Chemical Ecology and Management of Dengue Vectors.

Dengue, caused by the dengue virus, is the most widespread arboviral infectious disease of public health significance globally. This review explores the communicative function of olfactory cues that mediate host-seeking, egg-laying, plant-feeding, and mating behaviors in Aedes aegypti and Aedes albopictus, two mosquito vectors that drive dengue virus transmission. Aedes aegypti has adapted to live in close association with humans, preferentially feeding on them and laying eggs in human-fabricated water containers and natural habitats. In contrast, Ae. albopictus is considered opportunistic in its feeding habits and tends to inhabit more vegetative areas. Additionally, the ability of both mosquito species to locate suitable host plants for sugars and find mates for reproduction contributes to their survival. Advances in chemical ecology, functional genomics, and behavioral analyses have improved our understanding of the underlying neural mechanisms and reveal novel and specific olfactory semiochemicals that these species use to locate and discriminate among resources in their environment. Physiological status; learning; and host- and habitat-associated factors, including microbial infection and abundance, shape olfactory responses of these vectors. Some of these semiochemicals can be integrated into the toolbox for dengue surveillance and control.

Polymer Beads Increase Field Responses to Host Attractants in the Dengue Vector Aedes aegypti.

This study investigates the efficacy of three different olfactory cues - cyclohexanone, linalool oxide (LO), and 6-methyl-5-heptan-2-one (sulcatone) - in attracting Aedes aegypti, the primary vector of dengue, using BG sentinel traps in a dengue-endemic area (urban Ukunda) in coastal Kenya. Two experiments were conducted. Experiment 1 compared solid formulations of the compounds in polymer beads against liquid formulations with hexane as the solvent. CO2-baited traps served as controls. In Experiment 2, traps were baited with each compound in the polymer beads, commercial BG-Lure, and CO2. Our results indicate that CO2-baited traps recorded the greatest Ae. aegypti captures in both Experiment 1 and 2, whereas trap captures with polymer beads and solvent-based treatments were comparable. In experiment 2, polymer bead-based treatments yielded significantly greater female captures, each recording ~ 2-fold more captures than traps baited with the BG-Lure. There was no significant difference, however, between the treatments. Female Ae. aegypti captured in CO2-baited traps were mainly unfed (91%), with fewer gravid mosquitoes (6.4%) compared to traps with test compounds (range; 12.7-21.1%). Male captures were lower in LO and BG-Lure baited traps compared to other treatments. Gravimetric analysis showed LO had a slower release rate compared to other compounds. The findings suggest that host-associated compounds loaded on polymer beads are more effective in trapping Ae. aegypti than commercial BG-Lure and reveal sex-specific differences in mosquito responses. These results have implications for mosquito surveillance and control programs, highlighting the potential for selective trapping strategies.

Improved detection of flaviviruses in Australian mosquito populations via replicative intermediates.

Mosquito-borne flaviviruses are significant contributors to the arboviral disease burdens both in Australia and globally. While routine arbovirus surveillance remains a vital exercise to identify known flaviviruses in mosquito populations, novel or divergent and emerging species can be missed by these traditional methods. The MAVRIC (monoclonal antibodies to viral RNA intermediates in cells) system is an ELISA-based method for broad-spectrum isolation of positive-sense and double-stranded RNA (dsRNA) viruses based on detection of dsRNA in infected cells. While the MAVRIC ELISA has successfully been used to detect known and novel flaviviruses in Australian mosquitoes, we previously reported that dsRNA could not be detected in dengue virus-infected cells using this method. In this study we identified additional flaviviruses which evade detection of dsRNA by the MAVRIC ELISA. Utilising chimeric flaviviruses we demonstrated that this outcome may be dictated by the non-structural proteins and/or untranslated regions of the flaviviral genome. In addition, we report a modified fixation method that enables improved detection of flavivirus dsRNA and inactivation of non-enveloped viruses from mosquito populations using the MAVRIC system. This study demonstrates the utility of anti-dsRNA monoclonal antibodies for identifying viral replication in insect and vertebrate cell systems and highlights a unique characteristic of flavivirus replication.

