CELLS FOR DETECTING NEGATIVE-STRAND RNA VIRUSES

本提案的目标是开发用于检测和定量负链RNA病毒的生物测定的商业潜力。该生物测定法的基础（已用呼吸道合胞病毒原型化）是来自转化细胞细胞质内存在的人工病毒基因组的报告基因的感染依赖性表达。负链RNA病毒包括一些最臭名昭著的人类病原体，并且由于严重的呼吸道疾病、出血热综合征等而导致显著的全球发病率和死亡率。不幸的是，很少有治疗剂和疫苗可用于控制这些病毒。寻找新的抗病毒剂和开发疫苗的努力将需要检测和定量感染性病毒的方法，这些方法适用于高通量检测。 由于所有病毒都是专性细胞内微生物，因此任何检测感染性病毒和确定各种试剂对病毒复制的影响的方法都必须基于细胞培养。 即使与免疫测定或基于核酸的方法组合，传统的细胞培养测定通常也是劳动密集型的并且难以简化。近年来，在鉴定许多致病性负链RNA病毒复制过程中的顺式作用元件和反式作用因子方面取得了重大进展。这项建议所基于的技术利用这一信息来开发细胞系，在这些细胞系中，一种容易测量的酶的表达取决于特定病毒的感染。拟议的商业应用：负链RNA病毒包括一些最重要的新兴人类病原体。新的生物测定，以检测和定量的许多这些病毒中描述的这一建议将促进努力寻找新的抗病毒剂和开发疫苗来控制这些病毒。此外，如果敏感性和特异性问题得到解决，它有可能促进临床病毒学实验室对这些病毒的诊断。

The goal of this proposal is to develop the commercial potential of a bioassay to detect and guantitate negative-strand RNA viruses. The basis of this bioassay, which has been prototyped with respiratory syncytial virus, is infection-dependent expression of a reporter gene from an artificial viral genome present within the cytoplasm of transformed cells. Negative-strand RNA viruses include some of the most notorious human pathogens and cause significant worldwide morbidity and mortality due to severe respiratory disease, hemorrhagic fever syndromes, etc. Unfortunately very few therapeutic agents and vaccines are available to control these viruses. Efforts to find new antiviral agents and to develop vaccines will require methods to detect and guantitate infectious virus which are amenable to highthroughput assays. Since all viruses are obligate intracellular microbes, any method to detect infectious virus and to determine the effects of various agents on viral replication must be based on cell culture. Traditional cellculture assays even when combined with immunoassays or nucleic acid-based methods are generally labor intensive and difficult to streamline. Significant advances have been made in identifying the cisacting elements and trans-acting factors involved in replication of many pathogenic negative-strand RNA viruses. The technology on which this proposal is based uses this information to develop cell lines in which the expression of an easily measurable enzyme is dependent on infection with a specific virus. PROPOSED COMMERCIAL APPLICATION: Negative-strand RNA viruses include some of the most important emerging human pathogens. The novel bioassay to detect and quantitate many of these viruses described in this proposal will promote efforts to find new antiviral agents and develop vaccines to control these viruses. Also, if sensitivity and specificty issues are addressed, it has the potential to facilitate diagnosis of these viruses in clinical virology laboratories.