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

Seasonal population dynamics of the primary yellow fever vector Haemagogus leucocelaenus (Dyar & Shannon) (Diptera: Culicidae) is mainly influenced by temperature in the Atlantic Forest, southeast Brazil

BACKGROUND Southeast Brazil has recently experienced a Yellow Fever virus (YFV) outbreak where the mosquito Haemagogus leucocelaenus was a primary vector. Climatic factors influence the abundance of mosquito vectors and arbovirus transmission. OBJECTIVES We aimed at describing the population dynamics of Hg. leucocelaenus in a county touched by the recent YFV outbreak. METHODS Fortnightly egg collections with ovitraps were performed from November 2012 to February 2017 in a forest in Nova Iguaçu, Rio de Janeiro, Brazil. The effects of mean temperature and rainfall on the Hg. leucocelaenus population dynamics were explored. FINDINGS Hg. leucocelaenus eggs were continuously collected throughout the study, with a peak in the warmer months (December-March). The climatic variables had a time-lagged effect and four weeks before sampling was the best predictor for the positivity of ovitraps and total number of eggs collected. The probability of finding > 50% positive ovitraps increased when the mean temperature was above 24ºC. The number of Hg. leucocelaenus eggs expressively increase when the mean temperature and accumulated precipitation surpassed 27ºC and 100 mm, respectively, although the effect of rainfall was less pronounced. MAIN CONCLUSIONS Monitoring population dynamics of Hg. leucocelaenus and climatic factors in YFV risk areas, especially mean temperature, may assist in developing climate-based surveillance procedures to timely strengthening prophylaxis and control.

Detection of the Asian II genotype of dengue virus serotype 2 in humans and mosquitoes in Brazil

Abstract INTRODUCTION: DENV-2 is the cause of most dengue epidemics worldwide and is associated with severe cases. METHODS: We investigated arboviruses in 164 serum samples collected from patients presenting with clinical symptoms of dengue fever and 152 mosquito pools. RESULTS: We detected the Asian II genotype of DENV-2 in humans and mosquitoes. Our results confirmed the circulation of the Asian II genotype in Brazil, in addition to the prevalent Asian/American genotype. CONCLUSIONS: The detection of Asian II genotype of DENV-2 in mosquito pools collected in a forest park may be related to a spillback event of human dengue virus.

Cacipacore virus as an emergent mosquito-borne Flavivirus

Abstract INTRODUCTION: Cacipacore virus (CPCV), a possible bird-associated flavivirus, has yet to be detected in mosquitoes. Our purpose is examining CPCV in mosquitoes from the Amazon region of Brazil. METHODS: Approximately 3,253 Culicidae (grouped into 264 pools) were collected from the Amazon region during 2002-2006 and analyzed using a Flavivirus genus-specific reverse transcription- polymerase chain reaction followed by nested polymerase chain reaction assay and by nucleotide sequencing of amplicons. RESULTS: Nucleotide sequences from five mosquito samples showed high similarity to the those of CPCV originally isolated in the Amazon region. CONCLUSIONS: This is the first report of CPCV-infected mosquitoes which has implications on the arbovirus maintenance in nature and transmission to man.

Mosquito-transmitted viruses - the great Brazilian challenge

ABSTRACT Arboviruses pose a serious threat to public health worldwide, overloading the healthcare system and causing economic losses. These viruses form a very diverse group, and in Brazil, arboviruses belonging to the families Flaviviridae and Togaviridae are predominant. Unfortunately, the number of arboviruses increases in proportion with factors such as deforestation, poor sanitation, climate changes, and introduction of new viruses like Chikungunya virus and Zika virus. In Brazil, dengue is endemic, along with the presence of other arboviruses. The situation is complicated by the scarcity of diagnostic infrastructure and the absence of approved vaccines for these diseases. Disease control, thus, relies solely on vector control. Therefore, enhanced clinical knowledge and improved general awareness about these arboviruses are indispensable to tackle diagnostic inadequacies.

Molecular detection of flaviviruses and alphaviruses in mosquitoes (Diptera: Culicidae) from coastal ecosystems in the Colombian Caribbean

Arboviruses belonging to the genera Flavivirus and Alphavirus were detected in mosquitoes in a rural area of San Bernardo del Viento (Córdoba, Colombia). A total of 22,180 mosquitoes were collected, sorted into 2,102 pools, and tested by generic/nested reverse transcription-polymerase chain reaction. Venezuelan equine encephalitis virus, dengue virus, West Nile virus, St. Louis encephalitis virus, yellow fever virus, and Culex flavivirus were detected and identified by sequencing. The detection of arboviral pathogens in this zone represents possible circulation and indicates a human health risk, demonstrating the importance of virological surveillance activities.

