MicroRNAs and other small RNAs in Aedes aegypti saliva and salivary glands following chikungunya virus infection.

Mosquito saliva facilitates blood feeding through the anti-haemostatic, anti-inflammatory and immunomodulatory properties of its proteins. However, the potential contribution of non-coding RNAs to host manipulation is still poorly understood. We analysed small RNAs from Aedes aegypti saliva and salivary glands and show here that chikungunya virus-infection triggers both the siRNA and piRNA antiviral pathways with limited effects on miRNA expression profiles. Saliva appears enriched in specific miRNA subsets and its miRNA content is well conserved among mosquitoes and ticks, clearly pointing to a non-random sorting and occurrence. Finally, we provide evidence that miRNAs from Ae. aegypti saliva may target human immune and inflammatory pathways, as indicated by prediction analysis and searching for experimentally validated targets of identical human miRNAs. Overall, we believe these observations convincingly support a scenario where both proteins and miRNAs from mosquito saliva are injected into vertebrates during blood feeding and contribute to the complex vector-host-pathogen interactions.

Aedes mosquitoes in the emerging threat of urban yellow fever transmission.

This last decade has seen a resurgence of yellow fever (YF) in historical endemic regions and repeated attempts of YF introduction in YF-free countries such as the Asia-Pacific region and the Caribbean. Infected travellers are the main entry routes in these regions where competent mosquito vectors proliferate in appropriate environmental conditions. With the discovery of the 17D vaccine, it was thought that YF would be eradicated. Unfortunately, it was not the case and, contrary to dengue, chikungunya and Zika, factors that cotribute to YF transmission remain under investigation. Today, all the signals are red and it is very likely that YF will be the next pandemic in the YF-free regions where millions of people are immunologically naïve. Unlike COVID-19, YF is associated with a high case-fatality rate and a high number of deaths are expected. This review gives an overview of global YF situation, including the non-endemic Asia-Pacific region and the Caribbean where Aedes aegypti is abundantly distributed, and also proposes different hypotheses on why YF outbreaks have not yet occurred despite high records of travellers importing YF into these regions and what role Aedes mosquitoes play in the emergence of urban YF.

Transgenic refractory Aedes aegypti lines are resistant to multiple serotypes of dengue virus.

The areas where dengue virus (DENV) is endemic have expanded rapidly, driven in part by the global spread of Aedes species, which act as disease vectors. DENV replicates in the mosquito midgut and is disseminated to the mosquito's salivary glands for amplification. Thus, blocking virus infection or replication in the tissues of the mosquito may be a viable strategy for reducing the incidence of DENV transmission to humans. Here we used the mariner Mos1 transposase to create an Aedes aegypti line that expresses virus-specific miRNA hairpins capable of blocking DENV replication. These microRNA are driven by the blood-meal-inducible carboxypeptidase A promoter or by the polyubiquitin promoter. The transgenic mosquitoes exhibited significantly lower infection rates and viral titers for most DENV serotypes 7 days after receiving an infectious blood meal. The treatment was also effective at day 14 post infection after a second blood meal had been administered. In viral transmission assay, we found there was significantly reduced transmission in these lines. These transgenic mosquitoes were effective in silencing most of the DENV genome; such an approach may be employed to control a dengue fever epidemic.

Risk of yellow fever virus transmission in the Asia-Pacific region.

Historically endemic to Sub-Saharan Africa and South America, yellow fever is absent from the Asia-Pacific region. Yellow fever virus (YFV) is mainly transmitted by the anthropophilic Aedes mosquitoes whose distribution encompasses a large belt of tropical and sub tropical regions. Increasing exchanges between Africa and Asia have caused imported YFV incidents in non-endemic areas, which are threatening Asia with a new viral emergence. Here, using experimental infections of field-collected mosquitoes, we show that Asian-Pacific Aedes mosquitoes are competent vectors for YFV. We observe that Aedes aegypti populations from Singapore, Taiwan, Thailand, and New Caledonia are capable of transmitting YFV 14 days after oral infections, with a number of viral particles excreted from saliva reaching up to 23,000 viral particles. These findings represent the most comprehensive assessment of vector competence and show that Ae. aegypti mosquitoes from the Asia-Pacific region are highly competent to YFV, corroborating that vector populations are seemingly not a brake to the emergence of yellow fever in the region.