Arbovirus surveillance: first dengue virus detection in local Aedes albopictus mosquitoes in Europe, Catalonia, Spain, 2015.

Dengue has emerged as the most important viral mosquito-borne disease globally. The current risk of dengue outbreaks in Europe appeared with the introduction of the vector Aedes albopictus mosquito in Mediterranean countries. Considering the increasing frequency of dengue epidemics worldwide and the movement of viraemic hosts, it is expected that new autochthonous cases will occur in the future in Europe. Arbovirus surveillance started in Catalonia in 2015 to monitor imported cases and detect possible local arboviral transmission. During 2015, 131 patients with a recent travel history to endemic countries were tested for dengue virus (DENV) and 65 dengue cases were detected. Twenty-eight patients with a febrile illness were viraemic, as demonstrated by a positive real-time RT-PCR test for DENV in serum samples. Entomological investigations around the viraemic cases led to the detection of DENV in a pool of local Ae. albopictus captured in the residency of one case. The sequence of the DENV envelope gene detected in the mosquito pool was identical to that detected in the patient. Our results show how entomological surveillance conducted around viraemic travellers can be effective for early detection of DENV in mosquitoes and thus might help to prevent possible autochthonous transmission.

Reemergence of St. Louis Encephalitis Virus, California, 2015.

St. Louis encephalitis virus infection was detected in summer 2015 in southern California after an 11-year absence, concomitant with an Arizona outbreak. Sequence comparisons showed close identity of California and Arizona isolates with 2005 Argentine isolates, suggesting introduction from South America and underscoring the value of continued arbovirus surveillance.

Noninvasive detection of Zika virus in mosquito excreta sampled from wild mosquito populations in French Guiana.

Arboviruses can be difficult to detect in the field due to relatively low prevalence in mosquito populations. The discovery that infected mosquitoes can release viruses in both their saliva and excreta gave rise to low-cost methods for the detection of arboviruses during entomological surveillance. We implemented both saliva and excreta-based entomological surveillance during the emergence of Zika virus (ZIKV) in French Guiana in 2016 by trapping mosquitoes around households of symptomatic cases with confirmed ZIKV infection. ZIKV was detected in mosquito excreta and not in mosquito saliva in 1 trap collection out of 85 (1.2%). One female Ae. aegypti L. (Diptera: Culicidae) was found with a ZIKV systemic infection in the corresponding trap. The lag time between symptom onset in a ZIKV-infected individual living near the trap site and ZIKV detection in this mosquito was 1 wk. These results highlight the potential of detection in excreta from trapped mosquitoes as a sensitive and cost-effective method to non invasively detect arbovirus circulation.

Analyses of Mosquito Species Composition, Blood-Feeding Habits and Infection with Insect-Specific Flaviviruses in Two Arid, Pastoralist-Dominated Counties in Kenya.

Insect-specific flaviviruses (ISFs), although not known to be pathogenic to humans and animals, can modulate the transmission of arboviruses by mosquitoes. In this study, we screened 6665 host-seeking, gravid and blood-fed mosquitoes for infection with flaviviruses and assessed the vertebrate hosts of the blood-fed mosquitoes sampled in Baringo and Kajiado counties; both dryland ecosystem counties in the Kenyan Rift Valley. Sequence fragments of two ISFs were detected. Cuacua virus (CuCuV) was found in three blood-fed Mansonia (Ma.) africana. The genome was sequenced by next-generation sequencing (NGS), confirming 95.8% nucleotide sequence identity to CuCuV detected in Mansonia sp. in Mozambique. Sequence fragments of a potential novel ISF showing nucleotide identity of 72% to Aedes flavivirus virus were detected in individual blood-fed Aedes aegypti, Anopheles gambiae s.l., Ma. africana and Culex (Cx.) univittatus, all having fed on human blood. Blood-meal analysis revealed that the collected mosquitoes fed on diverse hosts, primarily humans and livestock, with a minor representation of wild mammals, amphibians and birds. The potential impact of the detected ISFs on arbovirus transmission requires further research.

