The Toll immune signaling pathway control conserved anti-dengue defenses across diverse Ae. aegypti strains and against multiple dengue virus serotypes

Abstract

Dengue virus has become one of the most important arboviral pathogens affecting the world today. The virus is transmitted among humans by the mosquitoes Aedes aegypti and Ae. albopictus. Like other vector-borne pathogens, this virus encounters innate immune defenses within the mosquito vector that limit infection. We have previously demonstrated the involvement of the Toll pathway in the anti-dengue defense at 7 days after infection. In the present study, we have investigated the activity of this immune signaling pathway against different dengue virus serotypes at the early stages of infection in laboratory and field-derived mosquito strains. Our studies corroborate the importance of the Toll pathway in the anti-dengue defense repertoire at 3 days after an infectious blood meal, when new virions are released from the midgut for dissemination and infection of other mosquito tissues. These immune defenses are furthermore conserved among different Ae. aegypti strains and can act against a broad range of dengue virus serotypes.

Figure 1

Dengue virus titers in mosquitoes with transient activation of the Toll pathway through cactus gene silencing (dsRNA CACT) and transientrepression of the Toll pathway (dsRNA MyD88) through MyD88 gene silencing. There is a detrimental effect of Cactus silencing on dengue virus replication in the mosquito midgut at 3 days post-infection. Virus titers were measured by plaque titration in the C6/36 cell line. (*, p<0.05 in Mann-Whitney U test. Error bars represent standard error of the mean).

Figure 2

Dengue virus titers in the midgut of MyD88-silenced mosquitoes challenged with DENV-4 (814669 strain) or DENV-2 (NGC strain). Repression of the Toll pathway led to a significant increase in dengue virus titers, demonstrating the importance of this innate immune pathway in the defense against different dengue virus serotypes at the early stages of infection. Virus titers and infection phenotype were assayed by plaque titration in C6/36 cells. (*,p<0.05 in Mann-Whitney U test. Error bars represent standard error of the mean).

Figure 3

Dengue virus titers in the midgut of two MyD88-silenced mosquito strains that were derived from field populations. As was seen for the lab strain of Ae. aegypti (Rockefeller strain), the two field-derived strains of Ae. aegypti used in these assays showed a significant increase in viral loads after the Toll pathway was disrupted (via MyD88 silencing), when compared to the GFP control. Virus titers and infection phenotype were assayed by plaque titration in C6/36 cells. (*, p<0.05 in Mann-Whitney U test. Error bars represent standard error of the mean).

Figure 4

Fold expression change in the expression of selected immune genes in the midgut (A) and abdominal fat body (B) tissues of DENV-2-infected mosquitoes, as compared to uninfected mosquitoes at 24 hr and 3 days post-blood meal. Dipt, diptericin; DefA, defensin A; DefC, defensin C; LysG, Lysozyme G; CecA, cecropin A; Gam, gambicin; Atta, attacin. (Error bars represent standard error of the mean)