Transgenic indicator cells for influenza virus

描述(申请人提供)：流感病毒引起一种呼吸道大流行疾病，导致严重的人类发病率和死亡率。冬季流感病毒感染的诊断构成了临床病毒学工作量的很大组成部分。这项应用的目标是开发转基因细胞系，以便于在临床标本中检测流感病毒(IV)。此前，我们开发并申请了检测和定量其他RNA病毒的方法，如正链RNA病毒Sindbis病毒和负链RNA病毒呼吸道合胞病毒(RSV)。RSV系统虽然作为一种研究工具非常有用，但其目前的形式过于复杂，不适用于临床病毒学实验室的诊断测试。最近，我们开始开发流感病毒检测系统。我们构建了一个由聚合酶I启动子驱动携带萤火虫荧光素酶基因的甲型流感病毒人工基因组RNA片段转录的质粒。有趣的是，我们发现，感染该Edna的细胞表现出高水平的荧光素酶，而未感染的细胞表达背景水平。我们计划在这一结果的基础上，制造等同于B型流感的人造基因组RNA。然后，我们将使用这些构建物来产生稳定的细胞系，这些细胞系仅在甲型和/或乙型流感病毒感染时表达报告基因。这些细胞系将构成流感检测测试的基础，该测试将在第二阶段与俄亥俄州雅典的诊断杂交公司合作开发，并仿照FDA批准的现有单纯疱疹病毒测试(Elvis(R)-HSV)。

DESCRIPTION (provided by applicant): Influenza virus causes a pandemic disease of the respiratory tract that results in significant human morbidity and mortality. Diagnosis of influenza virus infection during the winter months makes up a large component of the clinical virology workload. The goal of this application is to develop transgenic cell lines that facilitate the detection of influenza virus (IV) in clinical specimens. Previously, we developed and patented methods for detecting and quantitating other RNA viruses such as Sindbis virus, a positive-strand RNA virus and respiratory syncytial virus (RSV), a negative-strand RNA virus. The RSV system, while very useful as a research tool, is too complicated in its present format to be useful in a diagnostic test for clinical virology laboratories. Recently, we began working to develop an influenza virus detection system. We constructed a plasmid in which a polymerase I promoter drives transcription of an influenza A virus artificial genomic RNA segment that carries a firefly luciferase gene. Interestingly, we have found that infection of cells transfected with this eDNA exhibited a high level of luciferase whereas uninfected cells expressed background levels. We plan to build on this result and make equivalent influenza B artificial genomic RNA. We will then use these constructs to generate stable cell lines that express a reporter gene only in response to influenza A and/or influenza B virus infection. Such cell lines will form the basis of an influenza detection test which will be developed in Phase II in collaboration with Diagnostic Hybrids, Inc. Athens, OH, and modeled after an existing FDA approved test for herpes simplex virus (ELVIS(r)-HSV).