Full inactivation of alphaviruses in single particle and crystallized forms

Abstract

Inherent in the study of viruses is the risk of pathogenic exposure, which necessitates appropriate levels of biosafety containment. Unfortunately, this also limits the availability of useful research instruments that are located at facilities not equipped to handle infectious pathogens. Abrogation of viral infectivity can be accomplished without severely disrupting the physical structure of the virus particle. Virus samples that are verifiably intact but not infectious may be enabled for study at research facilities where they would otherwise not be allowed. Inactivated viruses are also used in the development of vaccines, where immunogenicity is sought in the absence of active infection. We demonstrate the inactivation of Sindbis alphavirus particles in solution, as well as in crystallized form. Inactivation was accomplished by two different approaches: crosslinking of proteins by glutaraldehyde treatment, and crosslinking of nucleic acids by UV irradiation. Biophysical characterization methods, including dynamic light scattering and transmission electron microscopy, were used to demonstrate that the glutaraldehyde and UV inactivated Sindbis virus particles remain intact structurally. SDS-PAGE was also used to show evidence of the protein crosslinking that was expected with glutaraldehyde treatment, but also observed with UV irradiation.

UVC inactivation of pathogenic samples suitable for cryo-EM analysis

“…UVC irradiation is a widespread technique used for disinfection 18 , 19 . Specifically, it has been used for inactivation of viruses 9 . In this paper, we demonstrate that UVC irradiation is also suitable for inactivating pathogens in cryogenic samples intended for structural analysis by cryo-EM.…”

“…We tested this by applying ultraviolet-C (UVC) irradiation to inactivate two pathogenic organisms. UVC irradiation was chosen because of its well-described ability to inactivate pathogens 7 – 9 . Inactivation occurs by altering the DNA of the organism, leading to the prevention of transcription and replication 10 , 11 .…”

Evaluation of the inactivation of Venezuelan equine encephalitis virus by several common methods

“…Both reagents are widely used in preparation for microscopy, and their ability to successfully inactivate VEEV in cells was expected, although low concentrations of formaldehyde have been shown ineffective against yellow fever virus (Goodman et al, 2014). Glutaraldehyde is a more reactive crosslinker than formaldehyde, and may be used at concentrations as low as 0.1% to inactivate very high titers of Sindbis virus , another Alphavirus (Lawrence et al, 2016). The use of SDS to inactivate many types of virus samples has been recommended in previous protocols (Haddock and Feldmann, 2017; Howett et al, 1999).…”

Fomite Transmission, Physicochemical Origin of Virus–Surface Interactions, and Disinfection Strategies for Enveloped Viruses with Applications to SARS-CoV-2

“… 27 , 154 The effectiveness varies depending on the virus inactivation mechanism. In general, there are three modes of inactivation by disinfectants: (1) disruption of the lipid layer of the envelope (e.g., ethanol and detergents), 83 , 155 (2) modification of important protein sites on the envelope or capsid (e.g., chlorine and glutaraldehyde), 83 , 148 , 149 , 156 , 157 and (3) reaction with the nucleotides and amino acids in the genetic material, leading to the degradation of the nucleic acids (e.g., chlorine). 83 , 148 …”