Terrestrial Bird Migration and West Nile Virus Circulation, United States.

Host migration and emerging pathogens are strongly associated, especially with regard to zoonotic diseases. West Nile virus (WNV), a mosquitoborne pathogen capable of causing severe, sometimes fatal, neuroinvasive disease in humans, is maintained in highly mobile avian hosts. Using phylogeographic approaches, we investigated the relationship between WNV circulation in the United States and the flight paths of terrestrial birds. We demonstrated southward migration of WNV in the eastern flyway and northward migration in the central flyway, which is consistent with the looped flight paths of many terrestrial birds. We also identified 3 optimal locations for targeted WNV surveillance campaigns in the United States-Illinois, New York, and Texas. These results illustrate the value of multidisciplinary approaches to surveillance of infectious diseases, especially zoonotic diseases.

The Use of Insecticide-Treated Curtains for Control of Aedes aegypti and Dengue Virus Transmission in "Fraccionamiento" Style Houses in México.

Dengue, chikungunya, yellow fever, and Zika viruses transmitted by Aedes aegypti mosquitoes are major public health threats in the tropical and subtropical world. In México, construction of large tracts of "fraccionamientos" high density housing to accommodate population growth and urbanization has provided fertile ground for Ae. aegypti-transmitted viruses. We investigated the utility of pyrethroid-treated window curtains to reduce both the abundance of Ae. aegypti and to prevent dengue virus (DENV) transmission in fraccionamiento housing. Windows and doors of fraccionamiento homes in urban/suburban areas, where Ae. aegypti pyrethroid resistance associated with the Ile1016 knock down resistance (kdr) mutation in the voltage gated sodium channel gene was high, and in rural areas, where kdr resistance was low, were fitted with either insecticide-treated curtains (ITCs) or non-treated curtains (NTCs). The homes were monitored for mosquito abundance and DENV infection. ITCs reduced the indoor abundance of Ae. aegypti and the number of DENV-infected mosquitoes in homes in rural but not in urban/suburban study sites. The presence of non-treated screens also was associated with reduced numbers of mosquitoes in homes. "Super-infested" homes, yielding more than 50 mosquitoes, including DENV-infected mosquitoes, provide a significant public health risk to occupants, visitors, and people in neighboring homes.

Bunyavirus Taxonomy: Limitations and Misconceptions Associated with the Current ICTV Criteria Used for Species Demarcation.

The International Committee on Taxonomy of Viruses (ICTV) has implemented numerous changes to the taxonomic classification of bunyaviruses over the years. Whereas most changes have been justified and necessary because of the need to accommodate newly discovered and unclassified viruses, other changes are a cause of concern, especially the decision to demote scores of formerly recognized species to essentially strains of newly designated species. This practice was first described in the seventh taxonomy report of the ICTV and has continued in all subsequent reports. In some instances, viruses that share less than 75% nucleotide sequence identity across their genomes, produce vastly different clinical presentations, possess distinct vector and host associations, have different biosafety recommendations, and occur in nonoverlapping geographic regions are classified as strains of the same species. Complicating the matter is the fact that virus strains have been completely eliminated from ICTV reports; thus, critically important information on virus identities and their associated biological and epidemiological features cannot be readily related to the ICTV classification. Here, we summarize the current status of bunyavirus taxonomy and discuss the adverse consequences associated with the reclassification and resulting omission of numerous viruses of public health importance from ICTV reports. As members of the American Committee on Arthropod-borne Viruses, we encourage the ICTV Bunyavirus Study Group to reconsider their stance on bunyavirus taxonomy, to revise the criteria currently used for species demarcation, and to list additional strains of public and veterinary importance.

Metabolomics-Based Discovery of Small Molecule Biomarkers in Serum Associated with Dengue Virus Infections and Disease Outcomes.

