Study On The MiRNA Expression Profiles Of DEF Infected With H5N1 Highly Pathogenic Avian Influenza Virus

Waterfowl are the natural host of avian influenza virus(AIV). Also, they are the main source of the fragment donor of the recombinant influenza virus and the main source of infection. Generally, H5N1-infected wild birds(including ducks) appear to cause very mild or no symptoms, but can shed virus to the environment. In 2002, the reemergence of the H5N1 stains are highly pathogenic to ducks, which cause the death and severe neurological signs. Thus, it is particularly important to carry out research on accelerating the pathogenic avian influenza virus of waterfowl. The expression profile of mi RNA could be influenced by viral infection. Host mi RNA plays an important role in the interaction between virus and host innate immune response, it mediates the avian influenza virus latency infection. However, few research focus on mi RNA encoded by Muscovy Duck. The exactly change of mi RNA profile induced by H5N1 highly pathogenic avian influenza virus remains unknown. Besides, the role of mi RNA in the interaction between virus and host is not fully explained yet.In this study, a duck origin avian influenza virus DK383 was studied. DK383 was highly pathogenic for Muscovy Ducks, it caused lung and brain injury with high mortality rate of 70%. Previous study shows that the improper regulation of DK383-induced cytokines was correlated with the lung and brain injury in Muscovy Duck infected with AIV. Likewise, DK383 infection in Duck Eembryo Fibroblast(DEF) could cause activation of cytokines, such as IL-6, IL-10, TNF-? and IL-1?. In this study, the changes of mi RNA expression profile of DEF infected with DK383 were analyzed by high throughput sequencing analysis technology. A network of HPAIV related mi RNAs /target gene was established through gene ontology(GO) analysis. The results showed that: compared with the control group, 44 and 45 differentially expressed were identified at 12 and 24 hours post infection, respectively. According to the GO analysis result, the target genes of differentially expressed mi RNAs are mainly involved in many biological processes,including immune response, negative regulation of transcription, DNA-templated, palate development and positive regulation of fat cell differentiation.Cytokines play an important role in the host innate immune against AIV. In order to explore the role of mi RNA encoded by Muscovy Duck in the process of AIV infection,interleukin-12 was chosen to research. Interleukin-12(IL-12) is a member of the interleukin family with multiple immune regulation activities. IL-12 plays an important role in early activation of the immune response during primary in?uenza virus infection through the early induction of IFN-?, the inhibition of early virus replication, and the contribution to the activation of CTLs. Until now, no mi RNA was identified as the regulator of IL-12. The expression of IL-12 was upregulated in the DEF infected with DK383. We use Targetscan to screen for the differently expressed mi RNA in response to DK383 infection. We found six down-regulated mi RNA included mi R-6125, mi R-2974, mi R-138 a, mi R-196, mi R-196 a and mi R-196a-5p. We utilized the mi RNA specific Real-Time PCR to validate the result,and we predicted mi RNAs targets and made validation by luciferase reporter system. We found that mi R-6125 negatively regulate the expression of IL-12. Mutagenesis analysis revealed the precise binding site for mi R-6125 in IL-12's 3'UTR region. Western blot analysis demonstrated that mi R-6125 negatively regulate IL-12 protein expression. RealTime PCR showed that mi R-6125 repress the expression of IL-12 with decreasing its m RNA stability. In addition, we also have a comprehensive analysis of the role of mi R-6125 in the interaction of the replication of AIV and the expression of cytokines in DEF.After transfection of mi R-6125 mimic, virus replication was promoted significantly, the expression of IFN-?, IFN-? and TNF-? were downregulated. In summary, we find that DEF of Muscovy Duck could regulate the abundance of mi RNA to influence the innate immune response against AIV medicated by cytokines.After analysis of mi RNA expression changes in DEF infected with H5N1 HPAIV, we characterize mi R-6125 as a regulator in the interaction between AIV infection and host innate immune response medicated by cytokines. It deepens the understanding of mi RNA encoded by waterfowl. This study can provide insight into the promising role of mi RNAs for treatment of AIV.