Fig 1.
EBIV infection rates of Ae. aegypti through oral feeding.
EBIV titers (A) and infection rates (B) of mosquitoes at 4, 7, 10 and 14 days after feeding on blood meal containing 3.7 × 106 PFU ml−1 EBIV. EBIV titers (C) and infection rates (D) of mosquitoes from six serial viral titer groups at 4 and 10 days after feeding on blood meal containing 102 to 107 PFU ml−1 EBIV. EBIV titers in gut (E), head (F), saliva (G) and ovary (H) samples of mosquitoes at 4, 7, 10 and 14 days after feeding on blood meal containing 6.4 × 106 PFU ml−1 EBIV. Infection rates (I), dissemination rates (J), transmission rates (K) and ovary infection rates (L) of mosquitoes at 4, 7, 10 and 14 days after feeding on blood meal containing 6.4 × 106 PFU ml−1 EBIV. Each dot represents an individual mosquito. The same letters indicate no significant differences (multiple comparisons using non-parametric Kruskal-Wallis analysis). Differences in the rates were analyzed with Fisher’s exact test (*: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.005 and ****: p ≤ 0.001).
Fig 2.
EBIV titers and infection rates of Ae. aegypti through intrathoracic inoculation.
EBIV titers (A) and infection rates (B) of mosquitoes at 4, 7, 10 and 14 days after injection with 340 PFU EBIV. EBIV titers (C) and infection rates (D) of mosquitoes injected with 0.34 to 340 PFU EBIV at 7 dpi. EBIV titers in gut (E), head (F), saliva (G) and ovary (H) samples of mosquitoes injected with 34 PFU EBIV at 2, 4, 7 and 10 dpi. Gut (I), head (J), saliva (K) and ovary (L) infection rates of mosquitoes injected with 34 PFU EBIV at 2, 4, 7 and 10 dpi. Each dot represents an individual mosquito. The same letters indicate no significant differences (multiple comparisons using non-parametric Kruskal-Wallis analysis). Differences in rates were analyzed with Fisher’s exact test (*: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.005 and ****: p ≤ 0.001).
Fig 3.
Immunohistochemical visualization and electron micrographs of EBIV in Ae. aegypti midgut and ovary.
Immunolocalization of EBIV antigen in midguts of mosquitoes with oral infection at 14 dpi (A) and intrathoracic inoculation at 7 dpi (B). Immunolocalization of EBIV antigen in ovaries of mosquitoes infected via intrathoracic inoculation at 7 dpi (C). Immunolocalization of EBIV antigen in midguts of mosquitoes fed blood without EBIV at 14 dpi (D) and mosquitoes injected with 100 nl RPMI 1640 at 7 dpi (E). Immunolocalization of EBIV antigen in ovaries from mock-injected mosquitoes at 7 dpi (F). F-actin was stained with phalloidin (green). The cell nucleus was stained with DAPI (blue). NC: nurse cells, FC: follicle cells. (G-J) Virions observed in gut are indicated by red arrows on electron micrographs. H and J are the enlarged insets of the boxes in G and I, respectively. (K-N) Virions observed in ovary are indicated by red arrows on electron micrographs. L and N are the enlarged insets of the boxes in K and M, respectively. EBIV virion clusters were detected using a mouse anti-EBIV polyclonal antibody and goat anti-mouse IgG labeled with red fluorescent secondary antibody.
Fig 4.
EBIV affects specific gene expression patterns in intrathoracically infected mosquitoes.
Significantly upregulated (orange) and downregulated (blue) genes in EBIV-infected compared with mock-infected mosquitoes at 2 dpi (A) and 7 dpi (B). (C) Genes displaying differential responses in EBIV-infected mosquitoes at 2 and 7 dpi were determined via a scatter plot of expression changes. KEGG pathway analyses of DEGs at 2 dpi and 7 dpi are presented in (D) and (E), respectively. Enriched GO categories associated with DEGs at 2 dpi and 7 dpi are shown in (F) and (G), respectively.