Evaluation of Aedes aegypti,Aedes albopictus,and Culex quinquefasciatus Mosquitoes Competence to Oropouche virus Infection

The emergence of new human viral pathogens and re-emergence of several diseases are of particular concern in the last decades. Oropouche orthobunyavirus (OROV) is an arbovirus endemic to South and Central America tropical regions, responsible to several epidemic events in the last decades. There is little information regarding the ability of OROV to be transmitted by urban/peri-urban mosquitoes, which has limited the predictability of the emergence of permanent urban transmission cycles. Here, we evaluated the ability of OROV to infect, replicate, and be transmitted by three anthropophilic and urban species of mosquitoes, Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus. We show that OROV is able to infect and efficiently replicate when systemically injected in all three species tested, but not when orally ingested. Moreover, we find that, once OROV replication has occurred in the mosquito body, all three species were able to transmit the virus to immunocompromised mice during blood feeding. These data provide evidence that OROV is restricted by the midgut barrier of three major urban mosquito species, but, if this restriction is overcome, could be efficiently transmitted to vertebrate hosts. This poses a great risk for the emergence of permanent urban cycles and geographic expansion of OROV to other continents.     

Tissue Barriers to Arbovirus Infection in Mosquitoes

Arthropod-borne viruses (arboviruses) circulate in nature between arthropod vectors and vertebrate hosts. Arboviruses often cause devastating diseases in vertebrate hosts, but they typically do not cause significant pathology in their arthropod vectors. Following oral acquisition of a viremic bloodmeal from a vertebrate host, the arbovirus disease cycle requires replication in the cellular environment of the arthropod vector. Once the vector has become systemically and persistently infected, the vector is able to transmit the virus to an uninfected vertebrate host. In order to systemically infect the vector, the virus must cope with innate immune responses and overcome several tissue barriers associated with the midgut and the salivary glands. In this review we describe, in detail, the typical arbovirus infection route in competent mosquito vectors. Based on what is known from the literature, we explain the nature of the tissue barriers that arboviruses are confronted with in a mosquito vector and how arboviruses might surmount these barriers. We also point out controversial findings to highlight particular areas that are not well understood and require further research efforts.     

Expressing the Pro-Apoptotic Reaper Protein via Insertion into the Structural Open Reading Frame of Sindbis Virus Reduces the Ability to Infect Aedes aegypti Mosquitoes

Arboviruses continue to threaten a significant portion of the human population, and a better understanding is needed of the determinants of successful arbovirus infection of arthropod vectors. Avoiding apoptosis has been shown to be one such determinant. Previous work showed that a Sindbis virus (SINV) construct called MRE/rpr that expresses the Drosophila pro-apoptotic protein Reaper via a duplicated subgenomic promoter had a reduced ability to orally infect Aedes aegypti mosquitoes at 3 days post-blood meal (PBM), but this difference diminished over time as virus variants containing deletions in the inserted reaper gene rapidly predominated. In order to further clarify the effect of midgut apoptosis on disseminated infection in Ae. aegypti, we constructed MRE/rprORF, a version of SINV containing reaper inserted into the structural open reading frame (ORF) as an in-frame fusion. MRE/rprORF successfully expressed Reaper, replicated similarly to MRE/rpr in cell lines, induced apoptosis in cultured cells, and caused increased effector caspase activity in mosquito midgut tissue. Mosquitoes that fed on blood containing MRE/rprORF developed significantly less midgut and disseminated infection when compared to MRE/rpr or a control virus up to at least 7 days PBM, when less than 50% of mosquitoes that ingested MRE/rprORF had detectable disseminated infection, compared with around 80% or more of mosquitoes fed with MRE/rpr or control virus. However, virus titer in the minority of mosquitoes that became infected with MRE/rprORF was not significantly different from control virus. Deep sequencing of virus populations from ten mosquitoes infected with MRE/rprORF indicated that the reaper insert was stable, with only a small number of point mutations and no deletions being observed at frequencies greater than 1%. Our results indicate that expression of Reaper by this method significantly reduces infection prevalence, but if infection is established then Reaper expression has limited ability to continue to suppress replication.     

