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Zika virus (ZIKV) is a Flavivirus (Flaviviridae) transmitted to humans mainly by the bite of an infected Aedes mosquitoes. Aedes aegypti is the primary epidemic vector of ZIKV and Ae. albopictus, the secondary one. However, the epidemiological role of both Aedes species in Central Africa where Ae. albopictus was recently introduced is poorly characterized. Field-collected strains of Ae. aegypti and Ae. albopictus from different ecological settings in Central Africa were experimentally infected with a ZIKV strain isolated in West Africa. Mosquitoes were analysed at 14- and 21-days post-exposure. Both Ae. aegypti and Ae. albopictus were able to transmit ZIKV but with higher overall transmission efficiency for Ae. aegypti (57.9%) compared to Ae. albopictus (41.5%). In addition, disseminated infection and transmission rates for both Ae. aegypti and Ae. albopictus varied significantly according to the location where they were sampled from. We conclude that both Ae. aegypti and Ae. albopictus are able to transmit ZIKV and may intervene as active Zika vectors in Central Africa. These findings could contribute to a better understanding of the epidemiological transmission of ZIKV in Central Africa and develop suitable strategy to prevent major ZIKV outbreaks in this region., Author summary Zika virus (ZIKV), isolated for the first time in Uganda in 1947, is transmitted to human beings mainly by the bite of an infected mosquitoes belonging to the Aedes genus notably Ae. aegypti and Ae. albopictus. Both Aedes species are present in Central Africa, however their epidemiological role is poorly characterized. Here, we assessed the ability of Ae. aegypti and Ae. albopictus collected in different ecological settings in Central Africa to transmit a ZIKV strain isolated in West Africa. Analysis showed that both Ae. aegypti and Ae. albopictus are able to transmit ZIKV but with higher overall transmission efficiency for Ae. aegypti compared to Ae. albopictus. In addition, disseminated infection and transmission rates for both Aedes species varied significantly according to the sampling location. Overall, our result suggests that in Central Africa, Ae. aegypti is more competent to transmit ZIKV than Ae. albopictus although parameters such as the feeding behaviour, longevity and mosquito densities can modulate pathogens transmission in nature. These findings could contribute to a better understanding of the epidemiological transmission of ZIKV in Central Africa and develop suitable strategy to prevent major ZIKV outbreaks in this region.

Dengue virus (DENV) causes more human infections than any other mosquito-borne virus. The current lack of antiviral strategies has prompted genome-wide screens for host genes that are required for DENV infectivity. Earlier transcriptomic studies that identified DENV host factors in the primary vector Aedes aegypti used inbred laboratory colonies and/or pools of mosquitoes that erase individual variation. Here, we performed transcriptome sequencing on individual midguts in a field-derived Ae. aegypti population to identify new candidate host factors modulating DENV replication. We analyzed the transcriptomic data using an approach that accounts for individual co-variation between viral RNA load and gene expression. This approach generates a prediction about the agonist or antagonist effect of candidate genes on DENV replication based on the sign of the correlation between gene expression and viral RNA load. Using this method, we identified 39 candidate genes that went undetected by conventional pairwise comparison of gene expression levels between DENV-infected midguts and uninfected controls. Only four candidate genes were detected by both methods, emphasizing their complementarity. We demonstrated the value of our approach by functional validation of a candidate agonist gene encoding a sterol regulatory element-binding protein (SREBP), which was identified by correlation analysis but not by pairwise comparison. We confirmed that SREBP promotes DENV infection in the midgut by RNAi-mediated gene knockdown in vivo. We suggest that our approach for transcriptomic analysis can empower genome-wide screens for potential agonist or antagonist factors by leveraging inter-individual variation in gene expression. More generally, this method is applicable to a wide range of phenotypic traits displaying inter-individual variation., Author summary Dengue virus (DENV) is transmitted among humans by mosquitoes, primarily Aedes aegypti. Despite their potential as targets to interrupt DENV transmission, mosquito genes that modulate infection in Ae. aegypti remain largely unknown. Using a field-derived Ae. aegypti population, we observed substantial variation in DENV load in the mosquito midgut. We hypothesized that this inter-individual variation contained valuable information to identify host factors modulating viral infection. We analyzed single-midgut transcriptomes using an approach that takes advantage of inter-individual variation among infected mosquitoes. We demonstrated the added value of this method by identifying novel host factors during early DENV infection of Ae. aegypti that went undetected by conventional pairwise comparison between DENV-infected and control groups. We confirmed the agonist role of a candidate gene encoding a sterol regulatory element-binding protein, which underlines the importance of lipid metabolism during DENV infection of the mosquito midgut. Our method for transcriptomic analysis can enhance genome-wide screens for host factors by taking advantage of inter-individual variation. It is also applicable to a wide range of phenotypic traits displaying inter-individual variation.