Experimental adaptation of dengue virus 1 to Aedes albopictus mosquitoes by in vivo selection.

In most of the world, Dengue virus (DENV) is mainly transmitted by the mosquito Aedes aegypti while in Europe, Aedes albopictus is responsible for human DENV cases since 2010. Identifying mutations that make DENV more competent for transmission by Ae. albopictus will help to predict emergence of epidemic strains. Ten serial passages in vivo in Ae. albopictus led to select DENV-1 strains with greater infectivity for this vector in vivo and in cultured mosquito cells. These changes were mediated by multiple adaptive mutations in the virus genome, including a mutation at position 10,418 in the DENV 3'UTR within an RNA stem-loop structure involved in subgenomic flavivirus RNA production. Using reverse genetics, we showed that the 10,418 mutation alone does not confer a detectable increase in transmission efficiency in vivo. These results reveal the complex adaptive landscape of DENV transmission by mosquitoes and emphasize the role of epistasis in shaping evolutionary trajectories of DENV variants.

A Review: Wolbachia-Based Population Replacement for Mosquito Control Shares Common Points with Genetically Modified Control Approaches.

The growing expansion of mosquito vectors has made mosquito-borne arboviral diseases a global threat to public health, and the lack of licensed vaccines and treatments highlight the urgent need for efficient mosquito vector control. Compared to genetically modified control strategies, the intracellular bacterium Wolbachia, endowing a pathogen-blocking phenotype, is considered an environmentally friendly strategy to replace the target population for controlling arboviral diseases. However, the incomplete knowledge regarding the pathogen-blocking mechanism weakens the reliability of a Wolbachia-based population replacement strategy. Wolbachia infections are also vulnerable to environmental factors, temperature, and host diet, affecting their densities in mosquitoes and thus the virus-blocking phenotype. Here, we review the properties of the Wolbachia strategy as an approach to control mosquito populations in comparison with genetically modified control methods. Both strategies tend to limit arbovirus infections but increase the risk of selecting arbovirus escape mutants, rendering these strategies less reliable.

Estimating the risk of arbovirus transmission in Southern Europe using vector competence data.

Arboviral diseases such as chikungunya, dengue, and Zika viruses have been threatening the European countries since the introduction in 1979 of the major vector Aedes albopictus. In 2017, more than three hundred of CHIKV autochthonous cases were reported in Italy, highlighting the urgent need for a risk assessment of arboviral diseases in European countries. In this study, the vector competence for three major arboviruses were analyzed in eight Ae. albopictus populations from Europe. Here we show that Southern European Ae. albopictus were susceptible to CHIKV, DENV-1 and ZIKV with the highest vector competence for CHIKV. Based on vector competence data and vector distribution, a prediction risk map for CHIKV was generated stressing the fear of CHIKV and to a lesser extent, of other arboviruses for Europe, calling us for new public health strategies.

Evolution and biological significance of flaviviral elements in the genome of the arboviral vector Aedes albopictus.

Since its genome details are publically available, the mosquito Aedes albopictus has become the central stage of attention for deciphering multiple biological and evolutionary aspects at the root of its success as an invasive species. Its genome of 1,967 Mb harbours an unusual high number of non-retroviral integrated RNA virus sequences (NIRVS). NIRVS are enriched in piRNA clusters and produce piRNAs, suggesting an antiviral effect. Here, we investigated the evolutionary history of NIRVS in geographically distant Ae. albopictus populations by comparing genetic variation as derived by neutral microsatellite loci and seven selected NIRVS. We found that the evolution of NIRVS was far to be neutral with variations both in their distribution and sequence polymorphism among Ae. albopictus populations. The Flaviviral elements AlbFlavi2 and AlbFlavi36 were more deeply investigated in their association with dissemination rates of dengue virus (DENV) and chikungunya virus (CHIKV) in Ae. albopictus at both population and individual levels. Our results show a complex association between NIRVS and DENV/CHIKV opening a new avenue for investigating the functional role of NIRVS as antiviral elements shaping vector competence of mosquitoes to arboviruses.

Assessing the Potential Interactions between Cellular miRNA and Arboviral Genomic RNA in the Yellow Fever Mosquito, Aedes aegypti.

