Mosquito pollination of plants: an overview of their role and an assessment of the possible contribution of disease vectors.

Mosquitoes visit flowers to obtain sugar or other nutrients and therefore possibly serve as major or minor pollinators of some plant species. They also often derive plant nutrients from other sources, such as extrafloral nectaries and honeydew. In a few cases, the plant-mosquito relationship is close, and mosquito pollination has been confirmed. Most plant species visited by mosquitoes, however, appear to depend on multiple means of pollination, particularly other flower-feeding insects. In addition, most mosquito species visit the flowers of many kinds of plants, possibly dispersing pollen in both biologically meaningful and irrelevant ways. This apparent lack of selectivity by both plants and mosquitoes liberates each of them from dependence on an unreliable pollen vehicle or nutrient source. A hypothetical pollinating role for the two top vectors of devastating human-disease pathogens, Anopheles gambiae or Aedes aegypti, relies on indirect evidence. So far, this evidence suggests that their participation in pollen transfer of native, introduced, or beneficial plants is negligible. The few plant species likely to be pollinated by these vectors are mostly invasive, harmful weeds associated with humans. That conclusion draws support from four characteristics of these vectors: (1) the numerous alternative potential pollinators of the flowers they visit; (2) their common use of diverse non-floral sources of nutrients; (3) the females' infrequent sugar feeding and heavy reliance on human blood for energy; and (4) their relatively low population densities. From these traits it follows that focused suppression or elimination of these two vectors, by whatever means, is highly unlikely to have adverse effects on pollination in endemic biotic communities or on ornamental plants or food crops.

Isolation and sequencing of Orthoflavivirus ilheusense from mosquitoes collected in the Brazilian cerrado, West-Central region, Brazil.

The Orthoflavivirus ilheusense (ILHV) is an arbovirus that was first isolated in Brazil in 1944 during an epidemiologic investigation of yellow fever. Is a member of the Flaviviridae family and it belongs to the antigenic complex of the Ntaya virus group. Psorophora ferox is the primary vector of ILHV and this study presents the isolation and phylogenetic analysis of ILHV in a pool of Ps. ferox collected in the state of Goiás in 2021. Viral isolation tests were performed on Vero cells and C6/36 clones. The indirect immunofluorescence test (IFI) was used to confirm the positivity of the sample. The positive sample underwent RT-qPCR, sequencing, and phylogenetic analysis. This is the first report of ILHV circulation in this municipality and presented close relationship between this isolate and another ILHV isolate collected in the city of Belém (PA).

Unveiling the Peptidase Network Orchestrating Hemoglobin Catabolism in Rhodnius prolixus.

Chagas disease is transmitted to humans by obligatory hematophagous insects of Triatominae subfamily, which feeds on various hosts to acquire their nutritional sustenance derived from blood proteins. Hemoglobin (Hb) digestion is a pivotal metabolic feature of triatomines, representing a key juncture in their competence toward Trypanosoma cruzi; however, it remains poorly understood. To explore the Hb digestion pathway in Rhodnius prolixus, a major Chagas disease vector, we employed an array of approaches for activity profiling of various midgut-associated peptidases using specific substrates and inhibitors. Dissecting the individual contribution of each peptidase family in Hb digestion has unveiled a predominant role played by aspartic proteases and cathepsin B-like peptidases. Determination of peptidase-specific cleavage sites of these key hemoglobinases, in conjunction with mass spectrometry-based identification of in vivo Hb-derived fragments, has revealed the intricate network of peptidases involved in the Hb digestion pathway. This network is initiated by aspartic proteases and subsequently sustained by cysteine proteases belonging to the C1 family. The process is continued simultaneously by amino and carboxypeptidases. The comprehensive profiling of midgut-associated aspartic proteases by quantitative proteomics has enabled the accurate revision of gene annotations within the A1 family of the R. prolixus genome. Significantly, this study also serves to illuminate a potentially important role of the anterior midgut in blood digestion. The expanded repertoire of midgut-associated proteases presented in this study holds promise for the identification of novel targets aimed at controlling the transmission of Chagas disease.

Molecular Identification, Multigenic Characterization and Phylogenetic Analysis of Ctenocephalides Canis Fleas from Humans and Domestic Animals in Iran.

