Fig. 3. ZE4B-36 is highly attenuated in mice and mosquitoes.

a–d Four-week-old AB6 mice were infected with 2.2 × 10⁵ FFU WT FSS13025ic and ZE4B-36 (n = 7 per group). The immunized mice were monitored for weight loss (a) survival (b). Weight loss is indicated by percentage, using the weight on the day before immunization to define 100%. ***P < 0.001 compared to the WT group (log-rank test). c, d Viremia was determined by using Q-PCR and FFA at days 2 and 4 post infection (pi). n = 3 to 5. Data are presented as means ± standard error of the mean (s.e.m). *** P < 0.001 or **P < 0.01 compared to the WT group. e–g Schematic diagram of mosquito membrane blood feeding (e). Infection of WT and ZE4B-36 in Aedes aegypti. Complement-inactivated sheep blood was inoculated with 1 × 10⁶ FFU/ml of WT ZIKV-FSS13025 and ZE4B-36 viruses. The mosquitoes were fed on the virus and blood mixture. Fully engorged mosquitoes were selected and reared for additional 8 days. Individual engorged, incubated mosquitoes were homogenized, and viral burden of each mosquito was assayed by Q-PCR and calculated into genomic copies (f). One dot represents one mosquito. The numbers of infected mosquitoes versus total mosquitoes are shown above each column. The threshold for distinguishing positive mosquitoes was 1 × 10⁴ genomic copies. Data were pooled from two independent biological replicates. g The infection ratio of each group was counted using the numbers of infected mosquitoes divided by the number of total mosquitoes. ***P < 0.001 compared to the WT group.