Washout Kinetics of Viral Vectors from Cultured Mammalian Cells

The handling of infectious viral vectors such as adenovirus type 5 (Ad5), lentivirus (HIV1), and vaccinia virus (VV) requires biosafety level 2 (BSL-2) containment. This also applies to modified vaccinia Ankara (MVA) and adeno-associated virus type 2 (AAV2) provided that the insert involves additional hazard. To transfer infected cells to the lower BSL-1 containment, viral particles have to be demonstrably cleared from the supernatant as specified by biosafety guidelines. With the objective to provide data about the washout kinetics using common culture techniques, the authors infected different adherent mammalian cells with the aforementioned viral vectors. The supernatant of the cell culture was subsequently monitored for the presence of viral nucleic acids during various steps of washing and cell passaging. Complete clearance could be demonstrated for Ad5 when infecting Hela cells and for lentivirus (HIV1) in HEK293T cells. No or an undefined clearance was detected for AAV2, VV, and MVA in Vero-B4 cells and Ad5 after infection of HEK293 cells. Results demonstrated that virus persistence in the supernatant was greatly influenced by the washing procedure, the number of passages, and the vector titer, as well as the type of host cell line. The authors therefore conclude that procedures for clearance cannot be predefined for given virus-host systems. An analysis of the supernatant should be performed for each individual experimental setup prior to downgrading the risk class and subsequently the containment level used.