Canine Influenza Virus Induction of TNF

描述(申请人提供)：这项研究的长期目标是以H3N8流感病毒为模型，确定甲型流感病毒单基因或多基因片段中有助于促进物种间传播和诱导严重肺部疾病的病毒基因突变。这些研究中的假设是：马流感病毒传播给狗并在新宿主中适应以导致严重呼吸道疾病的特征是一个或几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肿瘤坏死因子。这项初步研究的具体目的是：1)比较2003-2004年接种H3N8犬流感病毒后和2003-1991年接种H3N8马流感病毒分离株后，犬肺泡巨噬细胞中病毒复制和诱导肿瘤坏死因子-βmRNA和蛋白质合成及其他细胞因子的情况；2)确定经反向遗传系统(CANINE/FL/04-RG)衍生的犬流感病毒是否与野生型病毒(A/CAINE/FL/04-RG)在犬肺泡巨噬细胞中复制和诱导类似水平的TNF-βmRNA和蛋白水平；3)比较犬和马流感病毒分离株在原代呼吸道上皮细胞中复制和诱导趋化因子(IL-8和MCP-2)的能力。该项目将使用在体外接种1991年至2003年分离的马流感病毒和2003年至2004年分离的犬流感病毒的初步分离的狗肺泡巨噬细胞。用实时定量聚合酶链式反应和抗原捕获试验分别检测肿瘤坏死因子基因和蛋白的表达。病毒产量将通过感染空斑试验和病毒基质基因实时定量聚合酶链式反应进行检测。

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.