Mosquito Virus Research Articles

Aedes aegypti abundance, larval indices and risk for dengue virus transmission in Kinondoni district, Tanzania

BackgroundTanzania has experienced periodic dengue outbreaks with increased incidence since 2010. However, there is limited information on vector dynamics and transmission risk in most parts of the country. This study was conducted to determine Aedes mosquito abundance, larval indices and dengue virus infection rate as risk indicators for DENV transmission in Kinondoni district, Dar es Salaam, Tanzania.MethodsA cross-sectional study was conducted in three wards of Kinondoni district in Tanzania between December 2019 and January 2020. In each ward, three streets were randomly selected for adult and immature mosquito sampling. The adult mosquitoes were collected using Mosquito Magnet traps, while mosquito larvae and pupae were inspected in water-holding containers in the selected household compounds. The detection of dengue virus (DENV) in female Aedes mosquitoes was done using a one-step reverse transcription–polymerase chain reaction (RT–PCR) method.ResultsOf the 1416 adult female mosquitoes collected, Ae. aegypti accounted for 16.8% (n = 238). A total of 333 water-holding containers were inspected and 201 (60.4%) had at least an Aedes larvae or pupae. Water-holding containers supporting the breeding of Aedes larvae and pupae included discarded car tires, flowerpots and small and large plastic containers. The overall House Index, Container Index and Breteau Index were 55.1%, 60.4% and 114.2, respectively. None of the 763 female Aedes mosquitoes tested by RT–PCR was found to be infected with DENV.ConclusionThe presence and abundance Ae. aegypti mosquitoes and the large proportion of water-holding containers infested with the mosquito larvae and pupae put residents of Kinondoni district at high risk of DENV transmission. Our findings emphasize the need for continuous mosquito vector surveillance and control to prevent the possibility of future DENV outbreaks in Tanzania.

A validated triplex RT-qPCR protocol to simultaneously detect chikungunya, dengue and Zika viruses in mosquitoes.

Recently, the incidences of chikungunya, dengue and Zika infections have increased due to globalization and urbanization. It is vital that reliable detection tools become available to assess the viral prevalence within mosquito populations. Based on the previous publications on clinical diagnosis in human infections, for the first time, we described a customized triplex RT-qPCR protocol for simultaneous detection of chikungunya virus (CHIKV), dengue virus serotypes 1-4 (DENV1-4) and Zika virus (ZIKV) in mosquitoes. In preliminary assessment to determine the specificity and sensitivity of primers and probes, all six targets were detected individually with the following thresholds as indicated by calculated pfu equivalents: 3.96x100 for CHIKV, 3.80x101 for DENV1, 3.20x101 for DENV2, 8.00x104 for DENV3, 1.58x100 for DENV4, and 6.20x100 for ZIKV When tested in a full combination of six targets (CDZ mix), CHIKV, DENV1-4 mix or ZIKV were all detected with the thresholds of 1.32x100 for CHIKV, 3.79x100 for DENV1-4 and 2.06x100 for ZIKV All targets, individually or in full combination were detected in the mixtures of Aedes aegypti (L.) homogenate and viral lysates. A robust evaluation with three replicates in each of three plates for CHIKV, DENV1-4 and ZIKV individually or in full combination was conducted. In individual assays, CHIKV was detected to 3.96x10-1, DENV1-4 to 1.14x100 and ZIKV to 3.20x100. In full combination assays, CHIKV was detected to 1.32x104, DENV1-4 to 3.79x101 and ZIKV to 1.07x100. This triplex RT-qPCR assay appears to consistently detect all six targets and does not cross react with Ae. aegypti homogenate, making it a feasible, practical, and immediately adoptable protocol for use among vector control and other entities, particularly in the endemic areas of CHIKV, DENVs and ZIKV.

First report on the detection of Japanese encephalitis virus in fruit bats from India.

