Introduction: Inactivation of biological agents and particularly select agents has come under increased scrutiny since the US Army inadvertently shipped live anthrax both inside and outside the US, leading to more stringent regulations regarding inactivation.
Methods: Formalin and Trizol® LS were used to inactivate virus samples in complex matrices. Cytotoxic chemicals were removed using either desalting or concentrating columns or through dilution using HYPERFlasks. Efficacy of inactivation was evaluated either through plaque assay or immunofluorescence assay.
Results: All virus samples and tissue specimens were successfully inactivated using either formalin or Trizol® LS. Both the desalting columns and concentrating columns were able to remove cytotoxic chemicals to facilitate viral amplification in controls. Dilution of cytotoxic chemicals through HYPERFlasks was also successful provided that media was changed completely within 48 hours of first cell passage.
Discussion: All inactivation testing demonstrates that both formalin and Trizol® LS successfully inactivate virus-infected cell lines and tissues, which is consistent with previously published literature. Each sample cleanup method has its benefits and pitfalls. Desalting columns can process the largest sample size but are also susceptible to plugging and degradation, whereas concentrating columns are not as vulnerable but can only process 5% of the sample load per run.
Conclusion: Based on our results along with those of our colleagues, it is recommended that the regulatory authorities re-evaluate the requirements for each entity to validate well-established inactivation methods in house because there would be limited benefits despite the considerable resources required for this effort.
Keywords: BSL-3; BSL-4; HYPERFlask; Trizol; formalin; inactivation.
© ABSA International 2020.