Mayaro virus and dengue virus 1 and 4 natural infection in culicids from Cuiabá, state of Mato Grosso, Brazil

This study aimed to verify the diversity of Culicidae species and their frequency of infection with flaviviruses and alphaviruses in Cuiabá, state of Mato Grosso, Brazil. Mosquitoes were captured with Nasci aspirators and hand net in 200 census tracts, identified alive at species level and pooled in one-20 (11,090 mosquitoes, 14 species). Female pools (n = 610) were subjected to multiplex seminested-reverse transcription-polymerase chain reaction (RT-PCR) for 11 flavivirus and five alphavirus. Positive pools were tested by single RT-PCR followed by nucleotide sequencing, by RT-PCR for E1 gene [Mayaro virus (MAYV)] and by inoculation in Vero cells (MAYV) or C6/36 cells (flaviviruses). One/171 Aedes aegypti was positive for dengue virus (DENV)-1, 12/403 Culex quinquefasciatus, and four/171Ae. aegypti for MAYV, which was isolated from two pools containing two nonengorged females of Ae. aegypti and two ofCx. quinquefasciatus. DENV-4 was detected in 58/171 pools of Ae. aegytpi, 105/403 Cx. quinquefasciatus, two/five Psorophora sp., two/11 Psorophora varipes/Psorophora albigenu, one/one Sabethes chloropterus, two/five Culex bidens/Culex interfor, and one/one Aedes sp. DENV-4 was isolated from two pools containing three and 16 nonengorged Cx. quinquefasciatus females. Phylogenetic analysis revealed MAYV belongs to genotype L, clustering with human samples of the virus previously identified in the city. Cuiabá has biodiversity and ecosystem favourable for vector proliferation, representing a risk for arbovirus outbreaks.

Conhecimento sobre síndrome da imunodeficiência humana de idosos de uma unidade de atenção ao idoso / Conocimiento sobre el síndrome de la inmunodeficiencia humana entre los ancianos de una unidad de atención al anciano / Knowledge about the human immunodeficiency syndrome among elders in a unit for the care of the elderly

RESUMO Objetivo Análise do conhecimento de idosos acerca da síndrome e do vírus da imunodeficiência humana. Métodos Estudo descritivo, transversal e quantitativo envolvendo 457 idosos da Unidade de Atenção ao Idoso de Uberaba, Minas Gerais, durante 3 meses. Foram aplicados os instrumentos Mini Exame do Estado Mental e o questionário sobre o vírus da imunodeficiência humana para terceira idade e, para a análise, foram usadas medidas de frequência. Resultados A maioria dos participantes eram mulheres (74%), entre 60 a 69 anos (51%). O maior índice de acertos obteve a transmissão por agulhas 96,2% e o menor (45,3%), a transmissão pela picada de mosquito e se a pessoa com o vírus da imunodeficiência humana sempre apresenta sintomas (49,6%). 88,2% deles relataram nunca usar camisinha. Conclusões O nível geral de conhecimento pode ser considerado bom, respondendo ao nosso objetivo. No entanto, foram identificadas lacunas decorrentes da falta de campanhas voltadas exclusivamente para tal público, bem como na conscientização de profissionais.

RESUMEN Objetivo Analizar el conocimiento de ancianos sobre HIV/ SIDA. Métodos Estudio descriptivo, transversal, cuantitativo, con 457 ancianos de la Unidad de Atención al Anciano de Uberaba - Minas Gerais, por 3 meses. Se aplicó el Mini-examen del Estado Mental, el cuestionario sobre el virus de la inmunodeficiencia humana para los ancianos, y medidas de frecuencia para el análisis. Resultado La mayoría eran mujeres (74%), entre 60 y 69 años (51%). La pregunta sobre la transmisión por jeringas/agujas tuvo el mayor número de respuestas correctas (96,2%), mientras las otras (45.3%) fueron sobre la transmisión por picaduras de mosquito, y si alguien con el virus de la inmunodeficiencia humana siempre presenta síntomas (49,6%). El 88,2% de ellos nunca utiliza condones. Conclusión El nivel general de conocimiento puede ser considerado bueno, respondiendo a nuestra meta. Sin embargo, se identificaron brechas en el conocimiento, en consecuencia de la falta de campañas exclusivas para este público, así como de la sensibilización de los profesionales.