An epidemic of Zika virus (ZIKV) illness that occurred in July 2007 on Yap Island in the Federated States of Micronesia prompted entomological studies to identify both the primary vector(s) involved in transmission and the ecological parameters contributing to the outbreak. Larval and pupal surveys were performed to identify the major containers serving as oviposition habitat for the likely vector(s). Adult mosquitoes were also collected by backpack aspiration, light trap, and gravid traps at select sites around the capital city. The predominant species found on the island was Aedes (Stegomyia) hensilli. No virus isolates were obtained from the adult field material collected, nor did any of the immature mosquitoes that were allowed to emerge to adulthood contain viable virus or nucleic acid. Therefore, laboratory studies of the probable vector, Ae. hensilli, were undertaken to determine the likelihood of this species serving as a vector for Zika virus and other arboviruses. Infection rates of up to 86%, 62%, and 20% and dissemination rates of 23%, 80%, and 17% for Zika, chikungunya, and dengue-2 viruses respectively, were found supporting the possibility that this species served as a vector during the Zika outbreak and that it could play a role in transmitting other medically important arboviruses., Author Summary Arthropod-borne viruses (arboviruses) cause significant human morbidity and mortality throughout the world. Zika virus, which is reported to be transmitted by Aedes (Stegomyia) species mosquitoes, caused an outbreak on the island of Yap, in the Federated States of Micronesia in 2007. This was the first described outbreak of Zika in Oceania, which has had several arbovirus outbreaks in the past. Diagnosing the outbreak was difficult due to the similarity in clinical symptoms between disease caused by Zika virus and other viruses. This work describes the efforts to identify the mosquito species that were responsible for transmission of the virus. While no virus was isolated from any species of mosquito collected during the current study, the predominant species found was Aedes hensilli and through the complementary laboratory studies, this mosquito was implicated as a probable vector for Zika virus. In addition, this species was found to be susceptible to both the medically important dengue-2 and chikungunya viruses.

Background The chikungunya (CHIK) outbreak that struck La Reunion Island in 2005 was preceded by few human cases of Dengue (DEN), but which surprisingly did not lead to an epidemic as might have been expected in a non-immune population. Both arboviral diseases are transmitted to humans by two main mosquito species, Aedes aegypti and Aedes albopictus. In the absence of the former, Ae. albopictus was the only species responsible for viral transmission on La Reunion Island. This mosquito is naturally super-infected with two Wolbachia strains, wAlbA and wAlbB. While Wolbachia does not affect replication of CHIK virus (CHIKV) in Ae. albopictus, a similar effect was not observed with DEN virus (DENV). Methods/Principal Findings To understand the weak vectorial status of Ae. albopictus towards DENV, we used experimental oral infections of mosquitoes from La Reunion Island to characterize the impact of Wolbachia on DENV infection. Viral loads and Wolbachia densities were measured by quantitative PCR in different organs of Ae. albopictus where DENV replication takes place after ingestion. We found that: (i) Wolbachia does not affect viral replication, (ii) Wolbachia restricts viral density in salivary glands, and (iii) Wolbachia limits transmission of DENV, as infectious viral particles were only detected in the saliva of Wolbachia-uninfected Ae. albopictus, 14 days after the infectious blood-meal. Conclusions We show that Wolbachia does not affect the replication of DENV in Ae. albopictus. However, Wolbachia is able to reduce viral infection of salivary glands and limit transmission, suggesting a role of Wolbachia in naturally restricting the transmission of DENV in Ae. albopictus from La Reunion Island. The extension of this conclusion to other Ae. albopictus populations should be investigated., Author Summary Aedes albopictus is an invasive species that is expanding its natural range of geographic distribution. While it was previously considered a secondary vector of different arboviruses, this mosquito species is involved in the most recent outbreaks of chikungunya but contributes weakly to dengue outbreaks. Ae. albopictus naturally carries two strains of the bacterium Wolbachia, wAlbA and wAlbB. Present in 20% of insect species, Wolbachia is an obligate intracellular symbiont mainly transmitted through females. When inoculated into some mosquito hosts, Wolbachia is able to shorten the adult life span and to block arbovirus transmission. We have previously shown that Wolbachia is not capable of limiting chikungunya replication in the mosquito vector. In this study, we show that the native Wolbachia is able to limit dengue transmission by restricting the delivery of infectious viral particles from the mosquito saliva when biting. Therefore, our results might explain the low vector competence of Ae. albopictus for dengue, and thus its weak contribution as an epidemic dengue vector.