BACKGROUND: Fleas belonging to the Pulicidae are prevalent ectoparasites infesting mammals and birds in Iran. This study focused on genetically identifying and characterizing Ctenocephalides canis collected both off-host and infesting humans and various domestic animals in the country. METHODS: A total of 918 adult flea samples were collected from 10 sites in western and northwestern Iran between April 2018 and May 2019. Out of these, 71 specimens were found off-host, while the remaining fleas were collected from humans (121), sheep (126), goats (184), and dogs (416). Morphological identification at the genus level was performed on all fleas, and ten selected specimens selected based on the sampling sites and hosts were subjected to molecular detection at the species level by using partial amplification and sequencing of the internal transcribed spacer 1 and 2, as well as the cytochrome oxidase I (COXI) markers. RESULTS: The morphological identification confirmed all fleas as Ctenocephalides spp. Alignment and phylogenetic analysis of nuclear and mitochondrial partial sequences confirmed the presence of C. canis. However, molecular divergence was observed among the ten isolates based on the ITS1 and ITS2 with diversity rates estimated at 0.15% and 3.36%, respectively. Notably, the analysis of the COXI marker revealed no molecular divergence among the partial sequences representing the ten studied isolates from C. canis. CONCLUSIONS: This study explores the diversity of C. canis in the western and northwestern regions of Iran, providing insights into their molecular taxonomy and potential role as disease vectors in these areas.

Wolbachia Interactions with Diverse Insect Hosts: From Reproductive Modulations to Sustainable Pest Management Strategies.

Effective in a variety of insect orders, including dipteran, lepidopteran, and hemipteran, Wolbachia-based control tactics are investigated, noting the importance of sterile and incompatible insect techniques. Encouraging approaches for controlling Aedes mosquitoes are necessary, as demonstrated by the evaluation of a new SIT/IIT combination and the incorporation of SIT into Drosophila suzukii management. For example, Wolbachia may protect plants from rice pests, demonstrating its potential for agricultural biological vector management. Maternal transmission and cytoplasmic incompatibility dynamics are explored, while Wolbachia phenotypic impacts on mosquito and rice pest management are examined. The importance of host evolutionary distance is emphasised in recent scale insect research that addresses host-shifting. Using greater information, a suggested method for comprehending Wolbachia host variations in various contexts emphasises ecological connectivity. Endosymbionts passed on maternally in nematodes and arthropods, Wolbachia are widely distributed around the world and have evolved both mutualistic and parasitic traits. Wolbachia is positioned as a paradigm for microbial symbiosis due to advancements in multiomics, gene functional assays, and its effect on human health. The challenges and opportunities facing Wolbachia research include scale issues, ecological implications, ethical conundrums, and the possibility of customising strains through genetic engineering. It is thought that cooperative efforts are required to include Wolbachia-based therapies into pest management techniques while ensuring responsible and sustainable ways.

Nasal bots carry relevant titers of CWD prions in naturally infected white-tailed deer.

Chronic wasting disease (CWD) is a prion disease affecting farmed and free-ranging cervids. CWD is rapidly expanding across North America and its mechanisms of transmission are not completely understood. Considering that cervids are commonly afflicted by nasal bot flies, we tested the potential of these parasites to transmit CWD. Parasites collected from naturally infected white-tailed deer were evaluated for their prion content using the protein misfolding cyclic amplification (PMCA) technology and bioassays. Here, we describe PMCA seeding activity in nasal bot larvae collected from naturally infected, nonclinical deer. These parasites efficiently infect CWD-susceptible mice in ways suggestive of high infectivity titers. To further mimic environmental transmission, bot larvae homogenates were mixed with soils, and plants were grown on them. We show that both soils and plants exposed to CWD-infected bot homogenates displayed seeding activity by PMCA. This is the first report describing prion infectivity in a naturally occurring deer parasite. Our data also demonstrate that CWD prions contained in nasal bots interact with environmental components and may be relevant for disease transmission.

The influence of humidity and temperature on the vertical richness and abundance of blood-sucking flies (Culicidae and Ceratopogonidae) in a montane cloud forest in Mexico.

