Evaluating vector competence for Yellow fever in the Caribbean.

The mosquito-borne disease, Yellow fever (YF), has been largely controlled via mass delivery of an effective vaccine and mosquito control interventions. However, there are warning signs that YF is re-emerging in both Sub-Saharan Africa and South America. Imported from Africa in slave ships, YF was responsible for devastating outbreaks in the Caribbean. In Martinique, the last YF outbreak was reported in 1908 and the mosquito Aedes aegypti was incriminated as the main vector. We evaluated the vector competence of fifteen Ae. aegypti populations for five YFV genotypes (Bolivia, Ghana, Nigeria, Sudan, and Uganda). Here we show that mosquito populations from the Caribbean and the Americas were able to transmit the five YFV genotypes, with YFV strains for Uganda and Bolivia having higher transmission success. We also observed that Ae. aegypti populations from Martinique were more susceptible to YFV infection than other populations from neighboring Caribbean islands, as well as North and South America. Our vector competence data suggest that the threat of re-emergence of YF in Martinique and the subsequent spread to Caribbean nations and beyond is plausible.

First report of natural Wolbachia infections in mosquitoes from Cuba.

Mosquitoes are extensively responsible for the transmission of pathogens. Novel strategies using Wolbachia could transform that scenario, since these bacteria manipulate mosquito reproduction, and can confer a pathogen transmission-blocking phenotype in culicids. Here, we screened the Wolbachia surface protein region by PCR in eight Cuban mosquito species. We confirmed the natural infections by sequencing and assessed the phylogenetic relationships among the Wolbachia strains detected. We identified four Wolbachia hosts: Aedes albopictus, Culex quinquefasciatus, Mansonia titillans, and Aedes mediovittatus (first report worldwide). Knowledge of Wolbachia strains and their natural hosts is essential for future operationalization of this vector control strategy in Cuba.

Zika vector competence data reveals risks of outbreaks: the contribution of the European ZIKAlliance project.

First identified in 1947, Zika virus took roughly 70 years to cause a pandemic unusually associated with virus-induced brain damage in newborns. Zika virus is transmitted by mosquitoes, mainly Aedes aegypti, and secondarily, Aedes albopictus, both colonizing a large strip encompassing tropical and temperate regions. As part of the international project ZIKAlliance initiated in 2016, 50 mosquito populations from six species collected in 12 countries were experimentally infected with different Zika viruses. Here, we show that Ae. aegypti is mainly responsible for Zika virus transmission having the highest susceptibility to viral infections. Other species play a secondary role in transmission while Culex mosquitoes are largely non-susceptible. Zika strain is expected to significantly modulate transmission efficiency with African strains being more likely to cause an outbreak. As the distribution of Ae. aegypti will doubtless expand with climate change and without new marketed vaccines, all the ingredients are in place to relive a new pandemic of Zika.

Vector competence of Aedes aegypti from Havana, Cuba, for dengue virus type 1, chikungunya, and Zika viruses.

BACKGROUND: Like many countries from the Americas, Cuba is threatened by Aedes aegypti-associated arboviruses such as dengue (DENV), Zika (ZIKV), and chikungunya (CHIKV) viruses. Curiously, when CHIKV was actively circulating in the region in 2013-2014, no autochthonous transmission of this virus was detected in Havana, Cuba, despite the importation of chikungunya cases into this city. To investigate if the transmission ability of local mosquito populations could explain this epidemiological scenario, we evaluated for the first time the vector competence of two Ae. aegypti populations (Pasteur and Párraga) collected from Havana for dengue virus type 1 (DENV-1), CHIKV, and ZIKV. METHODOLOGY/PRINCIPAL FINDINGS: Mosquito populations were fed separately using blood containing ZIKV, DENV-1, or CHIKV. Infection, dissemination, and transmission rates, were estimated at 3 (exclusively for CHIKV), 7, and 14 days post exposure (dpe) for each Ae. aegypti population-virus combination. Both mosquito populations were susceptible to DENV-1 and ZIKV, with viral infection and dissemination rates ranging from 24-97% and 6-67% respectively. In addition, CHIKV disseminated in both populations and was subsequently transmitted. Transmission rates were low (<30%) regardless of the mosquito population/virus combination and no ZIKV was detected in saliva of females from the Pasteur population at any dpe. CONCLUSIONS/SIGNIFICANCE: Our study demonstrated the ability of Ae. aegypti from Cuba to transmit DENV, ZIKV, and CHIKV. These results, along with the widespread distribution and high abundance of this species in the urban settings throughout the island, highlight the importance of Ae. aegypti control and arbovirus surveillance to prevent future outbreaks.

