Isolation, Identiifcation, Genetic Mutation And Pathogenicity Analysis Of Highly Pathogenic Porcine Reproductive And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome (PRRS), caused by porcine reproductive andrespiratory syndrome virus (PRRSV), is one of the most important infectious diseases of pigsthroughout the world, and it is characterized by reproductive failure of gestating sows and gilts andsevere respiratory disorders in piglets. An emerged highly pathogenic porcine reproductive andrespiratory syndrome virus (HP-PRRSV), which occurred in the pigs in main swine industry provincesof mainland China in2006, caused a tremendous economic loss to the swine industry. The present studydescribes our research works on the HP-PRRSV, including field virus isolation, molecular geneticevaluations and pathogenicity of HP-PRRSV.The samples were collected from sick pigs suspected of having PRRS from some northernprovinces in China. The virus isolates were propagated in Marc-145cells and the virus propagation inMarc-145cell were determined by indirect fluorescent assay (IFA), serum neutralization (SN) assay andRT-PCR. The results revealed that six strains of PRRSV were isolated and grew well in Marc-145cellculture, and these isolates were genetically similar to American type PRRSV. Those six isolates weredesignated as PRRSV TJ, NM1, HLJ1, JL1, JL2and LN strains. The results of pathogenicity of TJstrain demonstrated that the TJ strain is a highly pathogenic for piglets. And the study established achallenge model HP-PRRSV TJ train to60-day-old conventional pigs.RT-PCR was amplified using primers based on VR-2332, and sub-sequence of whole PRRSVgenome was sequenced respectively. The results showed that the six PRRSV strains TJ, NM1, HLJ1,JL1, JL2and LN all had15320bp (not including the poly (A) tail) in their genomes, and had theidentical fragment length with JXA1, HUN4and other HP-PRRSV strains. The complete sequence ofthe six viruses were compared with the strains published in GenBank, results demonstrated that thesesix viruses were related to the North American PRRSV genotype and shared above98.0%identity withHP-PRRSV strains isolated in China after2006, and in nsp2region, the discontinuous30-aa deletionwas observed at position481and533⁵61.HP-PRRS virus TJ strain was attenuated by serial passages and plaque clone every5to10passages in Marc-145cells. The pathogenicity of HP-PRRSV TJ strains in the course of attenuationwas analyzed. It was found that the virulence of the TJ strain to piglets was decreased greatly afterpassage19. Complete genome analysis of different passages of HP-PRRSV TJ strain (F19, F46, F78and F92) showed that a continuous120amino acids deletion and48amino acids mutations occurred inF92, among these48amino acid mutations,31(64.6%),7(14.6%),7(14.6%) and3(6.3%) occurredin F19, F46, F78and F92, respectively. The120amino acid deletion occurred from F19to F92.Therefore, we hypothesized that the31amino acid mutations distributed in different regions of nsp1β,nsp2-nsp5, nsp7, nsp9, nsp10, GP4and GP5, and the continuous120amino acid deletion in the nsp2region from passage19(F19) provide a strong potential molecular basis for the observed attenuatedphenotype.Attenuated PRRSV TJ strain F92(passage92) was identified as PRRSV TJM strain, and it wasinoculated into the pigs for five passages to further test its stability and safety. No significant changes were observed in the pigs at the5^th passage (P5), without high body temperature or obvious clinicalsymptoms, no grossly pathology and histopathology were found in the lung, however the level ofviremia slightly increased. The results showed that the capacity for TJM virus to replicate in pigs ofenhanced as the passages went on. Sequence analysis of P5virus isolated from the passage5pigsshowed that the continuous120amino acids deletion of TJM strain still existed, suggesting the stablecharacteristic of the deletion in vivo. Compared to TJM strain, there were21aa mutations in the20proteins encoded by PRRSV in P5, and14of them were revertant. Therefore, we speculated that the14aa revertants located in non-structural protein in nsp2,nsp3and nsp9, and structure protein GP3, GP4and GP5, may be related with the replication level of the virus in pigs.