Study of Arboviruses in Philander opossum, Didelphis marsupialis and Nectomys rattus captured from forest fragments in the municipality of Belém, Pará, Brazil / Estudo de Arbovírus em Philander opossum, Didelphis marsupialis e Nectomys rattus capturados em fragmentos florestais no município de Belém, Pará

ABSTRACT: Arboviruses are viruses that maintain their life cycle in the wild and are transmitted to vertebrate hosts by hematophagous diptera. They are zoonotic and can establish an enzootic cycle in the urban areas; in humans, the infection can manifest from being encephalitogenic to hemorrhagic. This study aimed to report the occurrence of arboviruses in mammals of the order Didelphimorphia and Rodentia, captured from the Amazon. Serum samples were subjected to hemagglutination inhibition test using a viral panel of 19 species of arboviruses that are known to occur in the Amazon. Altogether, 14 wild mammals, 12 of Philander opossum, 1 of Didelphis marsupialis, and 1 of Nectomys rattus were captured. Eight of these were reported to be seropositive for arboviruses (57.14%) with monotypic seroprevalence for the Eastern Equine Encephalitis Virus (n=1), the Ilheus Virus (n=2), and the Catu virus (n=4); 4 heterotypic responses were observed for Flavivirus and Orthobunyavirus. In conclusion, several arbovirus species are in active circulation and maintenance, exhibiting enzootic characteristics in the wild mammals of the Amazon region; these animals prove to be potential hosts in the transmission of diseases to humans.

RESUMO: Os arbovírus são vírus que mantêm o seu ciclo de vida em ambiente silvestre. Eles são transmitidos aos hospedeiros vertebrados por dípteros hematófagos, tem caráter zoonótico podendo estabelecer um ciclo enzoótico no meio urbano, sendo que em humanos a infecção pode ter caráter encefalomiogênico a hemorrágico. Este estudo teve como objetivo relatar a ocorrência de arbovírus em mamíferos da ordem Didelphimorphia e Rodentia capturados na Amazônia. Os soros dos animais foram submetidos a testes de Inibição da Hemaglutinação utilizando um painel viral com as 19 principais espécies de arbovírus que ocorrem na Amazônia. Foram capturados 14 espécimes de mamíferos silvestres, 12 Philander opossum, 1 Didelphis marsupialis e 1 Nectomys rattus. A soropositividade para arbovírus foi observada em 57,14% (8/14) dos espécimes estudados, com soroprevalência monotípica para o vírus da Encefalite Equina do Leste (n = 1), o vírus Ilheus (n = 2) e o vírus Catu (n = 4) e quatro reações heterotípicos para Flavivírus e Orthobyavírus. Conclui-se que há manutenção e circulação de espécies de arbovírus com características enzoóticas em mamíferos silvestres da região amazônica, podendo ser hospedeiros em potenciais na transmissão da doença para humanos.

First isolation of West Nile virus in Brazil

BACKGROUND Serological evidence of West Nile virus (WNV) infection has been reported in different regions of Brazil from equine and human hosts but the virus had never been isolated in the country. OBJECTIVES We sought to identify the viral etiology of equine encephalitis in Espírito Santo state. METHODS We performed viral culture in C6/36 cells, molecular detection of WNV genome, histopathology and immunohistochemistry from horse cerebral tissue. We also carried out sequencing, phylogenetic analysis and molecular clock. FINDINGS Histopathologic analysis from horse cerebral tissue showed injury related to encephalitis and WNV infection was confirmed by immunohistochemistry. The virus was detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) from brain tissue and subsequently isolated in C6/36 cells. WNV full-length genome was sequenced showing the isolated strain belongs to lineage 1a. The molecular clock indicated that Brazilian WNV strain share the same common ancestor that were circulating in US during 2002-2005. MAIN CONCLUSIONS Here we report the first isolation of WNV in Brazil from a horse with neurologic disease, which was clustered into lineage 1a with others US WNV strains isolated in beginning of 2000's decade.

