Detecção de flavivirus e alphavirus em mosquitos (diptera: culicidae) capturados em região próxima à Três Lagoas ­ Ms / Detection of flavivirus and alphavirus in mosquitoes (diptera: culicidae) captured in a region close to Tres Lagoas ­ Ms / Detección de flavivirus y alphavirus en mosquitos (diptera: culicidae) capturados en una región cercana a Tres Lagoas ­ Ms

Introdução: Arbovírus são causadores de doenças humanas, sendo que mudança ecológicas e aumento do contato humano-vetor aumenta a possibilidade de surtos. Objetivo: Detectar, identificar e caracterizar arbovírus presentes em mosquitos vetores capturados em regiões de mata próximas a Três Lagoas, MS. Metodologia: Mosquitos foram capturados utilizando armadilhas de luz em regiões de mata circunvizinha a Três Lagoas. Os mosquitos capturados foram classificados por gênero (chave morfológica) e agrupados em pools com até 20 espécimes, e utilizados através da reação de RT-PCR com posterior sequenciamento e análise filogenética. Resultados: Foram capturados 851 dos gêneros: Culex spp. (11 pools); Aedes spp. (13 pools); Haemagogus spp. (7 pools) e outros gêneros não identificados. Sequencias de vírus Dengue (DENV) foram amplificadas de 2/13 (15,38%) pools de Aedes spp. e uma sequência de vírus Mayaro (MAYV) 1/7 (7,7%) foi amplificada de pools de Haemagogus spp. As análises filogenéticas mostraram que as sequências de DENV agrupava-se no clado de DENV1 e DENV2. A sequência de MAYV agrupou-se junto a sequências de amostras de infecções humana por MAYV do grupo L. Conclusão: Estes resultados reforçam a circulação de DENV, que é causador de surtos anuais de doenças febris agudas no município, e detecção, por primeira vez na região, a circulação de MAYV, reforçando a necessidade de monitoramento viral constante nessa região.

Introduction: Arboviruses cause human diseases, and ecological changes and increased human-vector contact increase the possibility of outbreaks. Objective: To detect, identify and characterize arboviruses present in mosquito vectors captured in forest regions close to Tres Lagoas, MS. Methodology: Mosquitoes were captured using light traps in forest regions surrounding Tres Lagoas. The captured mosquitoes were classified by gender (morphological key) and grouped into pools with up to 20 specimens and used through the RT-PCR reaction with subsequent sequencing and phylogenetic analysis. Results: 851 of the genera were captured: Culex spp. (11 pools); Aedes spp. (13 pools); Haemagogus spp. (7 pools) and other unidentified genera. Dengue virus (DENV) sequences were amplified from 2/13 (15.38%) pools of Aedes spp. and a Mayaro virus (MAYV) sequence 1/7 (7.7%) were amplified from pools of Haemagogus spp. Phylogenetic analyzes showed that one of the DENV sequences clustered in the DENV1 and DENV2 clade. The MAYV sequence was grouped together with sequences from samples of human MAYV infections of the L group. Conclusion: These results reinforce the circulation of DENV, which causes annual outbreaks of acute febrile illnesses in the municipality, and detection, for the first time in the region, the circulation of MAYV, reinforcing the need for constant viral monitoring in this region.

Introducción: Los arbovirus causan enfermedades humanas, y los cambios ecológicos y el mayor contacto humano-vector aumentan la posibilidad de brotes. Objetivo: Detectar, identificar y caracterizar arbovirus presentes en mosquitos vectores capturados en regiones de selva próximas a Tres Lagoas, MS. Metodología: Los mosquitos fueron capturados utilizando trampas de luz en las regiones forestales que rodean Tres Lagoas. Los mosquitos capturados fueron clasificados por género (clave morfológica) y agrupados en pools de hasta 20 ejemplares, y utilizados mediante la reacción RT-PCR con posterior secuenciación y análisis filogenético. Resultados: Se capturaron 851 de los géneros: Culex spp. (11 pools); Aedes spp. (13 pools); Haemagogus spp. (7 pools) y otros géneros no identificados. Las secuencias del virus del dengue (DENV) se amplificaron a partir de 2/13 (15,38 %) grupos de Aedes spp. y una secuencia de virus Mayaro (MAYV) 1/7 (7,7%) de pools de Haemagogus spp. Los análisis filogenéticos mostraron que una de las secuencias de DENV se agrupaba en el clado DENV1 y DENV2. La secuencia de MAYV se agrupó con secuencias de muestras de infecciones humanas de MAYV del grupo L. Conclusión: Estos resultados refuerzan la circulación de DENV, causante de brotes anuales de enfermedades febriles agudas en el municipio, y la detección, por primera vez en la región, la circulación de MAYV, reforzando la necesidad de un monitoreo viral constante en esta región.

Zika virus RNA detection in blood donors in São Paulo, Brazil

ABSTRACT Introduction: The Zika Virus (ZIKV) is a single-stranded RNA genome virus, belonging to the family Flaviviridae, genus Flavivirus. Outbreaks around the world have demonstrated that the presence of asymptomatic viremic blood donors provides an increase in the risk of transfusion transmission (TT) and nucleic acid test (NAT) screening has been proposed to ensure the blood safety. This study implemented an "in-house" method to detect ZIKV RNA in blood sample donations. Methods: Primary plasma tubes are submitted to nucleic acid extraction on an automated platform. After extraction, the NAT set-up is performed in the robotic pipettor, in which an amplification mixture containing primers and probes for ZIKV and Polio vaccine virus (PV) are added in duplex as an internal control. The real-time polymerase chain reaction is then performed in a thermocycler, using the protocol established by the supplier. Results: From May 2016 to May 2018, 3,369 samples were collected from 3,221 blood donors (confidence coefficient 95%), of which 31 were considered false positive (0.92%), as they did not confirm initial reactivity when repeated in duplicates and 14 (0.42%) had their results invalid due to repeat failure in the internal control, 4 (0.12%), due to insufficient sample volume and 2 (0.05%), due to automatic pipettor failures. No Zika RNA reactive sample was identified. Conclusion: The test showed feasible to be incorporated into the blood screening routine. Our data do not indicate the need to screen for ZIKV RNA in São Paulo during the evaluated period. However, a generic NAT system covering a group of flaviviruses which are circulating in the region, such as DENV and YFV, among others, could be a useful tool.

