Evaluation of a Field-Portable DNA Microarray Platform and Nucleic Acid Amplification Strategies for the Detection of Arboviruses,Arthropods, and Bloodmeals

Highly multiplexed assays, such as microarrays, can benefit arbovirus surveillance by allowing researchers to screen for hundreds of targets at once. We evaluated amplification strategies and the practicality of a portable DNA microarray platform to analyze virus-infected mosquitoes. The prototype microarray design used here targeted the non-structural protein 5, ribosomal RNA, and cytochrome b genes for the detection of flaviviruses, mosquitoes, and bloodmeals, respectively. We identified 13 of 14 flaviviruses from virus inoculated mosquitoes and cultured cells. Additionally, we differentiated between four mosquito genera and eight whole blood samples. The microarray platform was field evaluated in Thailand and successfully identified flaviviruses (Culex flavivirus, dengue-3, and Japanese encephalitis viruses), differentiated between mosquito genera (Aedes, Armigeres, Culex, and Mansonia), and detected mammalian bloodmeals (human and dog). We showed that the microarray platform and amplification strategies described here can be used to discern specific information on a wide variety of viruses and their vectors.     

Dogs as Sentinels for Flavivirus Exposure in Urban,Peri-Urban and Rural Hanoi,Vietnam

Diseases caused by flaviviruses, including dengue fever and Japanese encephalitis, are major health problems in Vietnam. This cross-sectional study explored the feasibility of domestic dogs as sentinels to better understand risks of mosquito-borne diseases in Hanoi city. A total of 475 dogs serum samples from 221 households in six districts of Hanoi were analyzed by a competitive enzyme-linked immunosorbent assay (cELISA) for antibodies to the pr-E protein of West Nile virus and other flaviviruses due to cross-reactivity. The overall flavivirus seroprevalence in the dog population was 70.7% (95% CI = 66.4–74.8%). At the animal level, significant associations between seropositive dogs and district location, age, breed and keeping practice were determined. At the household level, the major risk factors were rural and peri-urban locations, presence of pigs, coil burning and households without mosquito-borne disease experience (p < 0.05). Mosquito control by using larvicides or electric traps could lower seropositivity, but other measures did not contribute to significant risk mitigation of flavivirus exposure in dogs. These results will support better control of mosquito-borne diseases in Hanoi, and they indicate that dogs can be used as sentinels for flavivirus exposure.     

Detection of Insect-Specific Flaviviruses in Mosquitoes (Diptera: Culicidae) in Northeastern Regions of South Africa

Mosquitoes in the Aedes and Culex genera are considered the main vectors of pathogenic flaviviruses worldwide. Entomological surveillance using universal flavivirus sets of primers in mosquitoes can detect not only pathogenic viruses but also insect-specific ones. It is hypothesized that insect-specific flaviviruses, which naturally infect these mosquitoes, may influence their vector competence for zoonotic arboviruses. Here, entomological surveillance was performed between January 2014 and May 2018 in five different provinces in the northeastern parts of South Africa, with the aim of identifying circulating flaviviruses. Mosquitoes were sampled using different carbon dioxide trap types. Overall, 64,603 adult mosquitoes were collected, which were screened by RT-PCR and sequencing. In total, 17 pools were found positive for insect-specific Flaviviruses in the mosquito genera Aedes (12/17, 70.59%) and Anopheles (5/17, 29.41%). No insect-specific viruses were detected in Culex species. Cell-fusing agent viruses were detected in Aedes aegypti and Aedes caballus. A range of anopheline mosquitoes, including Anopheles coustani, An. squamosus and An. maculipalpis, were positive for Culex flavivirus-like and Anopheles flaviviruses. These results confirm the presence of insect-specific flaviviruses in mosquito populations in South Africa, expands their geographical range and indicates potential mosquito species as vector species.     

Occurrence of Japanese Encephalitis Virus Mosquito Vectors in Relation to Urban Pig Holdings

Japanese encephalitis virus (JEV) is transmitted to humans from pigs or birds by mosquitoes. In this study, the association between urban pig keeping and mosquito vectors was analyzed. A total of 7, 419 mosquitoes were collected overnight in urban households with and without pigs in Can Tho City, Vietnam. The most prevalent vectors were Culex tritaeniorhynchus (36%), Cx. gelidus (24%), and Cx. quinquefasciatus (15%), which were present in all parts of the city. Pigs were associated with increased numbers of Cx. tritaeniorhynchus. Traps close to pigs had higher numbers of Cx. tritaeniorhynchus and Cx. gelidus than traps close to humans. Increased number of persons in the household was associated with increased numbers of Cx. quinquefasciatus. We demonstrate that JEV vector species are present at urban households with and without pigs, and show that keeping pigs in an urban area increase the number of mosquitoes competent as vectors for JEV.     