Clinical and serological tests for arboviruses in free-living domestic pigeons (Columba livia)

BACKGROUND In this study, we evaluated the role of free-living domestic pigeons (Columba livia) as a reservoir of arboviruses in the city of Belém, state of Pará, Brazil. We investigated the presence of antibodies against the most prevalent arboviruses. OBJECTIVES This study was aimed at evaluating some clinical and physical parameters of domestic pigeons, including the presence of antibodies to Amazon-endemic arboviruses. METHODS Eighty-five healthy pigeons were captured in Mangal das Garças Park, in Belém, and were bled. Upon capture, the birds were subjected to a clinical examination in search of alterations that could indicate the presence of arboviruses. Blood samples were converted to serum and tested using the haemagglutination inhibition (HI) technique with a panel of 19 antigens of arboviruses circulating in the Amazon. The confirmation assay for the positive reactions to the viral species tested by HI was a neutralisation test in new-born Swiss albino mice (Mus musculus) [mouse neutralisation test (MNT)]. FINDINGS A total of 10 (11.8%) serum samples tested positive for antiflavivirus antibodies by HI. All the samples positive for the HI test were subjected to MNT for detection of viruses and yielded negative results (logarithmic neutralisation index < 1.7). MAIN CONCLUSION The results represent the first serological detection of antiarbovirus antibodies in domestic pigeons as potential hosts of arboviruses in Brazil. The detection of haemagglutination-inhibiting antibodies against genus Flavivirus indicated that there was recent contact between the analysed domestic pigeons and these arboviruses. Further studies are needed to evaluate the role of free-living pigeons in the maintenance cycle and spread of arboviruses in the Amazon.

Prevalence of antibodies to equine alphaviruses in the State of Pará, Brazil / Prevalência de anticorpos para alphavirus em equinos do estado do Pará, Brasil

The State of Pará comprises 26% of Brazilian Amazon region, where a large diversity of arboviruses has been described. This study sought to assess the prevalence and distribution of hemagglutination inhibition (HI) antibodies against antigens of four alphaviruses (Togaviridae: Alphavirus ) from the species: Eastern equine encephalitis (EEEV), Western equine encephalitis (WEEV), Mayaro virus (MAYV), and Mucambo virus (MUCV) in 753 serum samples of horses in Pará State, Brazil. All investigated arboviruses were detected and indicate that horses are susceptible to these alphaviruses, and show evidences of their active circulation in farm animals in the Brazilian Amazon.(AU)

O estado do Pará corresponde a 26% da Amazônia brasileira, onde uma grande diversidade de arbovírus foi descrita. Este estudo procurou avaliar a prevalência e a distribuição de anticorpos inibidores da hemaglutinação (IH) contra antígenos de quatro alfavirus (Togaviridae: Alphavirus ), das espécies: Vírus da encefalite equina do leste (EEEV), Vírus da encefalite equina do oeste (WEEV), Vírus mayaro (MAYV) e Vírus mucambo (MUCV), de 753 amostras de soro de equinos no estado do Pará, Brasil. Todos os arbovirus pesquisados foram detectados, indicando que os equinos são suscetíveis a esses Alphavirus e mostrando evidências de sua circulação ativa em animais de fazenda na Amazônia brasileira.(AU)

Detection of arboviruses of public health interest in free-living New World primates (Sapajus spp.; Alouatta caraya) captured in Mato Grosso do Sul, Brazil

