Figure 6.

Emergence and transmission of the SNV-8639A (NS5_357E) variant in mosquitoes, mice, and across species

To monitor the emergence of the late-epidemic signatures, mice (A and B) and mosquitoes (C and D) were inoculated with the parental Ia (II-EEE) virus. We infected AGB6 mice with 500 PFU of the Ia virus via tail vein injection (n = 5), and we determined virus titers in mouse sera collected at 3 and 4 d.p.i. using focus-forming assays (A). The proportions of the late SNVs in each mouse at 4 d.p.i. were analyzed by pyrosequencing (B). We inoculated Aedes Aegypti mosquitoes with 500 PFU of the Ia virus via thoracic injection (n = 10 at each time point). Viral RNA copies (C) and proportions of the late SNVs (D) were determined in mosquitoes collected at 7, 14, and 21 d.p.i. (E) To monitor the emergence and transmission of late-epidemic signatures during the mice-mosquito transmission chain, we infected AGB6 mice with 500 PFU of the Ia virus and used them to feed naive mosquitoes (n = 12 at each time point) at 4 d.p.i. Viral RNA copies (F) and proportions of the late SNVs (G) in infected mosquitoes were determined at 7, 14, and 21 d.p.i. Virus titers and Viral RNA titers are represented as mean ± SD, while proportions of the late SNVs are expressed as the mean. Statistical significance was determined using one-way ANOVA, with significance levels indicated as ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.