Mosquitoes and Mosquito-Borne Arboviruses in the Qinghai-Tibet Plateau��Focused on the Qinghai Area, China

An investigation was conducted to identify the distribution of mosquitoes and mosquito-borne arboviruses in the Qinghai-Tibet Plateau, China from July to August in 2007. A total of 8,147 mosquitoes representing six species from three genera (Aedes, Culex, and Anopheles) were collected in three locations (Geermu city, altitude of 2,780 m; Xining city, 2,200 m; Minhe county, 1,700 m). Six virus isolates were obtained including Tahyna virus (TAHV), Liaoning virus, and Culex pipiens pallens Densovirus. A serosurvey showed immunoglobulin G antibodies by immunofluorescence assay (IFA) against TAHV in residents of all three locations. The IFA-positive human samples were confirmed by 90% plaque-reduction neutralization tests (PRNT₉₀) against TAHV with titers ranging from 1:20 to 1:10,240. In addition, TAHV seropositive cows, sheep, and swine were found in these locations. This investigation represents the first isolation of TAHV from Ae. (Och.) detritus and the first evidence of TAHV infection in residents and livestock in the Qinghai-Tibet Plateau.     

云南景东县乙型脑炎流行状况调查

目的调查云南省景东县流行性乙型脑炎的流行现状。方法媒介蚊虫调查采用诱蚊灯通宵捕蚊方法,蚊虫乙脑病毒检测采取RT-PCR方法,健康人群血清乙脑病毒抗体检测采用ELISA。结果共采集到蚊虫4属15种6653只,其中三带喙库蚊和中华按蚊属于当地的优势种群,分别占总捕获蚊数的82.07%和10.46%。RT-PCR检测三带喙库蚊和中华按蚊共16批1310只,各有1株感染乙脑病毒;ELISA检测当地健康人群血清乙脑病毒抗体阳性率为72.96%,不同年龄组人群乙脑病毒抗体水平差异有统计学意义(χ2=51.589,P0.01)。结论云南景东县蚊虫种类较多,三带喙库蚊和中华按蚊有传播乙型脑炎的作用。     

Distribution of yellow fever vectors in Northwestern and Western Provinces,Zambia

ObjectiveTo determine the distribution of yellow fever (YF) vectors species in Northwestern and Western of Zambia, which sampled mosquitoes inside and outside houses in rural, urban, peri-urban and forest areas.MethodsBack-pack aspirators spray catches and CDC light traps collected adult mosquitoes including 405 Aedes, 518 Anopheles, 471 Culex and 71 Mansonia. Morphological vector identification and PCR viral determination were done at a WHO Regional Reference Centre (Institute Pasteur Dakar), Senegal.ResultsThe two main YF vectors were Aedes (Stegomyia) aegypti (Ae. aegypti) and Aedes (Stegomyia) africanus. The first was collected in peri-urban areas and the later was in forest areas, both sparsely distributed in Northwestern Province, where the 0.43 Breteau and 1.92 container indexes, respectively implied low risk to YF. Aedes (Aedimorphus) mutilus; Aedes (Aedimorphus) minutus and Aedes (Finlaya) wellmani were also found in Northwestern, not in Western Province. No Aedes were collected from rural peri-domestic areas. Significantly more Aedes species (90.7%, n=398) than Anopheles (9.1%, n=40) were collected in forest areas (P<0.001) or Culex species (0.2%, n=2) (P<0.001). Ae. aegypti was found only in a discarded container but not in flower pots, old tyres, plant axils, discarded shallow wells, disused container bottles and canoes inspected.ConclusionsAe. aegypti and Aedes africanus YF vectors were found in the study sites in the Northwestern Province of Zambia, where densities were low and distribution was sparse. The low Breteau index suggests low risk of YF in the Northwestern Province. The presence of Aedes in Northwestern Province and its absence in the Western Province could be due to differing ecological factors in the sampled areas. Universal coverage of vector control interventions could help to reduce YF vector population and the risk to arthropod-borne virus infections.     