Because the vertical distribution and diversity of blood-sucking flies are poorly known, we determined the diversity, structure, and composition of culicids between vertical vegetation strata. We evaluated the influence of microclimatic variables during different times of the day over a year. We used eight CDC traps baited with CO2 at a height of 1.5 m and 12-15 m. We conducted rank-abundance curves, similarity analysis (ANOSIM and SIMPER), and multivariate clustering with incidence and abundance data. We used GAM models to analyze the influence of strata (understory vs canopy), humidity, and temperature on insect richness and abundance. During the day, the difference between strata was mainly due to higher abundance of Wyeomyia arthrostigma and Wyeomyia ca. adelpha in the understory. During the night, the differences were mainly due to higher abundance of Culex stigmatosoma, Culex salinarius, and Aedes allotecnon in the canopy, and Wyeomyia arthrostigma in the understory. Seasonality played a role in the similarity between the strata. Diversity during the day was positively related to humidity and temperature, and nocturnal diversity increased with temperature but decreased with higher humidity. The effects of environmental factors on the spatiotemporal distribution of fly species are essential for epidemiological surveillance.

Land-cover, land-use and human hantavirus infection risk: a systematic review.

Previous studies suggest that the risk of human infection by hantavirus, a family of rodent-borne viruses, might be affected by different environmental determinants such as land cover, land use and land use change. This study examined the association between land-cover, land-use, land use change, and human hantavirus infection risk. PubMed and Scopus databases were interrogated using terms relative to land use (change) and human hantavirus disease. Screening and selection of the articles were completed by three independent reviewers. Classes of land use assessed by the different studies were categorized into three macro-categories of exposure ('Agriculture', 'Forest Cover', 'Urban Areas') to qualitatively synthesize the direction of the association between exposure variables and hantavirus infection risk in humans. A total of 25 articles were included, with 14 studies (56%) conducted in China, 4 studies (16%) conducted in South America and 7 studies (28%) conducted in Europe. Most of the studies (88%) evaluated land cover or land use, while 3 studies (12%) evaluated land use change, all in relation to hantavirus infection risk. We observed that land cover and land-use categories could affect hantavirus infection incidence. Overall, agricultural land use was positively associated with increased human hantavirus infection risk, particularly in China and Brazil. In Europe, a positive association between forest cover and hantavirus infection incidence was observed. Studies that assessed the relationship between built-up areas and hantavirus infection risk were more variable, with studies reporting positive, negative or no associations.

Towards an integrated vector management approach for sustainable control of schistosomiasis and malaria in Mwea, Kirinyaga County, Kenya: Baseline epidemiological and vector results.

Background: Vector control is an important approach in the control of most parasitic and vector-borne diseases including malaria, and schistosomiasis. Distribution of these two infections often overlaps and in such areas it's more economically viable to employ an integrated approach in the control of their vectors which largely shares the same breeding ecosystem. We carried out a baseline epidemiological and vector surveys for malaria and schistosomiasis in Mwea, Kirinyaga County, in preparation for the upscaling of integrated vector management (IVM) for the two diseases. Methods: This was a repeated cross sectional survey, where mosquito and snails were sampled during dry and wet seasons in three different ecological zones, Kiamaciri, Thiba and Murinduko to identify possible breeding sites. Mosquito larvae were collected using standard dippers, adults using CDC miniature light traps while snail vectors were sampled using standard snail scoops in different breeding habitats. A total of 1200 pupils from 12 primary schools were tested for malaria using rapid diagnostic tests (Malaria Pf/PAN Ag combo). Stool samples were processed using the Kato Katz technique for intestinal schistosomiasis. Results: The overall prevalence of intestinal schistosomiasis was 9.08 % (95 % CI: 07.00-11.00), with Kiamaciri zone recording the highest prevalence at 19 % (95%CI: 15.00-23.00) and Murinduko zone the least at 0.17 % (95%CI: 0.00-0.01). Majority of the infections were of light intensity 78.9 % (95%CI: 70.04-86.13). There was no positive malaria case detected in this study. Of the 3208 adult mosquitoes sampled during the dry season, 20.6 % (95 % CI: 19.25-22.08) were Anopheles gambiae s.l while 79.4 % (95 % CI: 77.92-80.75) were culicines. During the wet season, 3378 adult mosquitoes were collected, of which 14.7 % (95 % CI: 13.56-15.98) were Anopheles gambiae s.l and 85.3 % (95 % CI: 84.02-86.44) culicines. Overall, 4085 mosquito larvae were collected during the two seasons, of which, 57.3 % and 42.7 % were anopheles and culicine respectively. Majority of the larvae (85.1 % (95%CI: 84.01-86.10) were collected during the wet season, with only 14.9 % (95%CI: 14.10-16.00) being collected during the dry season. A total of 2292 fresh water vector snails were collected with a majority (69.6 % (95%CI: 68.00-71.10) being Biomphalaria pffeiferi responsible for transmission of intestinal schistosomiasis. Conclusion: This study demonstrates that intestinal schistosomiasis is prevalent in Kiamaciri and Thiba zones, and points to the possibility of active transmission of schistosomiasis in Murinduko zone. Malaria vectors were predominantly observed in all sites despite there being no malaria positive case. Culex quinquefaciatus responsible for the spread of several arboviruses was also observed. The presence of these vectors may lead to future disease outbreaks in the area if concerted control initiatives are not undertaken. The disease vectors shared the same breeding sites and thus its economical and feasible to adopt an integrated vector management approach in control efforts for these disease in the study area.

