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H3 N8流感病毒作为模型，以确定甲型流感病毒单个或多个基因片段中的病毒基因突变，这些突变有助于种间传播和诱导严重肺部疾病。在这些研究中所追求的假设是：马流感病毒传播给狗和适应新的主机，以诱导严重的呼吸道疾病的特点是突变的一个或几个基因片段，使病毒复制到更大程度上在巨噬细胞和诱导更高水平的TNF-α。本试验项目的具体目的是：1）比较2003-2004年接种H3 N8犬流感病毒与2003 - 1991年接种H3 N8马流感病毒分离株后，犬肺泡巨噬细胞中病毒复制和TNF-α mRNA和蛋白质合成以及其他细胞因子的诱导; 2）确定犬流感病毒是否通过反向遗传学系统获得（犬/FL/04-rg）复制程度相似，并在犬肺泡巨噬细胞中诱导与野生型病毒相当的TNF-α mRNA和蛋白水平（A/canine/佛罗里达/43/2004）进行;和3）比较犬和马流感病毒分离物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1991年至2003年分离的马甲型流感病毒和2003年和2004年的初始犬流感病毒分离株体外接种的原代分离犬肺泡巨噬细胞。将分别采用定量实时PCR和抗原捕获试验测量TNF-α mRNA和蛋白质。将通过感染性空斑试验和病毒基质基因定量实时PCR测定病毒产量。

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.