ABSTRACT Objective To analyze the knowledge of elders regarding HIV/AIDS. Methods Descriptive, cross-sectional, quantitative study, with 457 elders from the Unit for the Care of Elders in Uberaba – Minas Gerais, lasting 3 months. The instruments used were the Mental State Mini-exam and a questionnaire on the human immunodeficiency virus for elders, together with frequency measures for their analysis. Results Most participants were female (74%), between 60 and 69 y/o (51%). The question with the most correct answers was about syringe/needle transmission (96.2%) and the ones with the least (45.3% and 49.6%, respectively) were regarding AIDS transmission through mosquito bites and whether a person with the human immunodeficiency virus always presents symptoms (49.6%). 88.2% of participants never use condoms. Conclusion The general level of knowledge can be considered good, responding to our goal. However, gaps in knowledge were identified due to the lack of campaigns aimed exclusively at such public, as well as to the lack of awareness of professionals.

Molecular identification of Saint Louis encephalitis virus genotype IV in Colombia

Saint Louis encephalitis virus (SLEV) is a member of the Japanese-encephalitis virus serocomplex of the genus Flavivirus. SLEV is broadly distributed in the Americas and the Caribbean Islands, where it is usually transmitted by mosquitoes of the genus Culex and primarily to birds and mammalian-hosts. Humans are occasionally infected by the virus and are dead-end hosts. SLEV causes encephalitis in temperate regions, while in tropical regions of the Americas, several human cases and a wide biological diversity of SLEV-strains have been reported. The phylogenetic analysis of the envelope (E) protein genes indicated eight-genotypes of SLEV with geographic overlap. The present paper describes the genotyping of two SLEV viruses detected in mosquito-pools collected in northern Colombia (department of Cordoba). We used reverse transcription-polymerase chain reaction to amplify a fragment of theE-gene to confirm the virus identity and completeE-gene sequencing for phylogenetic analysis and genotyping of the two-SLEV viruses found circulating in Córdoba. This is the first report of SLEV genotype IV in Colombia (Córdoba) in mosquitoes from a region of human inhabitation, implicating the risk of human disease due to SLEV infection. Physicians should consider SLEV as a possible aetiology for undiagnosed febrile and neurologic syndromes among their patients who report exposure to mosquito-bites.

Ecological aspects of mosquitoes (Diptera: Culicidae) in the gallery forest of Brasilia National Park, Brazil, with an emphasis on potential vectors of yellow fever

Introduction We analyzed the vertical and monthly distributions of culicid species in the gallery forest of Brasília National Park, with an emphasis on the potential vectors of yellow fever (YF). Methods Between September 2010 and August 2011, mosquitoes were captured on the ground and in the canopy of the forest for five consecutive days per month, from nine to 15 hours. The mosquitoes were examined to verify natural infection with flaviviruses by isolation in Aedes albopictus Skuse, 1864 cells followed by indirect immunofluorescence. Results We identified 2,677 culicids distributed in 29 species. Most of the mosquitoes were captured at ground level (69%) during the rainy season (86%). The most abundant species were Sabethes (Sabethes) albiprivus Theobald, 1903; Limatus durhamii Theobald, 1901; Haemagogus (Conopostegus) leucocelaenus Dyar & Shannon, 1924; Haemagogus (Haemagogus) janthinomys Dyar, 1921; Aedes (Ochlerotatus) scapularis Rondani, 1848; Psorophora (Janthinosoma) ferox Von Humboldt, 1819; and Aedes (Ochlerotatus) serratus Theobald, 1901. Limatus durhamii, Limatus durhamii, Psorophora ferox, Aedes scapularis and Aedes serratus showed significant differences (p<0.05) in their habitat use. Limatus durhamii was found more often in the canopy, unlike the other species. During the rainy season, the most abundant species were Sa. albiprivus, Haemagogus leucocelaenus and Limatus durhamii. During the dry season, the potential YF vectors exhibited a very low frequency and abundance, except Aedes scapularis and Aedes serratus. No flavivirus was detected in the 2,677 examined mosquitoes. Conclusions We recommend continued and systematic entomological monitoring in areas vulnerable to the transmission of YF in the Federal District of Brazil. .

