The influence of culture-dependent native microbiota in Zika virus infection in Aedes aegypti.

BACKGROUND: Emerging and re-emerging vector-borne diseases (VBDs) pose a recurring threat to tropical countries, mainly due to the abundance and distribution of the Aedes aegypti mosquito, which is a vector of the Zika, dengue, chikungunya, and yellow fever arboviruses. METHODS: Female 3-5 day-old Ae. aegypti were distributed into two experimental groups: group I-survey of cultivable bacteria; sucrose group: fed only on sucrose, i.e., non-blood-fed (UF); blood-fed group: (i) fed with non-infected blood (BF); (ii) fed with blood infected with the Zika virus (BZIKV); (iii) pretreated with penicillin/streptomycin (pen/strep), and fed with non-infected blood (TBF); (iv) pretreated with pen/strep and fed blood infected with ZIKV, i.e., gravid with developed ovaries, (TGZIKV); group II-experimental co-infections: bacteria genera isolated from the group fed on sucrose, i.e., non-blood-fed (UF). RESULTS: Using the cultivable method and the same mosquito colony and ZIKV strain described by in a previous work, our results reveled 11 isolates (Acinetobacter, Aeromonas, Cedecea, Cellulosimicrobium, Elizabethkingia, Enterobacter, Lysinibacillus, Pantoea, Pseudomonas, Serratia, and Staphylococcus). Enterobacter was present in all evaluated groups (i.e., UF, BF, BZIKV, TBF, and TGZIKV), whereas Elizabethkingia was present in the UF, BZIKV, and TBF groups. Pseudomonas was present in the BZIKV and TBF groups, whereas Staphylococcus was present in the TBF and TGZIKV groups. The only genera of bacteria that were found to be present in only one group were Aeromonas, Lysinibacillus, and Serratia (UF); Cedacea, Pantoea and Acinetobacter (BF); and Cellulosimicrobium (BZIKV). The mosquitoes co-infected with ZIKV plus the isolates group fed on sucrose (UF) showed interference in the outcome of infection. CONCLUSIONS: We demonstrate that the distinct feeding aspects assessed herein influence the composition of bacterial diversity. In the co-infection, among ZIKV, Ae. aegypti and the bacterial isolates, the ZIKV/Lysinibacillus-Ae. aegypti had the lowest number of viral copies in the head-SG, which means that it negatively affects vector competence. However, when the saliva was analyzed after forced feeding, no virus was detected in the mosquito groups ZIKV/Lysinibacillus-Lu. longipalpis and Ae. aegypti; the combination of ZIKV/Serratia may interfere in salivation. This indicates that the combinations do not produce viable viruses and may have great potential as a method of biological control.

Brazilian Aedes aegypti as a Competent Vector for Multiple Complex Arboviral Coinfections.

BACKGROUND: Aedes aegypti is a highly competent vector in the transmission of arboviruses, such as chikungunya, dengue, Zika, and yellow fever viruses, and causes single and coinfections in the populations of tropical countries. METHODS: The infection rate, viral abundance (VA), vector competence (VC), disseminated infection, and survival rate were recorded after single and multiple infections of the vector with 15 combinations of chikungunya, dengue, Zika, and yellow fever arboviruses. RESULTS: Infection rates were 100% in all single and multiple infection experiments, except in 1 triple coinfection that presented a rate of 50%. The VC and disseminated infection rate varied from 100% (in single and quadruple infections) to 40% (in dual and triple infections). The dual and triple coinfections altered the VC and/or VA of ≥1 arbovirus. The highest viral VAs were detected for a single infection with chikungunya. The VAs in quadruple infections were similar when compared with each respective single infection. A decrease in survival rates was observed in a few combinations. CONCLUSIONS: A. aegypti was able to host all single and multiple arboviral coinfections. The interference of the chikungunya virus suggests that distinct arbovirus families may have a significant role in complex coinfections.

Dengue Infection Susceptibility of Five Aedes aegypti Populations from Manaus (Brazil) after Challenge with Virus Serotypes 1-4.

