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Field release of Wolbachia-infected Aedes aegypti has emerged as a promising solution to manage the transmission of dengue, Zika and chikungunya in endemic areas across the globe. Through an efficient self-dispersing mechanism, and the ability to induce virus-blocking properties, Wolbachia offers an unmatched potential to gradually modify wild Ae. aegypti populations turning them unsuitable disease vectors. Here we describe a proof-of-concept field trial carried out in a small community of Niterói, greater Rio de Janeiro, Brazil. Following the release of Wolbachia-infected eggs, we report here a successful invasion and long-term establishment of the bacterium across the territory, as denoted by stable high-infection indexes (> 80%). We have also demonstrated that refractoriness to dengue and Zika viruses, either thorough oral-feeding or intra-thoracic saliva challenging assays, was maintained over the adaptation to the natural environment of Southeastern Brazil. These findings further support Wolbachia’s ability to invade local Ae. aegypti populations and impair disease transmission, and will pave the way for future epidemiological and economic impact assessments.

The emergence of new human viral pathogens and re-emergence of several diseases are of particular concern in the last decades. Oropouche orthobunyavirus (OROV) is an arbovirus endemic to South and Central America tropical regions, responsible to several epidemic events in the last decades. There is little information regarding the ability of OROV to be transmitted by urban/peri-urban mosquitoes, which has limited the predictability of the emergence of permanent urban transmission cycles. Here, we evaluated the ability of OROV to infect, replicate, and be transmitted by three anthropophilic and urban species of mosquitoes, Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus. We show that OROV is able to infect and efficiently replicate when systemically injected in all three species tested, but not when orally ingested. Moreover, we find that, once OROV replication has occurred in the mosquito body, all three species were able to transmit the virus to immunocompromised mice during blood feeding. These data provide evidence that OROV is restricted by the midgut barrier of three major urban mosquito species, but, if this restriction is overcome, could be efficiently transmitted to vertebrate hosts. This poses a great risk for the emergence of permanent urban cycles and geographic expansion of OROV to other continents.

Field release of Wolbachia-infected Aedes aegypti has emerged as a promising solution to manage the transmission of dengue, Zika and chikungunya in endemic areas across the globe. Through an efficient self-dispersing mechanism, and the ability to induce virus-blocking properties, Wolbachia offers an unmatched potential to gradually modify wild Ae. aegypti populations turning them unsuitable disease vectors. Here in this work, a proof-of-concept field trial was carried out in a small community of Niterói, greater Rio de Janeiro. Following the release of Wolbachia-infected eggs, we reported a successful invasion and long-term establishment of the bacterium across the territory, as denoted by stable high-infection indexes (>80%). We have also demonstrated that refractoriness to dengue and Zika viruses, either thorough oral-feeding or intra-thoracic saliva challenging assays, were maintained over the adaptation to the natural environment of Southeastern Brazil. These findings further support Wolbachia’s ability to invade local Ae. aegypti populations and impair disease transmission, and shall pave the way for future epidemiological and economic impact assessments.