Mosquitos (Díptera: Culicidae) vectores potenciales de arbovirus en la región de Urabá, noroccidente de Colombia[

Introducción. Los estudios encaminados a conocer los parámetros ecológicos de las poblaciones de mosquitos selváticos, permiten establecer el riesgo de transmisión de arbovirus y aportar recomendaciones sobre prevención, vigilancia y control a las autoridades de salud. Objetivo. Determinar la diversidad y abundancia de mosquitos nocturnos y crepusculares, potenciales vectores de arbovirus en zonas rurales de Apartadó y Turbo, Antioquia. Materiales y métodos. Se realizaron muestreos trimestrales. Para la recolección de mosquitos se usaron trampas CDC, Shannon y cebo humano protegido, en fragmentos de bosque, entre las 18:00 y las 06:00 horas. Se estimaron los índices de diversidad y abundancia de especies. Resultados. Se capturaron 583 mosquitos de 10 génerosy 27 especies. Las especies más abundantes fueron Coquilletidia venezuelensis (14,6 %), Aedes scapularis (14,08 %), Psorophora ferox (10,82 %) y Culex quinquefasciatus (10,3 %). La riqueza específica y los índices ecológicos calculados fueron mayores en Turbo; el fragmento de bosque estudiado en Turbo se considera de mayor riqueza y uniformidad de especies. El hallazgo de Cx. pedroi, Ae. scapularis, Ae. angustivittatus, Cq. venezuelensis, Cx. nigripalpus, Cx. quinquefasciatus, Cx. declarator, Mansonia titillans, Ma. pseudotitillans, Ps. ferox y Trichiprosopon digitatum reportados como vectores de arbovirus, alerta sobre la posibilidad de transmisión en la zona. Conclusión. La diversidad y abundancia de mosquitos en la zona de estudio son altas. Los análisis ecológicos más los reportes previos de capacidad vectorial de algunas de las especies registradas, permiten concluir que en la zona se pueden presentar brotes de arbovirosis.

Mosquitoes (Diptera: Culiciadae) as potential vectors of arbovirused in the Urabá region, Northwest of Colombia.

Introduction. Studies directed to investigate ecological parameters of sylvatic mosquitoes populations permit the establishment of risk levels in the transmission of arboviruses and provide the basis for recommendations to health authorities about prevention, surveillance and control. Objective. To establish the diversity and abundance of mosquitoes vectors of arbovirus in rural areas of Apartadó and Turbo, Antioquia. Materials and methods. Quarterly sampling was done. For mosquito collections in forest fragments, CDC traps, Shannon traps and human landing methods were used. Diversity and abundance indices were calculated. Results. Five hundred eighty-three mosquitoes were collected and identified in 10 genera and 27 species. The most abundant species were as follows: Coquilletidia venezuelensis (14.6%), Aedes scapularis (14.1%), Psorophora ferox (10.8%) and Culex quinquefasciatus (10.3%). Species richness and ecological indexes were highest in Turbo municipality; where the forest fragment was considered highest in species richness and uniformity. The identification in the samples of Culex pedroi,Ae. scapularis, Aedes angustivittatus, Cq. venezuelensis, Culex nigripalpus, Cx. quinquefasciatus, Culex declarator, Mansonia titillans, Mansonia pseudotitillans, Ps. ferox andTrichoprosopon. Digitatum--allpreviously reported as arbovirus vectors--warns about the possibility of arbovirus transmission in the zone. Conclusions. Mosquito diversity and abundance in the study area was very high. The ecological analysis, plus previous reports about vector competence of several of the recorded species, permits the conclusion that arbovirus outbreaks can occur in the Urabá region.

Tipos del virus dengue identificados en pacientes del Centro Médico Dr. Rafael Guerra Méndez, Valencia, Estado Carabobo, Venezuela / Dengue virus types identified in patients attended at Medical Center Dr. Rafael Guerra Méndez, Valencia, Carabobo, Venezuela

En esta investigación se llevó a cabo un estudio descriptivo de 114 muestras serológicas de pacientes con sospecha clínica de dengue, según criterios establecidos por la Organización Mundial para la Salud (OMS), que se recibieron durante el mes de junio de 2010 en el Centro Médico Dr. Rafael Guerra Méndez, de la ciudad de Valencia, Estado Carabobo. Las muestras se trasladaron al Instituto de Investigaciones Biomédicas de la Universidad de Carabobo (BIOMED-UC) y se analizaron por la técnica de RT-PCR anidada. Los resultados indicaron positividad en 46 muestras y la presencia de los 4 tipos del virus dengue. Los tipos detectados con mayor frecuencia fueron DEN-4 (34,8%), seguido por el DEN-3 (32,6%), sobre todo en niños ≤ 12 años que representaron el 54% del total de las muestras.

