title: Development of real-time RT-PCR for evaluation of JEV clearance during purification of HPV type 16 L1 virus-like particles.
abstract: Insect cell culture has greatly increased in part due to the widespread use of insect virus-based vectors for efficient expression of foreign proteins. Insect cells such as Sf9 cells are susceptible to arboviruses which may pose a safety concern by adventitious introduction during the production process. The objective of this study was to establish techniques for viral clearance validation of insect cell-derived biotechnological products using Japanese encephalitis virus (JEV) as a model, since JEV is a member of arthropod-borne flaviviruses that are known to be infectious in insect cells. 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. The JEV specific primer was selected from the 3' untranslated region, and the expected band size was 323 base pairs (bp). The sensitivity of the assay was calculated to be approximately 15 TCID(50)per reaction. Highly reproducible standard curves were obtained from experiments performed on three different days. JEV clearance was determined during the purification process of rHPV-16 L1 VLPs by CsCl equilibrium density centrifugation. The comparative results obtained by real-time RT-PCR assay for JEV and infectivity titrations suggested that the real-time RT-PCR assay could have an additive effect on the interpretation and evaluation of virus clearance, especially during the virus removal process.
authors.affiliation: Division of Viral Products, Korea Food and Drug Administration, 5 Nokbun, Eunpyeong, 122-704, Seoul, Republic of Korea.
authors.forename: Hye-Sung
authors.initials: HS
authors.lastname: Jeong
authors.forename: Jin-Ho
authors.initials: JH
authors.lastname: Shin
authors.forename: Young-Nam
authors.initials: YN
authors.lastname: Park
authors.forename: Jung-Yun
authors.initials: JY
authors.lastname: Choi
authors.forename: Young-Lim
authors.initials: YL
authors.lastname: Kim
authors.forename: Byoung-Guk
authors.initials: BG
authors.lastname: Kim
authors.forename: Seung-Rel
authors.initials: SR
authors.lastname: Ryu
authors.forename: Sun-Young
authors.initials: SY
authors.lastname: Baek
authors.forename: Seok-Ho
authors.initials: SH
authors.lastname: Lee
authors.forename: Sue-Nie
authors.initials: SN
authors.lastname: Park
mesh_terms: D020413:3' Untranslated Regions; D000818:Animals; D052160:Benzothiazoles; D017931:DNA Primers; D003959:Diamines; D004664:Encephalitis Virus, Japanese / Q000235:genetics / Q000302:isolation & purification*; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D027383:Papillomaviridae / Q000302:isolation & purification*; D011804:Quinolines; D012367:RNA, Viral / Q000097:blood / Q000134:cerebrospinal fluid / Q000302:isolation & purification*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014771:Virion / Q000302:isolation & purification*
chemical_list: D020413:3' Untranslated Regions; D052160:Benzothiazoles; D017931:DNA Primers; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; D012367:RNA, Viral; C098022:SYBR Green I