Indirect impact of COVID-19 on the incidence rates of vector-borne diseases in Mexico.

INTRODUCTION: SARS-CoV-2 infection spatial and temporal distribution overlaps with endemic areas of vector-borne diseases (VBD), whose surveillance in Mexico has substantially changed since the first COVID-19 confirmed case. OBJECTIVES: To estimate and compare the incidence rates of VBDs before and after the introduction of SARS-CoV-2 in Mexico. METHODS: Retrospective study of VBD cases from 2014 to 2021. The incidence rates of each VBD in the period before (2014-2019) and after (2020-2021) the introduction of SARS-CoV-2 in Mexico were calculated and compared. RESULTS: Before the introduction of SARS-CoV-2, the incidence rates of VBDs were high and after the introduction of coronavirus there was a decrease in epidemiological indices; however, there was only statistically significant difference in the incidence rate of malaria (p ≤ 0.05) and other rickettsiae (p ≤ 0.05). CONCLUSIONS: Some measures to reduce COVID-19 cases, such as social distancing, home confinement, reductions in public transport and working at home (home office), probably temporarily decreased the number of VBD cases; however, there may be a resurgence of VBDs in the near future.

INTRODUCCIÓN: La distribución espacial y temporal de la infección por SARS-CoV-2 sobrepasa las áreas endémicas de enfermedades transmitidas por vector (ETV), cuya vigilancia en México ha cambiado sustancialmente a partir del primer caso confirmado de COVID-19. OBJETIVOS: Estimar y comparar las tasas de incidencia de las ETV antes y después de la introducción del SARS-CoV-2 en México. MÉTODOS: Estudio retrospectivo de casos de ETV de 2014 a 2021. Las tasas de incidencia de cada ETV en el periodo previo (2014-2019) y posterior (2020-2021) a la introducción del SARS-CoV-2 en México fueron calculadas y comparadas. RESULTADOS: Antes de la introducción del SARS-CoV-2, las tasas de incidencia de las ETV fueron altas y posterior a la introducción del coronavirus hubo un descenso en los índices epidemiológicos; sin embargo, solo se identificó diferencia estadística significativa en la tasa de incidencia de la malaria (p ≤ 0.05) y otras rickettsias (p ≤ 0.05). CONCLUSIONES: Algunas medidas para reducir los casos de COVID-19, como el distanciamiento social, el confinamiento domiciliario, la reducción en el aforo en el transporte público y el trabajo en casa, probablemente contribuyeron a disminuir temporalmente el número de casos de las ETV; sin embargo, puede haber rebrote de las ETV en el futuro cercano.

Evidence of Zika Virus Infection in Pigs and Mosquitoes, Mexico.

Evidence suggests that pigs seroconvert after experimental exposure to Zika virus and are potential sentinels. We demonstrate that pigs are also susceptible to natural Zika virus infection, shown by the presence of antibodies in domestic pigs in Yucatan, Mexico. Zika virus RNA was detected in 5 species of mosquitoes collected inside pigpens.

Rickettsia parkeri (Rickettsiales: Rickettsiaceae) detected in Amblyomma maculatum ticks collected on dogs in Tabasco, Mexico.

The present study was carried out to identify Rickettsia species with zoonotic potential in ticks collected from dogs in a rural area in Tabasco, Mexico. In total 197 Amblyomma maculatum ticks were collected from 40 domestic dogs. The collected specimens were pooled and subjected to DNA extraction. A fragment (380 bp) of citrate synthase gene (gltA) was amplified by polymerase chain reaction (PCR) using universal primers for Rickettsia. A second PCR was later performed to amplify a fragment (420 bp) of the outer membrane protein B gene (ompB). The PCR products were purified, sequenced and compared using the basic local alignment search tool (BLAST). Twenty out of 40 (50%) tick pools assayed were positive for rickettsial DNA using both primer pairs. The consensus sequence obtained from the ompB gene fragments showed 99.5-100% of identity with strains of Rickettsia parkeri. This study provides the first molecular evidence of the presence of R. parkeri in A. maculatum ticks infesting domestic dogs from southeastern Mexico. Close contact between dogs and humans should lead to consider the infection caused by this species of Rickettsia among the differential diagnoses for people of Tabasco, Mexico, who show acute febrile syndrome associated to inoculation eschar and have a clinical history of tick exposure.