Newly emerging or re-emerging arthropod-borne viruses (arboviruses) are important causes of human morbidity and mortality worldwide. Arboviruses such as Dengue (DENV), Zika (ZIKV), Chikungunya (CHIKV), and West Nile virus (WNV) have undergone extensive geographic expansion in the tropical and sub-tropical regions of the world. In the Americas the main vectors of DENV, ZIKV, and CHIKV are mosquito species adapted to urban environments, namely Aedes aegypti and Aedes albopictus, whereas the main vector of WNV is Culex quinquefasciatus. Given the widespread distribution in the Americas and high permissiveness to arbovirus infection, these mosquito species may play a key role in the epidemiology of other arboviruses normally associated with sylvatic vectors. Here, we test this hypothesis by determining the vector competence of Ae. aegypti, Ae. albopictus, and Cx. quinquefasciatus to Mayaro (MAYV) virus, a sylvatic arbovirus transmitted mainly by Haemagogus janthinomys that has been causing an increasing number of outbreaks in South America, namely in Brazil. Using field mosquitoes from Brazil, female mosquitoes were experimentally infected, and their competence for infection and transmission rates of MAYV was evaluated. We found consistent infection rate for MAYV in Ae. aegypti (57.5%) and Ae. albopictus (61.6%), whereas very low rates were obtained for Cx. quinquefasciatus (2.5%). Concordantly, we observed high potential transmission ability in Ae. aegypti and Ae. albopictus (69.5% and 71.1% respectively), in contrast to Cx. quinquefasciatus, which could not transmit the MAYV. Notably, we found that very low quantities of virus present in the saliva (undetectable by RT-qPCR) were sufficiently virulent to guarantee transmission. Although Ae. aegypti and Ae. albopictus mosquitoes are not the main vectors for MAYV, our studies suggest that these mosquitoes could play a significant role in the transmission of this arbovirus, since both species showed significant vector competence for MAYV (Genotype D), under laboratory conditions., Author summary The present study demonstrated that Ae. aegypti and Ae. albopictus mosquitoes can be competent laboratory vectors for MAYV. In contrast, Cx. quinquefasciatus mosquitoes were refractory to MAYV. Regarding the viral dilution and nanoinjection, a higher detection sensitivity was observed after virus nanoinjection into naïve mosquitoes, indicating that only a few viral particles are required to infect mosquitoes, and these particles may not be detected by RT-qPCR before the nanoinjection procedure.

Wolbachia, an intracellular endosymbiont present in up to 70% of all insect species, has been suggested as a sustainable strategy for the control of arboviruses such as Dengue, Zika and Chikungunya. As Mayaro virus outbreaks have also been reported in Latin American countries, the objective of this study was to evaluate the vector competence of Brazilian field-collected Ae. aegypti and the impact of Wolbachia (wMel strain) upon this virus. Our in vitro studies with Aag2 cells showed that Mayaro virus can rapidly multiply, whereas in wMel-infected Aag2 cells, viral growth was significantly impaired. In addition, C6/36 cells seem to have alterations when infected by Mayaro virus. In vivo experiments showed that field-collected Ae. aegypti mosquitoes are highly permissive to Mayaro virus infection, and high viral prevalence was observed in the saliva. On the other hand, Wolbachia-harboring mosquitoes showed significantly impaired capability to transmit Mayaro virus. Our results suggest that the use of Wolbachia-harboring mosquitoes may represent an effective mechanism for the reduction of Mayaro virus transmission throughout Latin America.

Various mouse models to study dengue have been described by different authors, some of them using immunodeficient or some using humanized mice. Our group reported previously a deadly murine model, which used the intracranial inoculum of highly virulent Dengue virus (DENV) in immune competent mouse. Here we present a model of immune competent mouse (C57BL/6), infected subcutaneously by the same highly virulent DENV (DENV3 genotype I). In this immunocompetent systemic mice model, the cytokine levels and hematological parameters such as total and differential leukocyte and platelets counts, together with weight loss, were considered important monitoring parameters, allowing a better understanding of the systemic human disease. Mice were inoculated subcutaneously and evaluated by the percentage weight variation as well as the clinical signs. Hematological parameters and cytokines levels were measured and viral titration in brain tissue or serum neutralization was performed to confirm mice infection. The subcutaneously DENV inoculated mice showed weight loss after infection, but they did not show any other clinical signs. The leukocytes and platelets decreased after subcutaneous inoculation. The cytokines TNF alpha and IFN gamma increased after infection in mice. The subcutaneous model provided scope for improved understanding of the dengue pathogenesis, as well as possible mechanism for protection to subsequent mouse infected by intracranial route in mice. This model could be used to study the vertebrate immune response and evaluation of drugs or vaccine against dengue virus.