Febre do Nilo Ocidental / West Nile fever

A Febre do Nilo Ocidental (FNO) é uma infecção viral transmitida por meio da picada de mosquitos, principalmente do gênero Culex (pernilongo) infectados pelo agente etiológico, cujos hospedeiros naturais são algumas espécies de aves silvestres, que atuam como amplificadoras do vírus e como fonte de infecção para os vetores. Tal doença pode também infectar humanos, equinos, primatas e outros mamíferos sendo que, homem e equídeos são considerados hospedeiros acidentais e terminais, uma vez que a contaminação pelo vírus se dá por um curto período de tempo e em níveis insuficientes para infectar mosquitos, encerrando o ciclo de transmissão (WHO, 2017; ECDC , 2022a; CDC, 2017; BRASIL, 2021)

West Nile Fever (WNF) is a viral infection transmitted through the bite of mosquitoes, mainly of the Culex genus (legged mosquito) infected by the etiological agent, whose natural hosts are some species of wild birds, which act as amplifiers of the virus and as source of infection for the vectors. Such a disease can also infect humans, horses, primates and other mammals, and humans and horses are considered accidental and terminal hosts, since contamination by the virus occurs for a short period of time and at levels insufficient to infect mosquitoes, ending the transmission cycle (WHO, 2017; ECDC, 2022a; CDC, 2017). ; BRAZIL, 2021)

Implementación de un sistema de detección de flavivirus en mosquitos / Implementation of a flavivirus detection system in mosquitoes

Los Flavivirus constituyen virus transmitidos por artrópodos, principalmente mosquitos. Pueden producir enfermedades en humanos y animales, también incluyen virus específicos de insectos que solo infectan y se replican en los insectos, no así en vertebrados. En Paraguay los virus dengue, fiebre amarilla y Zika fueron detectados en infecciones humanas, pero los estudios de flavivirus en mosquitos son aún escasos. Por ello, el objetivo del presente estudio fue implementar un sistema de detección de flavivirus en mosquitos en el IICS-UNA. Primero, se organizaron capacitaciones en colecta, preparación de pools y procesamiento por técnicas de RT-PCRs convencionales realizadas por expertos internacionales a profesionales locales (bioquímicos y biólogos). Además, se implementaron planillas de registro de datos y de control de transporte de muestras de los lugares de colectas hasta el IICS-UNA. Se prepararon en total 201 pools de 1 a 35 mosquitos cada uno agrupados por especie, localidad, entre otros criterios. Para asegurar la integridad del RNA extraído se realizó la detección de un control interno (Actina-1), siendo todos los pools positivos para el mismo, 91/201 pools fueron positivos para flavivirus. Se realizó la secuenciación de 19/91 pools positivos para flavivirus identificándose flavivirus de insectos (detectándose principalmente Culex Flavivirus, cell fusing agents Flavivirus y Kamiti river virus), evidenciando la elevada distribución de estos virus. Estos resultados demuestran que fue factible implementar el sistema de detección de flavivirus en mosquitos, lo cual podría contribuir a fortalecer la vigilancia y control de estas virosis, así como el conocimiento sobre la importancia ecológica de flavivirus de insectos

Flaviviruses are viruses transmitted by arthropods, mainly mosquitoes. They can cause diseases in humans and animals, they also include specific insect viruses that only infect and replicate in insects, not in vertebrates. In Paraguay, dengue, yellow fever, and Zika viruses were detected in human infections, but studies of flaviviruses in mosquitoes are still scarce. Therefore, the objective of the present study was the implementation of a flavivirus detection system in mosquitoes at IICS-UNA. First, trainings on collection, pool preparation and processing by conventional RT-PCR techniques were organized by international experts for local professionals (biochemists and biologists). In addition, data log sheets and sample transport control forms from the collection sites to the IICS were implemented. A total of 201 pools of 1 to 35 mosquitoes were prepared, each grouped by species, locality, among others. To ensure the integrity of the extracted RNA, an internal control (Actin-1) detection was performed, all pools being positive for it; 91/201 pools were positive for flaviviruses. The sequencing of 19/91 pools positive for flavivirus was carried out, identifying flavivirus in all cases of insects (mainly detecting Culex Flavivirus, cell fusing agents Flavivirus and Kamiti river virus), evidencing the high distribution of these viruses. These results demonstrate that it was feasible to implement the flavivirus detection system in mosquitoes, which could contribute to strengthen the detection, surveillance and control of these viruses, as well as, the knowledge about the ecological importance of insect flaviviruses

Respostas imunes induzidas por vacinas de DNA contra Zika e neuroadaptação de um isolado viral em camundongos para futuros testes de vacinas / Immune responses induced by DNA vaccines against Zika and neuroadaptation of a viral isolate in mice for future vaccine trials