Molecular detection of Dirofilaria immitis in dogs and mosquitoes in Tabasco, Mexico.

BACKGROUND & OBJECTIVES: Dirofilaria immitis is a filarial nematode that causes heartworm disease in domestic as well as wild canines and felines; and cutaneous or pulmonary infections in humans. The purpose of the study was to estimate the prevalence of D. immitis in domestic dogs in Tabasco, Mexico and to assay mosquitoes temporally and spatially associated with dogs for evidence of infection. METHODS: Blood was collected from 1050 dogs in 1039 houses during a random household survey performed in 2016 and 2017. Genomic DNA was extracted and assayed by polymerase chain reaction (PCR) using pan-filarial primers and various species-specific primers. Dog owners were interviewed using a structured questionnaire designed to collect information on factors that may impact the occurrence of filarial infection. The association between canine dirofilariasis prevalence and factors likely to impact infection was determined by univariate logistic regression analysis, followed by multivariate binomial logistic regression analysis. Indoor and outdoor resting mosquitoes were collected from houses by manual aspiration. Mosquitoes were identified according to species, homogenized and tested by PCR for filarial nematodes. RESULTS: A total of 84 (8%) dogs were positive for D. immitis DNA, while 3 (0.3%) dogs contained Acanthocheilonema reconditum DNA. Several factors were significantly associated with D. immitis infection. For example, dogs that lived <100 m from a large source of open standing water were significantly more likely (p = 0.002) to become infected with D. immitis than other dogs. Additionally, dogs with infrequent or no anthelmintic treatment were significantly more likely (p = 0.0) to become infected than dogs that were regularly treated. The entomologic investigation yielded 2618 female mosquitoes from 14 species. Four pools of Culex quinquefasciatus were positive for D. immitis DNA and the minimum infection rate, calculated as the number of positive pools per 1000 mosquitoes tested, was 2.9. INTERPRETATION & CONCLUSION: The study identified several factors positively associated with an increased risk of D. immitis infection in domestic dogs in Tabasco and provides evidence that Cx. quinquefasciatus is potentially an important vector in this region. This information can be used by local veterinarians and dog owners to reduce the burden of D. immitis on canine health.

Chikungunya Virus in Febrile Humans and Aedes aegypti Mosquitoes, Yucatan, Mexico.

Chikungunya virus (CHIKV) was isolated from 12 febrile humans in Yucatan, Mexico, in 2015. One patient was co-infected with dengue virus type 1. Two additional CHIKV isolates were obtained from Aedes aegypti mosquitoes collected in the homes of patients. Phylogenetic analysis showed that the CHIKV isolates belong to the Asian lineage.

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.

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.

Dataset of assembly and annotation of the mitogenomes of Triatoma dimidiata and Triatoma huehuetenanguensis captured from Yucatán, México.

Triatoma dimidiata is a species complex, and its members are responsible for the transmission of Trypanosoma cruzi, the causative agent of Chagas disease. We present the assembly and annotation of the mitogenome of the Triatoma dimidiata (Latreille, 1811) and Triatoma huehuetenanguensis Lima-Cordón & Justi, 2019. The mitochondrial genomes were successfully sequenced using the Illumina Nextseq 500 platform, 2×75 cycles, and 5 million reads per sample. Contigs were assembled and annotated using the reference genomes of T. dimidiata and T. huehuetenanguensis available in Genbank (NC_002609 and NC_050325.1, respectively). The mitogenomes of T. dimidiata have lengths of 17,008 bp, while those of T. huehuetenanguensis are 15,910 bp and 15,909 bp. The genome comprises 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and a control region. The mitogenomes will be valuable to scholars and students focused on integrative taxonomy, phylogeography, and evolutionary studies of the Triatoma dimidiata complex and the transmission of Chagas diseases.

Entomo-virological surveillance of Flavivirus in mosquitoes in Yucatan State, Mexico.

