3.4 Mosquito challenge on inoculated goats
To further test the environmental safety profile of DDVax, goats were inoculated with either DDVax or MP-12 viruses. Mosquitoes were allowed to directly feed on the goats at 1 and 2 days post-inoculation (Figure S1). On day 3, blood was collected into EDTA-tubes and transferred to water-jacketed feeders for mosquito challenge in the laboratory. Numbers of engorged mosquitoes from each daily goat feeding are listed in Table S3. Sera from all goat blood specimens were negative for DDVax or MP-12 by plaque assay at 1, 2, and 3 dpi (limit of detection 1 log10 PFU/ml). However, trace levels of viral RNA were detectable by RT-qPCR (Figure S4). After a 7-day extrinsic incubation period, Aedes and Culex bodies showed evidence of infectious MP-12 by plaque assay (Figure 2), indicative of midgut infections, as previously described(Crabtree et al., 2012; Kading et al., 2014). Viral prevalence was highest in Aedes (28%) exposed to goats at 1 day post vaccination with MP-12 strain; these Aedesmosquito infection rates decreased to 12% and 6% in mosquitoes that fed on goats 2 and 3 days post-vaccination, respectively. In contrast, 6% (day 1), 2% (day 2) and 5% (day 3) of Aedes mosquitoes that fed on DDVax-inoculated goats were positive for infectious virus by CPE assay after a 7-day incubation period. Across the time series,Aedes mosquitoes exposed to MP-12 vaccinated goats showed significantly higher rates of virus-positive bodies than those exposed goats inoculated with DDVax (χ2 test, p = 0.011). Culex showed low rates of MP-12 virus infection (< 10%) and no evidence of infection with DDVax. Four of 87 Culex mosquitoes that fed on goats vaccinated with MP-12, and 0/59 Culex mosquitoes that fed on goats inoculated with DDVax, showed evidence of infection after a 7-day incubation. The differences in Culex were not significant, possibly due to low feeding rates (Table S3). All mosquito bodies that were CPE-positive were assessed for the presence of disseminated live virus in legs/wings. However, none of the mosquitoes that became infected after feeding on inoculated goats showed evidence of infectious virus in disseminated infection (positive legs/wings).