Japanese encephalitis (JE) is a mosquito borne viral zoonotic disease and JE virus (JEV) is responsible for causing several children deaths every year in India. Since 1978, cases of JE have been reported from Gorakhpur district of Uttar Pradesh state annually. The knowledge on the role played by wildlife reservoirs in the sylvatic transmission and maintenance of JE virus remains limited. Bats are reservoir hosts for several emerging and re-emerging viral pathogens but their role in zoonotic cycle of JEV has not been elucidated yet. In Gorakhpur district of Uttar Pradesh, 52 fruit bats were found dead on 26 May 2020. The post-mortem report of the bat samples conducted at the Indian Veterinary Research Institute stated that the bats died due to brain hemorrhage, caused by excessive heat. The brain tissue samples of the bats were subjected to investigation using molecular techniques to determine the presence of JEV. The present work reports for the first time the detection of JEV in brain samples of bats from India. The viral load ranging from 8 to 18 copies/reaction was detected in brain samples by TaqMan real Time RT-PCR. The low viral load might be the reason for the absence of apparent clinical signs in bats and suggests the probable role of fruit bats in maintaining the JEV in nature.

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.

Identification of potential new mosquito-associated viruses of adult Aedes aegypti mosquitoes from Tocantins state, Brazil.

Medically important arboviruses such as dengue virus (DENV), Zika virus (ZIKV), and chikungunya virus (CHIKV) are primarily transmitted by the globally distributed mosquito Aedes aegypti. Increasing evidence suggests that the transmission of some viruses can be influenced by mosquito-specific and mosquito-borne viruses. Advancements in high-throughput sequencing (HTS) and bioinformatics have expanded our knowledge on the richness of viruses harbored by mosquitoes. HTS was used to characterize the presence of virus sequences in wild-caught adult Ae. aegypti from Tocantins (TO) state, Brazil. Samples of mosquitoes were collected in four cities of Tocantins state and submitted to RNA isolation, followed by sequencing at an Illumina HiSeq platform. Our results showed initially by Krona the presence of 3% of the sequenced reads belonging to the viral database. After further analysis, the virus sequences were found to have homology to two viral families found in insects Phenuiviridae and Metaviridae. Three possible viral strains including putative new viruses were detected and named Phasi Charoen-like phasivirus isolate To-1 (PCLV To-1), Aedes aegypti To virus 1 (AAToV1), and Aedes aegypti To virus 2 (AAToV2). The results presented in this work contribute to the growing knowledge about the diversity of viruses in mosquitoes and might be useful for future studies on the interaction between insect-specific viruses and arboviruses.

A mutation-mediated evolutionary adaptation of Zika virus in mosquito and mammalian host.

Zika virus (ZIKV) caused millions of infections during its rapid and expansive spread from Asia to the Americas from 2015 to 2017. Here, we compared the infectivity of ZIKV mutants with individual stable substitutions which emerged throughout the Asian ZIKV lineage and were responsible for the explosive outbreaks in the Americas. A threonine (T) to alanine (A) mutation at the 106th residue of the ZIKV capsid (C) protein facilitated the transmission by its mosquito vector, as well as infection in both human cells and immunodeficient mice. A mechanistic study showed that the T106A substitution rendered the C a preferred substrate for the NS2B-NS3 protease, thereby facilitating the maturation of structural proteins and the formation of infectious viral particles. Over a complete "mosquito-mouse-mosquito" cycle, the ZIKV C-T106A mutant showed a higher prevalence of mosquito infection than did the preepidemic strain, thus promoting ZIKV dissemination. Our results support the contribution of this evolutionary adaptation to the occasional widespread reemergence of ZIKV in nature.

Dengue virus in humans and mosquitoes and their molecular characteristics in northeastern Thailand 2016-2018.

Dengue is hyperendemic in most Southeast Asian countries including Thailand, where all four dengue virus serotypes (DENV-1 to -4) have circulated over different periods and regions. Despite dengue cases being annually reported in all regions of Thailand, there is limited data on the relationship of epidemic DENV infection between humans and mosquitoes, and about the dynamics of DENV during outbreaks in the northeastern region. The present study was conducted in this region to investigate the molecular epidemiology of DENV and explore the relationships of DENV infection in humans and in mosquitoes during 2016–2018. A total of 292 dengue suspected patients from 11 hospitals and 902 individual mosquitoes (at patient’s houses and neighboring houses) were recruited and investigated for DENV serotypes infection using PCR. A total of 103 patients and 149 individual mosquitoes were DENV -positive. Among patients, the predominant DENV serotypes in 2016 and 2018 were DENV-4 (74%) and DENV-3 (53%) respectively, whereas in 2017, DENV-1, -3 and -4 had similar prevalence (38%). Additionally, only 19% of DENV infections in humans and mosquitoes at surrounding houses were serotypically matched, while 81% of infections were serotypically mismatched, suggesting that mosquitoes outside the residence may be an important factor of endemic dengue transmission. Phylogenetic analyses based on envelope gene sequences showed the genotype I of both DENV-1 and DENV-4, and co-circulation of the Cosmopolitan and Asian I genotypes of DENV-2. These strains were closely related to concurrent strains in other parts of Thailand and also similar to strains in previous epidemiological profiles in Thailand and elsewhere in Southeast Asia. These findings highlight genomic data of DENV in this region and suggest that people’s movement in urban environments may result in mosquitoes far away from the residential area being key determinants of DENV epidemic dynamics.