Aedes aegypti, Ae. albopictus and Culex quinquefasciatus Adults Found Coexisting in Urban and Semiurban Dwellings of Southern Chiapas, Mexico.

Tapachula, Mexico, a tropical city, is an endemic area for dengue, in addition to several outbreaks in the last decade with chikungunya and zika. As part of the migratory corridor from Central to North America and the risks of scattered infectious diseases that this implies, the identification and distribution of potential disease vectors in and around residential areas are essential in terms of entomological surveillance for the prevention of disease outbreaks. The identification of mosquito species of medical importance coexisting in houses and cemeteries in Tapachula and two semiurban sites in southern Chiapas was investigated. Adult mosquitoes were collected from May to December 2018, resting inside and outside houses and in the tombstones and fallen tree leaves in cemeteries. A total of 10,883 mosquitoes belonging to three vector species were collected across 20 sites; 6738 were from neighborhood houses, of which 55.4% were Culex quinquefasciatus, 41.6% Aedes aegypti, and 2.9% Ae. albopictus. Aedes aegypti was the most common mosquito resting inside houses (56.7%), while Ae. albopictus and Cx. quinquefasciatus were mostly found resting outside houses (75.7%). In the cemeteries, Cx. quinquefasciatus (60.8%) and Ae. albopictus (37.3%) were the most abundant, while Ae. aegypti (1.9%) was the least abundant. This is the first report to identify adults of three major disease vector species coexisting in the domestic environment of urban and semiurban sites and Ae. albopictus adult resting inside of urban houses in Mexico. It would be opportune to consider comprehensive strategies that can be applied in this region to control the three species at the same time and avoid outbreaks of the diseases they transmit.

DNA Barcodes of Mansonia (Mansonia) Blanchard, 1901 (Diptera, Culicidae).

Females of the genus Mansonia feed on the blood of humans, livestock, and other vertebrates to develop their eggs. The females' biting behavior may cause severe disturbance to blood hosts, with a negative impact on public health and economics. Certain species have been identified as potential or effective disease vectors. The accurate species identification of field-collected specimens is of paramount importance for the success of monitoring and control strategies. Mansonia (Mansonia) morphological species boundaries are blurred by patterns of intraspecific heteromorphism and interspecific isomorphism. DNA barcodes can help to solve taxonomic controversies, especially if combined with other molecular tools. We used cytochrome c oxidase subunit I (COI) gene 5' end (DNA barcode) sequences to identify 327 field-collected specimens of Mansonia (Mansonia) spp. The sampling encompassed males and females collected from three Brazilian regions and previously assigned to species based on their morphological characteristics. Eleven GenBank and BOLD sequences were added to the DNA barcode analyses. Initial morphospecies assignments were mostly corroborated by the results of five clustering methods based on Kimura two-parameter distance and maximum likelihood phylogeny. Five to eight molecular operational taxonomic units may represent taxonomically unknown species. The first DNA barcode records for Mansonia fonsecai, Mansonia iguassuensis, and Mansonia pseudotitillans are presented.

Filtration of environmentally sourced aquatic media impacts laboratory-colonised Aedes albopictus early development and adult bacteriome composition.

