Impact of simultaneous exposure to arboviruses on infection and transmission by Aedes aegypti mosquitoes

Abstract

The recent emergence of both chikungunya and Zika viruses in the Americas has significantly expanded their distribution and has thus increased the possibility that individuals may become infected by more than one Aedes aegypti-borne virus at a time. Recent clinical data support an increase in the frequency of coinfection in human patients, raising the likelihood that mosquitoes could be exposed to multiple arboviruses during one feeding episode. The impact of coinfection on the ability of relevant vector species to transmit any of these viruses (that is, their vector competence) has not been determined. Thus, we here expose Ae. aegypti mosquitoes to chikungunya, dengue-2 or Zika viruses, both individually and as double and triple infections. Our results show that these mosquitoes can be infected with and can transmit all combinations of these viruses simultaneously. Importantly, infection, dissemination and transmission rates in mosquitoes are only mildly affected by coinfection.

Figure 1. Global distribution of three arboviruses.

This map depicts countries with past or current autochthonous transmission of CHIKV, DENV and ZIKV. It was generated using the free online tool https://mapchart.net/detworld.html and is based on data provided by the CDC, PAHO, WHO, the National Institute for Communicable Disease (NICD-NHLS), as well as a review of literature. Other countries are also at risk and may have had unreported cases.

Figure 2. Virus replication kinetics and experimental design.

Virus growth curves were used to determine GE per ml (a), PFU per ml (b) and GE/PFU ratio (c) after infection of Vero cells with CHIKV (blue), DENV-2 (black) and ZIKV (green). Arrows indicate the time points used to collect fresh virus stocks for bloodmeals. Data points represent the mean of five biological replicates; error bars represent s.d. The experimental design of mosquito infections/coinfections is shown in d.

Figure 3. Susceptibility of Aedes aegypti to arbovirus infection after single and dual exposure.

Ae. aegypti mosquitoes were exposed to CHIKV, DENV-2 or ZIKV either individually or in combination by infectious bloodmeal. Infection rates for individual viruses after single exposure (yellow triangle) with CHIKV (a,b), DENV-2 (a,c) or ZIKV (b,c), and parallel co-exposures (blue square) with CHIKV/DENV-2 (a), CHIKV/ZIKV (b) and DENV-2/ZIKV (c) are presented. These values represent infection rates for each virus independent of coinfection status. Coinfection rates (that is, mosquitoes positive for both viruses) after co-exposure are presented separately (red square). Since there was no significant difference in the infection rate between time points (Supplementary Fig. 1), the combined proportion (%) from all replicates and time points are presented with 95% confidence interval (error bars). Total mosquito numbers were as follows: (a) CHIKV (n=221), DENV-2 (n=221), CHIKV/DENV-2 (n=222); (b) CHIKV (n=279), ZIKV (n=261), CHIKV/ZIKV (n=280); and (c) DENV-2 (n=221), ZIKV (n=180), DENV-2/ZIKV (n=238). Statistical significance was determined by Fisher's exact test (*P<0.05).

Figure 4. Dissemination and transmission dynamics in single and double infections.

Dissemination rates (a,c,e) and transmission rates (b,d,f) for CHIKV (a,b), DENV-2 (c,d) and ZIKV (e,f) in single and double infected mosquitoes. The overall percentage and 95% confidence interval from two independent experiments is shown. Statistical significance as indicated (*P<0.05; **P<0.01) was calculated using Fisher's exact test.

Figure 5. RNA levels in saliva of single and coinfected Aedes aegypti.

GE/saliva are shown for samples positive for CHIKV (a,b), DENV-2 (c,d) and ZIKV (e,f) at both 7 d.p.i. (a,c,e) and 14 d.p.i. (b,d,f). The geometric mean is indicated and error bars represent the 95% confidence interval. Statistical significance was determined using one-way ANOVA with multiple comparisons.