Investigation of Biological Factors Contributing to Individual Variation in Viral Titer after Oral Infection of Aedes aegypti Mosquitoes by Sindbis Virus

The mechanisms involved in determining arbovirus vector competence, or the ability of an arbovirus to infect and be transmitted by an arthropod vector, are still incompletely understood. It is well known that vector competence for a particular arbovirus can vary widely among different populations of a mosquito species, which is generally attributed to genetic differences between populations. What is less understood is the considerable variability (up to several logs) that is routinely observed in the virus titer between individual mosquitoes in a single experiment, even in mosquitoes from highly inbred lines. This extreme degree of variation in the virus titer between individual mosquitoes has been largely ignored in past studies. We investigated which biological factors can affect titer variation between individual mosquitoes of a laboratory strain of Aedes aegypti, the Orlando strain, after Sindbis virus infection. Greater titer variation was observed after oral versus intrathoracic infection, suggesting that the midgut barrier contributes to titer variability. Among the other factors tested, only the length of the incubation period affected the degree of titer variability, while virus strain, mosquito strain, mosquito age, mosquito weight, amount of blood ingested, and virus concentration in the blood meal had no discernible effect. We also observed differences in culture adaptability and in the ability to orally infect mosquitoes between virus populations obtained from low and high titer mosquitoes, suggesting that founder effects may affect the virus titer in individual mosquitoes, although other explanations also remain possible.     

Superinfection Exclusion in Mosquitoes and Its Potential as an Arbovirus Control Strategy

The continuing emergence of arbovirus disease outbreaks around the world, despite the use of vector control strategies, warrants the development of new strategies to reduce arbovirus transmission. Superinfection exclusion, a phenomenon whereby a primary virus infection prevents the replication of a second closely related virus, has potential to control arbovirus disease emergence and outbreaks. This phenomenon has been observed for many years in plants, insects and mammalian cells. In this review, we discuss the significance of identifying novel vector control strategies, summarize studies exploring arbovirus superinfection exclusion and consider the potential for this phenomenon to be the basis for novel arbovirus control strategies.     

Infection of adult Aedes aegypti and Ae. albopictus mosquitoes with the entomopathogenic fungus Metarhizium anisopliae

This study describes a laboratory investigation on the use of the insect-pathogenic fungus Metarhizium anisopliae against adult Aedes aegypti and Ae. albopictus mosquitoes. At a dosage of 1.6 x 10(10)conidia/m(2), applied on material that served as a mosquito resting site, an average of 87.1+/-2.65% of Ae. aegypti and 89.3+/-2.2% of Ae. albopictus became infected with the fungus. The life span of fungus-contaminated mosquitoes of both species was significantly reduced compared to uninfected mosquitoes. LT(50)-values of fungus-contaminated mosquitoes ranged between 3.1+/-0.2 days (male Ae. aegypti) and 4.1+/-0.3 days (female Ae. aegypti). LT(50)-values of uncontaminated mosquitoes ranged from 17.7+/-0.4 days (female Ae. albopictus) to 19.7+/-0.6 days (male Ae. albopictus). These results indicate that both mosquito species are highly susceptible to infection with this entomopathogen. Requirements for developing and incorporating this biological control method into current strategies to control major diseases vectored by these species, such as dengue fever, are discussed.     