Response to An Outbreak of Locally Transmitted Dengue in Key Largo, FL, by The Florida Keys Mosquito Control District.

Dengue virus (DENV) is an ever-increasing threat to the residents of South Florida. Seventy-two cases of locally acquired dengue were contracted by residents and visitors of Key Largo, FL, in 2020. The primary vector, Aedes aegypti, has been a large focus of the Florida Keys Mosquito Control District's (FKMCD) control measures for over a decade. This paper recounts the 2020 outbreak of DENV in Key Largo, FL, and the FKMCD's Ae. aegypti operational response. The overall House Index (13.43%) during the outbreak was considered high (>5%) risk for local transmission. Larval habitat characterized from property inspections was similar to previous larval (τ = 0.78, P < 0.005) and pupal (τ = 0.745, P < 0.005) habitat studies. Adult surveillance of the active dengue transmission area provided 3 positive pools out of 1,518 mosquitoes tested resulting in a minimum infection rate of 1.976. Increased personnel response with long-term larvicide formulations and increased aerial, truck, and handheld ultra-low-volume adulticide control measures quickly reduced the Ae. aegypti surveillance numbers below the action threshold. No active cases of dengue have been reported since October 2020.

First Detection of West Nile Virus Lineage 2 in Mosquitoes in Switzerland, 2022.

West Nile virus (WNV) is one of the most widespread flaviviruses in the world, and in recent years, it has been frequently present in many Mediterranean and Eastern European countries. A combination of different conditions, such as a favourable climate and higher seasonal average temperatures, probably allowed its introduction and spread to new territories. In Switzerland, autochthonous cases of WNV have never been reported, and the virus was not detected in mosquito vectors until 2022, despite an entomological surveillance in place in Canton Ticino, southern Switzerland, since 2010. In 2022, 12 sites were monitored from July to October, using BOX gravid mosquito traps coupled with honey-baited FTA cards. For the first time, we could detect the presence of WNV in FTA cards and mosquitoes in 8 out of the 12 sampling sites monitored, indicating an unexpectedly widespread circulation of the virus throughout the territory. Positive findings were recorded from the beginning of August until mid-October 2022, and whole genome sequencing analysis identified a lineage 2 virus closely related to strains circulating in Northern Italy. The entomological surveillance has proved useful in identifying viral circulation in advance of possible cases of WNV infection in humans or horses.

Viral diversity and blood-feeding patterns of Afrotropical Culicoides biting midges (Diptera: Ceratopogonidae).

Introduction: Culicoides biting midges (Diptera: Ceratopogonidae) are vectors of arboviral pathogens that primarily affect livestock represented by Schmallenberg virus (SBV), epizootic hemorrhagic disease virus (EHDV) and bluetongue virus (BTV). In Kenya, studies examining the bionomic features of Culicoides including species diversity, blood-feeding habits, and association with viruses are limited. Methods: Adult Culicoides were surveyed using CDC light traps in two semi-arid ecologies, Baringo and Kajiado counties, in Kenya. Blood-fed specimens were analysed through polymerase chain reaction (PCR) and sequencing of cytochrome oxidase subunit 1 (cox1) barcoding region. Culicoides pools were screened for virus infection by generic RT-PCR and next-generation sequencing (NGS). Results: Analysis of blood-fed specimens confirmed that midges had fed on cattle, goats, sheep, zebra, and birds. Cox1 barcoding of the sampled specimens revealed the presence of known vectors of BTV and epizootic hemorrhagic disease virus (EHDV) including species in the Imicola group (Culicoides imicola) and Schultzei group (C. enderleni, C. kingi, and C. chultzei). Culicoides leucostictus and a cryptic species distantly related to the Imicola group were also identified. Screening of generated pools (11,006 individuals assigned to 333 pools) by generic RT-PCR revealed presence of seven phylogenetically distinct viruses grouping in the genera Goukovirus, Pacuvirus and Orthobunyavirus. The viruses showed an overall minimum infection rate (MIR) of 7.0% (66/333, 95% confidence interval (CI) 5.5-8.9). In addition, full coding sequences of two new iflaviruses, tentatively named Oloisinyai_1 and Oloisinyai_2, were generated by next-generation sequencing (NGS) from individual homogenate of Culicoides pool. Conclusion: The results indicate a high genetic diversity of viruses in Kenyan biting midges. Further insights into host-vector-virus interactions as well as investigations on the potential clinical significance of the detected viruses are warranted.

