Exploring The Tropism Of Pseudorabies Virus In Different Tissues Of The Host Based On The MiRNA Level

Pesudorabies virus?PRV?is a highly contagious disease that causes fever,itching,encephalomyelitis and neurological symptoms and bring huge losses to China's pig industry.MicroRNA which are encoded by endogenous gene are single stranded RNA molecules of length 23 nucleotides,and play a regulatory role in many biological processes by binding to the 3'-UTR of target mRNAs,including cancer,cardiovascular system and apoptosis,tumorigenesis and the balance of the body.Nucleotide-binding oligomerization domain?NOD?,Unlike the family of Toll-like receptors?TLRs?,is a class of pattern recognition receptors present in the cytoplasm,not only does it recognize pathogens and their derivatives that invade cells,they also recognize substances released by casualty cells.NOD-like receptors?NLRs?and Toll-like receptors?TLRs?can also synergistically produce immune responses to pathogens.To explore the role of host miRNAs in host tissue tropism when hosts were infected by PRVs.In this experiment,180 g rats were infected by pseudorabies virus(0.1 ml,10⁶ dilution;0.18×LD₅₀).110 hours after infection,the rats developed poor mental status,ataxia,circling in the cage,convulsions etc.Kill these rats to take spleen,lung,kidney,brain,olfactory bulb and other tissues.RNA was extracted and the miRNAs which cut the mustard was selected by HiSeq2500 high-throughput sequencing.The expression levels of miRNAs in different tissues were determined before and after PRV infection.By comparing differentially expressed miRNAs and tissue-specific differentially expressed miRNAs,the miRNAs related to PRV tissue tropism were discovered.Then we analyze the target genes,gene annotations and biological functions of differentially expressed miRNAs,and explore the role of these miRNAs in antiviral and viral replication in vivo after infected by pseudorabies virus.The results of the HiSeq2500 sequencing showed 29,61 and 199 differently expressed miRNAs were obtained in olfactory bulbs,lungs and spleens of the PRV infected and uninfected rats.And 15,42 and 171 tissue-specifically expressed miRNAs were obtained in the differentially expressed miRNAs of olfactory bulbs,lungs and spleens.15 tissue-specifically expressed miRNAs of olfactory bulbs were obtained in the differentially expressed miRNAs,which contain 6 down-regulated miRNAs,3 up-regulated miRNAs and 6 non conservative miRNAs.42 tissue-specifically expressed miRNAs of lung were obtained in the differentially expressed miRNAs which contain 10 down-regulated miRNAs,13 up-regulated miRNAs and 19 non conservative miRNAs.171 tissue-specifically differentiall expressed miRNAs of spleen were obtained in the differentially expressed miRNAs,which contained 58 down-regulated miRNAs,31 up-regulated miRNAs and 82 non-conservative miRNAs.The target gene prediction and function of these miRNAs were annotated by comparing GO and KEGG databases.They were involved in the regulation of related pathways of the nervous system,respiratory and immune system,which affect the tropism of different tissues of the virus and played a pivotal role in virus infection and proliferation.Through comparative analysis of miR-122-5p expression in the sequenced tissues,it was found that the expression of miR-122-5p was significantly different in the three tissues.And It was found that the miR-122-5p was upregulated by 46.6 folds in the lungs and up to 146.0folds in the spleen,compared with control group.The target gene prediction results show that miR-122-5p can target NOD2-like protein sequences.Q-PCR was used to detect changes in the expression of NOD2 protein mRNA compared with the control group after overexpression of miR-122-5p in 293T cell.The results showed that miR-112-5p could target NOD2 and resulted in differential expression of NOD2 protein.At the same time,it was found that the downstream protein RIP2,IL-6 and IL-8 also expressed differentially by compared with the control group.So we confirm that miR-122-5p inhibits NOD signaling pathway,this date may be useful for miRNA-mediated viral tissue tropism.