Although the role of exogenous small interfering RNA (siRNA) and P-element induced wimpy testis (PIWI)-interacting RNA (piRNA) pathways in mosquito antiviral immunity is increasingly better understood, there is still little knowledge regarding the role of mosquito cellular microRNA (miRNA). Identifying direct interactions between the mosquito miRNAs and the RNA genome of arboviruses and choosing the relevant miRNA candidates to explore resulting antiviral mechanisms are critical. Here, we carried out genomic analyses to identify Aedes aegypti miRNAs that potentially interact with various lineages and genotypes of chikungunya, dengue, and Zika viruses. By using prediction tools with distinct algorithms, several miRNA binding sites were commonly found within different genotypes/and or lineages of each arbovirus. We further analyzed those miRNAs that could target more than one arbovirus, required a low energy threshold to form miRNA-viralRNA (vRNA) complexes, and predicted potential RNA structures using RNAhybrid software. We predicted miRNA candidates that might participate in regulating arboviral replication in Ae. aegypti. Even without any experimental validation, which should be done as a next step, this study can shed further light on the role of miRNA in mosquito innate immunity and targets for future studies.

Synthetic miRNAs induce dual arboviral-resistance phenotypes in the vector mosquito Aedes aegypti.

Mosquito-borne arboviruses are responsible for recent dengue, chikungunya, and Zika pandemics. The yellow-fever mosquito, Aedes aegypti, plays an important role in the transmission of all three viruses. We developed a miRNA-based approach that results in a dual resistance phenotype in mosquitoes to dengue serotype 3 (DENV-3) and chikungunya (CHIKV) viruses. The target viruses are from two distinct arboviral families and the antiviral mechanism is designed to function through the endogenous miRNA pathway in infected mosquitoes. Challenge experiments showed reductions in viral transmission efficiency of transgenic mosquitoes. Several components of mosquito fitness were examined, and transgenic mosquitoes with the PUb promoter showed minor fitness costs at all developing stages. Further development of these strains with gene editing tools could make them candidates for releases in population replacement strategies for sustainable control of multiple arbovirus diseases.

Potential of Aedes aegypti populations in Madeira Island to transmit dengue and chikungunya viruses.

BACKGROUND: Aedes spp. mosquitoes mainly transmit the arboviruses dengue virus (DENV) and chikungunya virus (CHIKV) in urban areas, causing a severe public health problem. In 2012-2013, a major dengue outbreak occurred on Madeira Island where the mosquito Aedes aegypti was the only vector. Up to now, the competence of Ae. aegypti populations from Madeira to transmit DENV or CHIKV remains unknown. This study aimed to assess experimentally the ability of Ae. aegypti populations from Madeira to transmit these viruses. RESULTS: By orally exposing mosquitoes to CHIKV (NC/2011-568) and DENV-2 (Bangkok), the vector competence of two field-collected Ae. aegypti populations, i.e. Funchal and Paúl do Mar, was evaluated. We found that both populations were similarly infected and ensured the dissemination and transmission of CHIKV at the same rates. With DENV-2, viral dissemination was significantly higher in the Funchal population compared to Paúl do Mar. We found no significant differences in transmission rates between populations. CONCLUSIONS: To our knowledge, this study has demonstrated for the first time the ability of temperate European Ae. aegypti populations from Madeira to transmit DENV and CHIKV. As our results suggest, there is a potential risk for the local transmission of DENV and CHIKV if introduced to Madeira or continental Europe where Aedes albopictus is present. Our results highlight the need for continuing vector surveillance and control on Madeira Island to future-proof the Island against mosquito-borne epidemics.

Aedes aegypti mosquitoes from Guadeloupe (French West Indies) are able to transmit yellow fever virus.

The recent yellow fever epidemic in Brazil has raised the concern of outbreaks in neighboring countries, particularly in the Caribbean region where the vector Aedes aegypti is predominant. This threat comes from the past when in the Americas, this disease caused devastating urban epidemics. We report the vector competence of Ae. aegypti from Guadeloupe for yellow fever virus by determining different parameters describing virus infection, dissemination, and transmission. The results indicate that Ae. aegypti Guadeloupe are susceptible to yellow fever virus with viral particles detected in mosquito saliva at 14 and 21 days post-infection. Local authorities and more broadly, international organizations should maintain the active surveillance of Aedes mosquitoes and the spreading of human cases from South America.

Differential Transmission of Antiviral Drug-Resistant Chikungunya Viruses by Aedes Mosquitoes.