Vector Competence of the Invasive Mosquito Species Aedes koreicus for Arboviruses and Interference with a Novel Insect Specific Virus

The global spread of invasive mosquito species increases arbovirus infections. In addition to the invasive species Aedes albopictus and Aedes japonicus, Aedes koreicus has spread within Central Europe. Extensive information on its vector competence is missing. Ae. koreicus from Germany were investigated for their vector competence for chikungunya virus (CHIKV), Zika virus (ZIKV) and West Nile virus (WNV). Experiments were performed under different climate conditions (27 ± 5 °C; 24 ± 5 °C) for fourteen days. Ae. koreicus had the potential to transmit CHIKV and ZIKV but not WNV. Transmission was exclusively observed at the higher temperature, and transmission efficiency was rather low, at 4.6% (CHIKV) or 4.7% (ZIKV). Using a whole virome analysis, a novel mosquito-associated virus, designated Wiesbaden virus (WBDV), was identified in Ae. koreicus. Linking the WBDV infection status of single specimens to their transmission capability for the arboviruses revealed no influence on ZIKV transmission. In contrast, a coinfection of WBDV and CHIKV likely has a boost effect on CHIKV transmission. Due to its current distribution, the risk of arbovirus transmission by Ae. koreicus in Europe is rather low but might gain importance, especially in regions with higher temperatures. The impact of WBDV on arbovirus transmission should be analyzed in more detail.     

Larval environmental stress alters Aedes aegypti competence for Sindbis virus

Objective To evaluate how stress at the larval stage alters adult mosquito performance and susceptibility to viral infection. Methods We used a model system consisting of Sindbis virus (SINV) and the yellow fever mosquito Aedes aegypti. Larvae were either reared under optimal conditions (control) or exposed to one of four types of stressors; suboptimal nutrients, starvation, elevated temperature, and a low dose of the insecticide malathion and adult females were fed SINV infectious blood meal. Differential expressions of stress, immune‐specific and detoxification genes was measured in fourth instar larvae (HSP70, HSP83, cecropin, defensin, transferrin and CYP6Z6) and 3‐day‐old females (cecropin, defensin, transferrin) to identify plausible molecular mechanisms associated with mosquito response to stress. Results There were stress‐specific variations in mosquito performance (survival, development time, female size), but all stressors had a consistent effect of significantly increasing susceptibility to viral infection and dissemination relative to the controls. Three genes were up‐regulated in fourth instar larvae exposed to temperature stress (cecropin, defensin and CYP6Z6) compared to single genes in suboptimal nutrient (cecropin) and malathion (transferrin) stress treatments and down‐regulation of all the six genes in starvation treatments. In adult samples, transferrin was up‐regulated in all but starvation treatments while defensin was up‐regulated in starvation and temperature stress treatments. Conclusions Stress during larval development may cause alterations in adult mosquito phenotype and immunity that can increase their susceptibility to pathogens.     

Breeding habitats and larval indices of Aedes aegypti and Ae. albopictus in the residential areas of Calcutta City.

Aedes aegypti and Ae. albopictus larvae were found breeding in almost all indoor and outdoor, temporary and/or permanent collections of water, either alone or in association with each other in residential areas of the city, the former throughout the year (June'97-May'98) and the latter during monsoons and post-monsoons only. Ae. aegypti showed preference for breeding in chowbachhas, indoors and Ae. albopictus for collections of water in flower pots and discarded containers and outdoors. The larval indices of both the species were highest during monsoons and post-monsoons.     

Dengue Infection Susceptibility of Five Aedes aegypti Populations from Manaus (Brazil) after Challenge with Virus Serotypes 1–4

The successful spread and maintenance of the dengue virus (DENV) in mosquito vectors depends on their viral infection susceptibility, and parameters related to vector competence are the most valuable for measuring the risk of viral transmission by mosquitoes. These parameters may vary according to the viral serotype in circulation and in accordance with the geographic origin of the mosquito population that is being assessed. In this study, we investigated the effect of DENV serotypes (1–4) with regards to the infection susceptibility of five Brazilian Ae. aegypti populations from Manaus, the capital of the state of Amazonas, Brazil. Mosquitoes were challenged by oral infection with the DENV serotypes and then tested for the presence of the arbovirus using quantitative PCR at 14 days post-infection, which is the time point that corresponds to the extrinsic incubation period of Ae. aegypti when reared at 28 °C. Thus, we were able to determine the infection patterns for DENV-1, -2, -3 and -4 in the mosquito populations. The mosquitoes had both interpopulation and inter-serotype variation in their viral susceptibilities. All DENV serotypes showed a similar tendency to accumulate in the body in a greater amount than in the head/salivary gland (head/SG), which does not occur with other flaviviruses. For DENV-1, DENV-3, and DENV-4, the body viral load varied among populations, but the head/SG viral loads were similar. Differently for DENV-2, both body and head/SG viral loads varied among populations. As the lack of phenotypic homogeneity represents one of the most important reasons for the long-term fight against dengue incidence, we expect that this study will help us to understand the dynamics of the infection patterns that are triggered by the distinct serotypes of DENV in mosquitoes.     

