Development of a high-throughput rubella virus infectivity assay based on viral activation of caspases

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• 作者：Pulkit S. Bose ; Jennifer Naspinski ; Girish Kartha ; Philip S. Bennett ; Christopher J. Wang ; John I. Haynes II ; Luca Benetti
• 关键词：Rubella virus ; TCID₅₀ ; Caspase-3/7 ; Apoptosis ; RA 27/3
• 刊名：Journal of Virological Methods
• 出版年：2010
• 出版时间：August 2010
• 年：2010
• 卷：167
• 期：2
• 页码：199-204
• 全文大小：704 K

文摘

Rubella virus (RV, German measles) is a teratogenic agent that can lead to serious congenital defects after maternal infection during early pregnancy. Currently, the disease can be prevented effectively by available live attenuated vaccines. An important requisite for the manufacture and release of a safe and potent live virus vaccine is the measurement of the vaccine titer (potency), to ensure the correct dose and efficacy of the vaccine. One historical method for measuring potency is the endpoint dilution TCID₅₀ assay. Traditionally, RV TCID₅₀ titers are calculated after visual inspection of cells for presence of cytopathic effect (CPE). Such visual scoring is tedious and labor intensive. The development of a new TCID₅₀ readout method, based on a fluorescent molecular marker of RV-induced apoptosis, is described in this report. Further, in order to calculate TCID₅₀ potency a novel mathematical model was established to convert the numerical fluorescence measurements into categorical data. Finally, the assay parameters such as signal-to-noise ratio, robustness, variability and bias were optimized. This new readout method demonstrated strong concordance with the standard manual scoring of CPE, and therefore can provide a practical, objective and higher-throughput alternative to the traditional TCID₅₀ readout used for calculating titers of rubella virus.