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).