Detection of Aedes (Stegomyia) albopictus skus. Mosquitoes in the Russian Federation

Aedes (Stegomyia) albopictus Skus. mosquitoes were for the first time collected in the Bolshoi Sochi region in the Russian Federetion. 16 females and 1 male were morphologically and genetically identified as Ae. albopictus and Aedes aegypti, respectively.     

Vector Competence of Mosquitoes from Germany for Sindbis Virus

Transmission of arthropod-borne viruses (arboviruses) are an emerging global health threat in the last few decades. One important arbovirus family is the Togaviridae, including the species Sindbis virus within the genus Alphavirus. Sindbis virus (SINV) is transmitted by mosquitoes, but available data about the role of different mosquito species as potent vectors for SINV are scarce. Therefore, we investigated seven mosquito species, collected from the field in Germany (Ae. koreicus, Ae. geniculatus, Ae. sticticus, Cx. torrentium, Cx. pipiens biotype pipiens) as well as lab strains (Ae. albopictus, Cx. pipiens biotype molestus, Cx. quinquefasciatus), for their vector competence for SINV. Analysis was performed via salivation assay and saliva was titrated to calculate the amount of infectious virus particles per saliva sample. All Culex and Aedes species were able to transmit SINV. Transmission could be detected at all four investigated temperature profiles (of 18 ± 5 °C, 21 ± 5 °C, 24 ± 5 °C or 27 ± 5 °C), and no temperature dependency could be observed. The concentration of infectious virus particles per saliva sample was in the same range for all species, which may suggest that all investigated mosquito species are able to transmit SINV in Germany.     

Genetic structure of Aedes aegypti populations in Chiang Mai (Thailand) and relation with dengue transmission

We analysed the population genetic structure and differentiation regarding vector competence for a dengue virus of 15 Aedes aegypti samples collected from Chiang Mai in northern Thailand. Based on polymorphism of 10 isoenzyme loci, genetic differentiation was confirmed among samples collected in different subdistricts (high FST values and P < 0.05). Based on infection rate for a dengue 2 virus, susceptibilities were similar in mosquitoes collected in San Nuea subdistrict and in Choeng Doi subdistrict, and were heterogeneous in populations sampled in other subdistricts. These findings are discussed and related to insecticide treatments.     

Releasing incompatible males drives strong suppression across populations of wild and Wolbachia-carrying Aedes aegypti in Australia

Releasing sterile or incompatible male insects is a proven method of population management in agricultural systems with the potential to revolutionize mosquito control. Through a collaborative venture with the “Debug” Verily Life Sciences team, we assessed the incompatible insect technique (IIT) with the mosquito vector Aedes aegypti in northern Australia in a replicated treatment control field trial. Backcrossing a US strain of Ae. aegypti carrying Wolbachia wAlbB from Aedes albopictus with a local strain, we generated a wAlbB2-F4 strain incompatible with both the wild-type (no Wolbachia) and wMel-Wolbachia Ae. aegypti now extant in North Queensland. The wAlbB2-F4 strain was manually mass reared with males separated from females using Verily sex-sorting technologies to obtain no detectable female contamination in the field. With community consent, we delivered a total of three million IIT males into three isolated landscapes of over 200 houses each, releasing ∼50 males per house three times a week over 20 wk. Detecting initial overflooding ratios of between 5:1 and 10:1, strong population declines well beyond 80% were detected across all treatment landscapes when compared to controls. Monitoring through the following season to observe the ongoing effect saw one treatment landscape devoid of adult Ae. aegypti early in the season. A second landscape showed reduced adults, and the third recovered fully. These encouraging results in suppressing both wild-type and wMel-Ae. aegypti confirms the utility of bidirectional incompatibility in the field setting, show the IIT to be robust, and indicate that the removal of this arbovirus vector from human-occupied landscapes may be achievable.

