Aedes aegypti Molecular Responses to Zika Virus: Modulation of Infection by the Toll and Jak/Stat Immune Pathways and Virus Host Factors

Abstract

Zika (ZIKV) and dengue virus (DENV) are transmitted to humans by Aedes mosquitoes. However, the molecular interactions between the vector and ZIKV remain largely unexplored. In this work, we further investigated the tropism of ZIKV in two different Aedes aegypti strains and show that the virus infection kinetics, tissue migration, and susceptibility to infection differ between mosquito strains. We also compare the vector transcriptome changes upon ZIKV or DENV infection demonstrating that 40% of the mosquito's midgut infection-responsive transcriptome is virus-specific at 7 days after virus ingestion. Regulated genes included key factors of the mosquito's anti-viral immunity. Comparison of the ZIKV and DENV infection-responsive transcriptome data to those available for yellow fever virus and West Nile virus identified 26 genes likely to play key roles in virus infection of Aedes mosquitoes. Through reverse genetic analyses, we show that the Toll and the Jak/Stat innate immune pathways mediate increased resistance to ZIKV infection, and the conserved DENV host factors vATPase and inosine-5'-monophosphate dehydrogenase are also utilized for ZIKV infection.

Keywords: Aedes aegypti; Zika virus; dengue virus; innate immunity; vector competence.

FIGURE 1

Zika virus (ZIKV) tropism in A. aegypti. Orlando (Orl) and Rockefeller (Rock) A. aegypti mosquito strains were infected with ZIKV via an infectious-blood meal, and (A–C) midguts, (D–F) abdomens, and (G–I) salivary glands were assessed for infection intensity at different time points. (A) 4 days (N = 34), 7 days (N = 36), 10 days (N = 46), 14 days (N = 36). (B) 4 days (N = 37), 7 days (N = 40), 10 days (N = 42), 14 days (N = 42). (D) 4 days (N = 35), 7 days (N = 38), 10 days (N = 25), 14 days (N = 51). (E) 4 days (N = 56), 7 days (N = 61), 10 days (N = 40), 14 days (N = 55). (G) 10 days (N = 33), 14 days (N = 38), 21 days (N = 27). (H) 10 days (N = 40), 14 days (N = 46), 21 days (N = 34). Each dot represents the plaque-forming units (PFUs) per individual tissue from three independent experiments. Bars represents the median. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001; Kruskal-Wallis test. (C,F,I) show strain comparisons between Orl and Rock; each point represents the median PFU per strain at the given time point per replicate (N = 3). ^∗∗P < 0.01, ^∗∗∗P < 0.001, ^∗∗∗∗P < 0.0001; Mann–Whitney test. (J) The prevalence (percentage) of infected mosquitoes for Orl vs. Rock was compared at each time point. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001; chi square test.

FIGURE 2

Comparative transcriptomic analysis of A. aegypti responses to ZIKV and DENV infection. RNA-seq analysis was performed on ZIKV- and DENV-infected Rock A. aegypti midguts at 7 dpibm. (A) PFU per midgut; bars represent the median for three independent experiments (N = 20). The prevalence (percentage) is shown below the graph, ^∗P < 0.05; chi square test. (B) Total up- and downregulated genes. (C) Venn diagram showing the shared and uniquely regulated genes in ZIKV vs. DENV infected mosquitoes. (D) Venn diagram showing genes regulated in the same direction upon infection with YFV, WNV, DENV, or ZIKV (Colpitts et al., 2011). (E) List of the identified mosquito genes with known function (11 of 26): upregulated genes shown in orange, downregulated in blue. Upregulated genes with unknown function (AAEL004157, AAEL006305, AAEL000586, AAEL005986, AAEL009058, AAEL011110) and downregulated genes with unknown function (AAEL012644, AAEL009181, AAEL002046, AAEL007703, AAEL013812, AAEL002889, AAEL004022, AAEL008106, AAEL006834).

FIGURE 3

Infection-responsive immune genes. The fold change of putative immune gene transcript abundance upon midgut infection with (A) ZIKV or (B) DENV, as compared to non-infected blood-fed controls. Orange indicates upregulation, blue downregulation. (C) The fold difference in transcript abundance of immune genes between ZIKV- vs. DENV-infected midguts; black and gray indicates genes showing a higher transcript abundance in ZIKV and DENV infected midguts, respectively. (D) Schematic representation of the four main immune pathways (Toll, Imd, Jak/Stat, and RNAi). Boxes without lines represent ZIKV infection-responsive genes, boxes with solid lines represent DENV infection-responsive genes, and boxes with dashed lines represent genes regulated upon infection with either virus. (E) Immune pathway activation demonstrated through dsRNA-mediated silencing of the negative regulators of the Toll pathway (dsCactus, N = 65), Imd pathway (dsCaspar, N = 66), and Jak/Stat pathway (dsPIAS, N = 65). GFP dsRNA was used as a control (N = 62). ZIKV infection was measured as PFU in individual midguts at 7 days post-infection. The bars represent the median. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001; Mann-Whitney test.

FIGURE 4

Mosquito host factors influence ZIKV infection. Genes encoding two subunits of the vATPase (VoB and Ac39) (N = 41, N = 65) and IMPDH (N = 63) were silenced upon ZIKV infection. (A) Each dot represents the PFUs per midgut in three independent experiments. The bars represent the median. ^∗P < 0.05, ^∗∗P < 0.01; Kruskal–Wallis test. (B) Prevalence (percentage) of infected mosquitoes. ^∗P < 0.05; chi square test.