BACKGROUND: Epidemic dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) are overwhelming public health capacity for diagnosis and clinical care of dengue patients throughout the tropical and subtropical world. The ability to predict severe dengue disease outcomes (DHF/DSS) using acute phase clinical specimens would be of enormous value to physicians and health care workers for appropriate triaging of patients for clinical management. Advances in the field of metabolomics and analytic software provide new opportunities to identify host small molecule biomarkers (SMBs) in acute phase clinical specimens that differentiate dengue disease outcomes. METHODOLOGY/PRINCIPAL FINDINGS: Exploratory metabolomic studies were conducted to characterize the serum metabolome of patients who experienced different dengue disease outcomes. Serum samples from dengue patients from Nicaragua and Mexico were retrospectively obtained, and hydrophilic interaction liquid chromatography (HILIC)-mass spectrometry (MS) identified small molecule metabolites that were associated with and statistically differentiated DHF/DSS, DF, and non-dengue (ND) diagnosis groups. In the Nicaraguan samples, 191 metabolites differentiated DF from ND outcomes and 83 differentiated DHF/DSS and DF outcomes. In the Mexican samples, 306 metabolites differentiated DF from ND and 37 differentiated DHF/DSS and DF outcomes. The structural identities of 13 metabolites were confirmed using tandem mass spectrometry (MS/MS). Metabolomic analysis of serum samples from patients diagnosed as DF who progressed to DHF/DSS identified 65 metabolites that predicted dengue disease outcomes. Differential perturbation of the serum metabolome was demonstrated following infection with different DENV serotypes and following primary and secondary DENV infections. CONCLUSIONS/SIGNIFICANCE: These results provide proof-of-concept that a metabolomics approach can be used to identify metabolites or SMBs in serum specimens that are associated with distinct DENV infections and disease outcomes. The differentiating metabolites also provide insights into metabolic pathways and pathogenic and immunologic mechanisms associated with dengue disease severity.

Biodistribution and Toxicity Studies of PRINT Hydrogel Nanoparticles in Mosquito Larvae and Cells.

Mosquito-borne diseases continue to remain major threats to human and animal health and impediments to socioeconomic development. Increasing mosquito resistance to chemical insecticides is a great public health concern, and new strategies/technologies are necessary to develop the next-generation of vector control tools. We propose to develop a novel method for mosquito control that employs nanoparticles (NPs) as a platform for delivery of mosquitocidal dsRNA molecules to silence mosquito genes and cause vector lethality. Identifying optimal NP chemistry and morphology is imperative for efficient mosquitocide delivery. Toward this end, fluorescently labeled polyethylene glycol NPs of specific sizes, shapes (80 nm x 320 nm, 80 nm x 5000 nm, 200 nm x 200 nm, and 1000 nm x 1000 nm) and charges (negative and positive) were fabricated by Particle Replication in Non-Wetting Templates (PRINT) technology. Biodistribution, persistence, and toxicity of PRINT NPs were evaluated in vitro in mosquito cell culture and in vivo in Anopheles gambiae larvae following parenteral and oral challenge. Following parenteral challenge, the biodistribution of the positively and negatively charged NPs of each size and shape was similar; intense fluorescence was observed in thoracic and abdominal regions of the larval body. Positively charged NPs were more associated with the gastric caeca in the gastrointestinal tract. Negatively charged NPs persisted through metamorphosis and were observed in head, body and ovaries of adults. Following oral challenge, NPs were detected in the larval mid- and hindgut. Positively charged NPs were more efficiently internalized in vitro than negatively charged NPs. Positively charged NPs trafficked to the cytosol, but negatively charged NPs co-localized with lysosomes. Following in vitro and in vivo challenge, none of the NPs tested induced any cytotoxic effects.

Biodistribution and trafficking of hydrogel nanoparticles in adult mosquitoes.