Em geral, a doença causada pelo vírus Zika (ZIKV) é clinicamente semelhante às de outros arbovírus. No entanto, ocasionalmente ela é associada a síndromes neurológicas como a síndrome de Guillain-Barré. Além disso, durante a gravidez a infecção pode causar a síndrome congênita do zika, com danos cerebrais e sistêmicos no feto e/ou bebês, incluindo microcefalia. O genoma do ZIKV codifica três proteínas estruturais, capsídeo, pré-membrana/membrana e envelope (E), e sete proteínas não estruturais (NS1-5). As proteínas E e NS1 são indicadas como antígenos promissores, sendo consideradas alvos importantes no desenho de vacinas contra diversos flavivírus. Nosso grupo construiu dois plasmídeos recombinantes que codificam o ectodomínio da proteína E (pZKectoE) e a proteína NS1 (pZKNS1) do ZIKV. O objetivo deste trabalho foi investigar a capacidade desses plasmídeos de mediar a expressão de proteínas recombinantes e induzir respostas imunes. Células BHK-21 foram transfectadas com estes plasmídeos e a expressão das proteínas recombinantes foi avaliada. Como esperado, o pZKectoE e o pZKNS1 mediaram a expressão das proteínas recombinantes E e NS1, respectivamente. A ativação das respostas imunes humoral e celular em camundongos foi avaliada por ELISA e por ELISPOT. Ambos os plasmídeos induziram títulos de anticorpos específicos significativamente mais elevados do que o plasmídeo controle (pcTPA). Além disso,induziram respostas de células T com produção de IFN-γ após estimulação com duas bibliotecas de peptídeos derivadas de E e NS1. O mapeamento de peptídeos imunogênicos através de ensaios de ELISPOT identificou cinco epítopos imunodominantes na proteína E e três na proteína NS1. A maioria dos modelos murinos para estudos de ZIKV usa animais imunocomprometidos, que fornecem infecções robustas e letais, mas talvez não sejam ideais para testes de vacinas. Portanto, a fim de estabelecer um modelo murino imunocompetente suscetível à infecção pelo ZIKV, iniciamos experimentos de neuroadaptação de um vírus isolado de um paciente no Brasil, por sucessivas passagens no cérebro de camundongos recém-nascidos. Grupos de camundongos Swiss, com idades entre três e nove dias, foram infectados pela via intracerebral com ZIKV. No sétimo dia após a infecção, os animais foram eutanasiados e os cérebros foram coletados. As amostras foram tituladas por ensaio de plaques em células Vero. Em cada passagem, a amostra com o título viral mais alto foi usada para inoculação subsequente no cérebro de outros camundongos recém-nascidos. Nossos resultados mostraram uma alteração na morfologia do plaque produzido pela infecção com o vírus em comparação àquelas observadas com a amostra inicial de ZIKV. Em relação à carga viral, houve um aumento de 4 log durante as primeiras quatro passagens, seguido por uma ligeira redução e o sequenciamento do genoma viral revelou algumas mutações pontuais. De modo geral, as vacinas pZKEctoE e pZKNS1 foram capazes de mediar a expressão das proteínas recombinantes E ou NS1 com ativação das respostas imunes humoral e celular. Até o momento, não foi possível o estabelecimento de um modelo murino imunocompetente. Para obtenção do vírus neuroadaptado, com capacidade de causar sinais clínicos da infecção em animais adultos, deverão ser realizadas mais passagens em cérebros de camundogos Swiss. (AU)

Seroprevalence of zika virus in a cattle flock

The present research evaluated the seroprevalence of anti-zika virus (anti-ZIKV) antibodies by virus neutralization test (VNT) in 529 bovines from Andradina city, São Paulo state, Brazil. The reading was performed in an inverted optical microscope, considering reagents when the antibodies were capable to neutralize the ZIKV. Of the 529 samples, 53 (10.01%) were reagents. The animals were healthy at the time of collection. The samples were collected in February 2018, a favorable period for the multiplication of the vector and the highest risk of disease transmission. None of the animals showed anti-bovine viral diarrhea virus (anti-BVDV) antibodies, ruling out a possible cross-reaction, reinforcing the possible contact of the bovine with the ZIKV. In the herd, 88 pregnant females were evaluated; of these, 12 cows were reactive, with no history of reproductive problems or fetal malformations. This is the first research on the seroprevalence of ZIKV in cattle in Brazil, and studies should continue to evaluate cattle as a possible host of this arbovirus and its possible consequences for unique health and agribusiness.

Caracterización biológica de cepas del virus Encefalitis San Luis en células de la inmunidad innata / Biological characterization of St. Louis Encephalitis virus strains in cells of innate immunity

Los flavivirus transmitidos por mosquitos son una amenaza actual y emergente en todo el mundo. Dentro de este género, el virus Encefalitis San Luis (VESL) causa una forma severa de enfermedad neuroinvasiva donde la respuesta inmune es un componente crucial de la defensa del huésped. En este trabajo se investigó la interacción entre VESL y células de la inmunidad innata, en un modelo de infección in vitro de monocitos humanos (células U937) con cepas de distinta virulencia y condiciones epidemiológicas de aislamiento (CbaAr-4005 y 78V-6507). Se evaluó la capacidad de infectar y replicar del virus, como también el efecto citopático y la cinética de viabilidad de monocitos durante la infección. Los resultados demostraron la susceptibilidad de los monocitos a la infección, replicación y muerte por ambas cepas virales. Sin embargo, se hallaron diferencias significativas entre ellas. La cepa epidémica y de mayor virulencia CbaAr-4005 registró una tasa de infección y replicación superior a la de la cepa endémica y de menor virulencia 78V-6507. Se comprobó también que el VESL indujo la muerte de monocitos humanos, dependiendo del tiempo post-infección (pi) y de la cepa. Así, CbaAr-4005 provocó a partir del día 3 pi el doble de mortalidad celular que 78V-6507. Además, en los monocitos infectados se observaron alteraciones de parámetros morfológicos que podrían relacionarse con el tipo de mecanismo de muerte celular asociado a la infección por VESL.