Introduction A sero-epidemiological survey was undertaken to detect the circulation of arboviruses in free-living non-human primates. Methods Blood samples were obtained from 16 non-human primates (13 Sapajus spp. and three Alouatta caraya) that were captured using terrestrial traps and anesthetic darts in woodland regions in the municipalities of Campo Grande, Aquidauana, Jardim, Miranda and Corumbá in the State of Mato Grosso do Sul, Brazil. The samples were sent to the Instituto Evandro Chagas (IEC) in Ananindeua, Pará, Brazil, to detect antibodies against 19 species of arboviruses using a hemagglutination inhibition test (HI). Results Of the 16 primates investigated in the present study, five (31.2%) were serologically positive for an arbovirus. Of these five, two (12.5%) exhibited antibodies to the Flavivirus genus, one (6.2%) exhibited a monotypic reaction to Cacipacoré virus, one (6.2%) was associated with Mayaro virus, and one (6.2%) was positive for Oropouche virus. Conclusions Based on the positive serology observed in the present study, it was possible to conclude that arboviruses circulate among free-living primates. The viruses in the areas studied might have been introduced by infected humans or by primates from endemic or enzootic areas. Studies of this nature, as well as efficient and continuous surveillance programs, are needed to monitor viral activities in endemic and enzootic regions. .

Persistence of experimental Rocio virus infection in the golden hamster (Mesocricetus auratus)

Rocio virus (ROCV) is an encephalitic flavivirus endemic to Brazil. Experimental flavivirus infections have previously demonstrated a persistent infection and, in this study, we investigated the persistence of ROCV infection in golden hamsters (Mesocricetus auratus). The hamsters were infected intraperitoneally with 9.8 LD50/0.02 mL of ROCV and later anaesthetised and sacrificed at various time points over a 120-day period to collect of blood, urine and organ samples. The viral titres were quantified by real-time-polymerase chain reaction (qRT-PCR). The specimens were used to infect Vero cells and ROCV antigens in the cells were detected by immunefluorescence assay. The levels of antibodies were determined by the haemagglutination inhibition technique. A histopathological examination was performed on the tissues by staining with haematoxylin-eosin and detecting viral antigens by immunohistochemistry (IHC). ROCV induced a strong immune response and was pathogenic in hamsters through neuroinvasion. ROCV was recovered from Vero cells exposed to samples from the viscera, brain, blood, serum and urine and was detected by qRT-PCR in the brain, liver and blood for three months after infection. ROCV induced histopathological changes and the expression of viral antigens, which were detected by IHC in the liver, kidney, lung and brain up to four months after infection. These findings show that ROCV is pathogenic to golden hamsters and has the capacity to cause persistent infection in animals after intraperitoneal infection.

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.

Epidemiology of Saint Louis encephalitis virus in the Brazilian Amazon region and in the State of Mato Grosso do Sul, Brazil: elevated prevalence of antibodies in horses / Epidemiologia do vírus da encefalite Saint Louis na Amazônia brasileira e no Estado do Mato Grosso do Sul, Brasil: elevada prevalência de anticorpos em equinos

The immunity of horses (n = 1401) against Saint Louis encephalitis virus (SLEV) was investigated in the Brazilian Amazon region (Bragança/Pará, Salvaterra/Pará, Macapá/Amapá and Rio Branco/Acre) and Maracaju, State of Mato Grosso do Sul, by the hemagglutination inhibition (HI) and plaque reduction neutralization (PRNT) tests. HI and neutralizing antibodies specific (monotypic reactivity, MR) for SLEV and other flaviviruses included in the tests were detected, as was cross-reactivity (CR) against flaviviruses. In the HI test, MR was observed in 248 (17.7 percent) serum samples, 137 of which were (55.2 percent) againstSLEV; CR was detected in 380 (27.1 percent). The frequency of MR against SLEV was significantly higher in Macapá and CR was significantly higher in Salvaterra. In the PRNT, neutralization of SLEV was observed in 713 (50.9 percent) samples, and the prevalence of neutralizing antibodies was significantly higher in Macapá than in Salvaterra (p = 0.0083). This study adds new data regarding the immunity of horses against SLEV in Brazil, and it confirms the wide distribution of SLEV and the diversity of flaviviruses in the country, as well as the apparent absence of disease in SLEV-infected horses.