Background Wolbachia’s ability to restrict arbovirus transmission makes it a promising tool to combat mosquito-transmitted diseases. Wolbachia-infected Aedes aegypti are currently being released in locations such as Brazil, which regularly experience concurrent outbreaks of different arboviruses. A. aegypti can become co-infected with, and transmit multiple arboviruses with one bite, which can complicate patient diagnosis and treatment. Methodology/principle findings Using experimental oral infection of A. aegypti and then RT-qPCR, we examined ZIKV/DENV-1 and ZIKV/DENV-3 co-infection in Wolbachia-infected A. aegypti and observed that Wolbachia-infected mosquitoes experienced lower prevalence of infection and viral load than wildtype mosquitoes, even with an extra infecting virus. Critically, ZIKV/DENV co-infection had no significant impact on Wolbachia’s ability to reduce viral transmission. Wolbachia infection also strongly altered expression levels of key immune genes Defensin C and Transferrin 1, in a virus-dependent manner. Conclusions/significance Our results suggest that pathogen interference in Wolbachia-infected A. aegypti is not adversely affected by ZIKV/DENV co-infection, which suggests that Wolbachia-infected A. aegypti will likely prove suitable for controlling mosquito-borne diseases in environments with complex patterns of arbovirus transmission., Author summary Wolbachia is an endosymbiotic bacterium that infects insects. It has been artificially transferred into Aedes aegypti, a mosquito species that can transmit medically important viruses including dengue, chikungunya, and Zika. Wolbachia in A. aegypti limits infection with these viruses, making the mosquitoes much less capable of transmitting them to people. In tropical areas, where these viral pathogens are commonly found, it is not unusual for outbreaks of different viruses to occur at the same time, which can complicate diagnosis and treatment for those afflicted. Mosquitoes with Wolbachia are currently being released into these areas to reduce transmission of these diseases. In our study, we assessed whether Wolbachia infection in A. aegypti mosquitoes could still effectively inhibit the dengue and Zika viruses if the mosquitoes were fed both viruses at the same time. We found that Wolbachia was still very effective at inhibiting the replication of both viruses in the mosquito, and likewise still greatly reduced the chance of transmission of either virus. Our results suggest that Wolbachia-infected mosquitoes should be able to limit infection with more than one virus, should they encounter them in the field.

Background: Yellow fever outbreaks have re-emerged in Brazil during 2016-18, with mortality rates up to 30%. Although urban transmission has not been reported since 1942, the risk of re-urbanization of yellow fever is significant, as Aedes aegypti is present in most tropical and sub-tropical cities in the World and still remains the main vector of urban YFV. Although the YFV vaccine is safe and effective, it does not always reach populations at greatest risk of infection and there is an acknowledged global shortage of vaccine supply. The introgression of Wolbachia bacteria into Ae. aegypti mosquito populations is being trialed in several countries (www.worldmosquito.org) as a biocontrol method against dengue, Zika and chikungunya. Here, we studied the ability of Wolbachia to reduce the transmission potential of Ae. aegypti mosquitoes for Yellow fever virus (YFV). Methods: Two recently isolated YFV (primate and human) were used to challenge field-derived wild-type and Wolbachia-infected (wMel +) Ae. aegypti mosquitoes. The YFV infection status was followed for 7, 14 and 21 days post-oral feeding (dpf). The YFV transmission potential of mosquitoes was evaluated via nano-injection of saliva into uninfected mosquitoes or by inoculation in mice. Results: We found that Wolbachia was able to significantly reduce the prevalence of mosquitoes with YFV infected heads and thoraces for both viral isolates. Furthermore, analyses of mosquito saliva, through indirect injection into naïve mosquitoes or via interferon-deficient mouse model, indicated Wolbachia was associated with profound reduction in the YFV transmission potential of mosquitoes (14dpf). Conclusions: Our results suggest that Wolbachia introgression could be used as a complementary strategy for prevention of urban yellow fever transmission, along with the human vaccination program.

Summary The recent association of Zika virus with cases of microcephaly has sparked a global health crisis and highlighted the need for mechanisms to combat the Zika vector, Aedes aegypti mosquitoes. Wolbachia pipientis, a bacterial endosymbiont of insect, has recently garnered attention as a mechanism for arbovirus control. Here we report that Aedes aegypti harboring Wolbachia are highly resistant to infection with two currently circulating Zika virus isolates from the recent Brazilian epidemic. Wolbachia-harboring mosquitoes displayed lower viral prevalence and intensity and decreased disseminated infection and, critically, did not carry infectious virus in the saliva, suggesting that viral transmission was blocked. Our data indicate that the use of Wolbachia-harboring mosquitoes could represent an effective mechanism to reduce Zika virus transmission and should be included as part of Zika control strategies., Graphical Abstract, Highlights • Mosquitoes harboring Wolbachia were resistant to current circulating Zika virus isolates • Zika virus prevalence, intensity, and disseminated infection were reduced • Saliva from Wolbachia-harboring mosquitoes did not contain infectious Zika virus, Strategies to combat Zika virus (ZIKV) and its mosquito vector are urgently needed. Dutra et al. report that Wolbachia-carrying mosquitoes are highly resistant to ZIKV and display reduced virus prevalence and intensity. Saliva from Wolbachia-carrying mosquitoes did not contain infectious virus, suggesting the possibility to block ZIKV transmission.

