The tissue culture infectious dose 50 (TCID50) end-point dilution assay measures the concentration of infectious virus (titre) in a specimen. It is the gold-standard assay for viruses with negligible or ambiguous cytopathic effects, as it can be followed by an Enzyme-Linked Immunosorbent Assay (ELISA) to detect viral antigens and improve assay specificity. Paraformaldehyde (PFA) and acetone are two commonly used fixative agents. It is established that 4% PFA effectively inactivates the infectiousness of enveloped viruses [1, 2]. However, similar inactivation studies have not been reported for standard acetone fixation methods (20% acetone for 24h at 4°C).
This study evaluated the inactivation efficacy of acetone on enveloped viruses, specifically dengue virus (DENV) and Ross River virus (RRV), which represent flavivirus and alphavirus genera, respectively. Infected plates were treated with 10%, 20%, or 50% acetone for 24 hours at 4°C. Fixatives or control plate media were removed, and fresh media was added. The presence of residual infectious virus was assessed by transferring supernatants to naïve cells and incubating for 5 days. Infection was determined by qRT-PCR of intracellular viral RNA. Our results demonstrated that both 50% acetone and 4% PFA fully inactivated DENV and RRV, but 10% or 20% acetone did not reduce the infectivity of these viruses compared to the no-fixative control.
We next compared the sensitivity of ELISAs performed on cells infected with titrations of DENV or RRV, fixed with 20% or 50% acetone. Viruses were detected with mAb 4G2, and B10, respectively. Both buffers yielded comparable viral quantification for DENV and RRV. Together, our findings suggest that 20% acetone is insufficient to inactivate DENV and RRV and instead 50% acetone should be used to prepare infectious samples for downstream assays conducted outside biosafety cabinets. This highlights the importance of robust testing of inactivation protocols to protect laboratory personnel.