The genus Flavivirus (Family: Flaviviridae) comprises arboviruses with the capacity to infect humans and animals. It also integrates insect-specific viruses. This study aimed to identify Flavivirus in mosquitoes captured in 17 municipalities in Yucatan State, Mexico. The mosquitoes were caught in households from November 2021 to May 2022. A total of 4,321 adult mosquitoes from five species were caught. The most abundant were Culex quinquefasciatus (n = 3,563) and Aedes aegypti (n = 734). For molecular investigations, 600 female mosquitoes were split into groups of 10, mostly for species and site location. Reverse transcriptase polymerase chain reaction (RT-PCR) amplified a region of the NS5 gene to find the Flavivirus ribonucleic acids (RNA). A total of 24 pools that were positive for Flavivirus were detected in Ae. aegypti specimens and subsequently subjected to sequencing using the Sanger method. A total of 12 sequences matched the established quality criteria and were subsequently employed for sequence homology analysis. We found that one sequence corresponded to the Zika virus (ZIKV), and 11 sequences had sequence similarity with Phlebotomus-associated flavivirus (PAFV), an insect-specific virus (ISF). In conclusion, we found ZIKV in the Merida municipality, Yucatan State, which suggests that the virus is silently circulating. Phlebotomus-associated flavivirus is distributed in five municipalities in Yucatan State, Mexico. Future studies could focus on isolating this virus and studying its biological role within Ae. aegypti.

The mosquitoes (Diptera: Culicidae) of the Mexican Yucatan Peninsula: a comprehensive review on the use of taxonomic names.

The Yucatan Peninsula is a biogeographic province of the Neotropical region which is mostly encompassed by the 3 Mexican states of Campeche, Quintana Roo, and Yucatán. During the development of the International Joint Laboratory ELDORADO (Ecosystem, bioLogical Diversity, habitat mOdifications and Risk of emerging PAthogens and Diseases in MexicO), a French-Mexican collaboration between the IRD (Institut de Recherche pour le Développement) and UNAM (Universidad Nacional Autónoma de México) in Mérida, it became evident that many putative mosquito species names recorded in the Mexican Yucatan Peninsula were misidentifications/misinterpretations or from the uncritical repetition of incorrect literature records. To provide a stronger foundation for future studies, the mosquito fauna of the Mexican Yucatan Peninsula is here comprehensively reviewed using current knowledge of taxonomy, ecology, and distribution of species through extensive bibliographic research, and examination of newly collected specimens. As a result, 90 mosquito species classified among 16 genera and 24 subgenera are recognized to occur in the Mexican Yucatan Peninsula, including 1 new peninsula record and 3 new state records.

New State Record for Culex rejector and Cx. restrictor in Yucatan, Mexico.

Several known mosquito species occur in the Mexican state of Yucatan, including competent and suspected vectors responsible for transmitting zoonotic arboviruses. Between February and July 2022, mosquito collections were carried out in several forested areas in Yucatan. As part of the ongoing monitoring, we present the first reports of Culex (Microculex) rejector and Cx. (Anoedioporpa) restrictor. Another 14 species were identified during the monitoring: Aedes albopictus, Ae. bimaculatus, Ae. tormentor, Ae. cozumelensis, Anopheles albimanus, Cx. coronator s.l., Cx. erraticus, Cx. lactator, Cx. salinarius, Coquillettidia venezuelensis, Limatus durhamii, Psorophora ciliata, Toxorhynchites theobaldi, and Wyeomyia mitchellii. Currently, the mosquito fauna in Yucatan consists of 65 species. The subgenera Microculex and Anoedioporpa had not been documented in Yucatan State prior to the current investigation.

Mites associated with budgerigars Melopsittacus undulatus (Psittaciformes: Psittacidae) and the first report of Ornithonyssus bursa (Mesostigmata: Macronyssidae) in Mexico.

Background: Hematophagous mites affect numerous bird species, causing severe injuries to the budgerigars. Some species can cause dermatitis in humans. Aims: The purpose was to morphologically identify the mites related to budgerigars (Melopsittacus undulatus) and their nests in Yucatan, Mexico. Methods: In May 2022, a private budgerigar hatchery was visited and mites were collected from the bodies of the birds and their nests. The morphological traits of the mites were confirmed by scanning electron microscopy. Results: Four of 30 birds showed severe clinical signs of mite infestation. The Budgerigars revealed lesions in the cere, nostrils, eyelids, beak, and paws. The bird's skin showed signs of dryness and beige coloring. The birds with severe damage also presented anorexia and had deformed paws and beaks. The parasitosis was caused by the "burrowing mites," Knemidocoptes pilae. The burrowing mites and the Grallacheles bakeri were recovered and identified from paw scabs. To eliminate mites, a topical application of Ivermectin was administered to the necks of the birds. The dose was a single, which has a residuality of 21 days. Two drops (0.115 mg/ml) of ivermectin were applied to each bird. A gradual reduction in crusted lesions due to mite mortality was noted. The "tropical fowl mite" Ornithonyssus bursa was identified in the nests, which represents the first record in Mexico. Conclusions: Three species of mites were discovered in a single budgerigar hatchery. This emphasizes the importance of deworming birds and keeping a clean environment in their cages to reduce the potential for parasitic mite infestation.