BACKGROUND: Nanotechnology offers great potential for molecular genetic investigations and potential control of medically important arthropods. Major advances have been made in mammalian systems to define nanoparticle (NP) characteristics that condition trafficking and biodistribution of NPs in the host. Such information is critical for effective delivery of therapeutics and molecules to cells and organs, but little is known about biodistribution of NPs in mosquitoes. METHODOLOGY/PRINCIPAL FINDINGS: PRINT technology was used to construct a library of fluorescently labeled hydrogel NPs of defined size, shape, and surface charge. The biodistribution (organ, tissue, and cell tropisms and trafficking kinetics) of positively and negatively charged 200 nm x 200 nm, 80 nm x 320 nm, and 80 nm x 5000 nm NPs was determined in adult Anopheles gambiae mosquitoes as a function of the route of challenge (ingestion, injection or contact) using whole body imaging and fluorescence microscopy. Mosquitoes readily ingested NPs in sugar solution. Whole body fluorescence imaging revealed substantial NP accumulation (load) in the alimentary tracts of the adult mosquitoes, with the greatest loads in the diverticula, cardia and foregut. Positively and negatively charged NPs differed in their biodistribution and trafficking. Following oral challenge, negatively charged NPs transited the alimentary tract more rapidly than positively charged NPs. Following contact challenge, negatively charged NPs trafficked more efficiently in alimentary tract tissues. Following parenteral challenge, positively and negatively charged NPs differed in tissue tropisms and trafficking in the hemocoel. Injected NPs were also detected in cardia/foregut, suggesting trafficking of NPs from the hemocoel into the alimentary tract. CONCLUSIONS/SIGNIFICANCE: Herein we have developed a tool box of NPs with the biodistribution and tissue tropism characteristics for gene structure/function studies and for delivery of vector lethal cargoes for mosquito control.

Two Novel Bioassays to Assess the Effects of Pyrethroid-Treated Netting on Knockdown-Susceptible Versus Resistant Strains of Aedes aegypti.

We describe 2 new mosquito bioassays for use with insecticide-treated netting or other textiles. The 1st is a cylinder bioassay in which a mosquito is forced to contact treated material regardless of where it lands within the bioassay construct. The 2nd is a repellency/irritancy and biting-inhibition bioassay (RIBB) in which human arms and breath are used as attractants. Mosquitoes have the choice to pass through holes cut in untreated or treated netting to move from a center release chamber into side chambers to reach arms and potentially bite. Trials were conducted with pyrethroid-susceptible (New Orleans), moderately resistant (Hunucmá), and highly resistant (Vergel) strains of Aedes aegypti. Tests with netting treated with different pyrethroids demonstrated the utility of the cylinder bioassay to quantify knockdown and mortality following exposure to treated netting, and of the RIBB to quantify spatial repellency/contact irritancy of the treated netting and biting inhibition after females land on and then pass through holes in the treated netting. Both tested brands of pyrethroid-treated mosquitocidal netting (DuraNet® and NetProtect®) were effective against New Orleans but ineffective against Vergel strains. Mortality in the cylinder bioassay was 100% for New Orleans for all tested brands of treated netting, but only 10-14% for Vergel. Rates of passage through treated netting to reach a human arm in the RIBB were 10-15% for New Orleans versus 24-37% for Vergel. The reduction in biting after passage through treated netting, compared with untreated netting in the same trial replicates, was 12-39% for New Orleans versus ≤9% for Vergel.

Urban Mosquito Fauna in Mérida City, México: Immatures Collected from Containers and Storm-water Drains/Catch Basins.

We examined the species composition and temporal occurrence of immature mosquitoes in containers and storm-water drains/catch basins from November 2011 to June 2013 in Mérida City, México. A wide range of urban settings were examined, including residential premises, vacant lots, parking lots, and streets or sidewalks with storm-water drains/catch basins. In total, 111,776 specimens of 15 species were recorded. The most commonly collected species were Aedes (Stegomyia) aegypti (L.) (n = 60,961) and Culex quinquefasciatus Say (45,702), which together accounted for 95.4% of the immatures collected. These species were commonly encountered during both rainy and dry seasons, whereas most other mosquito species were collected primarily during the rainy season. Other species collected were Aedes (Howardina) cozumelensis Diaz Najera, Aedes (Ochlerotatus) taeniorhynchus (Wiedemann), Aedes (Ochlerotatus) trivittatus (Coquillett), Culex coronator Dyar and Knab, Culex interrogator Dyar and Knab, Culex lactator Dyar and Knab, Culex nigripalpus Theobald, Culex salinarius Coquillett, Culex tarsalis Coquillett, Culex thriambus Dyar, Haemagogus equinus Theobald, Limatus durhamii Theobald, and Toxorhynchites rutilus (Coquillett). The greatest number of species was recorded from vacant lots (n = 11), followed by storm-water drains/catch basins (nine) and residential premises (six). Our study demonstrated that the heterogeneous urban environment in Mérida City supports a wide range of mosquito species, many of which are nuisance biters of humans and/or capable of serving as vectors of pathogens affecting humans or domestic animals. We also briefly reviewed the medical importance of the encountered mosquito species.

