keeped 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, but unlike HP-PRRSV, the positions of Nsp2 G191,ORF2 S23, ORF3 A225, ORF4 G43, ORF5 R164 and ORF7 R48S49 were same as vaccine strains and traditional attenuated strains. PRRSV Shaanxi-2 from wild boar have no significant differences with domestic popular strains which indicated that virus in Shaanxi and other areas have same origin and we could assume that Wild boar as a host did not make obvious variation of the PRRSV.After phylogenetic analysis of Nsp2,ORF5 and complete genome of Shaanxi-2, a conclusioncan be made that Shaanxi-2 is a branch of HP-PRRSV, however, Some mutations made it possess new features. This standpoint can be confirmed by animal regression experiment we made that Shaanxi-2 could cause disease but did not make the pigs die, that is different from HP-PRRSV.PRRSV Shaanxi-2 strains derived from wild boar in Shaanxi Province was multiple aligned and analyzed in this study,which made us preliminary understanding the source, genetic background and evolutionary direction of PRRSV in Shaanxi Province and provided valuable reference to PRRSV immunization in Shaanxi. Meanwhile, this study 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.