Isolation, Identification And Genetic Variation Analysis Of Cell Passage Of Highly Pathogenic PRRSV WUH3 Strain

Porcine reproductive and respiratory syndrome (PRRS) is a serious infectious disease of swine, caused by porcine reproductive and respiratory syndrome virus (PRRSV). It is characterized by severe reproductive failure in sows, and respiratory distress in growing pigs and piglets. In 1987, it was first reported in the south of United States, PRRS has already been one of the most economically important diseases of the global swine industry.The "high fever syndrome" occurred in the pigs in main pig-producing provinces in China in 2006, which characterized by high and continuous fever, and high-mortality. There are many research confirmed that the highly pathogenic PRRSV mutants are the major pathogens of the "high fever syndrome". In this study, in view of the new PRRS, virus were isolated and identified, PRRSV WUH3 strain was passaged to passage 75 (P75) in Marc-145 cell. Different passage cell viruses (parental strain, P25, P50, P75) were used to whole-genome sequence determination. Using molecular biology software, the bioinformatics of different passage cell virus genomes were analysis. The phylogenetics was analyzed between the parental strain with different passage cell viruses. It can provide relative data for relationship between amino acid variation and virulence of highly pathogenic PRRSV WUH3 strain, It also can provide support of theory and technique to design new PRRSV live-attenuated vaccine. The main research works were as following:1. Isolation and identification of the highly pathogenic PRRSVPRRSV were isolated from the clinical samples of pigs on the Marc-145 cells during the "high fever syndrome". It showed that the viruses isolated in this study were PRRSV by identification of RT-PCR and IFA. It was named PRRSV WUH3.2. The highly pathogenic PRRSV WUH3 was passaged in vitroThe highly pathogenic PRRSV WUH3 strain was passaged in Marc-145 cell to passage 75 (P75). Focal lesion started to appear after 3 day and 70% CPE was appeared after 5 day. With the increase of the number of passage, cell lesions were appeared shorter and shorter. Time of cell lesion reduced from 3 days in p5 to 24h in p75, time of 70% CPE reduced from 5 days in p5 to 24h in p75. It showed a increase tendency on TCID50. Virus quantity increased. It showed WUH3 strain have adapted cell through continuous cultivation in cell.3. Complete genome analysis of the highly pathogenic PRRSV WUH3 Different passage cell viruses (parental strain, P25, P50, P75) were used to whole-genome sequence determination. The results indicated PRRSV WUH3 strain and the passage cell viruses have similar genetic markers with highly pathogenic PRRSV which is prevalent in China. These genetic markers are a discontinuous deletion of 30 amino acids in Nsp2, a deletion of one nucleotide in 5'UTR and 3'UTR respectively. Compared with parental strain, P75 have 149 nucleotide changes resulted in a missenes mutations of 65. The bioinformatics of four passages cell virus genomes were analyzed. The results show that most of the amino acid substitutions were located in functional regions of the PRRSV genome, such as in putative signal peptide sequences (P4SORF4), transmembrane region (L172FORF4, P173AORF4, S174IORF4), glycosylation sites (S133NORF3) and predicted antigenic sites (A181TNSP2).All of which are hypothesized to have some affect in virus replication, protein transport, signal transduction. Besides, there were 8 mutation sites in same direction during synchronous passage of PRRSV WUH3 strains and JXA1 strains. These are E303DNSP2, E296DNSP3, N162SNSP7, R204HNSP10, F25ISORF2, L172FORF4, P173AORF4, S174IORF4.We speculated that these sites have a certain effect in attenuation of virulence during passage of PRRSV.