Inactivation of Venezuelan Equine Encephalitis Virus Genome Using Two Methods.

Mahgol Behnia,Kylene Kehn-Hall,Alan Baer,Andrew M Skidmore,Caitlin W Lehman,Steven B Bradfute,Nicole Bracci

doi:10.3390/v14020272

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.

Highlights

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]
To further confirm the lack of production of the infectious virion postfragmentation of viral RNA, we tested supernatants from the cells transfected with the fragmented RNA purified from Venezuelan equine encephalitis virus (VEEV) TC-83 infected cells and those transfected with the unfragmented RNA from VEEV TC-83 infected cells in a plaque assay
Our result showed the formation of the plaques with an average viral concentration of 7 × 108 PFU/mL in the Vero E6 cells that were incubated with the supernatant from the cells transfected with unfragmented viral RNA (Figure 1D)

Summary

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. 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

Methods

Results

Conclusion