Canine Influenza Virus Induction of TNF

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，确定流感病毒a的单个或多个基因片段中促进种间传播和诱导严重肺部疾病的病毒基因突变。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以诱发严重呼吸道疾病的特征是一个或几个基因片段的突变，这些突变允许病毒在巨噬细胞中更大程度地复制并诱导更高水平的TNF-¿。本中试项目的具体目的是：1)比较2003-2004年接种H3N8犬流感病毒与2003- 1991年接种H3N8马流感病毒分离株后犬肺泡巨噬细胞的病毒复制和诱导TNF- mRNA、蛋白合成及其他细胞因子；2)确定由反向遗传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）复制程度相似，并诱导犬肺泡巨噬细胞中TNF-¿mRNA和蛋白水平相当；3)比较犬和马流感病毒分离株在原代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犬肺泡巨噬细胞体外接种1991年至2003年分离的马甲型流感病毒和2003年至2004年分离的犬流感病毒。TNF- mRNA和蛋白质将分别用实时定量PCR和抗原捕获法测定。病毒的产生将通过感染空斑试验和病毒基质基因实时定量PCR来测量。

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. PUBLIC HEALTH RELEVANCE: The long term goal of this research is to understand which genetic changes in influenza virus from horses allowed it to be able to infect dogs and cause serious respiratory disease that could be passed on to other dogs. Equine influenza viruses from 1991 through 2003 will be compared with canine influenza virus first identified in 2003-2004 for their ability to induce a protein (tumor necrosis factor or TNF) when lung cells become infected with virus. Increased tumor necrosis factor secretion by lung cells is believed to cause severe changes in the lung that result in death. Information from this study will be used in other studies to identify the specific gene changes that allowed the virus to become deadly in dogs. The information will help better understand how influenza viruses from animals evolve to become infectious to other animals and man and cause disease.