Mosquito Fauna in the Mangroves of Yucatan, Mexico, and Identification of Alphavirus RNA.

The surveillance of arboviruses in mangrove mosquitoes is a neglected topic in Mexico. The Yucatan State is part of a peninsula and, therefore, is rich in mangroves along its coast. The purpose of the study was to identify alphavirus in the mosquito fauna of mangroves. Mosquitoes were captured in mangrove settings in seven communities in Yucatan between June 2019 and August 2021. From 1900 to 2200 h and from 0500 to 0800 h, mosquitoes were captured with a backpack-mounted aspirator. In total, 3,167 female mosquitoes of five genera and nine species were captured. Aedes taeniorhynchus and Anopheles crucians were the most abundant mosquitoes collected. Mosquitoes were sorted into 210 pools and tested by reverse transcription-polymerase chain reaction for alphavirus ribonucleic acid (RNA). Alphavirus RNA was found in Ae. taeniorhynchus, An. pseudopunctipennis, and An. crucians collected in the Celestun Mangrove. The community is part of the Ria Celestun Biosphere Reserve, and the presence arbovirus-infected mosquitoes could pose a health risk to residents and visitors alike in the area.

Cluster Analysis of Dengue Morbidity and Mortality in Mexico from 2007 to 2020: Implications for the Probable Case Definition.

Dengue cases and deaths occur frequently in Mexico, although the trend is not uniform across the country. We performed a Spatio-temporal analysis of dengue cases and deaths in Mexico from 2007 to 2020, and clustered states according to whether there was a low, moderate, or high risk of dengue. A total of 501,600 confirmed dengue cases were registered from 2007 to 2020, with 378,122 cases classified as dengue fever (DF) and 123,478 cases classified as dengue hemorrhagic fever (DHF). For each confirmed case, there were 4.68 probable cases. There were 1,230 dengue deaths, with highest numbers reported in 2009, 2012, 2013, and 2019. The number of deaths had a significant correlation (P ≤ 0.01) with DF (r = 0.82), DHF (r = 0.94), and probable dengue cases (r = 0.84). States were clustered using Machine Learning technique according to select indices associated with dengue. Cluster 1 (low risk) primarily contained states in the northwest, northcentral, and east. Cluster 2 (moderate risk) includes states in the northeast. Cluster 3 (high risk) mostly contained coastal states in the southeast, southwest, and west. The generation of the clusters was supported by the Kruskal-Wallis test. A significant difference was found in the incidence, mortality rates, and case-fatality rates of dengue among the clusters (P ≤ 0.01). Notably, cluster 3 contributed 71.4% of the confirmed cases and 89.2% of the deaths. Public health and vector control strategies designed to mitigate the burden of dengue in Mexico should consider the states in cluster 3 as high priority areas.

Evaluation of the effectiveness of fluralaner against adult stages of Rhodnius prolixus in dogs.

Triatomines are vectors of American Trypanosomiasis also known as Chagas´ disease where several reservoirs including dogs are involved in the transmission cycle of the causal agent (Trypanosoma cruzi). Considering that the prevalence of American trypanosomiasis in dogs is higher than in humans and that dogs in addition are susceptible of this disease, and are involved in peridomestic transmission to humans, the search for new alternatives for vector control of the triatomines responsible for transmission in dogs is required. Over the 20 weeks the study lasted, 600 individual female, adult of Rhodnius prolixus were offered to the 15 dogs treated with a single oral dose of Fluralaner (Bravecto®, MSD). Feeding pattern of triatomines was not affected by the treatment during the whole study. The fluralaner-induced mortality of R. prolixus had a significant effect until week 12 at which time 100% mortality was observed. Mortality decreased to 67.5% at week 16 to practically nil 0.8% on week 20. Fluralaner achieved 100% mortality of triatomines between 12- and 48-h post-feeding. It was demonstrated that a single oral dose of fluralaner in dogs is highly effective in producing mortality in adult R. prolixus for the time guaranteed by the manufacturer for other blood-sucking insects, with a considerable effective residual effect for up to 16 weeks. Due to this high efficacy, fluralaner could be considered in strategies to control the transmission vectors of Chagas disease in dogs and in turn decrease the peri-domestic transmission cycle, particularly in hyperendemic areas.