Chikungunya, Zika, Mayaro, and Equine Encephalitis virus detection in adult Culicinae from South Central Mato Grosso, Brazil, during the rainy season of 2018.

INTRODUCTION: Several arboviruses causing human disease have been reported in Brazil. In nature, arboviruses maintain a lifecycle involving vertebrates and vectors, which may contribute for periodical reemergence of those of public health concern in tropical regions, as Mato Grosso State (MT). In this study, we searched for arboviruses in mosquito body pools sampled during the rainy season of 2018 in 21 bird watching points of Cuiabá and Varzea Grande, South Central MT. METHODS: In total, 2873 (57%) males and 2167 (43%) females belonging to six urban and sylvatic mosquito genera allocated to 398 pools were subjected to RNA extraction and RT-PCR for arboviruses. Positive pools were subjected to virus isolation in C6/36 cells. RESULTS: A total of 102/398 pools, 66/233 (29.6%) of females, and 36/165 (21.8%) of males, mostly sampled in May (31/102), were positive for arboviruses. Chikungunya virus (CHIKV) was distributed in 19 points, Zika virus (ZIKV) was found in 14 points, Mayaro virus (MAYV) in 10 points, and East Equine encephalitis virus (EEEV) in three points. Culex quinquefasciatus pools (39/89 of females and 24/99 of males) were positive for CHIKV, ZIKV, and MAYV; Aedes (Stg) aegypti pools (11/46 of females and 12/33 of males) for CHIKV, ZIKV, MAYV, and EEEV; Aedes albopictus female pools (8/29) for CHIKV, ZIKV, and EEEV; and Psorophora albigenu (2/12) and Psorophora ferox female pools (4/16) for CHIKV. CONCLUSIONS: Arbovirus molecular detection in mosquito populations varies considerable between geographical regions and epidemics, influenced by genetic characteristics and microbiome interference on virus replication. Although infected females are responsible for the transmission to vertebrates during bloodfeeding, male infection by CHIKV, ZIKV, and MAYV resultant from vertical route could lead to interepidemic maintenance of these arboviruses in their natural reservoirs.

Jingmen Tick Virus in Ticks from Kenya.

Jingmen tick virus (JMTV) is an arbovirus with a multisegmented genome related to those of unsegmented flaviviruses. The virus first described in Rhipicephalus microplus ticks collected in Jingmen city (Hubei Province, China) in 2010 is associated with febrile illness in humans. Since then, the geographic range has expanded to include Trinidad and Tobago, Brazil, and Uganda. However, the ecology of JMTV remains poorly described in Africa. We screened adult ticks (n = 4550, 718 pools) for JMTV infection by reverse transcription polymerase chain reaction (RT-PCR). Ticks were collected from cattle (n = 859, 18.88%), goats (n = 2070, 45.49%), sheep (n = 1574, 34.59%), and free-ranging tortoises (Leopard tortoise, Stigmochelys pardalis) (n = 47, 1.03%) in two Kenyan pastoralist-dominated areas (Baringo and Kajiado counties) with a history of undiagnosed febrile human illness. Surprisingly, ticks collected from goats (0.3%, 95% confidence interval (CI) 0.1-0.5), sheep (1.8%, 95% CI 1.2-2.5), and tortoise (74.5%, 95% CI 60.9-85.4, were found infected with JMTV, but ticks collected from cattle were all negative. JMTV ribonucleic acid (RNA) was also detected in blood from tortoises (66.7%, 95% CI 16.1-97.7). Intragenetic distance of JMTV sequences originating from tortoise-associated ticks was greater than that of sheep-associated ticks. Phylogenetic analyses of seven complete-coding genome sequences generated from tortoise-associated ticks formed a monophyletic clade within JMTV strains from other countries. In summary, our findings confirm the circulation of JMTV in ticks in Kenya. Further epidemiological surveys are needed to assess the potential public health impact of JMTV in Kenya.

