Author(s): Amaro, Fátima Isabel Falcão, 1974-
Date: 2010
Persistent ID: http://hdl.handle.net/10451/2297
Origin: Repositório da Universidade de Lisboa
Subject(s): Flebovírus; Flavivírus; Flebótomos; Reservatórios; Saúde pública; Teses de doutoramento - 2010
Tese de doutoramento, Biologia (Microbiologia), Universidade de Lisboa, Faculdade de Ciências, 2010
O vírus Toscana (género Phlebovirus, família Bunyaviridae) é um vírus neurotrópico que circula na Bacia do Mediterrâneo. Embora o reservatório na natureza seja desconhecido, está confirmada a sua transmissão por dípteros do género Phlebotomus. Recentemente foram também detectados vírus do género Flavivirus (família Flaviviridae) nestes vectores. O objectivo principal desta tese foi determinar a importância dos flebovírus em Saúde Pública em Portugal. Deste modo, no âmbito deste trabalho foram padronizadas e implementadas técnicas para o diagnóstico laboratorial de flebovírus, tendo sido desenvolvida uma técnica de imunofluorescência indirecta (IFI) e implementada uma técnica de Enzyme Linked Immunosorbent Assay (ELISA), como teste confirmatório, para o diagnóstico de vírus Toscana. O índice de concordância entre as duas técnicas foi considerado excelente, sendo a sensibilidade da IFI de 100%, e a especificidade superior a 80% na detecção dos anticorpos pesquisados (IgG e IgM). Inquéritos sero-epidemiológicos realizados na população humana para detectar a presença de anticorpos anti-vírus Toscana incluíram 924 indivíduos, distribuídos por três populações. Na população considerada de risco a seroprevalência foi 3,4% e na população controlo foi 2%. Em relação à população de indivíduos com sintomatologia e pedido de diagnóstico laboratorial, a seroprevalência mais elevada foi detectada no grupo de indivíduos com sintomas do sistema nervoso central (4,2%), na qual foram confirmados cinco casos de infecção recente (3%). No grupo de indivíduos com sintomatologia não neurológica a seroprevalência foi 1,3%. No total de 29 soros com anticorpos anti-vírus Toscana foram confirmados cinco por testes de neutralização por redução em placas com a estirpe ISS.Phl.3, demonstrando-se a presença de mais do que um genótipo de vírus Toscana em circulação em Portugal. No intuito de determinar os prováveis reservatórios de flebovírus na natureza foram realizados estudos serológicos em vertebrados selvagens e domésticos, nomeadamente em roedores e canídeos selvagens e domésticos. Um lobo e uma raposa apresentaram anticorpos IgG. Os resultados sugerem que, provavelmente, os canídeos selvagens têm um papel importante na manutenção do ciclo biológico dos flebovírus na natureza. Para identificar as espécies de flebótomos em circulação em Portugal procedeu-se à captura de flebótomos com armadilhas CDC em duas zonas do país (Algarve e Arrábida). Foram capturados 7578 flebótomos, designadamente 3330 machos e 4248 fêmeas. A espécie P. perniciosus apresentou uma distribuição geográfica mais ampla e o maior efectivo populacional. Além de P. perniciosus, foram encontradas P. ariasi, P. sergenti e Sergentomyia minuta. Os resultados indicam que P. sergenti se encontra, provavelmente, em expansão no nosso país, já que ocorreu em altitudes baixas Sergentomyia minuta, foi encontrada no Algarve a partir dos 11 metros de altitude, o que indica que a sua distribuição também está a alterar-se. A identificação dos vírus em circulação nos vectores foi baseada no método de nestedRT-PCR. A presença de flebovírus não foi detectada 269 pools de flebótomos pesquisados. No entanto, num pool em 90 estudados foi revelada, pela primeira vez em Portugal, a presença de flavivírus em flebótomos. A sequência obtida é idêntica às sequências de flavivírus detectadas em mosquitos do género Culex no nosso país e apresenta 98% de identidade com a sequência de um flavivírus encontrado em Ochlerotatus caspius em Itália. A circulação de flebovírus em Portugal foi comprovada nos seres humanos e em potenciais reservatórios. Foi demonstrada, pela primeira no nosso país, a circulação de flavivírus em flebótomos. Os resultados obtidos justificam a continuação da investigação na área dos vírus transmitidos por flebótomos pela sua importância em Saúde Pública. Flebovírus, vírus Toscana, flavivírus, flebótomos, reservatórios