Mosquitoes transmit parasites and viruses that infect hundreds of millions of humans annually. Globally, one invasive species—Aedes aegypti (Linnaeus)—is responsible for the greatest transmission of arboviruses, causing diseases including yellow fever, dengue, chikungunya, and Zika (1). The paucity of effective vaccines for most of these diseases and observed concerns for both growing insecticide resistance and insecticide’s adverse effects on other beneficial species have prompted renewed interest in species-specific biological control methods (2).Releasing sterile or incompatible male insects en masse is one method of insect population control currently under development (3). The sterile insect technique (SIT) uses the release of sterile male insects that search and mate with wild females to prevent subsequent production of offspring. The SIT has been successfully used to control various insect pest species (4), including the New World screwworm fly (5), the tsetse fly (6), the Mediterranean fruit fly (7), and the apple codling moth (8). Previous SIT technologies have been hampered by their need for radiation or chemical sterilents, which can compromise male fitness, requiring the release of more sterile males to compensate for their fitness decline (9, 10). Furthermore, the development of genetic modification to generate sterile male Ae. aegypti mosquitoes has been hampered by perceived environmental risks as well as strong regulatory issues and community acceptance (11).The first applications of SIT to mosquitoes encountered mixed successes during the 1970s and ’80s (12), as it proved difficult to produce sufficient numbers of competitive sterile males to suppress natural populations. More recently, elegant transgenic approaches have generated sterile male mosquitoes for the malaria vector Anopheles gambiae (13), the dengue vector Ae. aegypti (3, 14), and the Asian tiger mosquito Aedes albopictus (15). Releases of transgenic sterile Ae. aegypti by Oxitec Ltd in Grand Cayman have demonstrated an effective reduction of these mosquitoes (16).Reproductively incompatible males can now be produced using a maternally inherited gram-negative endosymbiotic bacterium Wolbachia pipientis (17, 18). Many of these strains carry a cytoplasmic incompatibility (CI) phenotype, where males of one Wolbachia strain can be reproductively incompatible with females that do not carry the strain; thus, females lay eggs that fail to hatch (18). This scenario provides an opportunity to reassess the SIT using what is now termed the incompatible insect technique [IIT (19)]. The SIT is generally more effective when females are not released (20), as sterile female insects can still damage crops, transmit disease, or simply distract sterile males from searching out wild mates. In the case of mosquito SIT and IIT, releasing males means only releasing mosquitoes which do not bite, have fewer risks, and encourage community acceptance.The primary aim for this study was to demonstrate the effectiveness of an Ae. aegypti IIT in a replicated treatment control study using males infected with a Wolbachia wAlbB strain from Ae. albopictus. Australia’s northern Queensland towns have endemic populations of Ae. aegypti (21), some of which have recently been transformed to carry the wMel Wolbachia strain (transfected from Drosophila melanogaster) in order to reduce arbovirus transmission risk (22): this phenotype provides arbovirus-blocking properties (23). We hypothesized that by selecting isolated towns and suburbs in tropical northern Queensland, we could demonstrate strong Ae. aegypti suppression over one season. By monitoring over the following season, we also sought to test whether suppression during one season would have a subsequent impact on populations the following season. At the outset of this study, all Ae. aegypti populations in the study site were wild type (no Wolbachia). However, during preparations for our IIT experiment—and at the request of the Queensland state health authority—all experimental landscape populations were transformed by high levels of wMel-infected Ae. aegypti released by Eliminate Dengue (now the World Mosquito Program [WMP]) (24). Subsequently, we released wAlbB2-infected males into a mosaic population of both wild type and wMel-Ae. aegypti to induce incompatible mating. We observed strong suppression over 20 wk, with bidirectional incompatibility in a field setting—and evidence of an ongoing suppression effect lasting into the next season.     