A imunidade de equinos (n = 1401) contra o vírus da encefalite Saint Louis (SLEV) foi investigada na Amazônia brasileira (Bragança/PA, Salvaterra/PA, Macapá/AP e Rio Branco/AC) e Maracaju, no Estado do Mato Grosso do Sul, por meio detestes de inibição da hemaglutinação (IH) e neutralização por redução de placas (PRNT). Foram detectados anticorpos IH e neutralizantes específicos (reações monotípicas – RM) para SLEV e outros flavivírus incluídos nos testes, assim como reações cruzadas para flavivírus. Pelo teste de IH, RM foram observadas em 248 (17,7 por cento) amostras de soro, 137 (55,2 por cento) para SLEV, e RC para flavivírus foram detectadas em 380 (27,1 por cento). A frequência de RM para SLEV e de RC foi significativamente maior em Macapá e Salvaterra, respectivamente. Pelo PRNT, foi observada a neutralização do SLEV em 713 (50,9 por cento) amostras, e a prevalência de anticorpos neutralizantes foi significativamente maior em Macapá, em comparação com Salvaterra (p = 0,0083). Este estudo traz novos dados a respeito da imunidade de equinos contra SLEV no Brasil, e confirma a ampla distribuição de SLEV e a diversidade de flavivírus no País, bem como a aparente ausência de doenças em equinos infectados por SLEV.

Vigilância sorológica para arbovírus em Juruti, Pará, Brasil / Serological survey for arboviruses in Juruti, Pará State, Brazil

O objetivo foi avaliar a prevalência de anticorpos para arbovírus na área de influência do Projeto Juruti antes da exploração mineral local. Foram examinados 1.597 soros humanos e 85 de animais silvestres. A pesquisa de anticorpos foi realizada pelo teste de inibição da hemaglutinação contra antígenos dos arbovírus mais prevalentes na Amazônia brasileira e IgM-ELISA para dengue e febre amarela. Soros humanos inibidores da hemaglutinação positivos para Mayaro e Oropouche foram também testados por IgM-ELISA. Anticorpos inibidores da hemaglutinação para alfavírus, orthobunyavirus e/ou flavivírus foram detectados, sendo 28,7 por cento reações monotípicas. Infecções recentes para Oropouche (n = 23), Mayaro (n = 5), e dengue (n = 20) foram confirmadas por IgM-ELISA. A prevalência de anticorpos inibidores da hemaglutinação em soros de animais silvestres foi de 8,2 por cento para flavivírus. Os resultados obtidos mostram que houve circulação ativa dos vírus Mayaro, Oropouche e dengue no período do estudo em humanos em Juruti, Pará, Brasil, e de diversos arbovírus em animais silvestres, indicando presença desses vírus em áreas do Município de Juruti.

This study aimed to measure the prevalence of antibodies to arboviruses in the area affected by the Juruti Project before local mining operations. A total of 1,597 human and 85 wild animal sera were examined, using the hemagglutination inhibition test against 19 antigens from the most prevalent arboviruses in the Brazilian Amazon and IgM-ELISA for dengue and yellow fever. Positive sera for Mayaro and Oropouche viruses were also tested by IgM-ELISA. Prevalence of hemagglutination inhibition antibodies to alphaviruses, flaviviruses, and orthobunyaviruses in humans was 28.7 percent, while for wild animals it was 8.2 percent for flaviviruses. Recent infections based on presence of IgM was confirmed for Mayaro (n = 5), Oropouche (n = 23), and dengue (n = 20). The results showed active circulation of the dengue, Mayaro, and Oropouche viruses in Juruti, as well as flaviviruses in wild animals, suggesting the circulation of these viruses in the Municipality of Juruti.

