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.

Keywords: RNA fragmentation; Venezuelan equine encephalitis virus; cDNA synthesis; encephalitis; viral genome inactivation.

Figure 1

Fragmentation of VEEV TC-83 RNA renders it non-infectious. Vero E6 cells were transfected with total RNA purified from uninfected or TC-83 infected primary mouse neurons at 72 h post-transfection. Three wells/sample transfected; (A) Vero E6 cells transfected with 50 ng/well of fragmented total RNA purified from TC-83 infected primary mouse neurons; (B) Vero E6 cells transfected with 50 ng/well of total RNA purified from uninfected primary mouse neurons (negative control); (C) Vero E6 cells transfected with 50 ng/well of total RNA purified from TC-83 infected primary mouse neurons. The results shown (A–C) are representatives of three wells per experiment and two separate experiments. (D) Viral titers of VEEV TC-83 measured in plaque assays using supernatants collected from cells 72 h after transfection with fragmented VEEV TC-83 RNA or unfragmented VEEV TC-83 RNA (positive control). Uninfected Vero E6 cell supernatants were used as the negative control. ND = none detected. Viral titer reported at 72 h p.i. and as the average of PFU/mL for two separate experiments. The dotted line indicates the limit of detection.

Figure 2

Fragmentation of VEEV TrD RNA renders it non-infectious. Vero E6 cells transfected with total RNA purified from uninfected or TrD infected primary mouse neurons at 72 h post-transfection; (A) Vero E6 cells transfected with 50 ng/well of fragmented total RNA purified from TrD infected primary mouse neurons; (B) Vero E6 cells transfected with 50 ng/well of total RNA purified from uninfected primary mouse neurons (negative control); (C) Vero E6 cells transfected with 50 ng/well of total RNA purified from TrD infected primary mouse neurons. The results shown are representative of three wells per experiments and three separate experiments.

Figure 3

Low concentrations of viral RNA cause cytopathic effect in Vero E6 cells. Total RNA purified from uninfected or TC-83 infected Vero cells transfected to Vero cells in 50 or 5 ng/well, images were taken at 72 h post-transfection. (A) 50 ng of total RNA from TC-83 infected Vero cells transfected to Vero cells; (B) 50 ng of total RNA from uninfected Vero cells transfected to Vero cells; (C) 5 ng of RNA from TC-83 infected Vero cells transfected to Vero cells; (D) 5 ng of RNA from uninfected Vero cells transfected to Vero cells. The results shown are representative of three wells in two separate experiments.

Figure 4

Low concentration of viral RNA causes cytopathic effect in Vero E6 cells. Total RNA purified from uninfected or TC-83 infected Vero E6 cells and transfected into Vero E6 cells in 0.5 ng/well; images were taken at 72 h post-transfection. (A,B) 0.5 ng of total RNA from TC-83 infected Vero cells transfected to Vero cells; (C,D) 5 ng of RNA from uninfected Vero cells transfected to Vero cells. The result shown is representative of four wells in two separate experiments.

Figure 5

Viral RNA does not cause CPE without artificial entry. Total RNA purified from uninfected or TC-83 infected Vero cells directly added or transfected to Vero cells, images taken at 72 h post-administration; (A) 1 μg/well of total RNA from TC-83 infected Vero cells directly added to Vero cells; (B) 1 μg/well of RNA from TC-83 infected Vero cells transfected to Vero cells (positive control); (C) 1 μg/well of total RNA from uninfected Vero cells directly added to Vero cells (negative control); (D) 1 μg/well of total RNA from uninfected Vero cells Transfected to Vero cells (negative control). The result shown is representative of three wells in two separate experiments.

Figure 6

cDNA synthesis followed by RNase treatment renders VEEV, EEEV and WEEV RNA non-infectious; (A,B) RNA isolated from uninfected Vero cells (neg. control), VEEV TC-83, VEEV TrD WEEV, or EEEV infected Vero cells was converted to cDNA and treated with RNase A and H. cDNA samples were then transfected into Vero cells. Images were taken at 4- and 7-days post-transfection; (C) Vero cells were infected with VEEV TC-83 (MOI 5), VEEV TrD (MOI 0.1), WEEV (MOI 0.1), or EEEV (MOI 0.1) as positive controls for CPE. Images were taken at 7-days post-transfection. Results shown are representative of two separate experiments. (D,E) Plaque assays were performed on Day 7 supernatants from VEEV TC-83 and EEEV cDNA transfected cells. Supernatants collected at 24 h p.i. from VEEV TC-83 and EEEV infected cells (MOI 5) were included as a positive control. ND = none detected. The dotted line indicates the limit of detection.