Isolation And Identification Of Porcine Reproductive And Respiratory Syndrome Virus TJ Strain And Sequence Analysis Of Its ORF5 Gene

Porcine reproductive and respiratory syndrome virus( PRRSV)is causative agent for porcine reproductive and respiratory syndrome (PRRS), which is characterized by reproductive failure in sows and gilts, and severe respiratory diseases, including pneumonia in neonatal piglets. Since the disease broke out at the first time in America in 1987, it has spread rapidly all over the world, resulting in great economic losses to the swine industry. Although nucleotide variations exist in each gene of PRRSV genome, the ORF5 gene is one of the most important genes with variations. Therefore, analysis of ORF5 gene could provide basic information of gene variations of PRRSV genome in the most cases.In this study, a porcine reproductive and respiratory syndrome virus (PRRSV) field strain, PRRSV TJ, was isolated from a sick pig with high fever in a pig farm, Tianjin, China. This isolate replicated well in Marc-145 cells and could cause cytopathic effect (CPE) on the cell culture, and the infected cells became rouding, adhered to each other and fell off from monolayer of cells. The virus was identified as a PRRSV strain (named as PRRSV TJ strain) by an indirect immunofluorescent assay (IFA) and identified to be a member of America genotype as analyzed by RT-PCR. The third passage of PRRSV-TJ (in Marc-145 cells) was studied for its virulence in five 25-day-old piglets. All 5 piglets were infected with the virus and 3 of the 5 died from the infection, suggesting that PRRSV TJ strain has a higher virulence.Primers were designed and synthesized according to the ORF5 gene sequence of ATCC VR-2332. Using the primers in RT-PCR, the F3, F51 and F92 ORF5 genes of PRRSV TJ strain were amplified and cDNAs were made., and then the cDNAs were cloned into vector pMD 18-T for sequencing. Comparison of the ORF5 gene of PRRSV TJ-F3 to American and European strains revealed that the homologies are 89.4% and 63.5%, respectively to American VR-2332 strain and European LV strain. Results also demonstrated 88.1%～100% of nucleotide homology of PRRSV TJ to other American strains and 62.5%～63.7% of nucleotide homology to other European strains identified either in China or in other countries. The phylogenetic tree created with the sequences of ORF5 gene illustrated that PRRSV TJ strain located in the branch of American type and it had the closer relationship with HB-1(sh)/2002, CH-1a, HB-2(sh)/2002 and high virulent PRRSV isolates HUB1,HUB2, HEB2,HUN4, NM1, GD,SHH, JXA1, and they belonged to the same subclade. Sequence analysis showed that there were five amino acid mutations, coded by ORF5 gene, among the 3 passages of PRRSV TJ strain. The changes located at 23aa, 80aa, 128aa, 151aa, and 196aa sites. On 151aa site, amino acid mutated from R in TJ-F3 to K in TJ-F51 and F92. There were three transmembrane regions for GP5 in TJ-F3, but only two in TJ-F51 and F92. The mutation on 151aa site and changes of transmembrane region might be responsible for the decrease of pathogenicity of PRRSV TJ strain after passages in cell cultures.