Vector-borne transmission and evolution of Zika virus.

Zika virus (ZIKV), discovered in the Zika Forest of Uganda in 1947, is a mosquito-borne flavivirus related to yellow fever, dengue and West Nile viruses. From its discovery until 2007, only sporadic ZIKV cases were reported, with mild clinical manifestations in patients. Therefore, little attention was given to this virus before epidemics in the South Pacific and the Americas that began in 2013. Despite a growing number of ZIKV studies in the past three years, many aspects of the virus remain poorly characterized, particularly the spectrum of species involved in its transmission cycles. Here, we review the mosquito and vertebrate host species potentially involved in ZIKV vector-borne transmission worldwide. We also provide an evidence-supported analysis regarding the possibility of ZIKV spillback from an urban cycle to a zoonotic cycle outside Africa, and we review hypotheses regarding recent emergence and evolution of ZIKV. Finally, we identify critical remaining gaps in the current knowledge of ZIKV vector-borne transmission.

Spatio-temporal distribution of vertically transmitted dengue viruses by Aedes aegypti (Diptera: Culicidae) from Arroyo Naranjo, Havana, Cuba.

OBJECTIVE: To study the distribution of vertical transmission of dengue viruses in field-collected Aedes aegypti larvae in the municipality of Arroyo Naranjo in Havana, Cuba. METHODS: Aedes aegypti larvae and pupae were collected monthly between September 2013 and July 2014 in the seven Municipal Health Areas of Arroyo Naranjo. Pools formed of 30-55 larvae were examined through PCR and sequencing to detect the presence of each serotype. RESULTS: We analysed 111 pools of larvae and pupae (4102 individuals) of which 37 tested positive for at least one DENV. More than one DENV type was observed in 10 of the 37 positive pools. Infected pools were detected every month, except in January, suggesting a sustained circulation of DENV in the vector populations. DENV-1 and DENV-3 were the most frequent and dispersed, though all four DENV types were detected. Nucleotide sequencing from positive pools confirmed RT-PCR results for DENV-1 (genotype V), DENV-3 (genotype III) and DENV-4 (genotype II). DENV-2 was detected by RT-PCR but could not be confirmed by nucleotide sequencing. CONCLUSION: Our study of the distribution of natural vertical transmission of dengue virus types highlights extrinsic virus activity patterns in the area and could be used as a new surveillance tool.

First record of natural vertical transmission of dengue virus in Aedes aegypti from Cuba.

While horizontal transmission (human-mosquito-human) of dengue viruses largely determines the epidemiology of the disease, vertical transmission (infected female mosquito- infected offspring) has been suggested as a mechanism that ensures maintenance of the virus during adverse conditions for horizontal transmission to occur. The purpose of this study was to analyze the natural infection of larval stages of Aedes aegypti (Diptera: Culicidae) with the dengue virus (DENV) in Cuba. Here, we report vertical transmission of DENV-3 genotype III in natural populations of Ae. aegypti through RT-PCR detection and serotyping plus sequencing. Our report constitutes the first record of vertical transmission of DENV in Ae. aegypti from Cuba with details of its serotype and genotype.

Actividad incrementada de las enzimas citocromo P450 monooxigenasas en cepas cubanas de Aedes aegypti de referencia, resistentes a insecticidas / Increased activity of cytochrome P450 monooxygenase enzymes in reference insecticide-resistant Aedes aegypti strains from Cuba

Introducción: las enzimas desintoxicadoras citocromo P450 monooxigenasas (MFO) constituyen uno de los principales mecanismos de resistencia de Aedes aegypti a insecticidas. En Cuba, aunque la presencia de estas enzimas se ha estudiado in vivo mediante el uso de sinergistas, su actividad no se ha determinado cuantitativamente in vitro, elemento que resulta imprescindible para abordar estudios de resistencia metabólica en los insectos. Objetivo: estandarizar un método para detectar la actividad de las citocromo P450 monooxigenasas in vitro, y determinarla entonces, en larvas y adultos de cepas de referencia de Aedes aegypti. Métodos: se utilizaron 3 cepas de laboratorio de Aedes aegypti seleccionadas por 14 o 15 generaciones con temefos, deltametrina o propoxur, respectivamente, y una cepa susceptible a los insecticidas. Resultados: se establecieron las condiciones para los ensayos de actividad enzimática (concentración de proteínas y de sustrato, 0,4 mg/mL y 12 mmol/L, respectivamente; y tiempo de reacción de 10 min). Hubo un incremento significativo de la actividad de las citocromo P450 monooxigenasas en las cepas resistentes, con una mayor frecuencia fenotípica de este carácter en el estadio larva. Conclusiones: las modificaciones a la técnica para la determinación de la actividad enzimática permitieron discriminar entre mosquitos de cepas susceptibles y resistentes en los estadios larva y adulto, por lo que se cuenta con otra herramienta para la detección de la resistencia metabólica en Cuba