Brazil has experienced several arbovirus outbreaks in recent years, among which yellow fever stands out. The state of Minas Gerais faced outbreaks of sylvatic yellow fever in 2017 and 2018, with 1002 confirmed cases and 340 deaths. This work presents the results of survey efforts to detect the yellow fever virus in mosquitoes from two conservation areas in the metropolitan region of Belo Horizonte, Brazil. A total of 867 mosquitoes of 20 species were collected between September 2017 and May 2018, the most abundant being Psorophora (Janthinosoma) ferox (von Humboldt, 1819) (31.3%), Limatus durhamii Theobald, 1901 (19.1%) and Haemagogus (Haemagogus) janthinomys Dyar, 1921 (18.2%). Total RNA was extracted from the mosquitoes for real-time PCR analysis for yellow fever, chikungunya, mayaro, Zika and dengue viruses. The yellow fever infection rate was 8.2% for Hg. janthinomys (13 mosquitoes), which is the main vector of sylvatic yellow fever in Brazil. In addition to surveying the mosquito fauna of these conservation units, this work demonstrates the importance of monitoring the circulation of viruses near large urban centers.

Background The leishmaniases are important neglected diseases caused by Leishmania spp. which are transmitted by sand flies, Lutzomyia longipalpis being the main vector of visceral leishmaniasis in the Americas. The methodologies for leishmaniasis control are not efficient, causing 1.5 million reported cases annually worldwide, therefore showing the need for development of novel strategies and interventions to control transmission of the disease. The bacterium Wolbachia pipientis is being used to control viruses transmitted by mosquitoes, such as dengue and Zika, and its introduction in disease vectors has been effective against parasites such as Plasmodium. Here we show the first successful establishment of Wolbachia into two different embryonic cell lines from L. longipalpis, LL-5 and Lulo, and analysed its effects on the sand fly innate immune system, followed by in vitro Leishmania infantum interaction. Results Our results show that LL-5 cells respond to wMel and wMelPop-CLA strains within the first 72 h post-infection, through the expression of antimicrobial peptides and inducible nitric oxide synthase resulting in a decrease of Wolbachia detection in the early stages of infection. In subsequent passages, the wMel strain was not able to infect any of the sand fly cell lines while the wMelPop-CLA strain was able to stably infect Lulo cells and LL-5 at lower levels. In Wolbachia stably infected cells, the expression of immune-related genes involved with downregulation of the IMD, Toll and Jak-Stat innate immune pathways was significantly decreased, in comparison with the uninfected control, suggesting immune activation upon Wolbachia transinfection. Furthermore, Wolbachia transinfection did not promote a negative effect on parasite load in those cells. Conclusions Initial strong immune responses of LL5 cells might explain the inefficiency of stable infections in these cells while we found that Lulo cells are more permissive to infection with Wolbachia causing an effect on the cell immune system, but not against in vitro L. infantum interaction. This establishes Lulo cells as a good system for the adaptation of Wolbachia in L. longipalpis. Electronic supplementary material The online version of this article (10.1186/s13071-018-3227-4) contains supplementary material, which is available to authorized users.