Neotropical Sylvatic Mosquitoes and Aedes aegypti Are Not Competent to Transmit 17DD Attenuated Yellow Fever Virus from Vaccinated Viremic New World Non-Human Primates

Beside humans, thousands of non-human primates (NHPs) died during the recent outbreak caused by the yellow fever virus (YFV) in Brazil. Vaccination of NHPs against YFV with the YF 17DD attenuated virus has emerged as a public health strategy, as it would reduce sylvatic transmission while also preserving endangered susceptible species. The hypothesis of establishing an uncontrolled transmission of this attenuated virus in nature was raised. We assessed vector competence of four sylvatic mosquito species, Haemagogus leucocelaenus, Haemagogus janthinomys/capricornii, Sabethes albiprivus, and Sabethes identicus, as well as the urban vector Aedes aegypti for YF 17DD attenuated vaccine virus when fed directly on eleven viremic lion tamarins or artificially challenged with the same virus. No infection was detected in 689 mosquitoes engorged on viremic lion tamarins whose viremia ranged from 1.05 × 10³ to 6.61 × 10³ FFU/mL, nor in those artificially taking ≤ 1 × 10³ PFU/mL. Low viremia presented by YF 17DD-vaccinated New World NHPs combined with the low capacity and null dissemination ability in sylvatic and domestic mosquitoes of this attenuated virus suggest no risk of its transmission in nature. Thus, vaccination of captive and free-living NHPs against YFV is a safe public health strategy.     

Transmission potential of two chimeric Chikungunya vaccine candidates in the urban mosquito vectors, Aedes aegypti and Ae. albopictus

Chikungunya virus (CHIKV) is an emerging, mosquito-borne alphavirus that has caused major epidemics in Africa and Asia. We developed chimeric vaccine candidates using the non-structural protein genes of either Venezuelan equine encephalitis virus (VEEV) attenuated vaccine strain TC-83 or a naturally attenuated strain of eastern equine encephalitis virus (EEEV) and the structural genes of CHIKV. Because the transmission of genetically modified live vaccine strains is undesirable because of the potentially unpredictable evolution of these viruses as well as the potential for reversion, we evaluated the ability of these vaccines to infect the urban CHIKV vectors, Aedes aegypti and Ae. albopictus. Both vaccine candidates exhibited significantly lower infection and dissemination rates compared with the parent alphaviruses. Intrathoracic inoculations indicated that reduced infectivity was mediated by midgut infection barriers in both species. These results indicate a low potential for transmission of these vaccine strains in the event that a vaccinee became viremic.     

First record of Ae. albopictus (Skuse 1894), in Central African Republic

Methods Mosquito aquatic stages were collected in domestic and peri‐domestic areas, and epidemic risk indexes (Breteau, Container) were calculated for each prospected location. Adult female mosquitoes were captured by human landing catches, while larvae were sampled by inspecting artificial and natural breeding sites in randomly selected premises. Results Seventy‐eight adults Aedes albopictus were collected in Bangui and Bayanga. Mosquito biting rate and abundance were, respectively, 0.33–1.70 bites/human/hour and 14.6% in Bangui and 0.04–0.16 and 0.4% in Bayanga. Larval sampling revealed a large diversity of water container harbouring the species in Bangui, Bayanga, Nola and Salo including unused containers, old tires, vehicle carcasses, buckets, barrels and stem of bamboo. The epidemic risk indices were erratic according to the location, ranging between 1.5–27.6 for Breteau and 1.3–47.1 for Container. Conclusion This is the first record of Ae. albopictus in two bioclimatic zones of CAR This observation emphasizes the need to further investigate its potential impact on dengue and chikungunya viruses transmission regarding their recent emergences in Africa (Côte d’Ivoire, Senegal, Mali, Somalia, Gabon, Cape Verde Islands).     

