Abstract
Complete inactivation of infectious Ebola virus (EBOV) is required before a sample may be removed from a Biosafety Level 4 laboratory. The United States Federal Select Agent Program regulations require that procedures used to demonstrate chemical inactivation must be validated in-house to confirm complete inactivation. The objective of this study was to develop a method for validating chemical inactivation of EBOV and then demonstrate the effectiveness of several commonly-used inactivation methods. Samples containing infectious EBOV (Zaire ebolavirus) in different matrices were treated, and the sample was diluted to limit the cytopathic effect of the inactivant. The presence of infectious virus was determined by assessing the cytopathic effect in Vero E6 cells. Crucially, this method did not result in a loss of infectivity in control samples, and we were able to detect less than five infectious units of EBOV (Zaire ebolavirus). We found that TRIzol LS reagent and RNA-Bee inactivated EBOV in serum; TRIzol LS reagent inactivated EBOV in clarified cell culture media; TRIzol reagent inactivated EBOV in tissue and infected Vero E6 cells; 10% neutral buffered formalin inactivated EBOV in tissue; and osmium tetroxide vapors inactivated EBOV on transmission electron microscopy grids. The methods described herein are easily performed and can be adapted to validate inactivation of viruses in various matrices and by various chemical methods.
Highlights
An increasing number of zoonotic pathogens, RNA viruses, are important targets for research because of their potential to emerge in new hosts or regions [1,2]
Part 73.3), any select agent must be rendered non-infectious prior to removal from Select Agent registered space. This requires that the agent no longer have any ability to grow or replicate. This can be achieved by a chemical inactivation procedure, many of which are well established, but the procedure must be appropriately validated in-house to confirm a lack of infectivity [4]
Mock infected samples of cell culture media were mixed at an appropriate ratio with each inactivation reagent in a total volume of 1 mL
Summary
An increasing number of zoonotic pathogens, RNA viruses, are important targets for research because of their potential to emerge in new hosts or regions [1,2]. Such research can be challenging when working with viruses that must be handled in maximum containment laboratories. Ebola virus (EBOV) is a highly lethal RNA virus that can cause Ebola virus disease (EVD) [3]. Within the United States (U.S.), these laboratories are regulated by the U.S. Centers for Disease Control and Prevention’s (CDC) Division of Select Agents and Toxins (DSAT) and the
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