Vector Competence of Culex (Melanoconion) taeniopus for Equine-Virulent Subtype IE Strains of Venezuelan Equine Encephalitis Virus

The mosquito Culex (Melanoconion) taeniopus is a proven vector of enzootic Venezuelan equine encephalitis virus (VEEV) subtype IE in Central America. It has been shown to be highly susceptible to infection by this subtype, and conversely to be highly refractory to infection by other VEEV subtypes. During the 1990s in southern coastal Mexico, two VEE epizootics in horses were attributed to subtype IE VEEV. These outbreaks were associated with VEEV strains with an altered infection phenotype for the epizootic mosquito vector, Aedes (Ochlerotatus) taeniorhynchus. To determine the infectivity for the enzootic vector, Culex taeniopus, mosquitoes from a recently established colony were orally exposed to VEEV strains from the outbreak. The equine-virulent strains exhibited high infectivity and transmission potential comparable to a traditional enzootic subtype IE VEEV strain. Thus, subtype IE VEEV strains in Chiapas are able to efficiently infect enzootic and epizootic vectors and cause morbidity and mortality in horses.     

Early start of seasonal transmission and co-circulation of West Nile virus lineage 2 and a newly introduced lineage 1 strain,northern Italy,June 2022

In spring 2022, Europe faced an unprecedented heatwave, increasing the risk of West Nile virus (WNV) outbreaks. As early as 7 June 2022, WNV was detected in Culex mosquitoes in northern Italy, and – in the following days – in two blood donors, a patient with encephalitis, wild birds and additional mosquito pools. Genome sequencing demonstrated co-circulation of WNV lineage 2 and a newly introduced WNV lineage 1, which was discovered in the region in 2021.     

Mercadeo Virus: A Novel Mosquito-Specific Flavivirus from Panama

Viruses in the genus Flavivirus (family Flaviviridae) include many arthropod-borne viruses of public health and veterinary importance. However, during the past two decades an explosion of novel insect-specific flaviviruses (ISFs), some closely related to vertebrate pathogens, have been discovered. Although many flavivirus pathogens of vertebrates have been isolated from naturally infected mosquitoes in Panama, ISFs have not previously been reported from the country. This report describes the isolation and characterization of a novel ISF, tentatively named Mercadeo virus (MECDV), obtained from Culex spp. mosquitoes collected in Panama. Two MECDV isolates were sequenced and cluster phylogenetically with cell-fusing agent virus (CFAV) and Nakiwogo virus (NAKV) to form a distinct lineage within the insect-specific group of flaviviruses.     

Vector competence of New Zealand mosquitoes for selected arboviruses

New Zealand (NZ) historically has been free of arboviral activity with the exception of Whataroa virus (Togaviridae: Alphavirus), which is established in bird populations and is transmitted by local mosquitoes. This naive situation is threatened by global warming, invasive mosquitoes, and tourism. To determine the threat of selected medically important arboviruses to NZ, vector competence assays were conducted using field collected endemic and introduced mosquito species. Four alphaviruses (Togaviridae): Barmah Forest virus, Chikungunya virus, Ross River virus, and Sindbis virus, and five flaviviruses (Flaviviridae): Dengue virus 2, Japanese encephalitis virus, Murray Valley encephalitis virus, West Nile virus, and Yellow fever virus were evaluated. Results indicate some NZ mosquito species are highly competent vectors of selected arboviruses, particularly alphaviruses, and may pose a threat were one of these arboviruses introduced at a time when the vector was prevalent and the climatic conditions favorable for virus transmission.     

Flaviviruses isolated from mosquitoes collected during the first recorded outbreak of Japanese encephalitis virus on Cape York Peninsula, Australia.

In response to an outbreak of Japanese encephalitis (JE) virus on Cape York Peninsula, Australia, in 1998, mosquitoes were collected using CO2 and octenol-baited Centers for Disease Control and Prevention light traps. A total of 35,235 adult mosquitoes, comprising 31 species, were processed for virus isolation. No isolates of JE virus were recovered from these mosquitoes. However, 18 isolates of Kokobera virus, another flavivirus were obtained from Culex annulirostris. Twelve isolates were from western Cape York (minimum infection rate (MIR) of 0.61: 1,000 mosquitoes) and 6 were from the Northern Peninsula Area (MIR of 1.0:1,000). Potential explanations for the failure to detect JE virus in mosquitoes collected from Cape York Peninsula include the timing of collections, the presence of alternative bloodmeal hosts, differences in pig husbandry, asynchronous porcine seroconversion, and the presence of other flaviviruses.     