COVID-19 and zoonoses in Brazil: Environmental scan of one health preparedness and response.

The emergence of the COVID-19 pandemic reinforced the central role of the One Health (OH) approach, as a multisectoral and multidisciplinary perspective, to tackle health threats at the human-animal-environment interface. This study assessed Brazilian preparedness and response to COVID-19 and zoonoses with a focus on the OH approach and equity dimensions. We conducted an environmental scan using a protocol developed as part of a multi-country study. The article selection process resulted in 45 documents: 79 files and 112 references on OH; 41 files and 81 references on equity. The OH and equity aspects are poorly represented in the official documents regarding the COVID-19 response, either at the federal and state levels. Brazil has a governance infrastructure that allows for the response to infectious diseases, including zoonoses, as well as the fight against antimicrobial resistance through the OH approach. However, the response to the pandemic did not fully utilize the resources of the Brazilian state, due to the lack of central coordination and articulation among the sectors involved. Brazil is considered an area of high risk for emergence of zoonoses mainly due to climate change, large-scale deforestation and urbanization, high wildlife biodiversity, wide dry frontier, and poor control of wild animals' traffic. Therefore, encouraging existing mechanisms for collaboration across sectors and disciplines, with the inclusion of vulnerable populations, is required for making a multisectoral OH approach successful in the country.

Laboratory Evaluation of the Rapid Analyte Measurement Platform Assay to Detect Dengue Virus in Mosquito Pools.

We report the results of a laboratory sensitivity and specificity evaluation of the Rapid Analyte Measurement Platform (RAMP®) Dengue Virus (DENV) antigen detection assay, which is designed to detect all serotypes of DENV in mosquito pools. The RAMP DENV assay was able to detect geographically distinct strains of all 4 DENV serotypes in virus-spiked mosquito pools that contained at least 4.3 log10 plaque forming units/ml, although discrete sensitivity limits varied slightly for each serotype. The RAMP DENV assay also detected DENV 1-4 in mosquito pools containing a single infected mosquito and 24 laboratory-reared uninfected mosquitoes. No false positives were detected in negative control mosquito pools or in samples containing high titers of nontarget arboviruses. We found that while the kit-supplied RAMP buffer reduced the infectious titer of DENV, it did not completely inactivate all serotypes. We recommend adding a detergent, Triton X-100, to the buffer to ensure complete inactivation of DENV if the assay is to be conducted at a lower biosafety level than required for DENV handling.

Detection of Cell-Fusing Agent virus across ecologically diverse populations of Aedes aegypti on the Caribbean island of Saint Lucia.

