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Avian influenza (AI) is a major respiratory disease of poultry caused by type A avian influenza viruses (AIV). Symptoms in AIV-infected birds range from unapparent or mild respiratory symptoms to respiratory failure and death, depending on both virus strain and host. Due to the limited availability of effective measure against AIV infection and the potential host jump from animal to humans, AI outbreaks in chickens could not only cause devastating economic losses to the poultry industry, but also threaten human health.
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The long-term goal of this project is to develop new strategies to treat AIV infection in chickens by evaluating chicken microRNAs (miRNA) that affect AI virus replication. The objectives of this study are to: 1) over-express and knock-down identified miRNAs in a chicken fibroblast cell line (DF-1); 2) determine the effects of over-expression and knock-down of miRNAs on the replication of AIV in DF-1 cells; 3) validate target AIV genes by these miRNAs using luciferase reporter assay. <P>
The expected outputs will be to identify critical miRNAs regulating AIV replication in chickens.The new knowledge generated will deepen our understanding on the mechanism of host-AIV interaction during viral replication. This, in turn, is expected to lead to identify new targets for controlling AI that will complement current approaches of control through vaccination.
NON-TECHNICAL SUMMARY: Avian influenza is a major respiratory disease of poultry caused by avian influenza viruses (AIV). Symptoms in AIV-infected birds range from unapparent or mild respiratory symptoms to respiratory failure and death. Due to the lack of effective measure against AIV infection and the potential host jump from animal to humans, AI outbreaks in chickens could not only cause dramatic economic losses to the poultry industry, but also threaten human health. MicroRNAs have been recognized as one of the essential regulators of gene expression in animals and viruses. Our recent microRNAs expression profiling by deep sequencing in AIV infected chickens demonstrated that gga-let-7 family and a few other chicken microRNAs play an important role in the regulation of AIV infection. The investigation of regulatory mechanisms of these chicken microRNAs on AIV replication will lay the foundation to develop new therapeutic methods to control AIV infection.
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APPROACH: Based on our preliminary results, identified chicken miRNAs will be selected for gain- and loss-of-function in chicken DF-1 cell line by lentiviral vector delivery system. Over-expression and knock-down effect will be measured using real-time PCR to examine gene expression of these target miRNAs. After the confirmation of gain- and loss-of-function of these miRNAs, stable cell lines will be established by antibiotics selection. After avian influenza virus (AIV) infection, AIV gene expression targeted by chicken miRNAs will be examined by real-time PCR, and AIV replication in DF-1 cells after gain- and loss-of-function will be determined by influenza matrix gene using real-time PCR. Plaque assay and virus titer will be measured to evaluate the effect of overexpression or knock-down of chicken miRNAs. Finally, A luciferase reporter assay will be used to confirm target AIV genes by chicken miRNAs.