Identification of bacteria and bacteria-associated chemical cues that mediate oviposition site preferences by Aedes aegypti

The yellow fever mosquito, Aedes aegypti, the global vector of dengue and yellow fever, is inexorably linked to water-filled human-made containers for egg laying and production of progeny. Oviposition is stimulated by cues from water containers, but the nature and origin of these cues have not been elucidated. We showed that mosquito females directed most of their eggs to bamboo and white-oak leaf infusions, and only a small fraction of the eggs were laid in plain water containers. In binary choice assays, we demonstrated that microorganisms in leaf infusions produced oviposition-stimulating kairomones, and using a combination of bacterial culturing approaches, bioassay-guided fractionation of bacterial extracts, and chemical analyses, we now demonstrate that specific bacteria-associated carboxylic acids and methyl esters serve as potent oviposition stimulants for gravid Ae. aegypti. Elucidation of these compounds will improve understanding of the chemical basis of egg laying behavior of Ae. aegypti, and the kairomones will likely enhance the efficacy of surveillance and control programs for this disease vector of substantial global public health importance.     

First Evidence of West Nile Virus Overwintering in Mosquitoes in Germany

Mosquitoes collected from mid-December 2020 to early March 2021 from hibernacula in northeastern Germany, a region of West Nile virus (WNV) activity since 2018, were examined for WNV-RNA. Among the 6101 mosquitoes tested in 722 pools of up to 12 specimens, one pool of 10 Culex pipiens complex mosquitoes collected in early March 2021 in the cellar of a medieval castle in Rosslau, federal state of Saxony-Anhalt, tested positive. Subsequent mosquito DNA analysis produced Culex pipiens biotype pipiens. The pool homogenate remaining after nucleic acid extraction failed to grow the virus on Vero and C6/36 cells. Sequencing of the viral NS2B-NS3 coding region, however, demonstrated high homology with virus strains previously collected in Germany, e.g., from humans, birds, and mosquitoes, which have been designated the East German WNV clade. The finding confirms the expectation that WNV can overwinter in mosquitoes in Germany, facilitating an early start to the natural transmission season in the subsequent year. On the other hand, the calculated low infection prevalence of 0.016–0.20%, depending on whether one or twelve of the mosquitoes in the positive pool was/were infected, indicates a slow epidemic progress and mirrors the still-hypoendemic situation in Germany. In any case, local overwintering of the virus in mosquitoes suggests its long-term persistence and an enduring public health issue.     

Brazilian Populations of Aedes aegypti Resistant to Pyriproxyfen Exhibit Lower Susceptibility to Infection with Zika Virus

Zika virus (ZIKV) infection has caused devastating consequences in Brazil as infections were associated with neurological complications in neonates. Aedes aegypti is the primary vector of ZIKV, and the evolution of insecticide resistance (IR) in this species can compromise control efforts. Although relative levels of phenotypic IR in mosquitoes can change considerably over time, its influence on vector competence for arboviruses is unclear. Pyriproxyfen (PPF)-resistant populations of Ae. aegypti were collected from five municipalities located in Northeast of Brazil, which demonstrated different resistance levels; low (Serrinha, Brumado), moderate (Juazeiro do Norte, Itabuna), and high (Quixadá). Experimental per os infection using ZIKV were performed with individuals from these populations and with an insecticide susceptible strain (Rockefeller) to determine their relative vector competence for ZIKV. Although all populations were competent to transmit ZIKV, mosquitoes derived from populations with moderate to high levels of IR exhibited similar or lower susceptibility to ZIKV infection than those from populations with low IR or the susceptible strain. These observations suggest an association between IR and arbovirus infection, which may be attributable to genetic hitchhiking. The use of PPF to control Brazilian Ae. aegypti may be associated with an indirect benefit of reduced susceptibility to infection, but no changes in disseminated infection and transmission of ZIKV among PPF-resistant phenotypes.