  Citation: Behnia, M.; Baer, A.; Skidmore, A.M.; Lehman, C.W.; Bracci, N.; Kehn-Hall, K.; Bradfute, S.B. Inactivation of Venezuelan Equine Encephalitis Virus Genome Using Two Methods. Viruses 2022, 14, 272. https://doi.org/10.3390/v14020272 Received: 14 January 2022 Accepted: 26 January 2022 Published: 28 January 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). viruses Communication Inactivation of Venezuelan Equine Encephalitis Virus Genome Using Two Methods Mahgol Behnia 1 , Alan Baer 2 , Andrew M. Skidmore 1 , Caitlin W. Lehman 3 , Nicole Bracci 3 , Kylene Kehn-Hall 2,3 and Steven B. Bradfute 1, * 1 Department of Internal Medicine, University of New Mexico Health Sciences Center, Albuquerque, NM 87131, USA; mabehnia@salud.unm.edu (M.B.); amskidmore@salud.unm.edu (A.M.S.) 2 National Center for Biodefence and Infectious Diseases, George Mason University, Manassas, VA 20110, USA; alangbaer@gmail.com (A.B.); kkehnhall@vt.edu (K.K.-H.) 3 Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, VA 24061, USA; woodsonc@vt.edu (C.W.L.); nbracci@vt.edu (N.B.) * Correspondence: sbradfute@salud.unm.edu; Tel.: +1-505-272-9368 Abstract: Venezuelan equine encephalitis virus (VEEV) is an Alphavirus in the Togaviridae family of positive-strand RNA viruses. The viral genome of positive-strand RNA viruses is infectious, as it produces infectious virus upon introduction into a cell. VEEV is a select agent and samples containing viral RNA are subject to additional regulations due to their infectious nature. Therefore, RNA isolated from cells infected with BSL-3 select agent strains of VEEV or other positive-strand viruses must be inactivated before removal from high-containment laboratories. In this study, we tested the inactivation of the viral genome after RNA fragmentation or cDNA synthesis, using the Trinidad Donkey and TC-83 strains of VEEV. We successfully inactivated VEEV genomic RNA utilizing these two protocols. Our cDNA synthesis method also inactivated the genomic RNA of eastern and western equine encephalitis viruses (EEEV and WEEV). We also tested whether the purified VEEV genomic RNA can produce infectious virions in the absence of transfection. Our result showed the inability of the viral genome to cause infection without being transfected into the cells. Overall, this work introduces RNA fragmentation and cDNA synthesis as reliable methods for the inactivation of samples containing the genomes of positive-strand RNA viruses. Keywords: encephalitis; Venezuelan equine encephalitis virus; RNA fragmentation; cDNA synthesis; viral genome inactivation 1. Introduction RNA viruses are grouped based on the types of RNA that serve as their genome. The genome of a positive-strand RNA virus has mRNA characteristics and can be used as a template for the production of infectious virions upon introduction into susceptible cells [1]. Among the highly pathogenic positive-strand RNA viruses that infect humans, some Flaviviruses and Alphaviruses are categorized as bioterrorism agents because of their infectivity in aerosolized form and ability to cause severe debilitating diseases in humans and livestock. For example, the Far Eastern subtype of Tick-borne encephalitis virus, a Flavivirus, and eastern equine encephalitic virus (EEEV), an Alphavirus, cause encephalitis in humans with high mortality rates and are select agents [2–4]. Currently, many laboratories use modern technologies such as RNA sequencing and qRT-PCR to study positive-strand RNA viruses and virus–host interactions. Many of these techniques rely upon RNA extraction, manipulation, and transfer to lower containment facilities for downstream analysis. The ability of the positive-strand RNA genome to initiate the viral life cycle upon introduction to the susceptible cells raises the concern about the production of the highly pathogenic positive-strand select agents in unauthorized facilities. Viruses 2022, 14, 272. https://doi.org/10.3390/v14020272 https://www.mdpi.com/journal/viruses