| Pseudorabies virus(PRV), an important member of the alpha herpes virus subfamily, being neurotropism and latent infection, has caused great economic losses to the pig industry. After infection, viral and cellular mi RNAs play important roles in regulation of the life cycle of the herpes virus and interactions between pathogen and host. At present PK-15 cells rather than nerve cells have mainly been used in PRV-related in vitro studies. The aim of this study was to establish PRV infection cell model and then identify mi RNA expression profile, and study the possible mechanism of interaction between the PRV and host.The proliferation of PRV on Neuro-2 cells was evaluated by cytopathic effect(CPE), TCID50, PCR and indirect immunofluorescence assay. The results showed that PRV-infected Neuro-2a cells exhibited typical CPE; the viral titer increased gradually with the time; PCR and indirect immunofluorescence assay also confirmed the replication of PRV on Neuro-2a cells. This study indicates that Neuro-2a cells were permissive to PRV, the cell line can be used for studying the mechanism of infection, latent infection and pathogenesis.A method of q RT-PCR was developed to detect the expression level of IE180, EP0 and VP16 in PRV infected cells. The expression levels of IE180, EP0 and VP16 were determined at 0 h, 2 h, 4 h and 6 h after PRV infected PK-15 cells and Neuro-2a cells(MOI = 5). Results showed that the expression patterns of PRV IE180, EP0 and VP16 in two cell lines were different, suggesting that PRV infected the epithelial cells or nerve cells with different “strategy".mi RNA expression profile in PRV-infected Neuro-2a cells were identified by Solexa sequencing technology and stem-loop q RT-PCR. The regulatory network diagram of host and PRV mi RNAs to viral target genes and the influence of virus infection on host mi RNA expression profile were analyzed by using bioinformatics methods. High throughput sequencing results showed that seven PRV micro RNAs were encoded in PRV-infected Neuro-2a cells at 4h post PRV infection, which were prv-mi R-LLT1, prv-mi R-LLT2, prv-mi R-LLT4, prv-mi R-LLT5, prv-mi R-LLT6, prv-mi R-LLT10 a and prv-mi R-LLT10 b respectively. The expression levels of these micro RNAs were low. However, three more PRV micro RNAs were encoded at 28 h p o st P RV i n fec t i on, w h i c h w e r e pr v- mi R- L LT 3, p r v- mi R- L LT 11 a a n d prv-mi R-LLT11 b respectively. The expression level of PRV micro RNAs at 28 h post PRV infection was higher. There are 13 PRV micro RNAs published in mi RBase database, prv-mi R-LLT7, prv-mi R-LLT8 and prv-mi R-LLT9 weren’t detected at 4 h and 28 h post infection. 4 micro RNAs were differentially expressed at 4 h between the experimental and control group, and the expression level of those micro RNAs(mi R-146b-5p, mi R-714, mi R-466 k, mi R-6538) were higher in the experimental group. Seven micro RNAs were differentially expressed at 28 h and the expression vaule of those micro RNAs(mi R-99b-5p, mi R-301a-3p, mi R-1249-3p, mi R-6538, mi R-714, mi R-19a-3p and mi R-101c) increased in the experimental group. Compared with the PRV micro RNAs at 4h in experimental group, only the expression level of prv-mi R-LLT1 was significantly higher at the 28 h.The reliability of high-throughput sequencing results was confirmed with stem-loop q RT-PCR method. The results of GO analysis of micro RNA-targeted genes indicated that the micro RNA-targeted genes mainly affected cell components, molecular and biological processes; Pathway analysis showed that the micro RNA-targeted genes were mainly involved in PI3K-Aktsignaling pathway, MAPK signaling pathway, neuroactive ligand-receptor interaction, regulation of actin cytoskeleton and focal adhesion. Network diagram made by micro RNAs differentially expression and virus genes showed that micro RNAs can target multiple viral genes to form a complex network. Multiple mice micro RNAs have been predicted, but further study is needed.In summary, above results laid a foundation for further study of the mechanism of micro RNA-regulated alpha herpes virus’ s neurotropism, latent infection and immune escape. |
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