Screening And Regulating Analysis Of The Genes Differentially Expressed In Marc-145 Cells Infected With High-virulent PRRSV Strain GD And The Attenuated Strain Derived From Strain GD By Cellular Passage

Porcine reproductive and respiratory syndrome is an epidemic disease caused by porcine reproductive and respiratory syndrome virus (PRRSV), and it is classified as a notifiable (previously List B) disease by the World Organization for Animal Health (OIE). In 2006, a wide-spread epidemic infection was catched in China, which was characterized as high fever and high mortality of infected pigs. After the pathogen isolation and characterization, this type disease was ascribed to the genetic variation in PRRSV NSP2 (non-structural protein 2), and named as highly pathogenic porcine reproductive and respiratory syndrome (HP-HRRS).In 2007, the HP-PRRSV inactivated vaccine was applied in China, however, the efficacy was not as good as expected, mainly because this type of vaccine was mainly able to stimulate the humoral immunity rather than the cellular immunity which plays an vital role in the defence of PRRSV, for these reason, the development of attenuated live vaccine against HP-PRRS is on agenda in recent years. In Oct 2006, one strain of HP-PRRSV was isolated by China Institute of Veterinary Drug Control in Guangdong province, termed as HP-PRRSV strain GD, and which was propagated and passaged in cell line Marc-145. In this study, the 5^thand the100^th generation GD strains from Marc-145 were used as the viruses materials of which the genetic variation, cellular adaptability and the virulence were determined. The gene differential expression in Marc-145 induced by these two generation viruses were characterized by Suppression Subtractive Hybridization and Dot blot assay, and also the RNA interference and over-expression assay were both employed to determine the function of the differentially expressed gene in the propagation of these two generation viruses. The results are as follows:1. A total of eleven generations nucleotide sequences between the 5^th and the100^th in PRRSV GD was amplifid by RT-PCR and sequenced. The above eleven generations of PRRSV GD were inoculated in Marc-145 cells with equivalent titers, respectively. Then the emergence time of cytopathic effects (CPE) between the eleven generations was observed, and the virus titers of 75% extent CPE were titrated, the virulence to the host animal swines both of the 5^th and 100^th generations were determined respectively. The results showed that 29 base mutations and 18 amino acid mutations were observed between the 5^th and the100^th generation in PRRSV GD; the emergence time was -10 h and the negatively logarithm value of virus titer was +2; the virulence of 100^th generation was decreased when compared to the 5^th.2. On the time of 24 h, 36 h, and 48 h, the differences of gene expression between Marc-145 genes infected with the 5^th generation strain of PRRSV GD and the100^th of PRRSV GD were detected by the SSH assay, and the results were also confirmed by Dot blot. We successfully screened 11 EST sequences, including S8, S29, RPL6, RPL26, and RPL 30; five ribosomal protein genes; four un-known genes and two metabolism-related enzymes, DEAD-BOX polypeptide and internally tangent Glucanase. The results showed that really differences are existed between the cell gene transcription in Marc-145 infected by 5^th and 100^th generations in PRRSV GD, and S8 may be associated with cell adaptability or virulence in PRRSV GD.3. Gene sequences of shRNA were designed according to S8, and transcription I sequences of ribosomal protein S8 were amplified and cloned into psiRNA-hH1neo and pcDNA3.1/V5-His-TOPO plasmids respectively. Then the recombinant plasmids were transfected into Marc 145 cell lines, and the stable expression cell lines were established by G418 selection. Real-time PCR and western-blot were employed to validate the gene inhibition efficacy and gene expression levels of ribosomal protein S8 in the stable cell lines. Two cell lines were inoculated with the 5^th and the100^th generations of PRRSV GD, with control and mock groups, which were detected by real-time PCR and immuno-fluorescence. The results showed when ribosomal protein S8 was inhibited, different degrees of proliferation decrease were observed in both the 5^th and the100^th generations; while ribosomal protein S8 was over-expressed, different degrees of proliferation increase were observed in both generations. In conclusion, the propagation of PRRSV strain GD was associated with the regulation on ribosomal protein S8 in Marc-145.