Virus-specific thermostability and heat inactivation profiles of alphaviruses

Serological diagnosis is a critical component for disease surveillance and is important to address the increase in incidence and disease burden of alphaviruses, such as the chikungunya (CHIKV) and Ross River (RRV) viruses. The gold standard for serological diagnosis is the plaque reduction neutralization test (PRNT), which demonstrates the neutralizing capacity of serum samples after the removal of complement activity and adventitious viruses. This procedure is normally performed following inactivation of the virus at 56°C for 30min. Although this protocol has been widely accepted for the inactivation of envelope RNA viruses, recent studies have demonstrated that prolonged heat inactivation is required to completely inactivate two alphaviruses, Western equine encephalitis virus and CHIKV. Incomplete inactivation of viruses poses a laboratory biosafety risk and can also lead to spurious test results. Despite its importance in ensuring the safety of laboratory personnel as well as test integrity, systematic investigation on the thermostability of alphaviruses has not been performed. In this study, the temperature tolerance and heat inactivation profiles of RRV, Barmah Forest, and o'nyong-nyong viruses were determined. Variations in thermostability were observed within the Semliki forest serocomplex. Therefore, evidence-based heat inactivation procedures for alphaviruses are recommended.