This research conducted a descriptive study of 114 serum samples from patients with clinical suspicion of dengue, according to criteria established by the World Health Organization (WHO) who were received during the month of June 2010 at the Medical Center Dr. Rafael Guerra Mendez in the city of Valencia, Carabobo State. Samples were transported to the Institute of Biomedical Research of the University of Carabobo (BIOMED-UC) and analyzed by RT-nested PCR. The results showed positivity in 46 samples and the presence of the 4 types of dengue virus. The most frequently detected types were DEN-4 (34.8%), followed by DEN-3 (32.6%) which were, especially in children ≤ 12 years and accounted for 54% of total samples.

Seroepidemiological monitoring in sentinel animals and vectors as part of arbovirus surveillance in the state of Mato Grosso do Sul, Brazil / Monitoramento soroepidemiológico em animais-sentinela e vetores como parte da vigilância de arbovírus, no estado de Mato Grosso do Sul, Brasil

INTRODUCTION: From February-September 2010, seroepidemiological surveys were conducted on non-human primates and transmitter vector capture was used to investigate the possible circulation of arboviruses in the municipalities of Bonito, Campo Grande, and Jardim, State of Mato Grosso do Sul, Brazil. METHODS: A total of 65 primates from the wild and captivity were used, and potential vectors were captured using Castro and dip nets. Serum samples were tested at the Instituto Evandro Chagas, State of Pará, using the hemagglutination inhibition test to detect total antibodies against 19 different arboviruses. Virus isolation was attempted from serum samples and arthropod suspensions using newborn mice and the C6/36 cell line clone. In addition, identification of the vector species was conducted. RESULTS: From the 19 serum samples from Campo Grande, 1 sample had a 1:20 titer for Flavivirus. From the 35 samples collected in Bonito, 17 samples had antibodies to arboviruses, 4 (11.4%) were positive for Alphavirus, and 5 (14.2%) were positive for Flavivirus. Monotypic reactions were observed for the Mayaro (n = 10) and Oropouche (n = 5) viruses, and 6 (17.1%) samples had titers for >1 virus. We captured 120 Culicidae individuals that were potential arbovirus transmitters in Jardim; however, all the samples were negative for the viruses. CONCLUSIONS: Mato Grosso do Sul has a variety of vertebrate hosts and transmission vectors, thereby providing ideal conditions for the emergence or reemergence of arboviruses, including some pathogenic to human beings.

INTRODUÇÃO: No período de fevereiro a setembro de 2010, foram realizados inquéritos soroepidemiológicos em primatas não humanos e captura de vetores transmissores, com o intuito de investigar a possível circulação de arbovírus nos municípios de Bonito, Campo Grande e Jardim, no Estado do Mato Grosso do Sul, Brasil. MÉTODOS: Foram utilizados 65 primatas de vida livre e de cativeiro, e potenciais vetores capturados por Castro e puçás. As amostras séricas foram testadas pelo teste de inibição da hemaglutinação para a detecção de anticorpos totais contra 19 diferentes arbovírus e a tentativa de isolamento viral (camundongo recém-nascido e linhagem celular-clone C6/36) nas amostras séricas e suspensões de artrópodes, bem como a identificação das espécies vetoriais foram realizadas no Instituto Evandro Chagas-IEC no Estado do Pará. RESULTADOS: Das 19 amostras séricas do município de Campo Grande, 1 apresentou titulo de 1:20 para Flavivirus. Das 35 amostras coletadas em Bonito, 17 apresentaram anticorpos para arbovírus, sendo 4 (11,4%) positivos para Alphavirus, e 5 (14,2%) positivos para Flavivirus. Reações monotipicas foram observados para o vírus Mayaro (n=10) e para o vírus Oropouche (n=5) e 6 (17,1%) amostras apresentaram títulos para mais de um dos vírus estudados. Foram capturados 120 Culicídeos potenciais transmissores de arbovírus no município de Jardim. Todas as amostras coletadas foram negativas para o isolamento viral. CONCLUSÕES: Por possuir uma variedade de hospedeiros vertebrados e vetores transmissores, o Mato Grosso do Sul apresenta condições propícias para a emergência ou reemergência de arbovírus, inclusive alguns patogênicos para os seres homem.