The successful spread and maintenance of the dengue virus (DENV) in mosquito vectors depends on their viral infection susceptibility, and parameters related to vector competence are the most valuable for measuring the risk of viral transmission by mosquitoes. These parameters may vary according to the viral serotype in circulation and in accordance with the geographic origin of the mosquito population that is being assessed. In this study, we investigated the effect of DENV serotypes (1-4) with regards to the infection susceptibility of five Brazilian Ae. aegypti populations from Manaus, the capital of the state of Amazonas, Brazil. Mosquitoes were challenged by oral infection with the DENV serotypes and then tested for the presence of the arbovirus using quantitative PCR at 14 days post-infection, which is the time point that corresponds to the extrinsic incubation period of Ae. aegypti when reared at 28 °C. Thus, we were able to determine the infection patterns for DENV-1, -2, -3 and -4 in the mosquito populations. The mosquitoes had both interpopulation and inter-serotype variation in their viral susceptibilities. All DENV serotypes showed a similar tendency to accumulate in the body in a greater amount than in the head/salivary gland (head/SG), which does not occur with other flaviviruses. For DENV-1, DENV-3, and DENV-4, the body viral load varied among populations, but the head/SG viral loads were similar. Differently for DENV-2, both body and head/SG viral loads varied among populations. As the lack of phenotypic homogeneity represents one of the most important reasons for the long-term fight against dengue incidence, we expect that this study will help us to understand the dynamics of the infection patterns that are triggered by the distinct serotypes of DENV in mosquitoes.

Tripartite interactions: Leishmania, microbiota and Lutzomyia longipalpis.

The microbial consortium associated with sandflies has gained relevance, with its composition shifting throughout distinct developmental stages, being strongly influenced by the surroundings and food sources. The bacterial components of the microbiota can interfere with Leishmania development inside the sandfly vector. Microbiota diversity and host-microbiota-pathogen interactions regarding New World sandfly species have yet to be thoroughly studied, particularly in Lutzomyia longipalpis, the primary vector of visceral leishmaniasis in Brazil.The native microbiota of different developmental stages and physiological conditions of Lu. longipalpis (Lapinha Cave), was described by culturing and 16s rRNA gene sequencing. The 16s rRNA sequencing of culture-dependent revealed 13 distinct bacterial genera (Bacillus, Enterococcus, Erwinia, Enterobacter, Escherichia, Klebsiella, Lysinibacillus, Pseudocitrobacter, Providencia, Pseudomonas, Serratia, Staphylococcus and Solibacillus). The in vitro and in vivo effects of each one of the 13 native bacteria from the Lu. longipalpis were analyzed by co-cultivation with promastigotes of L.i. chagasi, L. major, L. amazonensis, and L. braziliensis. After 24 h of co-cultivation, a growth reduction observed in all parasite species. When the parasites were co-cultivated with Lysinibacillus, all parasites of L. infantum chagasi and L. amazonensis died within 24 hours. In the in vivo co-infection of L.chagasi, L. major and L. amazonensis with the genera Lysinibacillus, Pseudocitrobacter and Serratia it was possible to observe a significant difference between the groups co-infected with the bacterial genera and the control group.These findings suggest that symbiont bacteria (Lysinibacillus, Serratia, and Pseudocitrobacter) are potential candidates for paratransgenic or biological control. Further studies are needed to identify the nature of the effector molecules involved in reducing the vector competence for Leishmania.

Vertical Transmission of Zika Virus (Flaviviridae, Flavivirus) in Amazonian Aedes aegypti (Diptera: Culicidae) Delays Egg Hatching and Larval Development of Progeny.

