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. 2020 Mar:277:113792.
doi: 10.1016/j.jviromet.2019.113792. Epub 2019 Nov 28.

A new inactivation method to facilitate cryo-EM of enveloped, RNA viruses requiring high containment: A case study using Venezuelan Equine Encephalitis Virus (VEEV)

Amar D Parvate  1 , Frank Vago  1 , S Saif Hasan  1 , Jasper Lee  2 , Evan P Williams  2 , Jason Lanman  1 , Colleen B Jonsson  3
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A new inactivation method to facilitate cryo-EM of enveloped, RNA viruses requiring high containment: A case study using Venezuelan Equine Encephalitis Virus (VEEV)

Amar D Parvate et al. J Virol Methods. 2020 Mar.

Abstract

The challenges associated with operating electron microscopes (EM) in biosafety level 3 and 4 containment facilities have slowed progress of cryo-EM studies of high consequence viruses. We address this gap in a case study of Venezuelan Equine Encephalitis Virus (VEEV) strain TC-83. Chemical inactivation of viruses may physically distort structure, and hence to verify retention of native structure, we selected VEEV strain TC-83 to develop this methodology as this virus has a 4.8 Å resolution cryo-EM structure. In our method, amplified VEEV TC-83 was concentrated directly from supernatant through a 30 % sucrose cushion, resuspended, and chemically inactivated with 1 % glutaraldehyde. A second 30 % sucrose cushion removed any excess glutaraldehyde that might interfere with single particle analyses. A cryo-EM map of fixed, inactivated VEEV was determined to a resolution of 7.9 Å. The map retained structural features of the native virus such as the icosahedral symmetry, and the organization of the capsid core and the trimeric spikes. Our results suggest that our strategy can easily be adapted for inactivation of other enveloped, RNA viruses requiring BSL-3 or BSL-4 for cryo-EM. However, the validation of inactivation requires the oversight of Biosafety Committee for each Institution.

Keywords: BSL-3; Cryo-EM; Glutaraldehyde-crosslinking; RNA virus; VEEV TC-83.

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Conflict of interest statement

Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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