Background. Outbreaks of mosquito-borne arboviral diseases including dengue virus (DENV), Zika virus (ZIKV), yellow fever virus (YFV) and chikungunya virus (CHIKV) have recently occurred in the Caribbean. The geographical range of the principal vectors responsible for transmission, Aedes (Ae.) aegypti and Ae. albopictus are increasing and greater mosquito surveillance is needed in the Caribbean given international tourism is so prominent. The island of Saint Lucia has seen outbreaks of DENV and CHIKV in the past five years but vector surveillance has been limited with the last studies dating back to the late 1970s. Natural disasters have changed the landscape of Saint Lucia and the island has gone through significant urbanisation. Methods. In this study, we conducted an entomological survey of Ae. aegypti and Ae. albopictus distribution across the island and analysed environmental parameters associated with the presence of these species in addition to screening for medically important arboviruses and other flaviviruses. Results. Although we collected Ae. aegypti across a range of sites across the island, no Ae. albopictus were collected despite traps being placed in diverse ecological settings. The number of Ae. aegypti collected was significantly associated with higher elevation, and semi-urban settings yielded female mosquito counts per trap-day that were five-fold lower than urban settings. Screening for arboviruses revealed a high prevalence of cell-fusing agent virus (CFAV). Conclusions. Outbreaks of arboviruses transmitted by Ae. aegypti and Ae. albopictus have a history of occurring in small tropical islands and Saint Lucia is particularly vulnerable given the limited resources available to undertake vector control and manage outbreaks. Surveillance strategies can identify risk areas for predicting future outbreaks. Further research is needed to determine the diversity of current mosquito species, investigate insect-specific viruses, as well as pathogenic arboviruses, and this should also be extended to the neighbouring smaller Caribbean islands.

High-Throughput Method for Detection of Arbovirus Infection of Saliva in Mosquitoes Aedes aegypti and Ae. albopictus.

Vector competence refers to the ability of a vector to acquire, maintain, and transmit a pathogen. Collecting mosquito saliva in medium-filled capillary tubes has become the standard for approximating arbovirus transmission. However, this method is time-consuming and labor-intensive. Here we compare the capillary tube method to an alternative high-throughput detection method the collection of saliva on paper cards saturated with honey, with (FTA card) and without (filter paper) reagents for the preservation of nucleic acid for Aedes aegypti and Aedes albopictus mosquitoes infected with two emerging genotypes of the chikungunya virus (CHIKV). Model results showed that the Asian genotype CHIKV dissemination in the harvested legs of both Ae. aegypti and Ae. albopictus increased the odds of females having a positive salivary infection and higher salivary viral titers, while for the IOL genotype the same effect was observed only for Ae. aegypti. Of the three tested detection methods, the FTA card was significantly more effective at detecting infected saliva of Ae. aegypti and Ae. albopictus females than the capillary tube and filter paper was as effective as the capillary tube for the Asian genotype. We did not find significant effects of the detection method in detecting higher viral titer for both Asian and IOL genotypes. Our results are discussed in light of the limitations of the different tested detection methods.

Isolation and Genome Phylogenetic Analysis of Arthropod-Borne Viruses, Including Akabane Virus, from Mosquitoes Collected in Hunan Province, China.

This study investigated the abundance of mosquitoes and circulation of mosquito-borne arboviruses from 16 villages in 8 cities of Hunan Province, China, in July-August of 2010 and in August of 2011. In total, 16,076 mosquitoes consisting of seven species from four genera were collected by ultraviolet-light trap. Culex quinquefasciatus was the most common species, accounting for 50.63% (8140/16,076) of the total. Anopheles sinensis (24.26%, 3900/16,076) made up the second most common species, followed by Culex tritaeniorhynchus (9.76%, 1569/16,076). The proportions of Culex pipiens pallens, Armigeres subalbatus, and Culex modestus were 6.7%, 5.2%, and 3.31%, respectively. Fourteen Aedes albopictus were detected. The mosquitoes were identified by morphologic characteristics and frozen in liquid nitrogen. The mosquitoes were pooled, triturated, and centrifuged. The clarified supernatant was used to inoculate monolayers of C6/36 and baby hamster kidney-21 cells. We obtained six virus isolates that caused cytopathic effects. Phylogenetic analysis revealed that two isolates were Akabane virus (AKAV, from A. sinensis and C. quinquefasciatus), two isolates were Japanese encephalitis virus (from C. pipiens pallens and C. quinquefasciatus), and two isolates were Tibet orbivirus (from C. quinquefasciatus and C. tritaeniorhynchus). This is the first report of AKAV isolated from A. sinensis and C. quinquefasciatus in nature in China. The detection of AKAV in these species confirms circulation of AKAV in Hunan province and suggests potential challenges to the prevention and control of arthropod-borne animal viruses in mainland China.

