Determination of the biological functions of undefined domains of VEEV nsP2.

Skidmore, Andrew M.

doi:10.18297/etd/3648

Cited by 1 publication

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“…A total of forty cycles were performed. Primers and probes were custom ordered from IDT and based upon previous work (Table 1) [9]. RNA Fragmentation: to inactivate the viral RNA, total RNA extracted from virally infected cells was fragmented using an Ion Total RNA-seq Kit v2 (ThermoFisher Scientific, Waltham, MA, USA, Cat# 4479789) according to the manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

Inactivation of Venezuelan Equine Encephalitis Virus Genome Using Two Methods

Behnia

Baer

Skidmore

et al. 2022

Viruses

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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.

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Section: Methodsmentioning

confidence: 99%