title: Vesivirus 2117: Cell line infectivity range and effectiveness of amplification of a potential adventitious agent in cell culture used for biological production.
abstract: Vesivirus 2117 (VV-2117) has been recently described as a contaminant of cell culture operations in several biologics manufacturing facilities. VV-2117 has been poorly studied and little information exists about its biology, pathogenicity and infectivity range in cell culture settings. In this study we evaluated the potential in vitro viral infectivity of VV-2117 using a range of established mammalian cell lines from various species, the effectiveness of virus amplification in CHO-K1 cells at differing infection levels, and the relative sensitivity of two test methods (cytopathic effect [CPE] and polymerase chain reaction [PCR]) to detect infection and viral amplification. Of eight cell culture systems studied, two originating from hamster (CHO-K1 and BHK-21) and one from canine (MDCK) were positive for CPE and also showed a marked increase of viral RNA in a reverse transcriptase quantitative PCR (RT-qPCR) test. CHO-K1 cell cultures inoculated at 10, 1 and 0.1 genome copies per cell (gc/cell) showed both CPE and amplification of VV-2117 RNA, indicating that infection had occurred in these cultures. CHO-K1 cultures inoculated at 0.01, 0.001, 0.0001 and 0.00001 gc per cell showed neither CPE nor VV-2117, indicating that infection had not occurred. Therefore, the minimum dose necessary for infection of CHO-K1 cells was approximately 0.1 genome copies per cell. At any infection level where VV-2117 amplification was observed by RT-qPCR, the cultures also showed CPE. There was no low-level infection that could be detected by RT-qPCR without developing signs of CPE. However, the RT-qPCR assay appeared more sensitive in that it detected VV-2117 infection earlier than the onset of observable CPE.
authors.affiliation: Genzyme a Sanofi Company, Manufacturing Sciences and Technology, One Research Road, Westborough, MA 01581, USA. Electronic address: zmplavsic@gmail.com.
authors.forename: Mark
authors.initials: M
authors.lastname: Plavsic
authors.affiliation: Eurofins Lancaster Laboratories, Inc., Molecular and Cell Biology Services, 2425 New Holland Pike, Lancaster, PA 17601, USA.
authors.forename: Klaudia
authors.initials: K
authors.lastname: Shick
authors.affiliation: Eurofins Lancaster Laboratories, Inc., Viral Safety and Clearance Services, 2425 New Holland Pike, Lancaster, PA 17601, USA.
authors.forename: Katherine F
authors.initials: KF
authors.lastname: Bergmann
authors.affiliation: Sanofi Pasteur, 1541 Avenue Marcel Merieux, 69280 Marcy L'Etoile, France.
authors.forename: Laurent
authors.initials: L
authors.lastname: Mallet
mesh_terms: D000818:Animals; D016466:CHO Cells; D018929:Cell Culture Techniques / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D004285:Dogs; D061985:Madin Darby Canine Kidney Cells; D020133:Reverse Transcriptase Polymerase Chain Reaction; D029342:Vesivirus / Q000235:genetics / Q000254:growth & development* / Q000302:isolation & purification
keywords: CHO cells; Cytopathic effect; Infectivity; Polymerase chain reaction; Vesivirus 2117