The chikungunya virus (CHIKV) is transmitted by female Aedes aegypti and Aedes albopictus mosquitoes, mostly present in (sub)tropical regions. No antivirals are available to treat CHIKV infections. If antiviral drugs are proven efficient to treat CHIKV-infected patients, it will be pivotal to determine whether drug-resistant viruses can be transmitted from one human to another by their mosquito vectors. We orally infected Aedes aegypti mosquitoes with a blood meal containing wild-type or drug-resistant CHIKV variants (i.e., MADTPres CHIKV, with mutation in the nsP1 gene, and T-705res CHIKV, with mutation in the RNA-dependent RNA polymerase [RdRp] gene). Viral loads were quantified in bodies (infection), heads (dissemination), and saliva (transmission) of individual mosquitoes. The infection rate of the resistant viruses was similar to that of the wild-type virus. However, the dissemination of T-705res CHIKV was markedly decreased compared to wild-type and MADTPres CHIKV. Furthermore, T-705res CHIKV was only transmitted in the saliva at day 20 postinfection (p.i.), whereas transmission of wild-type CHIKV was observed at day 3 p.i. The attenuated phenotype of the T-705res virus was confirmed in mosquito cell culture, whereas the replication fitness in Vero cells was similar to that of the wild type. In bodies and heads of mosquitoes infected with the resistant variants, the resistant phenotype and genotype were retained. Also in the saliva, the resistant genotype of MADTPres CHIKV was maintained. Our results illustrate that the fitness of drug-resistant variants should be evaluated in both hosts to be able to select antiviral drugs with a limited risk for the spread of drug resistance by mosquitoes.IMPORTANCE Because of its global reemergence and unusual morbidities associated with infection, the chikungunya virus (CHIKV) has become a substantial public health problem. However, no antivirals are currently available to treat CHIKV infections. If antiviral drugs will prove to be efficient to treat CHIKV-infected patients, it will be essential to understand if drug-resistant viruses can be transmitted from one human to another by the mosquito. We therefore orally infected Aedes mosquitoes with drug-resistant CHIKV variants and determined the replication and transmission levels. One of the antiviral drug-resistant CHIKV variants could efficiently replicate and disseminate in both laboratory and field-collected mosquitoes. In addition, this variant retained its drug-resistant genotype in the saliva. In contrast, the other drug-resistant variant was markedly attenuated in mosquitoes. Our results illustrate that extra caution for drug resistance should be considered when developing an antiarbovirus antiviral in order to minimize the risk of spreading drug resistance by mosquitoes.

Combining Wolbachia-induced sterility and virus protection to fight Aedes albopictus-borne viruses.

Among the strategies targeting vector control, the exploitation of the endosymbiont Wolbachia to produce sterile males and/or invasive females with reduced vector competence seems to be promising. A new Aedes albopictus transinfection (ARwP-M) was generated by introducing wMel Wolbachia in the ARwP line which had been established previously by replacing wAlbA and wAlbB Wolbachia with the wPip strain. Various infection and fitness parameters were studied by comparing ARwP-M, ARwP and wild-type (SANG population) Ae. albopictus sharing the same genetic background. Moreover, the vector competence of ARwP-M related to chikungunya, dengue and zika viruses was evaluated in comparison with ARwP. ARwP-M showed a 100% rate of maternal inheritance of wMel and wPip Wolbachia. Survival, female fecundity and egg fertility did not show to differ between the three Ae. albopictus lines. Crosses between ARwP-M males and SANG females were fully unfertile regardless of male age while egg hatch in reverse crosses increased from 0 to about 17% with SANG males aging from 3 to 17 days. When competing with SANG males for SANG females, ARwP-M males induced a level of sterility significantly higher than that expected for an equal mating competitiveness (mean Fried index of 1.71 instead of 1). The overall Wolbachia density in ARwP-M females was about 15 fold higher than in ARwP, mostly due to the wMel infection. This feature corresponded to a strongly reduced vector competence for chikungunya and dengue viruses (in both cases, 5 and 0% rates of transmission at 14 and 21 days post infection) with respect to ARwP females. Results regarding Zika virus did not highlight significant differences between ARwP-M and ARwP. However, none of the tested ARwP-M females was capable at transmitting ZIKV. These findings are expected to promote the exploitation of Wolbachia to suppress the wild-type Ae. albopictus populations.