Inactivation of Venezuelan Equine Encephalitis Virus Genome Using Two Methods

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.

Establishment of an Inactivation Method for Ebola Virus and SARS-CoV-2 Suitable for Downstream Sequencing of Low Cell Numbers

“…It should be noted that the genomes of positive sense RNA viruses can be used to transfect cells to generate infectious progeny virus. For this reason, inactivated samples of positive sense RNA viruses that are classified as Select Agents, such as Venezuelan equine encephalitis virus and SARS-CoV-1, are also classified as Select Agents as long as the RNA genome is intact [ 34 ]. However, rescue of SARS-CoV-2 (not a Select Agent) from RNA genomes requires the addition of plasmid-encoded nucleoprotein to be efficient [ 35 ].…”