Mosquitoes borne Flavivirus infections are an actual and emergent worldwide threat to human health. Within this genus, Saint Louis Encephalitis Virus (SLEV) causes a severe neuroinvasive disease where immune response is crucial for host survival. In this study the interaction between SLEV and innate immune cells was evaluated. An in vitro infection model with human monocytes (U937 cells) and strains with variations in virulence and isolation conditions (CbaAr-4005 and 78V-6507) were used. Infection capacity, replication capacity, cytopathic effect and monocyte viability kinetics were measured. The results showed susceptibility to infection and replication to both strains. However, significant differences were found among them. CbaAr-4005, the epidemic and more virulent strain, showed higher infection and replication ratios compared to 78V-6507. SLEV infection that induces cell death of human monocytes was also found in a post-infection time and in a strain dependent manner. Since day 3 post-infection, twice the mortality in CbaAr-4005 infected cells was observed. Furthermore, infected monocytes showed alterations in morphologic parameters that could be related with apoptosis mechanisms associated to SLEV infections.

Os Flavivírus transmitidos por mosquitos são uma ameaça atual e emergente no mundo todo. Nesse gênero, o vírus Encefalite Saint Louis (VESL) causa uma forma grave de doença neuroinvasiva onde a resposta imune é um componente crucial da defesa do hospedeiro. Neste trabalho nos investigamos a interação entre VESL e células de imunidade inata em um modelo de infecção in vitro de monócitos humanos (células U937) com estirpe de diferentes virulências e condições epidemiológicas de isolamento (CbaAr-4005 e 78V-6507). Foi avaliada a capacidade do vírus de infectar e replicar , assim como o efeito citopático e a viabilidade cinética dos monócitos durante a infecção. Os resultados demonstraram a suscetibilidade dos monócitos à infecção, replicação e morte por ambas as estirpes virais. No entanto, foram detectadas diferenças significativas entre eles. A estirpe epidémica e de maior virulenta CbaAr-4005 teve uma maior taxa de infecção e replicação do que a estirpe endémica e menos virulenta 78V-6507. Foi comprovado também que o VESL induziu a morte de monócitos humanos, dependendo do tempo pós-infecção (pi) e da estirpe. Assim, a CbaAr-4005 causou a partir do dia 3 pi o dobro da mortalidade celular o que a 78V- 6507. Além disso, alterações nos parâmetros morfológicos foram observadas nos monócitos infectados que poderiam estar relacionadas ao tipo de mecanismo de morte celular associado à infecção pelo VESL.

Vigilancia epidemiológica del virus Occidental del Nilo en las llanuras orientales de Colombia / Epidemiological surveillance of West Nile virus in the Eastern Plains of Colombia

RESUMEN Objetivo. Identificar la presencia del virus del Oeste del Nilo en equinos y mosquitos en ocho municipios del departamento del Meta. Materiales y métodos. La investigación contó con el aval del Comité de bioética de la Universidad de los Llanos. Se analizaron mediante pruebas serológicas y moleculares 613 muestras de equinos criollos y de raza cuarto de milla, destinados a actividades deportivas y de trabajo, con un rango de edad de 2 a 15 años, en los transectos: Villavicencio-Restrepo-Cumaral, San Martín - Castilla la Nueva-Granada y Puerto López-Puerto Gaitán, analizados en 62 pool y 213 mosquitos. Los pool de sueros de equinos y mosquitos fueron analizados por ELISA y PCR. Resultados. No se encontraron animales seropositivos mediante la prueba de ELISA y las pruebas moleculares también fueron negativas. Conclusiones. Aunque en este estudio no se evidenció la presencia de anticuerpos IgM por la técnica de Elisa y las pruebas moleculares (RT-PCR) también fueron negativas para circulación viral, en los municipios objeto de estudio, es importante indicar que la detección molecular en sueros, requiere unos niveles de viremia representativos y que el animal se encuentre en la fase aguda de la enfermedad. Aunque es posible que la población equina se mantenga libre de contacto con el virus, se debe mantener la vigilancia epidemiológica frente a este importante patógeno para la salud humana, especialmente por la presentación de brotes de otros virus zoonóticos como la Encefalitis Equina del Este y Encefalitis Equina Venezolana en los departamentos del Meta y Casanare, contiguo a este.

ABSTRACT Objective. Identify the presence of West Nile virus in horses and mosquitoes in eight municipalities of the department of Meta. Materials and methods. The research was supported by the Bioethics Committee of the University of Los Llanos. 613 samples of Creole and quarter-mile equine horses, intended for sports and work activities, with an age range of 2 to 15 years, were analyzed using serological and molecular tests in the transects: Villavicencio-Restrepo-Cumaral, San Martín- Castilla la Nueva-Granada and Puerto López-Puerto Gaitán, analyzed in 62 pools and 213 mosquitoes. The pool of sera of horses and mosquitoes were analyzed by ELISA and PCR. Results. No seropositive animals were found by the ELISA test and molecular tests were also negative. Conclusions. Although in this study the presence of IgM antibodies was not evidenced by the Elisa technique, and molecular tests (RT-PCR) were also negative for viral circulation, in the municipalities under study, it is important to indicate that the molecular detection in sera, it requires representative levels of viremia and that the animal is in the acute phase of the disease. Although it is possible that the equine population remains free of contact with the virus, epidemiological surveillance should be maintained against this important pathogen for human health, especially due to the outbreak of other zoonotic viruses such as Eastern Equine Encephalitis and Encephalitis Venezuelan Equine in the departments of Meta and Casanare, adjacent to this.