First detection of Rio Negro virus (Venezuelan equine encephalitis complex subtype VI) in Córdoba, Argentina

Rio Negro virus (RNV) (Venezuelan equine encephalitis subtype VI) circulates only in Argentina; in northern provinces, isolates have been obtained from mosquitoes and rodents since 1980 and have been associated with acute febrile illness in humans. However, no studies of RNV have been performed in the central area of the country. We carried out molecular and serological detection of RNV in Córdoba, a province of the central part of the country, in mosquitoes and humans, respectively. One mosquito pool tested positive for alphavirus RNA by reverse transcriptase-nested polymerase chain reaction (RT-nested PCR). Subsequent sequencing determined that this alphavirus grouped with RNV. Serological studies detected antibodies to RNV in one human serum sample, which was obtained during the same period that RNV was detected using the aforementioned molecular methods. This is the first report of RNV circulation in the central area of Argentina, indicating an expansion of its original distribution. These results highlight the importance of strengthening surveillance procedures in endemic areas, as well as in new regions where RNV may emerge.

Estudo prospectivo para avaliação das alterações clínicas e laboratoriais em casos de dengue / Prospective study to evaluate the clinical and laboratory changes in dengue cases

Realizamos dois estudos de coorte prospectivos em pacientes com dengue no município de Campos de Goytacazes-RJ, durante a epidemia de 2008. No primeiro estudo foi avaliada a evolução das manifestações clínicas e laboratoriais em 90 pacientes com dengue, em um período de dois meses. Vinte controles foram arrolados para a análise da função hepática. Em ambos os grupos foram realizadas coletas de dados e sangue nos primeiros cinco dias da doença, e posteriormente aos 8, 15, 30 e 60 dias após o início da doença. Foram excluídos pacientes com hepatite B, hepatite C, gestantes e aqueles sabidamente soropositivos para HIV. No final do primeiro e segundo mês após o início da dengue, 44.5 por cento e 32.2 por cento dos pacientes apresentavam por cento pelo menos um sinal ou sintoma devido à doença. Dos sinais e sintomas analisados, 57,7 por cento (15/26) estavam presentes no final do segundo mês. Os maiores percentuais de persistência foram: artralgia, adinamia, fraqueza, fadiga, anorexia, alteração do paladar e queda de cabelo. A anorexia e a alteração do paladar em pacientes com dengue grave apresentaram maior persistência quando comparada à dos pacientes com dengue com e sem sinais de alerta. A infecção prévia pelo vírus da dengue não predispôs a uma maior duração dos sinais e sintomas analisados. As transaminases hepáticas permaneceram elevadas em 40,6 por cento e 7,6 ppor cento dos pacientes no final do primeiro e segundo mês respectivamente. Não houve diferença significativa entre os níveis de aspartato aminotransferase (AST) e alanina aminotransferase (ALT) nos diferentes momentos da análise, exceto no dia 15 quando a ALT foi significativamente mais elevado. Na fase de convalescença, o percentual de pacientes com transaminases elevadas não diferiu entre homens e mulheres, no entanto, homens apresentaram níveis de ALT significativamente mais elevados nesse período. Não foram observadas alterações relevantes na análise das demais funções hepáticas. No segundo estudo avaliamos em 67 pacientes com dengue a associação e correlação das citocinas IP10, MIP1beta e VEGF com as alterações clínicas, hemograma e as transaminases hepáticas na fase aguda e nos dias 8, 15, 30 e 60 após o início da doença. Após o início da doença, o IP10 apresentou níveis significativamente aumentados por pelo menos 30 dias, o MIP1beta somente nos primeiros cinco dias e o VEGF nos dias 8 e 15. Não houve associação entre os níveis de citocinas e a gravidade dos casos. Dos sinais e sintomas estudados observamos uma associação entre níveis significativamente elevados do IP10 com a presença de artralgia. Níveis baixos do IP10 na fase aguda foram associados à persistência de artralgia no final do primeiro e segundo mês. Observamos uma correlação positiva entre os níveis de VEGF e o hematócrito. Portanto, os resultados encontrados neste estudo demonstram que a dengue pode apresentar persistência de alterações clínicas e elevação de transaminases hepáticas por pelo menos dois meses após o seu início. Estas alterações foram relacionadas com a elevação do IP10.

Culicídeos associados a bovinos de leite, no sul do Rio Grande do Sul, Brasil / Mosquitoes associated with dairy cattle in southern Rio Grande do Sul, Brazil

The occurrence of mosquitoes in the authoring environment of dairy cattle was verified in southern Rio Grande do Sul. For collection of specimens, it was installed a Magoon trap, in which during a couple of cattle-approximately six months of age was placed. Samples were collected during 24 hours, twice a month for 12 months. After 24 hours, the cattle were removed from the trap and the insects were collected inside the trap, with the aid of glass bottles, containing, cotton soaked in acetone. The specimens were taken to the Laboratory of Insect Biology, at the Universidade Federal de Pelotas, to sorting, counting, sexing and identification. Of the mosquitoes caught, specimens of Culex sp. Ochlerolatus scapularis and Mansonia titillans were present. Culex sp. had the largest number of individuals (39).

