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Characterization of Chicken Microrna and Their Regulatory Roles in the Pathogensis of H5N3 Avian Influenza Virus

Lupiani, Blanca
Texas A&M University
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Avian influenza (AI) is a major respiratory disease of poultry caused by type A avian influenza (AI) viruses. Although the mortality rate during infection with low pathogenic (LP) AI virus is low, it can reduce production efficiency due to concomitant environmental stress and secondary infections. In order to significantly reduce the risks of avian influenza virus infection in chickens, it is imperative to understand the cellular and molecular mechanisms of the pathogenesis of H5N3 avian influenza virus in birds. Our long-term goal is to understand the cellular and molecular mechanisms involved in pathogenesis of LPAI virus in chickens by analyzing gene expression profiling of chicken microRNAs (miRNAs) and virus miRNAs. The overall objective of the proposed study is to investigate the roles of chicken and AIV miRNAs in the regulation of pathogenesis of H5N3 strain of the LPAI in chickens. The central hypothesis of the current proposal is that certain miRNAs in chickens and/or virus play important roles in the modulation of H5N3 infection in chickens. The rationale behind the present proposal is that once we have determined the roles of miRNAs in the pathogenesis of H5N3 in chickens, this will lead to the development of new strategies for anti-viral drugs or vaccine in AI viruses infection in poultry. The specific objectives of this proposal are: Objective 1. Characterization of the miRNA gene expression profiling in chickens. The new chicken 44K Agilent array developed at Texas A&M university includes 150 chicken miRNAs. We will examine gene expression profiling of miRNAs in trachea epithelium and lung of uninfected controls and the H5N3 infected chickens using this chicken 44K array.

Objective 2. Identifying new miRNAs from chicken and H5N3 AI virus. Total RNA isolated from trachea epithelium and lung in the AI virus infected and non-infected chickens will be used to enrich chicken and virus miRNAs. These enriched RNAs will be isolated, sequenced, and predicted based on their sequences and secondary structure.

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APPROACH: SPF chickens will be randomly divided into 2 groups (24 chickens per group. At 4 weeks of age, the infected group will be inoculated with 0.2 ml/chicken containing 107 EID 50 of the H5N3 by the intra-tracheal route. The non-infected birds will be inoculated with the medium. Chickens will be observed daily for a total of 7 days. Cloacal and tracheal swabs will be collected at 2, 4, and 7 days PI to determine virus replication and shedding. Eight chickens in each group will be euthanized at 2, 4 and 7 days PI and tissue samples (tracheal epithelium and lung) taken for total RNA isolation. The labelled total RNA will be used for chicken 44K microarray hybridization to exame chicken miRNAs gene expression profiling. The small RNAs from both tissues will be separated, cloned, sequenced, and predicted based on their sequences and secondary structures.

PROGRESS: 2007/06 TO 2007/12 OUTPUTS: Commercial layer chickens was used to challenge with avian influenza virus H5N3. Total RNA were isolated from lung and trachea, and chicken microRNAs were enriched. Several chicken microRNA probes were designed to examine the differences between infected and non-infected birds at different tissues. The real-time PCR methods are still in the process of optimization. PARTICIPANTS: Dr. Huaijun Zhou and Dr. Blanca Lupiani serve as principal investigators. Ms. Ying Wang is a Ph.D. student who primarily works on this project. TARGET AUDIENCES: The target audience will be poultry industry, government agencies, students at universities and other professional through workshops, presentation, journal publication, reports and lab technique learning.

IMPACT: 2007/06 TO 2007/12 The findings in the study will help to increase genetic resistance to avian influenza virus infection and potential vaccine development in chickens to prevent AIV infection.

Funding Source
Nat'l. Inst. of Food and Agriculture
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Risk Assessment, Management, and Communication
Chemical Contaminants
Heavy Metals
Meat, Poultry, Game