Evaluation of Chikungunya Virus Infectious Events in Aedes Albopictus LR and Aedes Aegypti WE Mosquito Vectors

Arthropod-borne pathogens cause millions of human infections which are initiated by the feeding by an infected mosquito on a vertebrate, the interactions between the viruses and the mosquito vector that facilitate successful infection and transmission are still not fully understood. Chikungunya virus (CHIKV) is an alphavirus transmitted by Aedes albopictus and Aedes aegypti mosquitoes in tropical areas of Africa, Asia, and the islands of the Indian Ocean. In 2007 and 2009, CHIKV was transmitted outside these tropical areas to cause localized infections in people in Italy and France. Here we describe early CHIKV infectious events in Ae. albopictus derived C6/36 cells, in ex vivo Ae. albopictus LR mosquito midguts and in vivo in Ae. albopictus mosquitoes. As observed in mammalian cells, we demonstrate that CHIKV infection of mosquito cells depends on acidification of the endosome as indicated by significant inhibition following prophylactic treatment with the lysomotropic drugs chloroquine, ammonium chloride and monensin. While all three drugs inhibited CHIKV infection in C6/36 cells, ammonium chloride had less impact on cell mortality then the other drugs. These results demonstrate that inhibition of CHIKV infection in C6/36 cells is similar to that observed in mammalian cell lines and reaffirms the variability of entry pathways utilized by alphaviruses. Ex vivo infection of midguts after prophylactic treatment with these drugs also demonstrated inhibition of CHIKV infection with some correlation to our in vitro results. However, the highest concentration of chloroquine, ammonium chloride and monensin utilized in vitro and ex vivo did not inhibit CHIKV infection of in vivo Ae. albopictus LR mosquitoes. Tripling the concentrations of these drugs in the CHIKV infectious blood meal engorged by these mosquitoes also had no effect on inhibition of CHIKV. However, at 7dpi, increased viral titers in the treated mosquitoes suggest a potential mechanism of CHIKV infection enhancement in the mosquito midgut.
In order to temporally and spatially characterize CHIKV infection of Ae. albopictus midguts, a comparison of viral distribution in mosquitoes infected per os or by enema was conducted. Ae. albopictus infected with CHIKV LR 5’ GFP at a titer 106.95 TCID50/mL, were collected and analyzed for virus dissemination by visualizing GFP expression and titration up to 14 days post-infection. Additionally, midguts were dissected from the mosquitoes and imaged by fluorescent microscopy for comparison of midgut infection patterns between orally- and enema- infected mosquitoes. When virus was delivered via enema, mosquitoes displayed similar patterns of midgut infection to those observed in orally infected mosquitoes at 14 days post-infection. This work demonstrates that enema delivery of virus is a viable technique for infection of mosquitoes. Enema injection of mosquitoes may provide an alternative technique to intra-thoracic inoculation, because the enema delivery more closely models natural infection, and neither compromises midgut integrity nor involves a wound that can induce immune responses. Furthermore, unlike intra-thoracic delivery, the enema does not bypass midgut barriers to artificially infect tissues in the hemocoel of the mosquito. CHIKV infection of the midgut with CHIKV-GFP and CHIKV-Cherry viruses demonstrated homotypic exclusion of CHIKV occurs within the cells of the midgut of Ae. albopictus LR and Ae. aegypti WE mosquitoes, but not the midgut itself. These results correlate well with previously demonstrated homotypic exclusion of CHIKV in Ae. albopictus derived cell lines. Unlike VEEV and EEEV, CHIKV infection of these mosquito species presented with dispersed fluorophore expression; non-specific to any particular section of the midgut. Persistent and long lasting CHIKV infection of the midgut in both species of mosquito was indicated by the continued expression of GFP and Cherry fluorophores at 21 days post infection.
Reports of dually-infected patients have raised concern for the potential of dual virus transmission by mosquitoes. Heterotypic infection of Ae. albopictus LR and Ae. aegypti WE mosquitoes was performed with CHIKV and dengue virus serotype-2, New Guinea C strain (DENV-2 NGC) utilizing three approaches: 1) CHIKV infection with subsequent DENV-2 infection, 2) DENV-2 infection with subsequent CHIKV infection, and 3) simultaneous infection with CHIKV and DENV-2. Simultaneous infection of these mosquitoes with CHIKV and DENV-2 demonstrated limited infection of the mosquito heads and even lower rates of detection in the saliva collected from both mosquito species suggesting blood meals from a dual infected host offers little threat of continued transmission. Our results in Ae. aegypti WE mosquitoes, where the dual infection approach was the infection of CHIKV followed by DENV-2, demonstrated these mosquitoes had significantly higher (p<0.05) number of mosquitoes with DENV-2 infection of the head and saliva when compared to other Ae. aegypti WE mosquitoes infected with DENV-2 followed by CHIKV or when infected simultaneously with both viruses. The mechanism(s) by which this occurs has not been defined, but suggests CHIKV infection may compromise the mosquitoes immune response thereby enhancing DENV-2 dissemination and transmission. In Ae. albopictus LR mosquitoes infected with DENV-2 followed by CHIKV, significant higher (p<0.05) numbers of mosquitoes expressed DENV-2 in saliva when compared to other approaches of sequential infection in this species of mosquito. Additional comparison of Ae. albopictus LR mosquitoes infected with DENV-2 followed by CHIKV to the same species of mosquito simultaneously infected with CHIKV and DENV-2 resulted in a significantly higher (p<0.05) number of mosquitoes with both CHIKV and DENV-2 in their saliva.