Leishmaniasis is a disease caused by the protozoan Leishmania resulting in a variety of clinical manifestations, from self-healing skin lesions to fatal visceral disease. The development of polymerase chain reaction (PCR)-based techniques has made species identification easier, faster, and less labor intensive. The main targets for PCR amplification include kinetoplastid DNA (kDNA), miniexon, and conserved regions such as the internal transcribed spacer. The objective of this work was to evaluate 4 different PCR techniques designed to type Leishmania using laboratory strains. Parasites were subjected to 4 PCR procedures using specific Leishmania primers for miniexon (designated A1 and A2) and kDNA (designated B1 and B2, C1 and C2, and D1, D2 and D3). Discrimination between some species and the 2 main subgenera Leishmania and Viannia was achieved. Unweighted pair group method analysis resulted in the expected clustering of the 2 species from the subgenus Leishmania. However, some species in the subgenus Viannia could not be distinguished, representing a continued challenge for PCR-based protocols. Results are discussed in terms of advantages, limitations, and reproducibility of these 4 PCR-based techniques in the taxonomy of Leishmania.

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) National Institutes of Health Biomolecule Analysis Core Facility at the Border Biomedical Research Center/Biology/UTEP University of Texas at El Paso (National Institutes of Health) The glycosylphosphatidylinositol (GPI)-anchored mucins of Trypanosoma cruzi trypomastigotes play an important immunomodulatory role during the course of Chagas disease. Here, some biological activities of tGPI-mucins from four T cruzi isolates, including benznidazole-susceptible (BZS-Y), benznidazole-resistant (BZR-Y), CL, and Colombiana, were evaluated. GPI-mucins were able to differentially trigger the production of interleukin-12 and nitric oxide in BALB/c macrophages and modulate LLC-MK2 cell invasion. the significance of these variations was assessed after analysis of the terminal alpha-galactosyl residues. Enzymatic treatment with alpha-galactosidase indicated a differential expression of O-linked alpha-galactosyl residues among the strains, with higher expression of this sugar in BZS-Y and BZR-Y T cruzi populations followed by Colombiana and CL. Unweighted pair group method analysis of the carbohydrate anchor profile and biological parameters allowed the clustering of two groups. One group includes Y and CL strains (T cruzi II and VI), and the other group is represented by Colombiana strain (T. cruzi I). Fiocruz MS, Ctr Pesquisas Rene Rachou, BR-30190002 Belo Horizonte, MG, Brazil Universidade Federal de São Paulo, UNIFESP, Dept Ciencias Biol, Diadema, SP, Brazil Univ Texas El Paso, Dept Biol Sci, Border Biomed Res Ctr, El Paso, TX 79968 USA Universidade Federal de São Paulo, UNIFESP, Dept Ciencias Biol, Diadema, SP, Brazil CNPq: 305042/2010-6 CNPq: 552072/2009-5 CNPq: PAPES-IV-400138/2006-9 CNPq: PDJ-150880/2005-7 National Institutes of Health: 1R01AI070655-04 National Institutes of Health: 3R01AI070655-04S1 National Institutes of Health: 2G12RR008124-16A1 National Institutes of Health: 2G12RR008124-16A1S1 Biomolecule Analysis Core Facility at the Border Biomedical Research Center/Biology/UTEP University of Texas at El Paso (National Institutes of Health): 2G12RR008124-16A1 Biomolecule Analysis Core Facility at the Border Biomedical Research Center/Biology/UTEP University of Texas at El Paso (National Institutes of Health): 2G12RR008124-16A1S1 Biomolecule Analysis Core Facility at the Border Biomedical Research Center/Biology/UTEP University of Texas at El Paso (National Institutes of Health): G12MD007592 Web of Science

Background. Leishmaniases are diseases with a wide spectrum of clinical manifestations including cutaneous (CL) and visceral (VL) forms. Many factors may affect their occurrence and expansion including environmental, geographic, and social conditions. In the past two decades, Divinópolis, Minas Gerais State, Brazil, has exhibited the potential for a disease outbreak, with the appearance of CL, and VL cases (human and canine). Hence, this study was initiated to monitor public knowledge of the disease. Questionnaires were administered in four neighborhoods (Jardim Belvedere, Esplanada, Danilo Passos I and II) where most of the human and canine cases have been reported. The analyses demonstrated that public knowledge of the disease is sparse and fragmented. A strong perception of the dog as the main reservoir was observed. Five veterinary clinics were evaluated for the presence of canine VL using serological (RIFI and ELISA) and molecular (PCR-RFLP) techniques. This is the first study demonstrating the occurrence ofLeishmania infantumin Divinópolis, suggesting a possible urbanization of VL.