Analysis The Interaetion Between PRV/PRV Amutant Strain And PK-15 Cells Based On The MicroRNA Expression Profile

Pseudorabies virus (PRV)belonging to alphaherpes virus, as a major hazard of pigs, brings great economic losses to breeding industry every year. An important virulence gE gene of PRV, play an important role in virus spread in the cell and the infection of the nervous system,with the gI of PRV.Currently,The gene vaccine with gE gene deleted is widely used in the prevention and control of PRV, and combining with antibody detection of gE can also be used to distinguish between vaccination infected pigs and wild pigs.Micrornas (miRNA) is the length of about 20 to 24 nt single small RNA that control gene expression level after transcription.Numerous studies have demonstrated that the virus and the host can use micrornas to regulate itself and the transcription of each other, to implement the immune escape and self protection. Virus infection can lead to miRNA expression changes of host,in order to create favorable conditions for their survival, on the other hand, the host can also use the miRNA to achieve immune clearance.In this study, PRV and PRV-gEgI effected respectively with swine kidney cell line (PK-15), collected in three different time and extracting total RNA.Through solexa sequencing technology, the two strains infect PK-15 cells before and after the miRNA expression level analysis.Combined with bioinformatics method, comparative analysis the influence of virus infection to host miRNA expression and deleted gene associated miRNA.The high-throughput sequencing results showed that after the PRV infection with PRV gEgI infected and uninfected PK-15 cells were detected.In miRBase19.0 database,218,221, 221mature pig miRNA was obtained in the three samples.In addition, a large number of new pig miRNA and five new virus miRNA were identified in three samples. The qRT-PCR test result showed that change trends of randomly selected 12 miRNA are consistent with high throughput sequencing results, witch show the sequencing results believable and can be used for subsequent variance analysis.After PRV infection and infection of PRV-gEgI,PK-15 cells were identified 137 and 135significant different miRNA, respectively.By algorithm of TargetScan and miRanda two databases, their target gene were predicted.9 miRNA and their target gene were elected to build the miRNA-target control network,which showed that ssc-miR-24-3p controls most of the target genes, ssc-miR-30a-5p and ssc-miR-30d in PRV infection group regulated network in the core position, ssc-miR-10a-5p and ssc-miR-lOb in PRV-gEgI infection group regulated network in the core position and influence the stability of the whole network.PRV-gEgI infection relative to the PRV infection group has 85 (60 up 25 down) significant difference miRNA, shows that deleted genes have different effects on the expression of cell micrornas.In order to understand the function of abnormal expression miRNA in the virus infected cells and the virus, ssc-miR-34a, ssc-miR-16, ssc-miR-145-5p and ssc-miR-499-5p were randomly selected and transfecting cells respectively.After effected virus and cells were picked up in the 12 h and 36 poison, respectively.By fluorescence quantitative detection results show that the ssc-miR-34a, ssc-miR-16 have effects on virus replication.This study identified the miRNA expression profile in PRV infection with PRV-gEgI infection PK-15 cells before and after.The comparative analysis on them can deepen the understanding of the interactions mechanism between pathogen and host, also offer a new view of the gEgI role in the cell.Based on detailed miRNA analysis, the pathogen host interaction mechanism can build some basis for the prevention and control of PRV as well as a vaccine, carrier, its nerve conduction theory reference for the applications of tracer, etc.