Zika virus (ZIKV) has emerged as a globally important arbovirus and has been reported from all states of Brazil. The virus is primarily transmitted to humans through the bite of an infective Aedes aegypti (Linnaeus, 1762) or Aedes albopictus (Skuse, 1895). However, it is important to know if ZIKV transmission also occurs from Ae. aegypti through infected eggs to her offspring. Therefore, a ZIKV and dengue virus (DENV) free colony was established from eggs collected in Manaus and maintained until the third-fourth generation in order to conduct ZIKV vertical transmission (VT) experiments which used an infectious bloodmeal as the route of virus exposure. The eggs from ZIKV-infected females were allowed to hatch. The resulting F1 progeny (larvae, pupae, and adults) were quantitative polymerase chain reaction (qPCR) assayed for ZIKV. The viability of ZIKV vertically transmitted to F1 progeny was evaluated by cultivation in C6/36 cells. The effects of ZIKV on immature development of Ae. aegypti was assessed and compared with noninfected mosquitoes. AmazonianAe. aegypti were highly susceptible to ZIKV infection (96.7%), and viable virus passed to their progeny via VT. Moreover, eggs from the ZIKV-infected mosquitoes had a significantly lower hatch rate and the slowest hatching. In addition, the larval development period was slower when compared to noninfected, control mosquitoes. This is the first study to illustrate VT initiated by oral infection of the parental population by using mosquitoes, which originated from the field and a ZIKV strain that is naturally circulating in-country. Additionally, this study suggests that ZIKV present in the Ae. aegypti can modify the mosquito life cycle. The data reported here suggest that VT of ZIKV to progeny from naturally infected females may have a critical epidemiological role in the dissemination and maintenance of the virus circulating in the vector.

Coinfection with Zika Virus (ZIKV) and Dengue Virus Results in Preferential ZIKV Transmission by Vector Bite to Vertebrate Host.

Background: Several tropical cities are permissive to Aedes aegypti and dengue virus (DENV) endemicity and have allowed for invasion and circulation of Zika virus (ZIKV) in the same areas. People living in arbovirus-endemic regions have been simultaneously infected with ≥2 arboviruses. Methods: A. aegypti mosquitoes from Manaus, the capital city of Amazonas State in Brazil, were coinfected with circulating strains of DENV and ZIKV. The coinfected vectors were allowed to bite BALB/c mice. Results: A. aegypti from Manaus is highly permissive to monoinfection and coinfection with DENV and ZIKV and is capable of cotransmitting both pathogens by bite. Coinfection strongly influences vector competence, favoring transmission of ZIKV to the vertebrate host. Conclusions: This finding suggests that A. aegypti is an efficient vector of ZIKV and that ZIKV would be preferentially transmitted by coinfected A. aegypti. Coinfection in the vector population should be considered a new critical epidemiological factor and may represent a major public health challenge.

Bacterial diversity of wild-caught Lutzomyia longipalpis (a vector of zoonotic visceral leishmaniasis in Brazil) under distinct physiological conditions by metagenomics analysis.

BACKGROUND: The leishmaniases are a group of diseases caused by protozoans of the genus Leishmania, which are transmitted by the bite of phlebotomine sand flies. In the New World, Lutzomyia longipalpis is the most important vector of visceral leishmaniasis and is a proven vector for Leishmania infantum chagasi in Brazil. During development within the vector, Leishmania can interact with a variety of microorganisms such as fungi and bacteria. The presence of bacteria in the midgut of sand flies can influence the development and survival of the parasite. RESULTS: The bacteria-targeted metagenomic analysis revealed different community compositions between the distinct physiological stages of those tested. The amplicon-oriented metagenomic profiling revealed 64 bacterial genera and 46 families. By crossing the taxa indices from each experimental condition a core composed of 6 genera was identified (Enterobacter, Serratia, Stenotrophomonas, Enhydrobacter, Pseudomonas and Chryseobacterium). CONCLUSIONS: The observed dynamic nature of the bacterial community expands the knowledge pertaining to the tripartite host-microbiota-pathogen interactions. Further studies addressing how laboratory and field collected communities differ are critical to successfully develop control strategies based on bacterial symbionts and paratransgenesis, as already tested in other arthropod vectors.

Zika virus transmission to mouse ear by mosquito bite: a laboratory model that replicates the natural transmission process.

