Biological Safety Manual - University of Florida

and inoculated on to an indicator cell line. Cells/supernatants are analyzed for viral markers usingvarious tests e.g.: p24 gag antigen ELISA. Decrease in protein expression with successive passages indicatesabsence of RCL. A kit is available (Perkin Elmer) to detect protein expression. Positive controlstandard (purified p24 gag protein) is included in the kit. The test is easy, rapid and reproduciblebut the kits are expensive. Product-enhanced reverse transcriptase (PERT) assay to detect reverse transcriptase associatedwith the virus. This is reported to be a very sensitive assay. psi-gag and VSV-G envelope PCR assays using genomic DNA extracted from indicator cell line.Plasmid DNA containing the respective sequences is used as positive control. Marker Rescue assay: GFP expressing cell line (e.g. HeLa4G) is transduced with LVV vectorstock. The cells are passaged a few times after which the media is inoculated onto a GFPnegativecell line (e.g. 293T). GFP expression is studied by FACS/ immunofluorescence.Positive controls are required and must be included with the tests. At least 2 consecutive assays onindependently generated LVV preparations are usually recommended. Use of at least two separateassays that detect different viral genes or functions is advised.Risk Assessment: BSL-2+ is recommended for in vitro use of LVV; + emphasizes appropriate personal protectiveequipment, safety sharps and substitution of plastic for glass. Caution is always advised to takemeasures to avoid self-inoculation with LVV to prevent insertional dysregulation of cellular genesthat can occur at a frequent rate ABSL-2 is recommended for animal injections and housing for use of second generation or lowerLVV where RCL data is not available, lowered to ABSL-1+ with instructions for using safe sharps,safety needles, protection of mucous membranes, skin, eyes, and conducting work in certifiedbiosafety cabinets to minimize the risk to researchers in case of accidental exposure to thevector, with ABSL-1 for subsequent housing if the vector stock is tested for RCL and results of 3consecutive tests are reviewed by the Institutional Biosafety Committee (IBC). All LVV made using 3 rd and 4 th generation systems are considered replication incompetent andRCL testing is not required. These are generally recommended at ABSL1+ for animal handlingand ABSL-1 for housing ABSL-1+/ABSL-1 is also recommended if the vector backbone has tested negative for RCL inthe past (by UF PI) or if the documentation of RCL testing for vectors obtained from PIs outside ofUF, or from a commercial source is available and is reviewed by the IBC. Based on the risk assessment, biosafety levels maybe higher if LVV is cloned withoncogenic/toxic inserts. Large scale LVV preparation (>10L) is recommended at BSL-3 containment levelAdvantages of using Lentiviral Vectors: Can be used in non-dividing cells Used in clinical trialsDisadvantages of using Lentiviral Vectors: Can stably integrate into host genome Insertional mutagenesis is a point of concern. RCL can occur by combination between vector and the viral genesReferences: Stéphanie Durand and Andrea Cimarelli (2011). The inside out of Lentiviral vectors. Viruses 3,132-159 James N. Warnock, Claire Daigre, & Mohamed Al-Rubeai. Introduction to viral vectors. Methodsin Molecular Biology (2011) 737: 1-25.28