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Flaviviruses in mosquitoes from Southern Portugal, 2009-2010
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Os flavivírus são vírus pertencentes à família Flaviviridae, género Flavivirus. Estes formam um grande grupo caraterizado pela sua ampla distribuição e diversidade genética. Os flavivírus são, na sua maioria, transmitidos por artrópodes vectores incluíndo agentes patogénicos para humanos e animais que podem potencialmente provocar grandes epidemias e causar elevadas taxas de mortalidade e morbidade. Nos últimos anos, tem-se registado uma grande expansão a nível da distribuição geográfica dos flavivirus e diversidade dos seus hospedeiros. O vírus do Nilo Ocidental tem sido continuamente detectado em toda a Europa recentemente, e também isolado de mosquitos colhidos no Sul de Portugal, onde já foram registados casos humanos e animais.
O principal objectivo deste trabalho é o rastreio de flavivírus em mosquitos colhidos em duas regiões do Sul de Portugal, onde os mesmos foram anteriormente detectados.
As colheitas de mosquitos foram realizadas em 24 locais em zonas húmidas nos districtos de Faro e Setúbal, através de armadilhas luminosas tipo CDC com CO2 e aspiradores mecânicos manuais para colheita de mosquitos em repouso em abrigos de animais.
Os mosquitos colhidos foram agrupados por lotes contendo aproximadamente 50 espécimens cada, e rastreados para a presença de flavivírus por heminested RT-PCR, direccionado à amplificação de um pequeno fragmento do gene NS5 usando oligonucleótidos degenerados específicos para flavivírus.
Entre Abril e Outubro de 2009 e 2010 foram colhidos no total 36273 mosquitos pertencentes às seguintes espécies: Anopheles algeriensis, An.atroparvus, Aedes berlandi, Ae.caspius, Ae.detritus, Coquillettidia richiardii, Culex laticinctus, Cx.pipiens, Cx.theileri, Cx.univittatus, Culiseta annulata, Cs.longiareolata, Cs.subochrea, e Uranotaenia unguiculata. As espécies mais abundantes foram Ae.caspius, Cx.theileri e Cx.pipiens, respectivamente. Contudo, as densidades de mosquitos foram variáveis de acordo com o método de colheita e área de amostragem. As densidades de mosquitos colhidos em 2010 foram quatro vezes superior às registadas no ano anterior. No total foram analisados 745 lotes dos quais 31% testaram positivos para a presença de sequências de flavivirus.
As espécies que apresentaram taxas de positividade mais elevadas foram: An.algeriensis com uma Taxa Mínima de Infecção (TMI) de 56/1000 no Algarve em 2009, Cs.annulata TMI =22/1000 no Algarve em 2010, Cx.theileri e Cx.pipiens em Setúbal em 2010, TMI =20/1000. An. atroparvus, Ae. caspius, Ae. detritus e Cx. univittatus também produziram lotes positives. No geral, a positividade foi maior no Algarve.
Análise das sequências virais obtidas revelou homologia das nossas sequências virais com sequências de referência de flavivírus específicos de mosquitos depositadas em bases de dados de acesso livre. A análise filogenética reflectiu a variabilidade genética dos flavivírus e revelou a relação genética das nossas sequências com as de outros flavivírus, especialmente os específicos de insectos.
Tendo em consideração os anteriores isolamentos do vírus do Nilo Ocidental, o aumento acentuado nas densidades de mosquitos, o aumento de temperaturas que se tem vindo a registar, os casos recentes de transmissão de flavivírus por toda a Europa e o padrão desconhecido e imprevisível dos surtos destes vírus, os programas contínuos de vigilância epidemiológica têm-se revelado uma ferramenta indispensável para a Saúde Pública.