Temporal correlations between mosquito-based dengue virus surveillance measures or indoor mosquito abundance and dengue case numbers in Mérida City, México.

Surveillance of dengue virus (DENV) in Aedes (Stegomyia) aegypti (L.) females is of potential interest because human DENV infections are commonly asymptomatic, which decreases the effectiveness of dengue case surveillance to provide early warning of building outbreaks. Our primary aim was to examine if mosquito-based virological measures--monthly percentages of examined Ae. aegypti females infected with DENV or examined homes from which at least one DENV-infected Ae. aegypti female was collected--are correlated with reported dengue cases in the same or subsequent months within study neighborhoods in Méida City, México. The study encompassed approximately 30 neighborhoods in the southern and eastern parts of the city. Mosquitoes were collected monthly over a 15-mo period within study homes (average of 145 homes examined per month); this produced approximately 5,800 Ae. aegypti females subsequently examined for DENV RNA. Although monthly dengue case numbers in the study neighborhoods varied > 100-fold during the study period, we did not find statistically significant positive correlations between monthly data for mosquito-based DENV surveillance measures and reported dengue cases in the same or subsequent months. Monthly average temperature, rainfall, and indoor abundance of Ae. aegypti females were positively correlated (P < or = 0.001) with dengue case numbers in subsequent months with lag times of 3-5, 2, and 1-2 mo, respectively. However, because dengue outbreak risk is strongly influenced by serotype-specific susceptibility of the human population to DENV, the value of weather conditions and entomological indices to predict outbreaks is very limited. Potential ways to improve the sensitivity of mosquito-based DENV surveillance are discussed.

Household use of insecticide consumer products in a dengue-endemic area in México.

OBJECTIVES: To evaluate the household use of insecticide consumer products to kill mosquitoes and other insect pests, as well as the expenditures for using these products, in a dengue-endemic area of México. METHODS: A questionnaire was administered to 441 households in Mérida City and other communities in Yucatán to assess household use of insecticide consumer products. RESULTS: A total of 86.6% of surveyed households took action to kill insect pests with consumer products. The most commonly used product types were insecticide aerosol spray cans (73.6%), electric plug-in insecticide emitters (37.4%) and mosquito coils (28.3%). Mosquitoes were targeted by 89.7% of households using insecticide aerosol spray cans and >99% of households using electric plug-in insecticide emitters or mosquito coils. Products were used daily or every 2 days in most of the households for insecticide aerosol spray cans (61.4%), electric plug-in insecticide emitters (76.2%) and mosquito coils (82.1%). For all products used to kill insect pests, the median annual estimated expenditure per household that took action was 408 Mexican pesos ($MXN), which corresponded to approximately 31 $US. These numbers are suggestive of an annual market in excess of 75 million $MXN (>5.7 million $US) for Mérida City alone. CONCLUSION: Mosquitoes threaten human health and are major nuisances in homes in the study area in México. Households were found to have taken vigorous action to kill mosquitoes and other insect pests and spent substantial amounts of money on insecticide consumer products.

Vacant lots: productive sites for Aedes (Stegomyia) aegypti (Diptera: Culicidae) in Mérida City, México.