Identification of Essential Containers for Aedes Larval Breeding to Control Dengue in Dhaka,Bangladesh

Dengue fever (DF), one of the most important emerging arboviral diseases, is transmitted through the bite of container breeding mosquitoes Aedes aegypti and Aedes albopictus. A household entomological survey was conducted in Dhaka from August through October 2000 to inspect water-holding containers in indoor, outdoor, and rooftop locations for Aedes larvae. The objective of this study was to determine mosquito productivity of each container type and to identify some risk factors of households infested with Aedes larvae. Of 9,222 households inspected, 1,306 (14.2%) were positive for Aedes larvae. Of 38,777 wet containers examined, 2,272 (5.8%) were infested with Aedes larvae. Containers used to hold water, such as earthen jars, tanks, and drums were the most common containers for larval breeding. Tires in outdoor and rooftop locations of the households were also important for larval breeding. Although present in abundance, buckets were of less importance. Factors such as independent household, presence of a water storage system in the house, and fully/partly shaded outdoors were found to be significantly associated with household infestation of Aedes larvae. Identification and subsequent elimination of the most productive containers in a given area may potentially reduce mosquito density to below a level at which dengue transmission may be halted.     

Temperature and Dengue Virus Infection in Mosquitoes: Independent Effects on the Immature and Adult Stages

Temperature is one of the most important environmental factors affecting biological processes of mosquitoes, including their interactions with viruses. In these studies, we show independent effects of rearing temperature on the immature aquatic stages and holding temperature on the adult terrestrial stage in terms of alterations in adult survival and progression of dengue-1 virus infection in the Asian tiger mosquito Aedes (Stegomyia) albopictus. Our studies show that adult survival was determined by adult-holding temperature, regardless of rearing conditions of the immature stages. In contrast, spread of virus throughout the body of the mosquito, a pre-requisite for transmission, was reduced when the immature stages were reared in cool conditions. These results show that immature-rearing temperature selectively modified mosquito traits that influence competency for viruses, and they further our understanding of the nature of temperature effects on interactions between mosquitoes and virus pathogens and risk of disease transmission.     

Sindbis Virus Infection in Non-Blood-Fed Hibernating Culex pipiens Mosquitoes in Sweden

A crucial, but unresolved question concerning mosquito-borne virus transmission is how these viruses can remain endemic in regions where the transmission is halted for long periods of time, due to mosquito inactivity in, e.g., winter. In northern Europe, Sindbis virus (SINV) (genus alphavirus, Togaviridae) is transmitted among birds by Culex mosquitoes during the summer, with occasional symptomatic infections occurring in humans. In winter 2018–19, we sampled hibernating Culex spp females in a SINV endemic region in Sweden and assessed them individually for SINV infection status, blood-feeding status, and species. The results showed that 35 out of the 767 collected mosquitoes were infected by SINV, i.e., an infection rate of 4.6%. The vast majority of the collected mosquitoes had not previously blood-fed (98.4%) and were of the species Cx. pipiens (99.5%). This is the first study of SINV overwintering, and it concludes that SINV can be commonly found in the hibernating Cx. pipiens population in an endemic region in Sweden, and that these mosquitoes become infected through other means besides blood-feeding. Further studies on mosquito ecology and viral interactions are needed to elucidate the mechanisms of the persistence of these viruses over winter.     

Innate Immune Antagonism of Mosquito-Borne Flaviviruses in Humans and Mosquitoes

Mosquito-borne viruses of the Flavivirus genus (Flaviviridae family) pose an ongoing threat to global public health. For example, dengue, Japanese encephalitis, West Nile, yellow fever, and Zika viruses are transmitted by infected mosquitoes and cause severe and fatal diseases in humans. The means by which mosquito-borne flaviviruses establish persistent infection in mosquitoes and cause disease in humans are complex and depend upon a myriad of virus-host interactions, such as those of the innate immune system, which are the main focus of our review. This review also covers the different strategies utilized by mosquito-borne flaviviruses to antagonize the innate immune response in humans and mosquitoes. Given the lack of antiviral therapeutics for mosquito-borne flaviviruses, improving our understanding of these virus-immune interactions could lead to new antiviral therapies and strategies for developing refractory vectors incapable of transmitting these viruses, and can also provide insights into determinants of viral tropism that influence virus emergence into new species.     

