Canine Influenza Virus Induction of TNF

犬流感病毒诱导TNF

• 批准号: 8462907
• 负责人: WILLIAM L CASTLEMAN
• 金额: $ 7.33万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8462907
• 关键词: Aerosols; Alveolar; Alveolar Macrophages; Animals; Antigens; Biological Assay; Bronchitis; CCL8 gene; Canis familiaris; Cells; Centers for Disease Control and Prevention (U.S.); Cessation of life; Collaborations; Coughing; Data; Disease; Distant; Eligibility Determination; Epithelial Cells; Equine Influenza Virus; Equus caballus; Feasibility Studies; Florida; Funding; Future; Gene Mutation; Genes; Goals; Grant; Hemorrhage; Human; IL8 gene; Immune response; In Vitro; Infection; Inflammatory Response; Influenza; Influenza A virus; Injury; Kentucky; Letters; Lung; Lung diseases; Maps; Measures; Messenger RNA; Methods; Modeling; Mutation; National Institute of Allergy and Infectious Disease; Pathogenesis; Plaque Assay; Pneumonia; Production; Protein Biosynthesis; Proteins; Pulmonary Edema; Race; Research; Research Project Grants; Ruthenium Ben; System; TNF gene; Time; Tissues; Tracheitis; Tumor Necrosis Factor-alpha; Universities; Veterinary Medicine; Viral; Virus; Virus Activation; Virus Diseases; Virus Replication; chemokine; college; cytokine; influenzavirus; macrophage; man; overexpression; positional cloning; response; transmission process; virus genetics

DESCRIPTION (provided by applicant): The long term goal of this research is to use H3N8 influenza virus as a model to determine viral genetic mutations in single or multiple gene segments of influenza virus A that facilitate interspecies transmission and induction of severe pulmonary disease. The hypothesis being pursued in these studies is: Transmission of equine influenza virus to dogs and adaptation in the new host to induce severe respiratory disease was characterized by mutations in one or several gene segments that allowed the virus to replicate to a greater extent in macrophages and induce higher levels of TNF-¿. The specific aims of this pilot research project are: 1)To compare virus replication and induction of TNF-¿ mRNA and protein synthesis and other cytokines in canine alveolar macrophages following inoculation with H3N8 canine influenza viruses from 2003-2004 with those following inoculation with H3N8 equine influenza virus isolates ranging from 2003 to 1991; 2) To determine whether the canine influenza virus derived with a reverse genetics system (canine/FL/04-rg) replicates to similar extent and induces comparable levels of TNF-¿ mRNA and protein in canine alveolar macrophages as wild type virus (A/canine/Florida/43/2004) does; and 3) To compare the capacity of canine and equine influenza virus isolates to replicate in primary airway epithelial cells and induce chemokines (IL-8 and MCP-2). The project will use primary isolated dog alveolar macrophages inoculated in vitro with equine influenza A viruses isolated from 1991 through 2003 and initial canine influenza isolates from 2003 and 2004. TNF-¿ mRNA and protein will be measured with quantitative real-time PCR and antigen capture assays, respectively. Virus production will be measured by infectious plaque assay and virus matrix gene quantitative real-time PCR.

描述（由申请人提供）：本研究的长期目标是使用 H3N8 流感病毒作为模型，确定甲型流感病毒单个或多个基因片段的病毒基因突变，这些突变促进种间传播和诱发严重肺部疾病。这些研究中所追求的假设是：马流感病毒传播给狗并在新宿主中适应诱发严重呼吸道疾病的特征是一个或多个基因片段发生突变，使病毒能够在巨噬细胞中更大程度地复制并诱导更高水平的 TNF-¿。该试点研究项目的具体目标是： 1）比较2003-2004年接种H3N8犬流感病毒后与2003-1991年接种H3N8马流感病毒分离株后的病毒复制和TNF-¿ mRNA的诱导以及蛋白质合成和其他细胞因子在犬肺泡巨噬细胞中的情况； 2) 确定反向遗传学系统衍生的犬流感病毒（canine/FL/04-rg）是否与野生型病毒（A/canine/Florida/43/2004）一样，在犬肺泡巨噬细胞中复制到相似程度并诱导相似水平的TNF-¿ mRNA和蛋白质； 3) 比较犬和马流感病毒分离株在初级气道上皮细胞中复制和诱导趋化因子（IL-8 和 MCP-2）的能力。该项目将使用原代分离的狗肺泡巨噬细胞，并在体外接种 1991 年至 2003 年分离的马甲型流感病毒和 2003 年和 2004 年分离的初始犬流感病毒。TNF-¿ mRNA 和蛋白质将分别通过定量实时 PCR 和抗原捕获测定进行测量。病毒产量将通过感染斑测定和病毒基质基因定量实时PCR来测量。