Symbiotic Interactions Between Mosquitoes and Mosquito Viruses

Mosquitoes not only transmit human and veterinary pathogens called arboviruses (arthropod-borne viruses) but also harbor mosquito-associated insect-specific viruses (mosquito viruses) that cannot infect vertebrates. In the past, studies investigating mosquito viruses mainly focused on highly pathogenic interactions that were easier to detect than those without visible symptoms. However, the recent advances in viral metagenomics have highlighted the abundance and diversity of viruses which do not generate mass mortality in host populations. Over the last decade, this has facilitated the rapid growth of virus discovery in mosquitoes. The circumstances around the discovery of mosquito viruses greatly affected how they have been studied so far. While earlier research mainly focused on the pathogenesis caused by DNA and some double-stranded RNA viruses during larval stages, more recently discovered single-stranded RNA mosquito viruses were heavily studied for their putative interference with arboviruses in female adults. Thus, many aspects of mosquito virus interactions with their hosts and host-microbiota are still unknown. In this context, considering mosquito viruses as endosymbionts can help to identify novel research areas, in particular in relation to their long-term interactions with their hosts (e.g. relationships during all life stages, the stability of the associations at evolutionary scales, transmission routes and virulence evolution) and the possible context-dependent range of interactions (i.e. beneficial to antagonistic). Here, we review the symbiotic interactions of mosquito viruses considering different aspects of their ecology, such as transmission, host specificity, host immune system and interactions with other symbionts within the host cellular arena. Finally, we highlight related research gaps in mosquito virus research.

Zika virus outbreak in Brazil under current and future climate

IntroductionZika virus (ZIKV) is primarily transmitted byAedes aegypti and Aedes albopictus mosquitoes between humans and non-human primates. Climate change may enhance virus reproduction in Aedes spp. mosquito populations, resulting in intensified ZIKV outbreaks. The study objective was to explore how an outbreak similar to the 2016 ZIKV outbreak in Brazil might unfold with projected climate change. MethodsA compartmental infectious disease model that included compartments for humans and mosquitoes was developed to fit the 2016 ZIKV outbreak data from Brazil using least squares optimization. To explore the impact of climate change, published polynomial relationships between temperature and temperature-sensitive mosquito population and virus transmission parameters (mosquito mortality, development rate, and ZIKV extrinsic incubation period) were used. Projections for future outbreaks were obtained by simulating transmission with effects of projected average monthly temperatures on temperature-sensitive model parameters at each of three future time periods: 2011–2040, 2041–2070, and 2071–2100. The projected future climate was obtained from an ensemble of regional climate models (RCMs) obtained from the Co-Ordinated Regional Downscaling Experiment (CORDEX) that used Representative Concentration Pathways (RCP) with two radiative forcing values, RCP4.5 and RCP8.5. A sensitivity analysis was performed to explore the impact of temperature-dependent parameters on the model outcomes. ResultsClimate change scenarios impacted the model outcomes, including the peak clinical case incidence, cumulative clinical case incidence, time to peak incidence, and the duration of the ZIKV outbreak. Comparing 2070–2100 to 2016, using RCP4.5, the peak incidence was 22,030 compared to 10,473; the time to epidemic peak was 12 compared to 9 weeks, and the outbreak duration was 52 compared to 41 weeks. Comparing 2070–2100 to 2016, using RCP8.5, the peak incidence was 21,786 compared to 10,473; the time to epidemic peak was 11 compared to 9 weeks, and the outbreak duration was 50 compared to 41weeks. The increases are due to optimal climate conditions for mosquitoes, with the mean temperature reaching 28 °C in the warmest months. Under a high emission scenario (RCP8.5), mean temperatures extend above optimal for mosquito survival in the warmest months. ConclusionOutbreaks of ZIKV in locations similar to Brazil are expected to be more intense with a warming climate. As climate change impacts are becoming increasingly apparent on human health, it is important to quantify the effect and use this knowledge to inform decisions on prevention and control strategies.