Isolation of yellow fever virus (YFV) from naturally infectied Haemagogus (Conopostegus) leucocelaenus (diptera, cukicudae) in São Paulo State, Brazil, 2009 / Isolamento do vírus da Febre Amarela de mosquitos naturalmente infectados Haemagogus (Conopostegus) leucocelaenus (Diptera, Culicidae), São Paulo, Brasil, 2009

After detecting the death of Howlers monkeys (genus Alouatta) and isolation of yellow fever virus (YFV) in Buri county, São Paulo, Brazil, an entomological research study in the field was started. A YFV strain was isolated from newborn Swiss mice and cultured cells of Aedes albopictus - C6/36, from a pool of six Haemagogus (Conopostegus) leucocelaenus (Hg. leucocelaenus) mosquitoes (Dyar & Shannon) collected at the study site. Virus RNA fragment was amplified by RT-PCR and sequenced. The MCC Tree generated showed that the isolated strain is related to the South American I genotype, in a monophyletic clade containing isolates from recent 2008-2010 epidemics and epizootics in Brazil. Statistical analysis commonly used were calculated to characterize the sample in relation to diversity and dominance and indicated a pattern of dominance of one or a few species. Hg. leucocelaenus was found infected in Rio Grande do Sul State as well. In São Paulo State, this is the first detection of YFV in Hg. leucocelaenus.

Após a detecção de morte de macacos Bugios (gênero Alouatta) e isolamento do vírus da Febre Amarela (YFV) no município de Buri, Estado de São Paulo, Brasil, foi iniciada uma investigação entomológica em campo. Uma cepa de YFV foi isolada em camundongos recém-nascidos e cultura de células de Aedes albopictus - C6/36, a partir de um lote de seis mosquitos Haemagogus (Conopostegus) leucocelaenus (Hg leucocelaenus) Dyar & Shannon coletados no local de estudo. RNA do vírus foi amplificado por RT-PCR e seqüenciado. A topologia gerada indica que a cepa isolada está relacionada ao genótipo South American I, em clado monofilético englobando isolados recentes de epidemias e epizootias entre 2008 e 2009. Análises estatísticas geralmente usadas caracterizaram a amostra em relação à diversidade e dominância, indicando dominância relativa de uma ou poucas espécies. Hg. leucocelaenus foi detectado infectado também no Rio Grande do Sul. No Estado de São Paulo trata-se da primeira detecção do YFV em Hg leucocelaenus.

Preliminary investigation of Culicidae species in South Pantanal, Brazil and their potential importance in arbovirus transmission / Investigação preliminar das espécies de culicídeos do Pantanal Sul brasileiro e sua potencial importância na transmissão de arbovírus

In view of the high circulation of migratory birds and the environmental and climatic conditions which favor the proliferation of arthropods, the Brazilian Pantanal is susceptible to circulation of arboviruses. However, the amount of data concerning arbovirus vectors in this area is scarce; therefore the aim of this study was to conduct a preliminary investigation of Culicidae species in the Nhecolândia Sub-region of South Pantanal, Brazil and their potential importance in the arbovirus transmission. A total of 3684 specimens of mosquitoes were captured, 1689 of which caught in the rainy season of 2007, were divided into 78 pools and submitted to viral isolation, Semi-Nested RT-PCR and Nested RT-PCR, with a view to identifying the most important arboviruses in Brazil. Simultaneously, 70 specimens of ticks found blood-feeding on horses were also submitted to the same virological assays. No virus was isolated and viral nucleic-acid detection by RT-PCR was also negative. Nevertheless, a total of 22 Culicidae species were identified, ten of which had previously been reported as vectors of important arboviruses. The diversity of species found blood-feeding on human and horse hosts together with the arboviruses circulation previously reported suggest that the Nhecolândia Sub-region of South Pantanal is an important area for arbovirus surveillance in Brazil.