Molecular survey of Zika virus in the animal-human interface in traditional farming.

Backyard animal husbandry is common in rural communities in developing countries and, given the conditions in which it occurs, it can increase the risk of disease transmission, such as arboviruses. To determine the presence of the Zika virus (ZIKV) and abundance of its arthropod vectors we evaluated the socioeconomic implications involved in its transmission in two highly vulnerable Mayan communities in the state of Yucatan that practice backyard farming. An analytical cross-sectional study was carried out throughout 2016 to understand socioeconomic variables and seasonal patterns in mosquito populations. We selected 20 households from each community. Social exclusion indicators were analyzed, human and domestic animals were sampled, and mosquitoes were collected and identified. Four out of eight indicators of social exclusion were higher than the reported national averages. We captured 5,825 mosquitoes from 16 species being Culex quinquefasciatus and Aedes aegypti the most abundant. The presence of chickens and human overcrowding in dwellings were the most significant factors (P = 0.026) associated with the presence of Ae. aegypti. Septic tanks (odds ratio = 6.64) and chickens (odds ratio = 27.41) in backyards were the main risk factors associated with the presence of immature states of Ae. aegypti in both communities. Molecular analysis to detect ZIKV was performed in blood samples from 416 humans, 1,068 backyard animals and 381 mosquito pools. Eighteen humans and 10 pig pools tested positive for ZIKV. Forty-three mosquito pools tested positive for flavivirus. Ten of the 43 pools of positive mosquitoes were sequenced, corresponding 3/10 to ZIKV and 1/10 to Dengue virus type 2. The findings obtained indicate the continuous circulation of Flavivirus (including ZIKV) in backyard environments in vulnerable communities, highlighting the importance of studying their transmission and maintenance in these systems, due that backyard animal husbandry is a common practice in these vulnerable communities with limited access to health services.

An Updated Review of the Invasive Aedes albopictus in the Americas; Geographical Distribution, Host Feeding Patterns, Arbovirus Infection, and the Potential for Vertical Transmission of Dengue Virus.

Aedes (Stegomyia) albopictus is a mosquito native to Southeast Asia. Currently, it has a wide distribution in America, where natural infection with arboviruses of medical and veterinary importance has been reported. In spite of their importance in the transmission of endemic arbovirus, the basic information of parameters affecting their vectorial capacity is poorly investigated. The aim of the work was to update the distribution range of Ae. albopictus in the Americas, review the blood-feeding patterns, and compare the minimum infection rate (MIR) of the Dengue virus (DENV) between studies of vertical and horizontal transmission. The current distribution of Ae. albopictus encompasses 21 countries in the Americas. An extensive review has been conducted for the blood-feeding patterns of Ae. albopictus. The results suggest that the mosquito is capable of feeding on 16 species of mammals and five species of avian. Humans, dogs, and rats are the most common hosts. Eight arboviruses with the potential to infect humans and animals have been isolated in Ae. albopictus. In the United States of America (USA), Eastern equine encephalitis virus, Keystone virus, La Crosse Virus, West Nile virus, and Cache Valley virus were isolated in the Asian mosquito. In Brazil, Mexico, Colombia, and Costa Rica, DENV (all serotypes) has been frequently identified in field-caught Ae. albopictus. Overall, the estimated MIR in Ae. albopictus infected with DENV is similar between horizontal (10.95) and vertical transmission (8.28). However, in vertical transmission, there is a difference in the MIR values if the DENV is identified from larvae or adults (males and females emerged from a collection of eggs or larvae). MIR estimated from larvae is 14.04 and MIR estimated in adults is 4.04. In conclusion, it has to be highlighted that Ae. albopictus is an invasive mosquito with wide phenotypic plasticity to adapt to broad and new areas, it is highly efficient to transmit the DENV horizontally and vertically, it can participate in the inter-endemic transmission of the dengue disease, and it can spread zoonotic arboviruses across forest and urban settings.

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

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