BACKGROUND: Zika disease has transformed into a serious global health problem due to the rapid spread of the arbovirus and alarming severity including congenital complications, microcephaly and Guillain-Barré syndrome. Zika virus (ZIKV) is primarily transmitted to humans through the bite of an infective mosquito, with Aedes aegypti being the main vector. METHODS: We successfully developed a ZIKV experimental transmission model by single infectious Ae. aegypti bite to a laboratory mouse using circulating Brazilian strains of both arbovirus and vector. Mosquitoes were orally infected and single Ae. aegypti were allowed to feed on mouse ears 14 days post-infection. Additionally, salivary gland (SG) homogenates from infected mosquitoes were intrathoracically inoculated into naïve Ae. aegypti. Mosquito and mouse tissue samples were cultured in C6/36 cells and processed by quantitative real-time PCR. RESULTS: A total of 26 Ae. aegypti were allowed to feed individually on mouse ears. Of these, 17 mosquitoes fed, all to full engorgement. The transmission rate of ZIKV by bite from these engorged mosquitoes to mouse ears was 100%. The amount of virus inoculated into the ears by bites ranged from 2 × 102-2.1 × 1010 ZIKV cDNA copies and was positively correlated with ZIKV cDNA quantified from SGs dissected from mosquitoes post-feeding. Replicating ZIKV was confirmed in macerated SGs (2.45 × 107 cDNA copies), mouse ear tissue (1.15 × 103 cDNA copies, and mosquitoes 14 days post-intrathoracic inoculation (1.49 × 107 cDNA copies) by cytopathic effect in C6/36 cell culture and qPCR. CONCLUSIONS: Our model illustrates successful transmission of ZIKV by an infectious mosquito bite to a live vertebrate host. This approach offers a comprehensive tool for evaluating the development of infection in and transmission from mosquitoes, and the vertebrate-ZIKV interaction and progression of infection following a natural transmission process.

Transovarial transmission of DENV in Aedes aegypti in the Amazon basin: a local model of xenomonitoring.

BACKGROUND: Transovarial transmission of dengue virus in Aedes spp. mosquitoes is considered an important mechanism for the maintenance of the virus in nature and may be implicated in the occurrence of outbreaks and epidemics of the disease. However, there are few studies involving transovarial transmission and viral vector monitoring as a surveillance tool and control strategy. The present study evaluated transovarial transmission of dengue virus in Aedes aegypti populations as a xenomonitoring strategy in municipalities of the Amazonas state. RESULTS: Aedes sp. eggs (13.164) were collected, with 30% viability of third- and fourth-instar larvae. Transovarial transmission of DENV was detected in all municipalities. The transovarial infection rate (TOR) in the municipalities was 46% of the DENV positive samples. The minimum infection rate (MIR) was 17.7 in the state, varying from 11.4 to 24.1 per 1,000 larvae tested in the respective municipalities. Four DENV serotypes were identified, with DENV I and IV being present in all municipalities investigated. The number of reported dengue fever cases varied during this period. CONCLUSIONS: Our results suggest that transovarial transmission may be an important mechanism for the maintenance and spreading of the disease in Amazonas municipalities. Using qRT-PCR, it was possible to identify the four DENV serotypes in larval samples. The methodology used in the present study proved suitable as a DENV xenomonitoring model in immature mosquitoes, contributing to the development of systems for early detection of viral circulation and predictive models for the occurrence of outbreaks and epidemics of this disease. TRIAL REGISTRATION: CAAE34025414200005015 .

A ação da microbiota nativa de Lutzomyia longipalpis no desenvolvimento de Leishmania spp

As leishmanioses são doenças causadas por protozoários do gênero Leishmania e transmitidas pela picada dos flebotomíneos. O estudo da microbiota intestinal de Lutzomyia longipalpis é importante para determinar uma possível influência na competência vetorial desse inseto. In vitro, a ação da microbiota sobre o parasito foi avaliada demonstrando a lise na parede celular de L. infantum chagasi e L. braziliensis, causada por Serratia marcescens. Em estudos realizados in vivo, autores observaram uma redução no número de flebotomíneos infectados com Leishmania mexicana pré-alimentados com Pseudozyma sp., Asaia sp. e Ochrobactrum intermedium. Estes estudos sugerem que a microbiota do vetor pode modular a sua infecção por Leishmania, porém o seu papel ainda não esta claro. O trabalho aqui apresentado teve como objetivo caracterizar e avaliar o papel da comunidade bacteriana intestinal do Lu. longipalpis no desenvolvimento e diferentes espécies de Leishmania spp. Métodos morfológicos e moleculares foram utilizados para identificar e mapear a microbiota intestinal. O DNA das bactérias isoladas foram extraídos, a região 16S amplificada, utilizando-se um iniciador específico para esta região do DNA bacteriano. Em seguida, foram purificados, sequenciados e analisados em bancos de dados. Foram obtidas 60 UFCs (Unidades Formadoras de Colônias) e classificadas taxonomicamente em 10 gêneros bacterianos