Characterization of the virome associated with Haemagogus mosquitoes in Trinidad, West Indies

Currently, there are increasing concerns about the possibility of a new epidemic due to emerging reports of Mayaro virus (MAYV) fever outbreaks in areas of South and Central America. Haemagogus mosquitoes, the primary sylvan vectors of MAYV are poorly characterized and a better understanding of the mosquito’s viral transmission dynamics and interactions with MAYV and other microorganisms would be important in devising effective control strategies. In this study, a metatranscriptomic based approach was utilized to determine the prevalence of RNA viruses in field-caught mosquitoes morphologically identified as Haemagogus janthinomys from twelve (12) forest locations in Trinidad, West Indies. Known insect specific viruses including the Phasi Charoen-like and Humaiata-Tubiacanga virus dominated the virome of the mosquitoes throughout sampling locations while other viruses such as the avian leukosis virus, MAYV and several unclassified viruses had a narrower distribution. Additionally, assembled contigs from the Ecclesville location suggests the presence of a unique uncharacterized picorna-like virus. Mapping of RNA sequencing reads to reference mitochondrial sequences of potential feeding host animals showed hits against avian and rodent sequences, which putatively adds to the growing body of evidence of a potentially wide feeding host-range for the Haemagogus mosquito vector.

Interactions of the Insect-Specific Palm Creek Virus with Zika and Chikungunya Viruses in Aedes Mosquitoes.

Palm Creek virus (PCV) is an insect-specific flavivirus that can interfere with the replication of mosquito-borne flaviviruses in Culex mosquitoes, thereby potentially reducing disease transmission. We examined whether PCV could interfere with arbovirus replication in Aedes (Ae.) aegypti and Ae. albopictus mosquitoes, major vectors for many prominent mosquito-borne viral diseases. We infected laboratory colonies of Ae. aegypti and Ae. albopictus with PCV to evaluate infection dynamics. PCV infection was found to persist to at least 21 days post-infection and could be detected in the midguts and ovaries. We then assayed for PCV–arbovirus interference by orally challenging PCV-infected mosquitoes with Zika and chikungunya viruses. For both arboviruses, PCV infection had no effect on infection and transmission rates, indicating limited potential as a method of intervention for Aedes-transmitted arboviruses. We also explored the hypothesis that PCV–arbovirus interference is mediated by the small interfering RNA pathway in silico. Our findings indicate that RNA interference is unlikely to underlie the mechanism of arbovirus inhibition and emphasise the need for empirical examination of individual pairs of insect-specific viruses and arboviruses to fully understand their impact on arbovirus transmission.

Uncovering the Worldwide Diversity and Evolution of the Virome of the Mosquitoes Aedes aegypti and Aedes albopictus.

Aedes aegypti, the yellow fever mosquito, and Aedes albopictus, the Asian tiger mosquito, are the most significant vectors of dengue, Zika, and Chikungunya viruses globally. Studies examining host factors that control arbovirus transmission demonstrate that insect-specific viruses (ISVs) can modulate mosquitoes’ susceptibility to arbovirus infection in both in vivo and in vitro co-infection models. While research is ongoing to implicate individual ISVs as proviral or antiviral factors, we have a limited understanding of the composition and diversity of the Aedes virome. To address this gap, we used a meta-analysis approach to uncover virome diversity by analysing ~3000 available RNA sequencing libraries representing a worldwide geographic range for both mosquitoes. We identified ten novel viruses and previously characterised viruses, including mononegaviruses, orthomyxoviruses, negeviruses, and a novel bi-segmented negev-like group. Phylogenetic analysis suggests close relatedness to mosquito viruses implying likely insect host range except for one arbovirus, the multi-segmented Jingmen tick virus (Flaviviridae) in an Italian colony of Ae. albopictus. Individual mosquito transcriptomes revealed remarkable inter-host variation of ISVs within individuals from the same colony and heterogeneity between different laboratory strains. Additionally, we identified striking virus diversity in Wolbachia infected Aedes cell lines. This study expands our understanding of the virome of these important vectors. It provides a resource for further assessing the ecology, evolution, and interaction of ISVs with their mosquito hosts and the arboviruses they transmit.