Toscana virus (Phlebovirus genus, family Bunyaviridae) is a neurotropic virus which circulates in the Mediterranean Basin. Although the reservoir in nature is unknown, it is a fact that this virus is transmitted by sandflies of Phlebotomus genus. Viruses of the genus Flavivirus (family Flaviviridae) were recently detected in these vectors. The main objective of this thesis was to determine the importance of Phleboviruses in Public Health in Portugal. Within the aim of this work an in-house indirect immunofluorescence assay (IFI) was developed and an enzyme-linked immunosorbent assay (ELISA) with a commercial kit was also implemented for the laboratory diagnosis of Toscana virus. The agreement index between the two techniques was excellent, the sensitivity of IFI was 100% and the specificity exceeded 80% for both antibodies (IgG and IgM). Sero-epidemiological surveys were conducted in the human population, in 924 individuals, distributed in three populations, for the detection of antibodies anti Toscana virus. In the population considered at risk the prevalence was 3.4% and in the control population was 2%. In what concerns to symptomatic individuals with request for laboratorial diagnosis, the highest seroprevalence was detected in the population of individuals with neurological symptoms (4.2%), were there were five confirmed cases of recent infection (3%). In the population of individuals with no neurological symptoms, the seroprevalence was 1.3%. Five of the 29 positive sera were confirmed by plaque reduction neutralization tests with the strain ISS.Phl.3. This shows the presence of more than one genotype of Toscana virus circulating in Portugal. To determine the probable phleboviruses reservoirs in nature serological studies were performed on 538 samples of wild and domestic vertebrates, including rodents and wild canids. IgG antibodies were detected in two animals, namely a wolf and a fox. Toscana virus (Phlebovirus genus, family Bunyaviridae) is a neurotropic virus which circulates in the Mediterranean Basin. Although the reservoir in nature is unknown, it is a fact that this virus is transmitted by sandflies of Phlebotomus genus. Viruses of the genus Flavivirus (family Flaviviridae) were recently detected in these vectors. The main objective of this thesis was to determine the importance of Phleboviruses in Public Health in Portugal. Within the aim of this work an in-house indirect immunofluorescence assay (IFI) was developed and an enzyme-linked immunosorbent assay (ELISA) with a commercial kit was also implemented for the laboratory diagnosis of Toscana virus. The agreement index between the two techniques was excellent, the sensitivity of IFI was 100% and the specificity exceeded 80% for both antibodies (IgG and IgM). Sero-epidemiological surveys were conducted in the human population, in 924 individuals, distributed in three populations, for the detection of antibodies anti Toscana virus. In the population considered at risk the prevalence was 3.4% and in the control population was 2%. In what concerns to symptomatic individuals with request for laboratorial diagnosis, the highest seroprevalence was detected in the population of individuals with neurological symptoms (4.2%), were there were five confirmed cases of recent infection (3%). In the population of individuals with no neurological symptoms, the seroprevalence was 1.3%. Five of the 29 positive sera were confirmed by plaque reduction neutralization tests with the strain ISS.Phl.3. This shows the presence of more than one genotype of Toscana virus circulating in Portugal. To determine the probable phleboviruses reservoirs in nature serological studies were performed on 538 samples of wild and domestic vertebrates, including rodents and wild canids. IgG antibodies were detected in two animals, namely a wolf and a fox.The results suggest that probably wild canids have an important role in maintaining the biological cycle of phleboviruses in nature. For the identification of sandfly species in circulation in Portugal, captures of these vectors were performed with CDC light traps, in two areas of the country, namely Algarve and Arrabida. In the two prospection periods 7578 sandflies were captured, of which 3330 were male and 4248 were females. The species P. perniciosus showed a wider distribution and the greater effective population size. The obtained results during this work indicate that the geographic distribution of P. Sergenti is getting wider since it has occurred at lower altitudes. Sergentomyia minuta was also found at lower altitudes then previously expected, since it was present in the Algarve in an altitude as low as 11 meters. The identification of circulating virus in the vectors was based on nested RT-PCR method. The presence of phleboviruses was not detected in 269 screened pools. However, the presence of Flavivirus genus in sandflies was detected, for the first time in Portugal, in one of 90 pools surveyed. The obtained sequence is identical to the sequences of flaviviruses detected in mosquitoes of the genus Culex in our country and shows 98% identity with the sequence of a flaviviruses found in Ochlerotatus caspius in Italy. The circulation of phleboviruses in Portugal was proven in humans and in potential reservoirs. The presence of flaviviruses in sandflies was revealed for the first time in Portugal. These results justify the continuation of research on viruses transmitted by sandflies due to their importance in what concerns to Public health. Phleboviruses, Toscana virus, flaviviruses, sandflies, reservoirs