We assessed the potential for vacant lots and other nonresidential settings to serve as source environments for Aedes (Stegomyia) aegypti (L.) (Diptera: Culicidae) in Mérida City, México. Mosquito immatures were collected, during November 2011-June 2013, from residential premises (n = 156 site visits) and nonresidential settings represented by vacant lots (50), parking lots (18), and streets or sidewalks (28). Collections totaled 46,025 mosquito immatures of 13 species. Ae. aegypti was the most commonly encountered species accounting for 81.0% of total immatures, followed by Culex quinquefasciatus Say (12.1%). Site visits to vacant lots (74.0%) were more likely to result in collection of Ae. aegypti immatures than residential premises (35.9%). Tires accounted for 75.5% of Ae. aegypti immatures collected from vacant lots. Our data suggest that vacant lots should be considered for inclusion in mosquito surveillance and control efforts in Mérida City, as they often are located near homes, commonly have abundant vegetation, and frequently harbor accumulations of small and large discarded water-holding containers that we now have demonstrated to serve as development sites for immature mosquitoes. In addition, we present data for associations of immature production with various container characteristics, such as storage capacity, water quality, and physical location in the environment.

Stormwater drains and catch basins as sources for production of Aedes aegypti and Culex quinquefasciatus.

We present data showing that structures serving as drains and catch basins for stormwater are important sources for production of the mosquito arbovirus vectors Aedes aegypti and Culex quinquefasciatus in Mérida City, México. We examined 1761 stormwater drains - located in 45 different neighborhoods spread across the city - over dry and wet seasons from March 2012 to March 2013. Of the examined stormwater drains, 262 (14.9%) held water at the time they were examined and 123 yielded mosquito immatures. In total, we collected 64,560 immatures representing nine species. The most commonly encountered species were Cx. quinquefasciatus (n=39,269) and Ae. aegypti (n=23,313). Ae. aegypti and Cx. quinquefasciatus were collected during all 11 months when we found water-filled stormwater drains, and both were found in stormwater drains located throughout Mérida City. We also present data for associations between structural characteristics of stormwater drains or water-related characteristics and the abundance of mosquito immatures. In conclusion, stormwater drains produce massive numbers of Ae. aegypti and Cx. quinquefasciatus across Mérida City, both in the wet and dry seasons, and represent non-residential development sites that should be strongly considered for inclusion in the local mosquito surveillance and control program.

Towards a Casa Segura: a consumer product study of the effect of insecticide-treated curtains on Aedes aegypti and dengue virus infections in the home.

The home, or domicile, is the principal environment for transmission of dengue virus (DENV) between humans and mosquito vectors. Community-wide distribution of insecticide-treated curtains (ITCs), mimicking vector control program-driven interventions, has shown promise to reduce DENV infections. We conducted a Casa Segura consumer product intervention study in Mérida, Mexico to determine the potential to reduce intradomicillary DENV transmission through ITC use in individual homes. Dengue virus infections in mosquitoes and in humans were reduced in homes with ITCs in one of two study subareas. Overall, ITCs reduced intradomicillary DENV transmission; ITC homes were significantly less likely to experience multiple DENV infections in humans than NTC homes. Dengue virus-infected Aedes aegypti females were reduced within the ITC homes where curtain use was highest. Some homes yielded up to nine infected Ae. aegypti females. This study provides insights regarding best practices for Casa Segura interventions to protect homes from intradomicillary DENV transmission.

Detection of dengue virus NS1 antigen in infected Aedes aegypti using a commercially available kit.

Epidemic dengue has emerged throughout the tropical world. In the continued absence of a vaccine against dengue virus (DENV), mosquito vector surveillance and control programs are essential to reduce human infections. An effective test to detect DENV in infected mosquitoes would be a valuable addition to the surveillance effort. We investigated DENV detection in infected Aedes aegypti using a commercially available DENV non-structural protein 1 (NS1) ELISA kit (Platelia Dengue NS1 Ag), and by reverse transcription-polymerase chain reaction (RT-PCR) and virus isolation assays. The DENV-infected mosquitoes were subjected to field-relevant conditions and assayed individually and pooled with uninfected mosquitoes. Overall, DENV NS1 antigen was detected in 98% of infected mosquitoes/pools versus 79% for RT-PCR and 29% for virus isolation. Our results indicate that NS1 is an excellent analyte for detection of DENV in Ae. aegypti and that the tested NS1 antigen kit provides a sensitive, rapid, and convenient test for DENV surveillance in mosquitoes.

