Title : Improved techniques for validation of inactivated virus and RNA of positive sense RNA viruses for containment research
Abstract:
Alphaviruses, such as eastern equine encephalitis virus (EEEV), are genetically encoded by a single positive sense RNA genome. Following glycoprotein-assisted entry into the cells, fusion with the endosomal membrane, and capsid release, translation of the non-structural proteins is rapidly initiated by the host cell ribosome. With initiation of viral replication being driven by the host cell, functional alphavirus RNA that successfully enters a permissive host cell can result in production of progeny virions. This presents a challenge when there is a desire to remove samples from containment spaces as proof of viral RNA inactivation is required in addition to inactivation of infectious virus. Many laboratories validate inactivation of alphavirus RNA by merely adding the viral RNA to a media overlay on cells. However, this methodology does not provide entry of the RNA into cells where replication occurs, therefore, it does not validate that the RNA is inactivated rather that the RNA is not entering into cells. Alphaviruses are responsible for numerous severe human diseases that can cause neurological complications (new world alphaviruses) or debilitating arthritis (old world alphaviruses). Therefore, it is essential from both a biodefense and public health perspective to validate that viral RNA present in samples removed from the containment environment is inactivated and unable to produce infectious virions. The research proposed in this abstract aims to demonstrate these points using methods that ensure viral RNA enters into cells during a series of experiments aimed at validating inactivation of EEEV RNA by chemical means.