Microorganisms form close associations with metazoan hosts forming symbiotic communities, known as microbiomes, that modulate host physiological processes. Mosquitoes are of special interest in exploring microbe-modulated host processes due to their oversized impact on human health. However, most mosquito work is done under controlled laboratory conditions where natural microbiomes are not present and inferences from these studies may not extend to natural populations. Here we attempt to assemble a wild-resembling bacteriome under laboratory conditions in an established laboratory colony of Aedes albopictus using aquatic media from environmentally-exposed and differentially filtered larval habitats. While we did not successfully replicate a wild bacteriome using these filtrations, we show that these manipulations alter the bacteriomes of mosquitoes, generating a unique composition not seen in wild populations collected from and near our source water or in our laboratory colony. We also demonstrate that our filtration regimens impact larval development times, as well as impact adult survival on different carbohydrate diets.

Triatoma pallidipennis (Stål, 1872) (Hemiptera: Reduviidae) and its potential for infestation in Tecozautla, Hidalgo state, Mexico.

Triatoma pallidipennis is an exclusive and widely distributed species in Mexico and one of the three main vectors that transmit Chagas disease in the country. The state of Hidalgo is an endemic area for Chagas disease where the presence of several species of triatomines has been reported. The objective of our work was to describe the morphology, colonization process, and reproductive behavior of T. pallidipennis in Guadalupe, Tecozautla, two years after the first collection of a specimen in this region. A total of 28 specimens was collected at both domicile and peridomicile, showing a 17.8% infection rate. The main collection site was a woodshed, and despite the collection of adults in the dwelling, we did not find eggs, exuviae, or nymphs. One female monitored from collection day until death laid 566 eggs, with a hatching rate of 95%, showing an increase of oviposition when cohabited with a male. The results showed the capacity that T. pallidipennis has to infest areas (mainly human dwellings) when it settles down, which would imply a risk for the population that lives in the locality.

Exploring the Hidden World of Vectors of Chagas Disease: A Fascinating Look at the Taxonomic Aspects of the Psammolestes Genus (Hemiptera, Triatominae).

Chagas disease (CD) is a neglected illness affecting approximately seven million individuals, with vector transmission occurring via triatomine bugs. The Rhodniini tribe comprises 24 species, grouped into the Rhodnius and Psammolestes genera. Given the importance of accurately identifying CD vectors, the taxonomy of Psammolestes spp. was revisited using morphological and morphometric data. Specimens of P. tertius, P. coreodes, and P. arthuri were collected, and the morphological characteristics of the head, thorax, abdomen, and eggs were analyzed. Morphometric studies of eggs were also conducted. Dichotomous keys allowing for the differentiation of Psammolestes spp. were elaborated based on adult insect and egg morphological characteristics. Through these studies, it was possible to differentiate the three Psammolestes species and confirm that this genus should not be classified under the Rhodnius genus, contributing to Rhodniini taxonomy.

Development of an automated mosquito pupae counter.

The production of mosquitoes for control programs or basic research is facilitated by the standardization of rearing conditions allowing the daily manipulation of thousands of individuals. It is crucial to develop mechanical or electronic systems for controlling the density of mosquitoes at each development stage with the aim of reducing costs, time, and human errors. We present herein an automatic mosquito counter using a recirculating water system allowing rapid and reliable counting of pupae without detectable increased mortality. Using Aedes albopictus pupae, we determined the density of pupae and the time of counting for which the device is most accurate, and evaluated the time saved using this device. Lastly, we discuss how this mosquito pupae counter can be useful in small-scale or mass-rearing contexts enabling a number of applications for research purposes as well as operational mosquito control programs.

Climate and Environmental Changes and Their Potential Effects on the Dynamics of Chagas Disease: Hybridization in Rhodniini (Hemiptera, Triatominae).

Chagas disease affects about eight million people. In view of the issues related to the influence of anthropogenic changes in the dynamics of the distribution and reproductive interaction of triatomines, we performed experimental crosses between species of the Rhodniini tribe in order to evaluate interspecific reproductive interactions and hybrid production capacity. Reciprocal crossing experiments were conducted among Rhodnius brethesi × R. pictipes, R. colombiensis × R. ecuadoriensis, R. neivai × R. prolixus, R. robustus × R. prolixus, R. montenegrensis × R. marabaensis; R. montenegrensis × R. robustus, R. prolixus × R. nasutus and R. neglectus × R. milesi. With the exception of crosses between R. pictipes ♀ × R. brethesi ♂, R. ecuadoriensis ♀ × R. colombiensis ♂ and R. prolixus ♀ × R. neivai ♂, all experimental crosses resulted in hybrids. Our results demonstrate that both allopatric and sympatric species produce hybrids, which can generate concern for public health agencies in the face of current anthropogenic events. Thus, we demonstrate that species of the Rhodniini tribe are capable of producing hybrids under laboratory conditions. These results are of great epidemiological importance and raise an important discussion about the influence of climatic and environmental interactions on Chagas disease dynamics.