Flaviviruses are viruses belonging to the Flaviviridae family, genus Flavivirus. They comprise a large group of widely spread and genetically diverse arthropod-borne viruses including human and animal pathogens that can potentially cause large-scale epidemics and high mortality and morbidity. In the past few years, flaviviruses have largely expanded their geographical distribution and host range. West Nile virus has been continuously detected throughout Europe lately and has been isolated from mosquitoes in Southern Portugal, where human and animal cases have been reported. The main aim of this work was to search for flaviviruses in mosquitoes collected from two areas in Southern Portugal where West Nile virus and other flaviviruses have previously been detected. Mosquito surveys were carried out in 24 locations in the wetlands of the Faro and Setúbal districts, by CDC-CO2 light-traps and indoors resting collections. Pools containing approximately 50 mosquitoes were screened for flaviviruses by heminested RT-PCR, directed at the amplification of a small fragment of the viral NS5 gene, using degenerated flavivirus-specific primers. A total of 36273 mosquitoes were collected during 2009 and 2010 from April through October, from the following species: Anopheles algeriensis, An.atroparvus, Aedes berlandi, Ae. caspius, Ae. detritus, Coquillettidia richiardii, Culex laticinctus, Cx. pipiens, Cx. theileri, Cx. univittatus, Culiseta annulata, Cs. longiareolata, Cs. subochrea, and Uranotaenia unguiculata. Most abundant species were Ae. caspius Cx. theileri and Cx. pipiens, respectively. However, mosquito densities varied according to collection method and sampling area. A fourfold increase in mosquito density was registered in 2010 compared to 2009. A total of 745 pools were analysed of which 31% tested positive for flaviviral sequences. The species with higher positivity rates were An. algeriensis with Minimum infection rate (MIR) of 56/1000 in the Algarve 2009, Cs. annulata MIR =22/1000 in the Algarve 2010, Cx.theileri and Cx.pipiens in Setúbal 2010, MIR =20/1000. An. atroparvus, Ae. caspius, Ae. detritus and Cx. univittatus also yielded positive pools. Overall, positivity was higher in the Algarve. Viral sequences obtained from positive pools showed homology with insect-specific flavivirus (ISF) sequences deposited in free access public databases. Phylogenetic analysis reflected the genetic variability of flaviviruses and revealed the relatedness of our sequences with other known flaviviruses, especially the insect-specific. In view of previous WNV isolations and assessing from the four-fold increase in mosquito density, the increasing temperatures, the recent cases throughout Europe and the unknown and unpredictable pattern of flaviviruses outbreaks, continuous epidemiological surveillance programmes are quickly becoming indispensable tools for Public Health.
Flaviviruses are viruses belonging to the Flaviviridae family, genus Flavivirus. They comprise a large group of widely spread and genetically diverse arthropod-borne viruses including human and animal pathogens that can potentially cause large-scale epidemics and high mortality and morbidity. In the past few years, flaviviruses have largely expanded their geographical distribution and host range. West Nile virus has been continuously detected throughout Europe lately and has been isolated from mosquitoes in Southern Portugal, where human and animal cases have been reported. The main aim of this work was to search for flaviviruses in mosquitoes collected from two areas in Southern Portugal where West Nile virus and other flaviviruses have previously been detected. Mosquito surveys were carried out in 24 locations in the wetlands of the Faro and Setúbal districts, by CDC-CO2 light-traps and indoors resting collections. Pools containing approximately 50 mosquitoes were screened for flaviviruses by heminested RT-PCR, directed at the amplification of a small fragment of the viral NS5 gene, using degenerated flavivirus-specific primers. A total of 36273 mosquitoes were collected during 2009 and 2010 from April through October, from the following species: Anopheles algeriensis, An.atroparvus, Aedes berlandi, Ae. caspius, Ae. detritus, Coquillettidia richiardii, Culex laticinctus, Cx. pipiens, Cx. theileri, Cx. univittatus, Culiseta annulata, Cs. longiareolata, Cs. subochrea, and Uranotaenia unguiculata. Most abundant species were Ae. caspius Cx. theileri and Cx. pipiens, respectively. However, mosquito densities varied according to collection method and sampling area. A fourfold increase in mosquito density was registered in 2010 compared to 2009. A total of 745 pools were analysed of which 31% tested positive for flaviviral sequences. The species with higher positivity rates were An. algeriensis with Minimum infection rate (MIR) of 56/1000 in the Algarve 2009, Cs. annulata MIR =22/1000 in the Algarve 2010, Cx.theileri and Cx.pipiens in Setúbal 2010, MIR =20/1000. An. atroparvus, Ae. caspius, Ae. detritus and Cx. univittatus also yielded positive pools. Overall, positivity was higher in the Algarve. Viral sequences obtained from positive pools showed homology with insect-specific flavivirus (ISF) sequences deposited in free access public databases. Phylogenetic analysis reflected the genetic variability of flaviviruses and revealed the relatedness of our sequences with other known flaviviruses, especially the insect-specific. In view of previous WNV isolations and assessing from the four-fold increase in mosquito density, the increasing temperatures, the recent cases throughout Europe and the unknown and unpredictable pattern of flaviviruses outbreaks, continuous epidemiological surveillance programmes are quickly becoming indispensable tools for Public Health.
Descrição
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Parasitologia médica Entomologia médica Vírus do Nilo ocidental Mosquitos Arboviroses Flavivirus Portugal Algarve
Contexto Educativo
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Instituto de Higiene e Medicina Tropical