Regiões como o Pantanal brasileiro, que apresentam fatores como riqueza de fauna silvestre incluindo circulação de aves migratórias e condições ambientais e climáticas favoráveis à proliferação de artrópodes estão potencialmente sujeitas à circulação de arbovírus. Entretanto, poucos trabalhos foram realizados acerca da presença de arbovírus em potenciais vetores no Pantanal. Neste sentido o principal objetivo deste trabalho foi conduzir uma investigação preliminar para presença de arbovírus em amostragens de culicídeos capturados na Sub-região da Nhecolândia no Pantanal Sul. Um total de 3684 mosquitos foi capturado, dos quais 78 grupos compondo uma amostragem de 1789 espécimes foram submetidos às técnicas de isolamento viral e RT-PCR para os mais importantes arbovírus no Brasil. Simultaneamente, 70 espécimes de carrapatos capturados durante hematofagia em cavalos também foram submetidos à pesquisa viral. Não houve isolamento viral em nenhuma amostra analisada e os resultados de detecção de ácido nucléico viral foram também negativos. Entretanto, foram identificadas 22 espécies de culicídeos, dez das quais previamente reportadas como vetores de importantes arbovírus. A competência vetorial de espécies capturadas durante hematofagia em humanos e cavalos aliada ao relato prévio de circulação de arbovírus sugerem a Sub-região da Nhecolândia como uma importante área de vigilância para arbovírus no Centro-Oeste do Brasil.

Ecologia de Haemagogus e Sabethes (Diptera: Culicidae) em áreas epizoóticas do vírus da febre amarela, Rio Grande do Sul, Brasil / Ecology of Haemagogus and Sabethes (Diptera: Culicidae) Mosquitoes in epizootic yellow regions of Rio Grande do Sul State, Brazil

Objetivou-se com este estudo investigar característica ecológica e o hábito alimentar de Haemagogus e Sabethes em área epizoótica da febre amarela silvestre em duas localidades do Rio Grande do Sul, capturados com aspirador, armadilha de Shannon e puçá. Para identificação do sangue ingerido pelas fêmeas foi utilizada a técnica imunoenzimática ELISA de captura. Obteve-se maior atividade de Hg. leucocelaenus na primavera e outono, entre 12 e 17 horas, enquanto Sabethes albiprivus e Sa. quasycianus durante todo o ano. Para fêmeas de Hg. leucocelaenus em Santo Antônio das Missões, houve predomínio de sangue humano e para as demais espécies destaca-se a atratividade para bovinos nos dois municípios. A capacidade vetorial do Hg. leucocelaenus fundamenta vigilância entomológica em seu papel de estratificar áreas problemas ou a sustentabilidade do programa de imunização da população.

The study was conducted in epizootic areas of sylvatic yellow fever in two municipalities in Rio Grande do Sul State, with the objective to investigate ecological characteristics of Haemagogus and Sabethes mosquitoes. Mosquitoes were collected using Shannon traps and insect nets. The host-feeding pattern was tested by an enzyme-linked immunosorbent assay. The highest activity levels were observed for Hg. leucocelaenus during spring and autumn, between 12 and17 hours, while Sabethes albiprivus and Sa. quasycianus were common throughout the year. The blood of females of Hg. leucocelaenus at one municipality, Santo Antonio das Missoes, was of human origin. Considering the other species, inboth municipalities of the study, bovine blood was predominant. The vectorial capacity of Hg. leucocelaenus justifies the entomological surveillance in order to stratify problematic areas or the sustainability of an immunization program.

Molecular characterization of full-length genome of Japanese encephalitis virus (KV1899) isolated from pigs in Korea

We have determined the complete nucleotide and deduced amino acid sequences of the Japanese encephalitis virus (JEV) strain KV1899, isolated from a fattening pig in Korea. In comparison with 22 fully sequenced JEV genomes currently available, we found that the 10,963-nucleotide RNA genome of KV1899 has a 13-nucelotide deletion in the 3' non-translated variable region and 53 unique nucleotide sequences including 3' non-translated region (NTR). Its single open reading frame has a total of 28 amino acid substitutions. Comparison of the KV1899 genomic sequence with those of the 21 fully sequenced JEV strains in published databases showed nucleotide homology ranging from 97.4% (Ishikawa strain) to 87.0% (CH2195 strain). Amino acid homology with KV1899 strain ranged from 96.4% (K94P05) to 91.0% (GP78). The KV1899 showed the highest nucleotide homology with Ishikawa strain and the highest amino acid homology with K94P05. We performed an extensive E gene based phylogenetic analysis on a selection of 41 JEV isolates available from the GenBank. Compared with Anyang strain, isolated from a pig in 1969, that is current live vaccine strain for swine in Korea, the homology of nucleotide sequence in envelope gene was only 87.1%. The prM gene of the isolate was closely related with those of Ishikawa and K94P05 strains, which were grouped into genotype I of JEV.