Background Since the major outbreak in 2007 in the Yap Island, Zika virus (ZIKV) causing dengue-like syndromes has affected multiple islands of the South Pacific region. In May 2015, the virus was detected in Brazil and then spread through South and Central America. In December 2015, ZIKV was detected in French Guiana and Martinique. The aim of the study was to evaluate the vector competence of the mosquito spp. Aedes aegypti and Aedes albopictus from the Caribbean (Martinique, Guadeloupe), North America (southern United States), South America (Brazil, French Guiana) for the currently circulating Asian genotype of ZIKV isolated from a patient in April 2014 in New Caledonia. Methodology/Principal Findings Mosquitoes were orally exposed to an Asian genotype of ZIKV (NC-2014-5132). Upon exposure, engorged mosquitoes were maintained at 28°±1°C, a 16h:8h light:dark cycle and 80% humidity. 25–30 mosquitoes were processed at 4, 7 and 14 days post-infection (dpi). Mosquito bodies (thorax and abdomen), heads and saliva were analyzed to measure infection, dissemination and transmission, respectively. High infection but lower disseminated infection and transmission rates were observed for both Ae. aegypti and Ae. albopictus. Ae. aegypti populations from Guadeloupe and French Guiana exhibited a higher dissemination of ZIKV than the other Ae. aegypti populations examined. Transmission of ZIKV was observed in both mosquito species at 14 dpi but at a low level. Conclusions/Significance This study suggests that although susceptible to infection, Ae. aegypti and Ae. albopictus were unexpectedly low competent vectors for ZIKV. This may suggest that other factors such as the large naïve population for ZIKV and the high densities of human-biting mosquitoes contribute to the rapid spread of ZIKV during the current outbreak., Author Summary Zika virus (ZIKV) is an emerging mosquito-borne arbovirus causing dengue-like symptoms. This virus was commonly detected in Africa and Asia. Since its emergence in Yap Island in Micronesia in 2007, ZIKV reemerged in the South Pacific region in 2013 and ultimately reached the American continent in 2015. The human biting mosquito Aedes aegypti and the less anthropophilic Aedes albopictus have been incriminated as vectors of ZIKV. Our study showed that American populations of Ae. aegypti and Ae. albopictus were able to become infected and disseminate ZIKV within the mosquito general cavity at early days (4, 7) post-infection (dpi). Nevertheless, transmission was unexpectedly low and only detected at 14 dpi. Our findings will help in designing more adapted vector control strategies and limiting the impact of a new emerging threat on human health in the Americas as did the chikungunya in 2014.

Background Environmental factors such as temperature can alter mosquito vector competence for arboviruses. Results from recent studies indicate that daily fluctuations around an intermediate mean temperature (26°C) reduce vector competence of Aedes aeygpti for dengue viruses (DENV). Theoretical predictions suggest that the mean temperature in combination with the magnitude of the diurnal temperature range (DTR) mediate the direction of these effects. Methodology/Principal Findings We tested the effect of temperature fluctuations on Ae. aegypti vector competence for DENV serotype-1 at high and low mean temperatures, and confirmed this theoretical prediction. A small DTR had no effect on vector competence around a high (30°C) mean, but a large DTR at low temperature (20°C) increased the proportion of infected mosquitoes with a disseminated infection by 60% at 21 and 28 days post-exposure compared to a constant 20°C. This effect resulted from a marked shortening of DENV extrinsic incubation period (EIP) in its mosquito vector; i.e., a decrease from 29.6 to 18.9 days under the fluctuating vs. constant temperature treatment. Conclusions Our results indicate that Ae. aegypti exposed to large fluctuations at low temperatures have a significantly shorter virus EIP than under constant temperature conditions at the same mean, leading to a considerably greater potential for DENV transmission. These results emphasize the value of accounting for daily temperature variation in an effort to more accurately understand and predict the risk of mosquito-borne pathogen transmission, provide a mechanism for sustained DENV transmission in endemic areas during cooler times of the year, and indicate that DENV transmission could be more efficient in temperate regions than previously anticipated., Author Summary Mosquitoes in the wild are exposed to daily fluctuations in temperature, but in the laboratory, the effect of temperature on vector competence is generally assessed using constant temperatures. Recent studies demonstrate that realistic fluctuations in temperature around an intermediate mean (26°C) can alter life-history traits, population dynamics, and the ability of a mosquito to become infected with and transmit dengue virus (DENV). Here we tested how fluctuations around high and low mean temperatures influence vector competence and the extrinsic incubation period. Small fluctuations around a high mean temperature (∼8°C swings around 30°C) had no detectable effect on vector competence. Large fluctuations around a low mean (∼18°C swings around 20°C) demonstrate that only 18.9 days were required for 50% of DENV-exposed mosquitoes to develop a disseminated infection, compared to 29.6 days at constant 20°C. Twenty-eight days post-exposure to the infectious blood meal, 100% of mosquitoes tested had a disseminated infection under fluctuating temperatures, but under a constant temperature this proportion was only 42%. Reduced duration of extrinsic incubation increases the potential for pathogen transmission. Results indicate that the rate of dengue transmission by mosquitoes in temperate regions with natural fluctuations may be underestimated by experiments conducted under constant temperatures.