Figure 1.
Schematic representation of the recombinant VEEV TC-83/IRES constructs.
Alternative positions of the EMCV IRES are indicated. Arrows represent functional subgenomic promoters.
Figure 2.
RNA synthesis and replication of VEEV TC-83 and TC-83/IRES constructs in BHK cells after electroporation.
(A) Analysis of 3[H]-labeled viral RNA production and (B) viral replication after transfection of 4 µg of in vitro-synthesized RNA into BHK cells. Dark dashed line indicates the limit of detection for the experiment.
Figure 3.
Replication of recombinant TC-83/IRES viruses and parental TC-83 on Vero cells.
(A) Cells were infected with VEEV TC-83, VEEV/mutSG/IRES/1 and VEEV/IRES/C at an MOI of 0.1 then viral titers in collected supernatants over time were determined by plaque assay. Bars indicate standard deviation for duplicate infections. (B) Vero cells were fixed with 10% formaldehyde 48 h post-infection and stained with crystal violet to observed plaque size and morphology.
Figure 4.
Survival in mice after infection with VEEV/IRES/C or VEEV TC-83 passaged in-vivo.
Six-day-old CD1 pups received a 5×104 PFU dose SC of viruses passaged 10 times in CD1 mice (mp10). Parent unpassaged VEEV TC-83 and VEEV/IRES/C were injected at the same dose as controls. Animals were monitored daily for survival for 14 days. No deaths occurred after day 11 post-infection. *** = P<0.0001.
Figure 5.
Replication of recombinant TC-83/IRES viruses in mosquito cells.
(A) Five serial passages were performed on C6/36 cells at an initial MOI of 0.1 with VEEV/IRES/C, as well as VEEV TC-83 as a control. Supernatants were analyzed by plaque assay for detection of viral replication. Dark dashed line indicates the limit of detection for the experiment. (B) Supernatants were subjected to RT-PCR analysis to detect presence of viral RNA. Supernatant from non-infected cells (NI) was used as negative control.
Table 1.
Virus replication in Ae. aegypti mosquitoes.
Figure 6.
Virulence in mice after injection of VEEV TC-83 and IRES-based viruses.
Six-day-old CD1 mice received 5×104 PFU of indicated viruses SC, and were monitored 2 weeks for survival (A) and weight change (B). No deaths were recorded after day 14 post-infection.
Figure 7.
Neurovirulence in 6-day-old mice following IC injection of VEEV TC-83 and IRES-based viruses.
Animals received 1×106 PFU of indicated viruses, and were monitored for survival (A) and weight change (B) for 2 weeks, with no deaths recorded after day 14 post-infection.
Figure 8.
Protection against challenge following vaccination of infant mice.
Six-day-old mice were inoculated SC with 5×104 PFU of VEEV TC-83 or IRES-based viruses. Animals were challenged 6 weeks post-vaccination with 104 PFU SC of VEEV IC strain 3908 and monitored daily for survival (A) and weight change (B), with no deaths recorded after day 8 post-challenge. *** = P<0.0001.
Table 2.
Seroconversion of infant CD1 mice.
Figure 9.
Survival following vaccination and challenge of adult mice.
Five-week-old CD1 mice were vaccinated with 105 PFU of VEEV TC-83 or IRES-based viruses. Challenge was performed 6 weeks post-vaccination by SC inoculation of 104 PFU of VEEV IC strain 3908, with daily monitoring of animals. No deaths occurred after day 11 post-challenge.
Table 3.
Seroconversion of adult mice after vaccination.
Table 4.
Viremia in vaccinated adult mice after challenge.