A febre do Oropouche: uma revisão dos aspectos epidemiológicos e moleculares na Amazônia brasileira / Oropouche fever: an overview of the epidemiological and molecular aspects in the brazilian Amazon region

O vírus Oropouche (VORO Bunyaviridae, Orthobunyavirus) é um dos mais importantes arbovírus que infectam humanos na Amazônia Brasileira, sendo o agente causador da febre do Oropouche. Entre os anos de 1961 e 2006, um grande número de epidemias foi registrado em diferentes centros urbanos do estado do Pará (Belém, Santa Isabel, Castanhal, Santarém, Oriximiná, Serra Pelada, zona Bragantina - Igarapé Açu, Maracanã e Magalhães Barata), do Amazonas (Manaus e Barcelos), Acre (Xapuri), Amapá (Mazagão), Maranhão (Porto Franco), Tocantins (Tocantinópolis) e Rondônia (Ariquemes e Oro Preto D'Oeste). Estudos moleculares têm demonstrado a presença de pelo menos três linhagens distintas do VORO na Amazônia Brasileira (genótipos I, II e III), sendo os genótipos I e II os mais frequentemente encontrados em regiões da Amazônia ocidental e oriental, respectivamente. O genótipo III do VORO, previamente encontrado somente no Paraná, foi recentemente descrito na região Sudeste do Brasil. A associação de dados epidemiológicos e moleculares vêm contribuindo substancialmente para a caracterização das cepas do VORO isoladas durante epidemias, no período de pelo menos quatro décadas, bem como permitindo um melhor entendimento a respeito da epidemiologia molecular do VORO no que tange à emergência de novas linhagens genéticas e à dinâmica evolutiva desse arbovírus nas Américas e principalmente na Amazônia Brasileira. Este trabalho tem por objetivo apresentar uma revisão dos aspectos epidemiológicos e moleculares do VORO enfatizando sua distribuição, a dinâmica das epidemias ocorridas entre 1961 e 2006, bem como a dispersão de diferentes genótipos no Brasil.

Dengue virus type 3 in Boa Vista, Northern Brazil

Casos de doença febril aguda ocorridos em Boa Vista, estado de Roraima, Brasil foram investigados em julho de 2005. Amostras de sangue (n igual a 142) foram obtidas de pacientes clinicamente suspeitos de febre do dengue (FD). A tentativa de isolamento viral foi realizada em células C6/36 para pacientes com menos de cinco dias de doença (n igual a 96). As cepas isoladas foram identificadas pelo teste de imunofluorescência indireta (IFI) utilizando anticorpos monoclonais, bem como pela técnica de RT-PCR. Amostras de soro foram testadas pelo método de inibição da hemaglutinação (IH) e confirmadas pelo IgM-ELISA. O vírus dengue 3 (VDEN-3) foi isolado e detectado po RT-PCR em 41 pacientes. A região E de sete cepas foi sequenciada, sendo as mesmas identificadas como pertencentes ao genótipo III. Pelo teste de IH, 98 amostras de soros foram positivas para anticorpos IH para o vírus da Dengue, sendo os resultados confirmados pela detecção de anticorpos IgM para dengue. Clinicamente, todos os pacientes mostraram quadro de FD, sendo todas as faixas etárias afetadas independentes do sexo. A epidemia de dengue ocorrida em Roraima foi causada pelo VDEN-3, genótipo III, genótipo este que circula nas Américas desde 1994.

The Acre Project: the epidemiology of malaria and arthropod-borne virus infections in a rural Amazonian population

The authors describe the baseline malaria prevalence and arbovirus seroprevalence among 467 subjects in an ongoing cohort study in rural Amazonia. Most subjects (72.2 percent) reported one or more previous episodes of malaria, and 15.6 percent had been hospitalized for malaria, but only 3.6 percent of individuals five years or older had malaria parasites detected by microscopy (10 with Plasmodium vivax and 4 with P. falciparum). Antibodies to Alphavirus, Orthobunyavirus, and/or Flavivirus were detected by hemagglutination inhibition (HI) in 42.6 percent of subjects aged five years or older, with a higher seropositivity rate among males (49.2 percent) than females (36.2 percent). Since 98.9 percent of subjects had been immunized for yellow fever, the presence of cross-reactive antibodies to dengue and other Flaviviruses cannot be ruled out, but at least 12 subjects (3.3 percent) with IgM antibodies to dengue virus detected by ELISA had a putative recent exposure to this virus.