New records of California serogroup viruses in Aedes mosquitoes and first detection in simulioidae flies from Northern Canada and Alaska

An expected consequence of climate warming is an expansion of the geographical distribution of biting insects and associated arthropod-borne viruses (arboviruses). Emerging and re-emerging arboviruses that can affect human and animal health are likely to pose significant consequences for Northern communities where access to health resources is limited. In the North American Arctic, little is known about arboviruses. Thus, in 2019, we sampled biting insects in Nunavik, Northern Québec (Kuujjuaq), Nunavut (Igloolik, Karrak Lake and Cambridge Bay), Northwest Territories (Igloolik and Yellowknife) and Alaska (Fairbanks). The main objective was to detect the presence of California serogroup (CSG) viruses– a widespread group of arboviruses across North America and that are known to cause a wide range of symptoms, ranging from mild febrile illness to fatal encephalitis. Biting insects were captured twice daily for a 7-day period in mid-summer, using a standardised protocol consisting of 100 figure-eight movements of a sweep net. Captured specimens were separated by genus (mosquitoes) or by superfamily (other insects) and then grouped into pools of 75 by geographical locations. In total, 5079 Aedes mosquitoes and 1014 Simulioidae flies were caught. We report the detection of CSG viruses RNA in mosquitoes captured in Nunavut (Karrak Lake) and Nunavik (Kuujjuaq). We also report, for the first time in North America, the presence of CSG viruses RNA in Simulioidae flies. These results highlight the use of biting insects for tracking any future emergence of arboviruses in the North, thereby providing key information for public health in Northern communities.

Optimal pooled testing design for prevalence estimation under resource constraints

Accurate estimation of disease prevalence is essential for mitigation efforts. Due to limited testing resources, prevalence estimation is often conducted via pooled testing, in which multiple specimens are combined and tested via a single test. The pool design, i.e., the number and sizes of testing pools, has a substantial impact on estimation accuracy. Determining an optimal pool design is challenging, especially for emerging or seasonal diseases for which information on the status of the disease is unreliable or unavailable prior to testing. We develop novel optimization models for testing pool design under uncertainty and limited resources, and characterize structural properties of optimal pool designs. We apply our models to estimate the prevalence of West Nile virus in mosquitoes (the main vector of transmission to humans). Our findings suggest that estimation accuracy can be substantially improved over the status quo through the proposed optimal pool designs.

Spatio-temporal modelling of the first Chikungunya epidemic in an intra-urban setting: The role of socioeconomic status, environment and temperature.

Three key elements are the drivers of Aedes-borne disease: mosquito infestation, virus circulating, and susceptible human population. However, information on these aspects is not easily available in low- and middle-income countries. We analysed data on factors that influence one or more of those elements to study the first chikungunya epidemic in Rio de Janeiro city in 2016. Using spatio-temporal models, under the Bayesian framework, we estimated the association of those factors with chikungunya reported cases by neighbourhood and week. To estimate the minimum temperature effect in a non-linear fashion, we used a transfer function considering an instantaneous effect and propagation of a proportion of such effect to future times. The sociodevelopment index and the proportion of green areas (areas with agriculture, swamps and shoals, tree and shrub cover, and woody-grass cover) were included in the model with time-varying coefficients, allowing us to explore how their associations with the number of cases change throughout the epidemic. There were 13627 chikungunya cases in the study period. The sociodevelopment index presented the strongest association, inversely related to the risk of cases. Such association was more pronounced in the first weeks, indicating that socioeconomically vulnerable neighbourhoods were affected first and hardest by the epidemic. The proportion of green areas effect was null for most weeks. The temperature was directly associated with the risk of chikungunya for most neighbourhoods, with different decaying patterns. The temperature effect persisted longer where the epidemic was concentrated. In such locations, interventions should be designed to be continuous and to work in the long term. We observed that the role of the covariates changes over time. Therefore, time-varying coefficients should be widely incorporated when modelling Aedes-borne diseases. Our model contributed to the understanding of the spatio-temporal dynamics of an urban Aedes-borne disease introduction in a tropical metropolitan city.