Zika virus serological diagnosis: commercial tests and monoclonal antibodies as tools

Zika virus (ZIKV), an emerging arthropod-borne virus (arbovirus) of the Flaviviridae family, is a current issue worldwide, particularly because of the congenital and neurological syndromes associated with infection by this virus. As the initial clinical symptoms of all diseases caused by this group are very similar, clinical diagnosis is difficult. Furthermore, laboratory diagnostic efforts have failed to identify specific and accurate tests for each virus of the Flaviviridae family due to the cross-reactivity of these viruses in serum samples. This situation has resulted in underreporting of the diseases caused by flaviviruses. However, many companies developed commercial diagnostic tests after the recent ZIKV outbreak. Moreover, health regulatory agencies have approved different commercial tests to extend the monitoring of ZIKV infections. Considering that a specific and sensitive diagnostic method for estimating risk and evaluating ZIKV propagation is still needed, this review aims to provide an update of the main commercially approved serological diagnostics test by the US Food and Drug Administration (FDA) and Brazilian National Health Surveillance Agency (ANVISA). Additionally, we present the technologies used for monoclonal antibody production as a tool for the development of diagnostic tests and applications of these antibodies in detecting ZIKV infections worldwide.(AU)

Superinfection exclusion studies using West Nile virus and Culex flavivirus strains from Argentina

In Argentina, many Flavivirus were recognised including West Nile virus (WNV). During 2009 several strains of Culex Flavivirus (CxFV), an insect-specific flavivirus, were isolated in the same region where circulation of WNV was detected. Hence, the objective of this study was to analyse the effect of co-infection in vitro assays using CxFV and WNV Argentinean strains in order to evaluate if CxFV could affect WNV replication. Our results showed that WNV replication was suppressed when multiplicity of infection (MOI) for CxFV was 10 or 100 times higher than WNV. Nevertheless, in vivo assays are necessary in order to evaluate the superinfection exclusion potential.

Molecular survey of flaviviruses and orthobunyaviruses in Amblyomma spp. ticks collected in Minas Gerais, Brazil / Investigação molecular de flavivírus e orthobunyaviruses em carrapatos do gênero Amblyomma spp. coletados em Minas Gerais, Brasil

Abstract Due to anthropic environmental changes, vector-borne diseases are emerging worldwide. Ticks are known vectors of several pathogens of concern among humans and animals. In recent decades, several examples of tick-borne emerging viral diseases have been reported (Crimean Congo hemorrhagic fever virus, Powassan virus, encephalitis virus, heartland virus, severe fever with thrombocytopenia syndrome virus). Unfortunately, few studies addressing the presence of viruses in wild ticks have been carried out in South America. With the aim of detecting flaviviruses and orthobunyaviruses in ticks, we carried out molecular detection in wild ticks collected in the state of Minas Gerais, Brazil. No Flavivirus-positive ticks were detected; however, we detected activity of Orthobunyavirus in 8 Amblyomma tick specimens. One of those individuals was positive for Bunyamwera orthobunyavirus, which represents the first report of this virus among ticks in South America. Further studies related to the ecology of zoonotic diseases are needed to increase knowledge of this topic, including attempts at viral isolation, full genome sequencing and biological characterization. In this way, we will obtain a better picture of the real risk of ticks as a vector for viral diseases for humans and animals on our continent, where no tick-borne viral disease is known to occur.

Resumo Alterações ambientais causadas pelo homem têm levado à emergência de doenças transmitidas por vetores no mundo. Carrapatos são vetores conhecidos de vários patógenos de importância médica e veterinária, tendo sido reportado nas últimas décadas um grande número de enfermidades virais emergentes transmitidas por eles (vírus da Febre Hemorrágica da Crimeia-Congo, vírus Powassan, vírus da Encefalite, vírus Heartland e vírus da Síndrome da Febre Trombocitopênica Severa). Infelizmente, poucos estudos envolvendo a pesquisa de vírus em carrapatos foram conduzidos na América do Sul até o momento, e nas últimas décadas um elevado número de enfermidades virais emergentes transmitidas por estes artrópodes foi relatado. Com o objetivo de investigar a presença de flavivírus e orthobunyavírus em carrapatos, foi conduzida uma análise molecular em espécimes coletados no estado de Minas Gerais, Brasil. Em nenhum carrapato foi detectada a presença de Flavivirus, no entanto, em 8 espécimes do gênero Amblyomma, foi detectada a presença de Orthobunyavirus, dos quais um espécime foi positivo para Bunyamwera orthobunyavirus. Novos estudos relacionados à ecologia de doenças zoonóticas, incluindo tentativas de isolamento viral, sequenciamento completo do genoma e caracterização biológica, são necessários. Desta forma, será possível ter uma base sobre os riscos da transmissão de vírus patogênicos por carrapatos em nosso continente, uma vez que até agora isso é desconhecido.

West Nile virus in Ecuador / Virus del Nilo Occidental en Ecuador

ABSTRACT Several studies have been carried out to determine the presence and circulation of West Nile Virus (WNV) in several species that interact in important ecosystems of Ecuador, such as the Galapagos Islands, where presence and surveillance studies of WNV have been carried out in wild and migratory birds (2003) (2008 to 2010), penguins (2003 to 2004). Studies have also been carried out on birds from different locations in Guayaquil (2011), and on Jauneche horses (2007), but no virus has been demonstrated in any of them. Nevertheless, in the Abras de Mantequilla wetland, two studies were conducted in equines aged between 3 months to 12 years, all of them mixed race, male and female, with no previous vaccination history and with presence of symptoms only in the first study. In the two studies the serum analysis was performed by the ELISA technique (reactivity determination) and Plaque Reduction Neutralization Test (PRNT). In the first study, 8.12% (13/160) of reactivity was determined in 13 horses and 22.22% of reactivity in 2 of 9 people; and only 3.12% (5/160 horses) of the presence of IgM antibodies against WNV. In relation to the second study, 12.6% (52/412) reactivity and 10.4% (43/412 horses) confirmed the serological evidence of WNV, with a final prevalence of 6.76%. Consequently, the WNV is present and circulating in the equines of the Ecuadorian coastal zone, which is a potential risk to the public health, nevertheless there is no updated information on investigations conducted in this regard.