First Isolation and Genome Sequence Analysis of West Nile Virus in Mosquitoes in Brazil.

West Nile virus is a flavivirus transmitted by mosquitoes, mainly of the genus Culex. In Brazil, serological studies have already indicated the circulation of the virus since 2003, with the first human case detected in 2014. The objective of the present paper is to report the first isolation of WNV in a Culex (Melanoconion) mosquito. Arthropods were collected by protected human attraction and CDC light bait, and taxonomically identified and analyzed by viral isolation, complement fixation and genomic sequencing tests. WNV was isolated from samples of Culex (Melanoconion) mosquitoes, and the sequencing analysis demonstrated that the isolated strain belonged to lineage 1a. The finding of the present study presents the first evidence of the isolation and genome sequencing of WNV in arthropods in Brazil.

Eugenol and menthol synergize the toxicity of permethrin in the blood-sucking bug, Triatoma infestans.

The blood-sucking bug, Triatoma infestans, is one of the main vectors of Chagas disease in America. It is usually controlled with pyrethroids, but the emergence of resistance to these insecticides creates the need to look for alternative products. Eugenol, menthol and menthyl acetate are botanical monoterpenes, which produce lethal and sublethal effects on insects. The purpose of this work was to determine what type of toxicological interactions occur when binary mixtures, formed by the pyrethroid permethrin and sublehtal doses of eugenol, menthol or menthyl acetate, are applied to T. infestans. First instar nymphs were exposed to filter papers impregnated with the insecticides. The number of knocked down insects was registered at different times and Knock Down Time 50% (KT50) values were calculated. The following KT50 values with their corresponding 95% Confidence Intervals were obtained: permethrin, 47.29 (39.92 - 56.32) min; permethrin + eugenol, 34.08 (29.60 - 39.01) min; permethrin + menthol, 27.54 (23.28 - 32.55) min; permethrin + menthyl acetate, 43.62 (39.99 - 47.59) min. Eugenol and menthol increased the speed of action of permethrin (synergism), but menthyl acetate had no effect on it (additivity). These results provide the basis to further explore interactions between conventional insecticides and plant monoterpenes as potential tools for controlling T. infestans.

Making the Genome Huge: The Case of Triatoma delpontei, a Triatominae Species with More than 50% of Its Genome Full of Satellite DNA.

The genome of Triatoma delpontei Romaña & Abalos 1947 is the largest within Heteroptera, approximately two to three times greater than other evaluated Heteroptera genomes. Here, the repetitive fraction of the genome was determined and compared with its sister species Triatoma infestans Klug 1834, in order to shed light on the karyotypic and genomic evolution of these species. The T. delpontei repeatome analysis showed that the most abundant component in its genome is satellite DNA, which makes up more than half of the genome. The T. delpontei satellitome includes 160 satellite DNA families, most of them also present in T. infestans. In both species, only a few satellite DNA families are overrepresented on the genome. These families are the building blocks of the C-heterochromatic regions. Two of these satellite DNA families that form the heterochromatin are the same in both species. However, there are satellite DNA families highly amplified in the heterochromatin of one species that in the other species are in low abundance and located in the euchromatin. Therefore, the present results depicted the great impact of the satellite DNA sequences in the evolution of Triatominae genomes. Within this scenario, satellitome determination and analysis led to a hypothesis that explains how satDNA sequences have grown on T. delpontei to reach its huge genome size within true bugs.

Survey of West Nile and Banzi Viruses in Mosquitoes, South Africa, 2011-2018.

We collected >40,000 mosquitoes from 5 provinces in South Africa during 2011-2018 and screened for zoonotic flaviviruses. We detected West Nile virus in mosquitoes from conservation and periurban sites and potential new mosquito vectors; Banzi virus was rare. Our results suggest flavivirus transmission risks are increasing in South Africa.