A Quantitative Assay of Japanese Encephalitis Virus as a Model Virus for Viral Clearance Validation in Insect Cell-Derived Biotechnology Products

ABSTRACT

Commercial interests towards insect cell cultures have greatly been increased recently, in part due to the widespread use of insect virus-based vectors for efficient expressions of foreign proteins. Insect cell-derived biotechnology products should be free of adventitious agents such as arboviruses and mycoplasmas. The objective of this study was to establish techniques for the viral safety evaluation of insect cell-derived biotechnology products using Japanese encephalitis virus (JEV) as a model, since JEV is a member of arthropod-borne flaviviruses which has been known to be infectious to insect cells such as Sf9 cells. Quantitative assays for viral infectivity, concentrations of an antigen or a genome are a prerequisite for the studies of viral clearance validation. Here, we report the development of a quantitative assay for JEV RNA using real-time reverse transcription-polymerase chain reaction (RT-PCR). The assay was performed using LightCycler and RNA amplification kit SYBR Green I. Viral RNA extracted from stock suspension of JEV with known infectivity titer was made to 10-fold serial dilution, and each dilution was subjected to the assay for the generation of a standard curve. A JEV specific primer was selected from 3' untranslated region with the expected band size of 323 base pairs. The real-time RT-PCR assay resulted in a successful amplification within 5 log dilution ranges of JEV RNA samples, and the sensitivity of the assay was calculated to be approximately 15 TCID50 per reaction. Highly reproducible standard curves were obtained from experiments performed on three different days. The real-time RT-PCR assay for the quantification of JEV will be an efficient alternative tool for viral clearance validation studies of insect cell-derived biotechnology products.