RESUMEN El presente es una revisión bibliográfica sobre estudios realizados para determinar la presencia y circulación del Virus del Nilo Occidental (VNO) en diversas especies que interactúan en importantes ecosistemas del Ecuador, como son las Islas Galápagos, en donde, se han realizado estudios de presencia y vigilancia del VNO en aves silvestres y migratorias (2003) (2008 al 2010) y pingüinos (2003 al 2004). También, se ha realizado estudios en aves de diversos lugares de Guayaquil (2011), y en equinos de Jauneche (2007) pero en ninguno de los lugares se evidenció la presencia del virus. Por otro lado, en el humedal Abras de Mantequilla, Coello y colaboradores realizaron dos estudios en equinos de edades entre 3 meses a 12 años, todos de raza mestiza, sexo machos y hembras, sin antecedentes de vacunación y con presencia de síntomas solo en el primer estudio. El análisis de los sueros en los dos estudios se realizó mediante la técnica de ELISA (determinación de reactividad) y la confirmación a través de Neutralización por Reducción del Número de Placas (NTRP). En el primer estudio se determinó el 8.12% (13/160) de reactividad en 13 equinos y el 22.22% de reactividad en 2 de 9 personas (no se confirmaron); del total de muestras reactivas en equinos, solo se confirmó el 3.12% (5 equinos/160) de la presencia de anticuerpos IgM contra VNO. Respecto al segundo estudio estableció el 12.6% (52/412) de reactividad y el 10.4% (43/412 equinos) se confirmó la evidencia serológica del VNO, con una prevalencia final del 6.76%. Por lo consiguiente, el VNO está presente y circulando en los equinos de la zona costera ecuatoriana, lo cual es un riesgo potencial para la salud pública, sin embargo no hay información actualizada de investigaciones realizadas al respecto.

Zika Virus Impairs Host NLRP3-mediated Inflammasome Activation in an NS3-dependent Manner

Zika virus (ZIKV) is a mosquito-borne flavivirus associated with severe neurological disorders including Guillain-Barré syndrome and microcephaly. The host innate immune responses against ZIKV infection are essential for protection; however, ZIKV has evolved strategies to evade and antagonize antiviral responses via its nonstructural (NS) proteins. Here, we demonstrated that ZIKV infection unexpectedly inhibits NLRP3-dependent inflammasome activation in bone marrow-derived macrophages and mixed glial cells from mouse brain. ZIKV infection led to increased transcript levels of proinflammatory cytokines such as IL-1β and IL-6 via activating NF-κB signaling. However, ZIKV infection failed to trigger the secretion of active caspase-1 and IL-1β from macrophages and glial cells even in the presence of LPS priming or ATP costimulation. Intriguingly, ZIKV infection significantly attenuated NLRP3-dependent, but not absent in melanoma 2-dependent caspase-1 activation and IL-1β secretion from both cells. ZIKV infection further blocked apoptosis-associated speck-like protein containing a caspase recruitment domain oligomerization in LPS/ATP-stimulated macrophages. Interestingly, expression of ZIKV NS3 protein reduced NLRP3-mediated caspase-1 activation and IL-1β secretion in macrophages, whereas NS1 and NS5 proteins showed no effects. Furthermore, NLRP3 was found to be degraded by the overexpression of ZIKV NS3 in 293T cells. Collectively, these results indicate that ZIKV evades host NLRP3 inflammasome-mediated innate immune responses in macrophages and glial cells; this may facilitate ZIKV's ability to enhance the replication and dissemination in these cells.

Spatial distribution of Aedes aegypti (Diptera: Culicidae) in vulnerable areas for the transmission of arboviruses

Abstract INTRODUCTION: Areas at risk of transmission of arboviruses have been monitored using ovitraps. This study aimed to evaluate the spatial distribution of Aedes aegypti in vulnerable areas for the transmission of arboviruses and assess the influence of climatic conditions on the infestation of these culicids. METHODS: Ovitraps were installed in Agrestina, Pernambuco, Northeastern Brazil. RESULTS: Overall, 44,936 eggs were collected, and the indexes of infestation varied. Relative humidity was significantly associated with the infestations. CONCLUSIONS: Using ovitraps, entomologic indexes and analysis of climatic factors might be good strategies for monitoring vulnerable areas for the transmission of arboviruses.

Previous dengue or Zika virus exposure can drive to infection enhancement or neutralisation of other flaviviruses

BACKGROUND Dengue virus (DENV) has circulated in Brazil for over 30 years. During this time, one serotype has cyclically replaced the other, until recently, when all four distinct serotypes began to circulate together. Persistent circulation of DENV for long time periods makes sequential infections throughout a person's life possible. After primary DENV infection, life-long immunity is developed for the infecting serotype. Since DENV and Zika virus (ZIKV) are antigenically similar, the possibility of cross-reactions has attracted attention and has been demonstrated in vitro. OBJECTIVE The aim of this study was to investigate whether immune-sera from DENV and ZIKV infected patients would cross-react in vitro with other Flaviviridae family members. METHODS Cross-reaction of the studied samples with yellow fever virus (YFV), West Nile virus (WNV), Rocio virus (ROCV), Saint Louis virus (SLEV) and Ilheus virus (ILHV) has been investigated by plaque reduction neutralisation test (PRNT) and the antibody-dependent enhancement (ADE) by flow-cytometry. FINDINGS Antibodies against ZIKV and DENV virus cross-reacted with other flaviviruses either neutralising or enhancing the infection. Thus, viral entrance into FcRFcɣRII-expressing cells were influenced by the cross-reactive antibodies. ZIKV or DENV immune sera enhanced cellular infection by WNV, ILHV, ROCV and SLEV. Finally, DENV immune sera presented higher neutralising activity for YFV and SLEV. While ZIKV immune sera neutralised WNV, ILHV and ROCV with high frequencies of positivity. MAIN CONCLUSIONS The co-circulation of those viruses in the same area represents a risk for the development of severe infections if they spread throughout the country. Successive flavivirus infections may have an impact on disease pathogenesis, as well as on the development of safe vaccine strategies.

