Differential Adaption On Marc-145 Cell Between High And Low Generations Of Highly Pathogenic Porcine Reproductive And Respiratory Syndrome Virus

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is the causative agent of Porcine Reproductive and Respiratory Syndrome which is characterized by reproductive failure in pregnant sows and respiratory problems in piglets. PRRSV possesses a restricted cell tropism and only several cell lines provide adaptable conditions for infection and proliferation of PRRSV. Several factors, including cell receptors and intracellular macromolecules, affect the process of virus'infection. TheTCID50 of each generation of PRRSV strain GD increased during the passage. The time of appearance of CPE between PRRSV strain GD 5th generation and 100th generation passaged in vitro differed greatly after inoculating into Marc-145 cells.34h and 24h were needed for PRRSV strain GD 5th generation and 100th generation in vitro respectively. Sequencing the complete genomes of selected passages of PRRSV strain GD,29 nucleic acid mutations leading to 18 missense mutations were founded.77.8% of amino acid mutations were found before 50 passages and nearly 66.7% amino acid mutations were found in the ORF which encode nonstructural protein. Analyzed by UTRScan, no functional elements in 5'UTR altered. Differential gene structure among serial passages of PRRSV strain GD in vitro was postulated as the reason causing the differential adaption on Marc-145 cells.Because mutations of amino acids were found in both ORFs encoding nonstructural protein and ORFs encoding structural protein, it was hard to speculate which mutation cause the difference of virus' cell adaption. The difference of regulation on the transcription of cell genome by PRRSV might be the most vital reason. Based on the above discussion, Suppression Subtractive Hybridization was employed to construct a differential expressed cDNA liberary for mRNA transcription of Marc-145 cell infected by the high passage and the low passage of PRRSV strain GD. Three times,12hPI,24hPI and 36hPI, were chosen to show the differential genes at different time. Sequencing and blasting the differential genes,11 genes derived from Marc-145 cells which were highly homologous with the genes of primate and 4 novel genes were founded. Six of known eleven genes were ribosomal protein. However, the function of these differential expressed genes was not available as fewer studies on them. The result suggested that the differential transcription of Marc-145 cell genome infected by the high generation and the low generation of passaged PRRSV strain GD did exist, which may contributing to the difference of cell adaption.