Aedes (Stegomyia) aegypti and Aedes (Howardina) cozumelensis in Yucatán State, México, with a summary of published collection records for Ae. cozumelensis.

We collected mosquito immatures from artificial containers during 2010-2011 from 26 communities, ranging in size from small rural communities to large urban centers, located in different parts of Yucatán State in southeastern México. The arbovirus vector Aedes (Stegomyia) aegypti was collected from all 26 examined communities, and nine of the communities also yielded another container-inhabiting Aedes mosquito: Aedes (Howardina) cozumelensis. The communities from which Ae. cozumelensis were collected were all small rural communities (<6,000 inhabitants) in the north-central part of Yucatán State. These new collection records for Ae. cozumelensis demonstrate that this mosquito has a far broader geographic range in the Yucatán Peninsula than previously known. Ae. cozumelensis immatures were collected from both residential premises and cemeteries, with specimens recovered from rock holes as well as various artificial containers including metal cans, flower vases, buckets, tires, and a water storage tank. The co-occurrence with Ae. aegypti in small rural communities poses intriguing questions regarding linkages between these mosquitoes, including the potential for direct competition for larval development sites. Additional studies are needed to determine how commonly Ae. cozumelensis feeds on human blood and whether it is naturally infected with arboviruses or other pathogens of medical or veterinary importance. We also summarize the published records for Ae. cozumelensis, which are restricted to collections from México's Yucatán Peninsula and Belize, and uniformly represent geographic locations where Ae. aegypti can be expected to occur.

Rapid intraspecific evolution of miRNA and siRNA genes in the mosquito Aedes aegypti.

RNA silencing, or RNA interference (RNAi) in metazoans mediates development, reduces viral infection and limits transposon mobility. RNA silencing involves 21-30 nucleotide RNAs classified into microRNA (miRNA), exogenous and endogenous small interfering RNAs (siRNA), and Piwi-interacting RNA (piRNA). Knock-out, silencing and mutagenesis of genes in the exogenous siRNA (exo-siRNA) regulatory network demonstrate the importance of this RNAi pathway in antiviral immunity in Drosophila and mosquitoes. In Drosophila, genes encoding components for processing exo-siRNAs are among the fastest evolving 3% of all genes, suggesting that infection with pathogenic RNA viruses may drive diversifying selection in their host. In contrast, paralogous miRNA pathway genes do not evolve more rapidly than the genome average. Silencing of exo-siRNA pathway genes in mosquitoes orally infected with arboviruses leads to increased viral replication, but little is known about the comparative patterns of molecular evolution among the exo-siRNA and miRNA pathways genes in mosquitoes. We generated nearly complete sequences of all exons of major miRNA and siRNA pathway genes dicer-1 and dicer-2, argonaute-1 and argonaute-2, and r3d1 and r2d2 in 104 Aedes aegypti mosquitoes collected from six distinct geographic populations and analyzed their genetic diversity. The ratio of replacement to silent amino acid substitutions was 1.4 fold higher in dicer-2 than in dicer-1, 27.4 fold higher in argonaute-2 than in argonaute-1 and similar in r2d2 and r3d1. Positive selection was supported in 32% of non-synonymous sites in dicer-1, in 47% of sites in dicer-2, in 30% of sites in argonaute-1, in all sites in argonaute-2, in 22% of sites in r3d1 and in 55% of sites in r2d2. Unlike Drosophila, in Ae. aegypti, both exo-siRNA and miRNA pathway genes appear to be undergoing rapid, positive, diversifying selection. Furthermore, refractoriness of mosquitoes to infection with dengue virus was significantly positively correlated for nucleotide diversity indices in dicer-2.

Productive container types for Aedes aegypti immatures in Mérida, México.