Shifting From Sectoral to Integrated Surveillance by Changing Collaborative Practices: Application to West Nile Virus Surveillance in a Small Island State of the Caribbean.

After spreading in the Americas, West Nile virus was detected in Guadeloupe (French West Indies) for the first time in 2002. Ever since, several organizations have conducted research, serological surveys, and surveillance activities to detect the virus in horses, birds, mosquitoes, and humans. Organizations often carried them out independently, leading to knowledge gaps within the current virus' situation. Nearly 20 years after the first evidence of West Nile virus in the archipelago, it has not yet been isolated, its impact on human and animal populations is unknown, and its local epidemiological cycle is still poorly understood. Within the framework of a pilot project started in Guadeloupe in 2019, West Nile virus was chosen as a federative model to apply the “One Health” approach for zoonotic epidemiological surveillance and shift from a sectorial to an integrated surveillance system. Human, animal, and environmental health actors involved in both research and surveillance were considered. Semi-directed interviews and a Social Network Analysis were carried out to learn about the surveillance network structure and actors, analyze information flows, and identify communication challenges. An information system was developed to fill major gaps: users' needs and main functionalities were defined through a participatory process where actors also tested and validated the tool. Additionally, all actors shared their data, which were digitized, cataloged, and centralized, to be analyzed later. An R Shiny server was integrated into the information system, allowing an accessible and dynamic display of data showcasing all of the partners' information. Finally, a series of virtual workshops were organized among actors to discuss preliminary results and plan the next steps to improve West Nile Virus and vector-borne or emerging zoonosis surveillance. The actors are willing to build a more resilient and cooperative network in Guadeloupe with improved relevance, efficiency, and effectiveness of their work.

Assessment of Mosquito Collection Methods for Dengue Surveillance.

Several methods exist to collect and assess the abundance of dengue vector mosquitoes, i.e., morning adult collection, pupal collection, ovitraps, human landing, and larval collection. Several of these methods are officially implemented to monitor mosquito density and make decisions on treatments for dengue control. This monitoring is also constrained by the need to conduct this assessment on a “one point/one day” process, meaning that once the threshold of 100 households is reached, the assessment is made, and the collectors teams move to another place, thus preventing the use of long-term sampling methods. This diversity of methods might be a source of variability and lack of statistical significance. There is also a lack of published data regarding the efficacy of these methods. Furthermore, the Stegomyia indices are shown to be not reliable for assessing the risk of dengue outbreaks. A mosquito survey was, thus, conducted in 39 locations corresponding to 15 dengue endemic provinces in Indonesia by using the different adult and larval collection methods recommended nationwide. A total of 44,675 mosquitoes were collected. The single larva method was the most efficient. Out of a total of 89 dengue-positive pools, the most frequently encountered virus was DENV2, which made up half of the positive samples, followed by DENV3 and DENV1, respectively. Factor analysis of mixed data showed that no correlation could be found between any methods and the presence of dengue virus in mosquitoes. Moreover, no correlation could be found between any methods and the incidence of dengue. There was no consistency in the efficacy of a given method from one site to another. There was no correlation between any of the parameters considered, i.e., method, incidence of dengue, location, and the presence of dengue virus in mosquitoes.

Entomovirological Surveillance in Schools: Are They a Source for Arboviral Diseases Transmission?

Surveillance and control activities for virus-transmitting mosquitoes have primarily focused on dwellings. There is little information about viral circulation in heavily trafficked places such as schools. We collected and analyzed data to assess the presence and prevalence of dengue, chikungunya, and Zika viruses in mosquitoes, and measured Aedes indices in schools in Medellín (Colombia) between 2016–2018. In 43.27% of 2632 visits we collected Aedes adults, creating 883 pools analyzed by RT-PCR. 14.27% of pools yielded positive for dengue or Zika (infection rates of 1.75–296.29 for Aedes aegypti). Ae. aegypti was more abundant and had a higher infection rate for all studied diseases. Aedes indices varied over time. There was no association between Aedes abundance and mosquito infection rates, but the latter did correlate with cases of arboviral disease and climate. Results suggest schools are important sources of arbovirus and health agencies should include these sites in surveillance programs; it is essential to know the source for arboviral diseases transmission and the identification of the most population groups exposed to these diseases to research and developing new strategies.