A atividade lítica ou efeito in vitro dessas bactérias nativas isoladas de Lu. longipalpis (Gruta da Lapinha) foi analisada por co-cultura utilizando promastigotas de Leishmania (L.) infantum chagasi,Leishmania (L.) amazonensis, Leishmania (V.) braziliensis e Leishmania (L.) major na concentração de 4 x 106 parasitas / mL e incubadas com diferentes gêneros de bactérias na concentração de 1 x 108 UFC / mL. Quando os parasitas foram co-cultivados com o gênero bacteriano Lysinibacillus todos os parasitas de L. infantum chagasi e L. amazonensis morreram em até 24h. L. braziliensis e L. major em até 48h de co-cultivo. Entre 96 a 144 horas, não observou-se a lise de todos os parasitas de L. infantum chagasi e L. amazonensis co-cultivados com Pseudomonas e Enterobacter. As bactérias Pseudomonas, Enterobacter e Erwinia quando co-cultivadas com L. braziliensis e de L. major co-cultivados com Pseudomonas, Enterobacter e Enterococcus. Os gêneros Lysinibacillus e Serratia apresentaram efeitos lítico sobre todas as quatros espécies de Leishmania testadas, abrindo a perspectiva de serem utilizadas em experimentos in vivo

Bacterial diversity of the American sand fly Lutzomyia intermedia using high-throughput metagenomic sequencing.

BACKGROUND: Parasites of the genus Leishmania cause a broad spectrum of diseases, collectively known as leishmaniasis, in humans worldwide. American cutaneous leishmaniasis is a neglected disease transmitted by sand fly vectors including Lutzomyia intermedia, a proven vector. The female sand fly can acquire or deliver Leishmania spp. parasites while feeding on a blood meal, which is required for nutrition, egg development and survival. The microbiota composition and abundance varies by food source, life stages and physiological conditions. The sand fly microbiota can affect parasite life-cycle in the vector. METHODS: We performed a metagenomic analysis for microbiota composition and abundance in Lu. intermedia, from an endemic area in Brazil. The adult insects were collected using CDC light traps, morphologically identified, carefully sterilized, dissected under a microscope and the females separated into groups according to their physiological condition: (i) absence of blood meal (unfed = UN); (ii) presence of blood meal (blood-fed = BF); and (iii) presence of developed ovaries (gravid = GR). Then, they were processed for metagenomics with Illumina Hiseq Sequencing in order to be sequence analyzed and to obtain the taxonomic profiles of the microbiota. RESULTS: Bacterial metagenomic analysis revealed differences in microbiota composition based upon the distinct physiological stages of the adult insect. Sequence identification revealed two phyla (Proteobacteria and Actinobacteria), 11 families and 15 genera; 87 % of the bacteria were Gram-negative, while only one family and two genera were identified as Gram-positive. The genera Ochrobactrum, Bradyrhizobium and Pseudomonas were found across all of the groups. CONCLUSIONS: The metagenomic analysis revealed that the microbiota of the Lu. intermedia female sand flies are distinct under specific physiological conditions and consist of 15 bacterial genera. The Ochrobactrum, Bradyrhizobium and Pseudomonas were the common genera. Our results detailing the constituents of Lu. intermedia native microbiota contribute to the knowledge regarding the bacterial community in an important sand fly vector and allow for further studies to better understand how the microbiota interacts with vectors of human parasites and to develop tools for biological control.