Investigação do vírus zika e outros flavivírus em animais silvestres no centro-oeste do Brasil / Investigation of the Zika virus and other flaviviruses in wild animals in central-western Brazil

À exceção dos estudos sobre o vírus da febre amarela em primatas não humanos (PNH) no Brasil, estudos sobre outros flavivírus em diferentes espécies silvestres de vida livre no país, são escassos. Neste sentido, como parte de um estudo de ecologia do vírus Zika (Zika virus ou ZIKV) do Centers for Disease Control and Prevention (CDC) na América do Sul, uma investigação sobre o ZIKV e outros flavivírus foi conduzida em animais silvestres capturados entre 2017 e 2018, em área metropolitana de Cuiabá e Campo Grande, na Região Centro-Oeste do Brasil. Amostras de sangue foram coletadas de 685 indivíduos hígidos de vinte espécies, comumente observadas em parques locais, incluindo mamíferos (N = 372), répteis (N = 213) e anfíbios (N = 100). Amostras de sangue total foram submetidas à extração de ácido ribonucleico (RNA), seguida de reação em cadeia da polimerase em transcrição reversa (RT-PCR) em tempo real para o gênero flavivírus. As amostras positivas foram então confirmadas através de RT-PCR específica para ZIKV e sequenciamento nucleotídico. Um total de 43 (6,3%) amostras foi positivo no RT-PCR para flavivírus e negativo no RT-PCR específico para ZIKV. Todas as 43 amostras positivas para RT-PCR de flavivírus foram submetidas ao sequenciamento nucleotídico, mas nenhuma apresentou similaridade às sequências de nucleotídeos ou proteínas de flavivírus, disponíveis na ferramenta básica de busca de alinhamento local (BLAST).

Dos 685 animais testados por RT-PCR, 174 (25,4%) tiveram amostras de plasma também submetidas ao teste de neutralização por redução de placas (PRNT90) para detecção de anticorpos neutralizantes para ZIKV. Das 174 amostras testadas, sete (4%) foram sororeativas (PRNT90 título ≥10), incluindo três capivaras x (Campo Grande), um morcego (Cuiabá), um quati (Campo Grande), um gambá (Cuiabá) e um macacoprego (Campo Grande), todos capturados em 2017. Os resultados encontrados no presente estudo sugerem ausência de infecção aguda, mas potencial exposição ao ZIKV de animais silvestres de vida livre capturados em área metropolitana de Cuiabá e Campo Grande, entre 2017 e 2018. Mais estudos são necessários, incluindo análises de diferentes tipos de amostras de cada indivíduo, bem como a pesquisa de anticorpos neutralizantes para outros flavivírus para descartar reações heterólogas e confirmar exposição ao ZIKV de animais silvestres da Região Centro-Oeste do Brasil. (AU)

Estudo da fauna de mosquitos (Diptera: Culicidae) com simultânea investigação de infecção natural por Flavivirus em duas Unidades de Conservação da Mata Atlântica, Estado de São Paulo / Not available

Os Flavivirus são transmitidos por mosquitos (Diptera: Culicidae) que se refugiam em remanesctentes de Mata Atlântica. Essas áreas verdes correspondem às Unidades de Conservação e parques urbanos, que estão espalhados pela região metropolitana de São Paulo. Este estudo foi realizado com o intuito de conhecer as espécies de culicídeos que circulam na Área de Proteção Ambiental (APA) Capivari-Monos, na zona Sul do município de São Paulo, e no Parque Estadual da Cantareira (PEC), na zona Norte do mesmo município, e de investigar infecção natural por Flavivirus na fauna de culicídeos amostrada. Também foi proposto relacionar a variedade, a quantidade e identidade dos Flavivirus detectados com os padrões de riqueza, abundância e diversidade das assembleias de mosquitos. Foram realizadas 14 coletas, mensalmente, em quatro pontos de coleta na APA e três no PEC, todos com diferentes níveis de intervenção antrópica, no período de março de 2016 a abril de 2017. Armadilhas automáticas luminosastipo CDC (com atração de CO2 e ácido lático) foram instaladas na copa das árvores e no nível do solo. O esforço amostral foi equivalente para os todos os pontos, sendo que foram instaladas duas armadilhas em cada ponto (uma na copa e outra no solo), com 18 horas de coleta, permitindo amostragem de culicídeos de hábitos diurnos, crepusculares e noturnos. Os espécimes foram transportados com vida para o Laboratório de Saúde Pública da Faculdade de Saúde Pública da Universidade de São Paulo, criopreservados a temperatura -70ºC, identificados morfologicamente e agrupados em pools (com até 10 indivíduos). Os pools foram submetidos à técnica de isolamento viral em cultura de células (C6/36), seguida do teste de imunofluorescência indireta. Os pools positivos foram submetidos à reação de RT-qPCR e, posteriormente, sequenciados. Duas árvores de similaridade foram construídas para confirmação dos Flavivirus. No total, 1216 exemplares de culicídeos foram amostrados (13 gêneros), cuja riqueza foi de 42 táxons. A APA registrou a maior abundância (878 espécimes) e maior riqueza (37 táxons). A Cachoeira foi o ponto de coleta na APA que amostrou a maior riqueza e abundância, contudo, com a mais baixa diversidade. Entretanto, a Borracharia obteve alta riqueza, baixa abundância e a maior diversidade. O PEC amostrou 338 indivíduos e a riqueza foi de 23 táxons. Dentre os pontos do PEC, a Trilha do Pinheirinho amostrou a maior riqueza e abundância. An. (Ker.) cruzii, Cx. (Cux.) sp, Cx. (Mel.) vaxus, Li. durhami, Wy. (Prl.) confusa e Wy. (Pho.) theobaldi foram detectadas com infecção natural por Flavivirus. O sequenciamento revelou infecção por ZIKV em An. (Ker.) cruzii, Li. durhami e Wy. (Prl.) confusa, e infecção por DENV-2 em Cx. (Cux.) sp e Cx. (Mel.) vaxus. Concluiu-se que a riqueza, abundância e diversidade estão relacionadas entre si e, juntas, influenciaram na detecção de espécies de culicídeos naturalmente infectadas por Flavivirus, sendo que estes foram detectados em espécies provenientes de pontos de coleta cuja riqueza e abundância foram altas, e a diversidade baixa. A quantidade e a variedade dos Flavivirus também foram influenciadas por esses três fatores, para ocorrer na natureza. Não foi possível correlacionar a identidade dos Flavivirus com os três fatores uma vez que as espécies detectadas com infecção natural por esses vírus não são apontadas como potenciais vetoras. Além disso, a abundância e a diversidade pareceram ter uma relação inversa entre si