A survey of mosquito-borne and insect-specific viruses in hospitals and livestock markets in western Kenya.

Aedes aegypti and Culex pipiens complex mosquitoes are prolific vectors of arboviruses that are a global threat to human and animal health. Increased globalization and ease of travel have facilitated the worldwide dissemination of these mosquitoes and the viruses they transmit. To assess disease risk, we determined the frequency of arboviruses in western Kenyan counties bordering an area of high arboviral activity. In addition to pathogenic viruses, insect-specific flaviviruses (ISFs), some of which are thought to impair the transmission of specific pathogenic arboviruses, were also evaluated. We trapped mosquitoes in the short and long rainy seasons in 2018 and 2019 at livestock markets and hospitals. Mosquitoes were screened for dengue, chikungunya and other human pathogenic arboviruses, ISFs, and their blood-meal sources as determined by high-resolution melting analysis of (RT-)PCR products. Of 6,848 mosquitoes collected, 89% were trapped during the long rainy season, with A. aegypti (59%) and Cx. pipiens sensu lato (40%) being the most abundant. Most blood-fed mosquitoes were Cx. pipiens s.l. with blood-meals from humans, chicken, and sparrow (Passer sp.). We did not detect dengue or chikungunya viruses. However, one Culex poicilipes female was positive for Sindbis virus, 30 pools of Ae. aegypti had cell fusing agent virus (CFAV; infection rate (IR) = 1.27%, 95% CI = 0.87%-1.78%); 11 pools of Ae. aegypti had Aedes flavivirus (AeFV; IR = 0.43%, 95% CI = 0.23%-0.74%); and seven pools of Cx. pipiens s.l. (IR = 0.23%, 95% CI = 0.1%-0.45%) and one pool of Culex annulioris had Culex flavivirus. Sindbis virus, which causes febrile illness in humans, can complicate the diagnosis and prognosis of patients with fever. The presence of Sindbis virus in a single mosquito from a population of mosquitoes with ISFs calls for further investigation into the role ISFs may play in blocking transmission of other arboviruses in this region.

Infection, dissemination, and transmission efficiencies of Zika virus in Aedes aegypti after serial passage in mosquito or mammalian cell lines or alternating passage in both cell types

BackgroundZika virus (ZIKV) is an arthropod-borne virus (arbovirus) with an urban transmission cycle that primarily involves humans and Aedes aegypti. Evidence suggests that the evolution of some arboviruses is constrained by their dependency on alternating between disparate (vertebrate and invertebrate) hosts. The goals of this study are to compare the genetic changes that occur in ZIKV after serial passaging in mosquito or vertebrate cell lines or alternate passaging in both cell types and to compare the replication, dissemination, and transmission efficiencies of the cell culture-derived viruses in Ae. aegypti.MethodsAn isolate of ZIKV originally acquired from a febrile patient in Yucatan, Mexico, was serially passaged six times in African green monkey kidney (Vero) cells or Aedes albopictus (C6/36) cells or both cell types by alternating passage. A colony of Ae. aegypti from Yucatan was established, and mosquitoes were challenged with the cell-adapted viruses. Midguts, Malpighian tubules, ovaries, salivary glands, wings/legs and saliva were collected at various times after challenge and tested for evidence of virus infection.ResultsGenome sequencing revealed the presence of two non-synonymous substitutions in the premembrane and NS1 regions of the mosquito cell-adapted virus and two non-synonymous substitutions in the capsid and NS2A regions of both the vertebrate cell-adapted and alternate-passaged viruses. Additional genetic changes were identified by intrahost variant frequency analysis. Virus maintained by continuous C6/36 cell passage was significantly more infectious in Ae. aegypti than viruses maintained by alternating passage and consecutive Vero cell passage.ConclusionsMosquito cell-adapted ZIKV displayed greater in vivo fitness in Ae. aegypti compared to the other viruses, indicating that obligate cycling between disparate hosts carries a fitness cost. These data increase our understanding of the factors that drive ZIKV adaptation and evolution and underscore the important need to consider the in vivo passage histories of flaviviruses to be evaluated in vector competence studies.Graphical abstract