Evaluation of honey-baited FTA cards in combination with different mosquito traps in an area of low arbovirus prevalence.

BACKGROUND: The threat of mosquito-borne diseases is increasing in continental Europe as demonstrated by several autochthonous chikungunya, dengue and West Nile virus outbreaks. In Switzerland, despite the presence of competent vectors, routine surveillance of arboviruses in mosquitoes is not being carried out, mainly due to the high costs associated with the need of a constant cold chain and laborious processing of thousands of mosquitoes. An alternative approach is using honey-baited nucleic acid preserving cards (FTA cards) to collect mosquito saliva that may be analysed for arboviruses. Here, we evaluate whether FTA cards could be used to detect potentially emerging viruses in an area of low virus prevalence in combination with an effective mosquito trap. METHODS: In a field trial in southern Switzerland we measured side-by-side the efficacy of the BG-Sentinel 2, the BG-GAT and the Box gravid trap to catch Aedes and Culex mosquitoes in combination with honey-baited FTA cards during 80 trapping sessions of 48 hours. We then screened both the mosquitoes and the FTA cards for the presence of arboviruses using reverse-transcription PCR. The efficacy of the compared trap types was evaluated using generalized linear mixed models. RESULTS: The Box gravid trap collected over 11 times more mosquitoes than the BG-GAT and BG-Sentinel 2 trap. On average 75.9% of the specimens fed on the honey-bait with no significant difference in feeding rates between the three trap types. From the total of 1401 collected mosquitoes, we screened 507 Aedes and 500 Culex females for the presence of arboviruses. A pool of six Cx. pipiens/Cx. torrentium mosquitoes and also the FTA card from the same Box gravid trap were positive for Usutu virus. Remarkably, only two of the six Culex mosquitoes fed on the honey-bait, emphasising the high sensitivity of the method. In addition, two Ae. albopictus collections but no FTA cards were positive for mosquito-only flaviviruses. CONCLUSIONS: Based on our results we conclude that honey-baited FTA cards, in combination with the Box gravid trap, are an effective method for arbovirus surveillance in areas of low prevalence, particularly where resources are limited for preservation and screening of individual mosquitoes.

ZIKA virus isolated from mosquitoes: a field and laboratory investigation in China, 2016.

A field investigation of arboviruses was conducted in Dejiang, Guizhou Province in the summer of 2016. A total of 8,795 mosquitoes, belonging to four species of three genera, and 1,300 midges were collected. The mosquito samples were identified on site according to their morphology, and the pooled samples were ground and centrifuged in the laboratory. The supernatant was incubated with mosquito tissue culture cells (C6/36) and mammalian cells (BHK-21) for virus isolation. The results indicated that 40% (3,540/8,795) were Anopheles sinensis, 30% (2,700/8,795) were Culex pipiens quinquefasciatus, and 29% (2,530/8,795) were Armigeres subbalbeatus. Furthermore, a total of eight virus isolates were obtained, and genome sequencing revealed two Zika viruses (ZIKVs) isolated from Culex pipiens quinquefasciatus and Armigeres subbalbeatus, respectively; three Japanese encephalitis viruses (JEVs) isolated from Culex pipiens quinquefasciatus; two Banna viruses (BAVs) isolated from Culex pipiens quinquefasciatus and Anopheles sinensis, respectively; and one densovirus (DNV) isolated from Culex pipiens quinquefasciatus. The ZIKVs isolated from the Culex pipiens quinquefasciatus and Armigeres subbalbeatus mosquitoes represent the first ZIKV isolates in mainland China. This discovery presents new challenges for the prevention and control of ZIKV in China, and prompts international cooperation on this global issue.