Flaviviruses are transmitted by mosquitoes (Diptera: Culicidae) that take refuge in remnants of the Atlantic Forest. These green areas correspond to Conservation Units and urban parks which are spread throughout the metropolitan region of São Paulo. This study was carried out in order to identify the Culicidae fauna that circulate in Capivari-Monos Environmental Protection Area (APA), located in the South area of the city of São Paulo, and in Cantareira State Park (PEC), North area of the same municipality and to investigate natural Flaviviruses infection in this sampled Culicidae fauna. It was also proposed to relate the variety, quantity and identity of the Flaviviruses detected with patterns of richness, abundance and diversity of mosquito assemblages. Fourteen collections were carried out monthly at four collection sites in the APA and three in the PEC, all sites with different levels of anthropogenic intervention, during March 2016 to April 2017. CDC automatic traps (with attraction of CO2 and lactic acid) were installed in the canopy and on ground. The sampling effort was equivalent for all the points, and two traps were installed at each point (one in the canopy and the other on ground), with 18 hours of sampling, allowing sampling culicidae of daytime, morning and evening twilight, and nightlyl habits. The specimens were carried alive to the Public Health Laboratory of the School of Public Health of the University of São Paulo, were cryopreserved at a -70ºC temperature, identified morphologically and grouped in pools (with up to 10 individuals). The pools were submitted to the virus isolation technique in cell culture tissue (C6 / 36), followed by the indirect immunofluorescence test. The positive pools were submitted to the RT-qPCR reaction and, subsequently, sequenced. Two similarity trees were made only to confirm Flaviviruses infection. In total, 1216 specimens of culicidae were sampled (13 genera), and the richness was 42 taxa. In addition to APA recorded the highest abundance (878 specimens) and also highest richness (37 taxa). Cachoeira was the collection site in APA that showed the greatest richness and abundance as well, however, with the lowest diversity. In addition, Borracharia obtained high richness, low abundance and highest diversity. PEC sampled 338 specimens and the richness was 23 taxa. Among the collection sites of the PEC, Pinheirinho Trail showed the highest richness and also abundance. An. (Ker.) cruzii, Cx. (Cux.) sp, Cx. (Mel.) vaxus, Li. durhami, Wy. (Prl.) confusa and Wy. (Pho.) theobaldi were detected with natural Flaviviruses infection. The sequencing analyzes revealed ZIKV infection in An. (Ker.) cruzii, Li. durhami and Wy. (Prl.) confusa, and DENV-2 infection in Cx. (Cux.) sp and Cx. (Mel.) vaxus. It has concluded that the richness, abundance and also diversity are related to each other and, together, influenced the detection of species of culicidae naturally infected by Flaviviruses, which were detected in species from collection sites whose richness and abundance were high. About quantity and variety of Flaviviruses, these were also influenced by the three factors on nature. It was not possible to correlate the identity of the Flaviviruses with the three factors since the species detected with natural infection by these viruses are not indicated as potential vectors. Moreover, abundance and diversity appeared to have an inverse relation.

Development of a Rapid Diagnostic Test Kit to Detect IgG/IgM Antibody against Zika Virus Using Monoclonal Antibodies to the Envelope and Non-structural Protein 1 of the Virus

We developed a Rapid Diagnostic Test (RDT) kit for detecting IgG/IgM antibodies against Zika virus (ZIKV) using monoclonal antibodies to the envelope (E) and non-structural protein 1 (NS1) of ZIKV. These proteins were produced using baculovirus expression vector with Sf9 cells. Monoclonal antibodies J2G7 to NS1 and J5E1 to E protein were selected and conjugated with colloidal gold to produce the Zika IgG/IgM RDT kit (Zika RDT). Comparisons with ELISA, plaque reduction neutralization test (PRNT), and PCR were done to investigate the analytical sensitivity of Zika RDT, which resulted in 100% identical results. Sensitivity and specificity of Zika RDT in a field test was determined using positive and negative samples from Brazil and Korea. The diagnostic accuracy of Zika RDT was fairly high; sensitivity and specificity for IgG was 99.0 and 99.3%, respectively, while for IgM it was 96.7 and 98.7%, respectively. Cross reaction with dengue virus was evaluated using anti-Dengue Mixed Titer Performance Panel (PVD201), in which the Zika RDT showed cross-reactions with DENV in 16.7% and 5.6% in IgG and IgM, respectively. Cross reactions were not observed with West Nile, yellow fever, and hepatitis C virus infected sera. Zika RDT kit is very simple to use, rapid to assay, and very sensitive, and highly specific. Therefore, it would serve as a choice of method for point-of-care diagnosis and large scale surveys of ZIKV infection under clinical or field conditions worldwide in endemic areas.

Serological and molecular epidemiology of Japanese encephalitis virus infections in swine herds in China, 2006–2012

Japanese encephalitis virus (JEV) is a mosquito-borne, zoonotic flavivirus causing viral encephalitis in humans and reproductive disorder in swine. JEV is prevalent throughout China in human; however, spatiotemporal analysis of JEV in Chinese swine herds has not been reported previously. Herein, we present serological and molecular epidemiological results and estimates of prevalence of JEV infections among swine herds in various regions of China. The results suggest that JEV infections are widespread and genotype I and III strains co-exist in the same regions. Therefore, there is an urgent need to monitor JEV infection status among swine herds in China.