Introduction: cytochrome P450 monooxygenase detoxifying enzymes (MFO) are one of the main resistance mechanisms of Aedes aegypti to insecticides. In vivo studies of the presence of these enzymes have been conducted in Cuba with the use of synergists. However, their activity has not been quantitatively determined in vitro, an indispensable step in studies about metabolic resistance in insects. Objective: standardize a method to detect the activity of cytochrome P450 monooxygenase in vitro, and then determine such activity in larvae and adults of Aedes aegypti reference strains. Methods: the study was based on three laboratory strains of Aedes aegypti selected for 14 or 15 generations with temephos, deltamethrin or propoxur, respectively, and a strain susceptible to insecticides. Results: the conditions for enzyme activity assays were established (protein and substrate concentration: 0.4 mg/mL and 12 mmol/L, respectively, and reaction time: 10 min). There was a significant increase in cytochrome P450 monooxygenase activity in resistant strains, with a higher phenotypic frequency in the larval stage. Conclusions: modifications to the technique used for determination of enzymatic activity made it possible to distinguish between mosquitoes from susceptible and resistant strains in larval and adult stages, providing a new tool for the detection of metabolic resistance in Cuba

Actividad incrementada de las enzimas citocromo P450 monooxigenasas en cepas cubanas de Aedes aegypti de referencia, resistentes a insecticidas / Increased activity of cytochrome P450 monooxygenase enzymes in reference insecticide-resistant Aedes aegypti strains from Cuba

Introducción: las enzimas desintoxicadoras citocromo P450 monooxigenasas (MFO) constituyen uno de los principales mecanismos de resistencia de Aedes aegypti a insecticidas. En Cuba, aunque la presencia de estas enzimas se ha estudiado in vivo mediante el uso de sinergistas, su actividad no se ha determinado cuantitativamente in vitro, elemento que resulta imprescindible para abordar estudios de resistencia metabólica en los insectos. Objetivo: estandarizar un método para detectar la actividad de las citocromo P450 monooxigenasas in vitro, y determinarla entonces, en larvas y adultos de cepas de referencia de Aedes aegypti. Métodos: se utilizaron 3 cepas de laboratorio de Aedes aegypti seleccionadas por 14 o 15 generaciones con temefos, deltametrina o propoxur, respectivamente, y una cepa susceptible a los insecticidas. Resultados: se establecieron las condiciones para los ensayos de actividad enzimática (concentración de proteínas y de sustrato, 0,4 mg/mL y 12 mmol/L, respectivamente; y tiempo de reacción de 10 min). Hubo un incremento significativo de la actividad de las citocromo P450 monooxigenasas en las cepas resistentes, con una mayor frecuencia fenotípica de este carácter en el estadio larva. Conclusiones: las modificaciones a la técnica para la determinación de la actividad enzimática permitieron discriminar entre mosquitos de cepas susceptibles y resistentes en los estadios larva y adulto, por lo que se cuenta con otra herramienta para la detección de la resistencia metabólica en Cuba(AU)

Introduction: cytochrome P450 monooxygenase detoxifying enzymes (MFO) are one of the main resistance mechanisms of Aedes aegypti to insecticides. In vivo studies of the presence of these enzymes have been conducted in Cuba with the use of synergists. However, their activity has not been quantitatively determined in vitro, an indispensable step in studies about metabolic resistance in insects. Objective: standardize a method to detect the activity of cytochrome P450 monooxygenase in vitro, and then determine such activity in larvae and adults of Aedes aegypti reference strains. Methods: the study was based on three laboratory strains of Aedes aegypti selected for 14 or 15 generations with temephos, deltamethrin or propoxur, respectively, and a strain susceptible to insecticides. Results: the conditions for enzyme activity assays were established (protein and substrate concentration: 0.4 mg/mL and 12 mmol/L, respectively, and reaction time: 10 min). There was a significant increase in cytochrome P450 monooxygenase activity in resistant strains, with a higher phenotypic frequency in the larval stage. Conclusions: modifications to the technique used for determination of enzymatic activity made it possible to distinguish between mosquitoes from susceptible and resistant strains in larval and adult stages, providing a new tool for the detection of metabolic resistance in Cuba(AU)