During 2007-2010, we examined which container types in Mérida, México, are most productive for Aedes aegypti (L.) immatures. Surveys for mosquito immatures followed routine surveillance methodology and container type classifications used by Servicios de Salud de Yucatán. Our main findings were that (1) small and larger discarded containers that serve no particular purpose and therefore can be removed from the environment contribute strongly to larval and pupal production in Mérida, and (2) the importance of different container types can vary among sets of residential premises as well as between dry and wet periods. These results may help to guide future implementation in Mérida of control efforts that target the most productive container types for Ae. aegypti immatures. Furthermore, if the Patio Limpio cleanup campaign that currently is ongoing in Mérida proves successful in removing discarded containers as important immature development sites, then we should see dramatic changes in the most productive container types in the future as the mosquito is forced to switch to other container types, which perhaps also will be easier to include in highly targeted mosquito control interventions.

The distribution of potential West Nile virus vectors, Culex pipiens pipiens and Culex pipiens quinquefasciatus (Diptera: Culicidae), in Mexico City.

BACKGROUND: Culex spp. mosquitoes are considered to be the most important vectors of West Nile virus (WNV) detected in at least 34 species of mosquitoes in the United States. In North America, Culex pipiens pipiens, Culex pipiens quinquefasciatus, and Culex tarsalis are all competent vectors of WNV, which is considered to be enzootic in the United States and has also been detected in equines and birds in many states of Mexico and in humans in Nuevo Leon. There is potential for WNV to be introduced into Mexico City by various means including infected mosquitoes on airplanes, migrating birds, ground transportation and infected humans. Little is known of the geographic distribution of Culex pipiens complex mosquitoes and hybrids in Mexico City. Culex pipiens pipiens preferentially feed on avian hosts; Culex pipiens quinquefasciatus have historically been considered to prefer mammalian hosts; and hybrids of these two species could theoretically serve as bridge vectors to transmit WNV from avian hosts to humans and other mammalian hosts. In order to address the potential of WNV being introduced into Mexico City, we have determined the identity and spatial distribution of Culex pipiens complex mosquitoes and their hybrids. RESULTS: Mosquito larvae collected from 103 sites throughout Mexico City during 2004-2005 were identified as Culex, Culiseta or Ochlerotatus by morphological analysis. Within the genus Culex, specimens were further identified as Culex tarsalis or as belonging to the Culex pipiens complex. Members of the Culex pipiens complex were separated by measuring the ratio of the dorsal and ventral arms (DV/D ratio) of the male genitalia and also by using diagnostic primers designed for the Ace.2 gene. Culex pipiens quinquefasciatus was the most abundant form collected. CONCLUSIONS: Important WNV vectors species, Cx. p. pipiens, Cx. p. quinquefasciatus and Cx. tarsalis, are all present in Mexico City. Hybrids of Cx. p. pipiens and Cx. p. quinquefasciatus were also collected and identified. The presence and abundance of these WNV competent vectors is a cause for concern. Understanding the distribution of these vectors can help improve viral surveillance activities and mosquito control efforts in Mexico City.

Mosquito infestation and dengue virus infection in Aedes aegypti females in schools in Merida, Mexico.

We determined abundance of Aedes aegypti mosquitoes and presence of dengue virus (DENV) in females collected from schools in Mérida, México, during 2008 and 2009. Backpack aspiration from 24 schools produced 468 females of Ae. aegypti and 1,676 females of another human biter, Culex quinquefasciatus. Ae. aegypti females were collected most commonly from classrooms followed by offices and bathrooms. Of these females, 24.7% were freshly fed. Examination of 118 pools of Ae. aegypti females (total of 415 females) for presence of DENV RNA produced 19 positive pools (16.1%). DENV-infected pools were detected from 11 (45.8%) of 24 schools and came from different room types, including classrooms, offices, and bathrooms. The overall rate of DENV infection per 100 Ae. aegypti females was 4.8. We conclude that schools in Mérida present a risk environment for students, teachers, and other personnel to be exposed to mosquitoes and bites of DENV-infected Ae. aegypti females.