Assessment of anti mosquito measures in households and resistance status of Culex species in urban areas in southern Ghana: Implications for the sustainability of ITN use

ObjectiveTo determine resistance status of Culex species to different class of insecticides and assess the major anti–mosquito strategies employed by urban households and their possible effects on malaria acquisition.MethodsStructured questionnaires were randomly administered to obtain information on demographic characteristics, measures that people use to prevent mosquito bites and their perception of where mosquitoes breed in their communities. Adult susceptibility assays were also carried out using WHO discriminating dosages of four insecticides from different chemical classes against Culex species.ResultsMajority of respondents (54.75%) preferred the use of domestic insecticides in the form of aerosols and coils. Among households that used domestic insecticides, the use of coil was most frequent (62.9%) with a mean (95% CI) of 9.18 (8.99, 9.37) coils per week. Strong level of pyrethroid–resistance and multiple insecticide resistance in Culex species were also detected in some of the study sites.ConclusionsThe excessive use of domestic insecticides and high level of resistance in Culex species observed in the study area has implications for the ITN component of the nation's malaria control program in more subtle ways. People will lose interest in the use of ITN when it fails to protect users from bites of resistant Culex species. Excessive use of domestic insecticides may also select resistance in both malaria vectors and Culex species. On this account we recommend that nuisance mosquitoes must be controlled as part of malaria control programs to improve acceptance and utilization of ITN.     

Detection of host virus-reactive antibodies in blood meals of naturally engorged mosquitoes

Although serosurvey in human or animals is a useful and straightforward strategy routinely used for public health, it often faces different types of impediments: ethics, beliefs, limitation by animal owners, hazard of access to wild animals. To survey virus circulation, we applied the enzyme-linked immunosorbent assay (ELISA) technique to detect Dengue and Japanese encephalitis (JE) virus-reactive antibodies in blood meals collected from mosquitoes without regard to the potential of mosquito species to be a virus vector. ELISA was performed on mosquito colonies and wild specimens collected from farms and urban areas. Blood meals from Aedes aegypti freshly fed on naturally infected volunteers showed the same levels of dengue immunoglobulin (Ig)G and IgM as the sera directly collected from volunteers. A significant clearance of antibodies during the digestion process started from 13 hours after blood meal, and a negative baseline was reached after 30 hours. The ELISA test performed on wild mosquitoes showed that 37% of Culex quinquefasciatus mosquitoes that engorged on humans in a dengue urban endemic area tested positive for dengue IgG, and in a JE virus-endemic area, 88% of Culex tritaeniorhynchus mosquitoes that engorged on pigs from a large pig farm tested positive for JE virus antibodies versus 11% in a small farm. The main limitation of the ELISA method is the antibody cross-reactivity among flaviviruses; also, sampling strategy should be adjusted to take into account that the actual host from which the blood meal was taken may not be determined. Nevertheless, ELISA performed on recently (1-2 days) engorged mosquito, or any other hematophagous arthropod species, could potentially be used as a "wild phlebotomist" to monitor the prevalence or emergence of a variety of pathogens, with less of the practical, ethical, or risk limitations due to direct blood collection from humans and wild or domestic animals.     

2019—2020年哈密市蚊虫种类分布及携带病毒调查

目的　了解新疆维吾尔自治区哈密市蚊虫种类及病毒携带情况,并鉴定该市尖音库蚊所属亚种。方法　分别于2019、2020年7月中旬,在新疆维吾尔自治区哈密市伊州区、伊吾县、巴里坤县采用诱蚊灯法捕捉蚊虫,鉴定所捕获蚊虫种属及尖音库蚊种属。采用逆转录PCR技术检测捕获蚊虫携带黄病毒属、甲病毒属、布尼亚病毒属、流行性乙型脑炎病毒、辽宁病毒、Tahyna病毒、蜱传脑炎病毒及西尼罗病毒情况。结果　在哈密市伊州区、伊吾县、巴里坤县共捕获蚊虫1 496只,分属于3属3种。尖音库蚊为优势蚊种,占所捕获蚊虫总数的65.91%（986只）;其次为里海伊蚊,占30.55%（457只）;阿拉斯加脉毛蚊最少,仅占3.54%（53只）。所捕获尖音库蚊经雄蚊尾器鉴定为尖音库蚊指名亚种。经逆转录PCR法检测,所捕获蚊虫黄病毒属、甲病毒属、布尼亚病毒属、流行性乙型脑炎病毒、辽宁病毒、Tahyna病毒、蜱传脑炎及西尼罗病毒均为阴性。结论　2019—2020年哈密市存在尖音库蚊、里海伊蚊、阿拉斯加脉毛蚊3种蚊虫,以尖音库蚊为优势蚊种;所捕获尖音库蚊均为指名亚种,未发现蚊类携带相关虫媒病毒。