Evaluation of TRIzol LS inactivation of viruses

Abstract

Prior to the release from BSL-3 or -4 laboratories, materials from which nucleic acids are to be extracted must be renderednoninfectious. Typically, phenol/guanidine thiocyanate reagents such as TRIzol LS have been used to extract and inactivate RNAfrom infectious viruses. Because of the toxicity of this reagent to the tissue culture cells used to propagate viruses, it has beendifficult to fully evaluate the sterility of extracted viral RNA using tissue culture. In this study, we have demonstrated the use ofsucrose cushion centrifugation to remove TRIzol LS from extracted viral suspensions. The inactivated viral suspensions were thensterility tested without confounding cell toxicity from TRIzol LS. This method allowed a clear demonstration of the use of TRIzolLS to inactivate a broad group of viral families that include Togaviridae, Arenaviridae, Bunyaviridae, Coronaviridae, Filoviridae,Flaviviridae, and Paramyxoviridae at titers ranging from a high of 106.1 to 108.3 tissue culture infectious dose 50% (TCID50/mL) to thelower limit of virus detection in cell culture. TRIzol LS used in a ratio of 4 parts TRIzol LS to 1 part viral suspension inactivatedrepresentative viruses of all families tested.