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Sequênciação e análise do genoma de um presumível flavivírus isolado de Aedes (Ochlerotatus) caspius
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O género Flavivirus (Flaviviridae) inclui mais de setenta vírus com genoma a RNA de cadeia simples, muitos dos quais são importantes agentes patogénicos para o Homem e os outros animais. A maioria dos flavivírus pode ser transmitidos por carraças, mosquitos ou, aparentemente, restringir-se a vertebrados (Cook e Holmes, 2006). No entanto, um grupo de flavivírus designados “não clássicos”, não parece ter hospedeiro vertebrado conhecido. Estes últimos são comumente colocados junto à raiz de árvores filogenéticas do género Flavivirus, sendo frequentemente isolados em mosquitos, justificando a sua designação de vírus específicos de insectos (ISF, do inglês insect-specific flaviviruses) (Farfan-Ale et al., 2009). A classificação dos ISF como flavivírus tem sido suportada por semelhanças ao nível da sua organização genómica, perfil de hidropatia proteica, locais de clivagem conservados da sequência da poliproteína que codificam, e domínios enzimáticos. No entanto, são distintos em termos antigénicos, partilhando o mesmo nível de distância genética quando comparados com outros membros do género que quando comparados com outros dois outros géneros da família Flaviviridae (Cook e Holmes, 2006; Gould et al., 2003).
Esta tese apresenta uma caracterização inicial, que inclui a obtenção da sequência genómica quase completa, de um novo ISF. Este vírus, com a designação proposta de OCFVPt, foi isolado de mosquitos adultos classificados como Aedes (Ochlerotatus) caspius (Pallas, 1771), os quais são encontrados em densidades elevadas nas zonas costeiras estuarinas dos distritos de Faro e Setúbal (Almeida et al., 2008).
Este vírus replica rapidamente na linha celular C6/36 (derivada de Aedes albopictus), e, como esperado, não replica em células Vero. Contrariamente a outros ISF, o OCFVPt aparentemente causa efeito citopático óbvio em células C6/36, as quais, depois de infectadas, rapidamente se separam do suporte sólido da placa de crescimento, ficando pequenas e redondas. Análises por microscopia electrónica de secções finas de células C6/36 48h após infecção com OCFVPt revelaram uma hiperplasia nuclear acentuada com aumento do espaço entre as cisternas da membrana nuclear, no qual podem ainda ser encontradas vesículas de várias dimensões.
O genoma do OCFVPt tem, no mínimo, 9.839 nt e codifica para uma única poliproteína com as caraterísticas normalmente associadas aos membros do género Flavivirus. As árvores filogenéticas geradas após alinhamento de sequências virais mostram que o OCFVPt forma, juntamente com HANKV (Huhtamo et al., 2012) um grupo monofilético distinto dentro da radiação dos ISF.
The genus Flavivirus (Flaviviridae) includes over seventy viruses with ssRNA genomes, many of which are important pathogens of humans and other animals. Flaviviruses comprehend tick-borne, mosquito-borne and no known vector viral agents, as well as so-called “non-classical flaviviruses” (Cook and Holmes, 2006) with no known vertebrate host. They are commonly placed at the roots of phylogenetic trees of the genus, are frequently found in mosquitoes, and have been tentatively designated insect-specific flaviviruses (ISF) (Farfan-Ale et al., 2009). While classification of ISF as flaviviruses is supported by their genetic organization, protein hydropathy plots, conserved polyprotein cleavage sites and enzyme domains, they are antigenically distinct from other flaviviruses, and share approximately the same level of nucleotide sequence identity with other members of the genus as when compared to the members of the two other established genera in the Flaviviridae family (Cook and Holmes, 2006; Gould et al., 2003). This work reports the initial characterization, including near full-length sequence and genome analysis, of an new ISF, tentatively designated OCFVPt, that was isolated from Aedes (Ochlerotatus) caspius (Pallas, 1771) adult mosquitoes, found in high densities in the coastal, and estuarine, districts of Setúbal and Faro (Almeida et al., 2008). This virus replicates rapidly in the Ae. albopitus-derived C6/36 cell line and, as expected for an ISF, does not replicate in Vero cells. Unexpectedly, unlike most ISF, OCFVPt seems to cause pronounced cytophatic effect in C6/36 cells, which round-up and rapidly detach from a solid support soon after infection. Electron microscope analysis of thin sections of C6/36 cells at 48h post-infection with OCFVPt revealed nuclear hyperplasy, and evident enlargement of the intercisternal space of the nuclear envelope, which is also filled with multiple sized vesicles. The OCFVPt genome is, at least, 9,839 nt long and encodes a single polyprotein showing all the features expected for a flaviviruses. The phylogenetic trees, based on alignment of viral sequences, resulted in similar topologies, in which OCFVPt always seems to form, along with the recently reported HANKV (Huhtamo et al., 2012), a divergent genetic line within the ISF radiation.
The genus Flavivirus (Flaviviridae) includes over seventy viruses with ssRNA genomes, many of which are important pathogens of humans and other animals. Flaviviruses comprehend tick-borne, mosquito-borne and no known vector viral agents, as well as so-called “non-classical flaviviruses” (Cook and Holmes, 2006) with no known vertebrate host. They are commonly placed at the roots of phylogenetic trees of the genus, are frequently found in mosquitoes, and have been tentatively designated insect-specific flaviviruses (ISF) (Farfan-Ale et al., 2009). While classification of ISF as flaviviruses is supported by their genetic organization, protein hydropathy plots, conserved polyprotein cleavage sites and enzyme domains, they are antigenically distinct from other flaviviruses, and share approximately the same level of nucleotide sequence identity with other members of the genus as when compared to the members of the two other established genera in the Flaviviridae family (Cook and Holmes, 2006; Gould et al., 2003). This work reports the initial characterization, including near full-length sequence and genome analysis, of an new ISF, tentatively designated OCFVPt, that was isolated from Aedes (Ochlerotatus) caspius (Pallas, 1771) adult mosquitoes, found in high densities in the coastal, and estuarine, districts of Setúbal and Faro (Almeida et al., 2008). This virus replicates rapidly in the Ae. albopitus-derived C6/36 cell line and, as expected for an ISF, does not replicate in Vero cells. Unexpectedly, unlike most ISF, OCFVPt seems to cause pronounced cytophatic effect in C6/36 cells, which round-up and rapidly detach from a solid support soon after infection. Electron microscope analysis of thin sections of C6/36 cells at 48h post-infection with OCFVPt revealed nuclear hyperplasy, and evident enlargement of the intercisternal space of the nuclear envelope, which is also filled with multiple sized vesicles. The OCFVPt genome is, at least, 9,839 nt long and encodes a single polyprotein showing all the features expected for a flaviviruses. The phylogenetic trees, based on alignment of viral sequences, resulted in similar topologies, in which OCFVPt always seems to form, along with the recently reported HANKV (Huhtamo et al., 2012), a divergent genetic line within the ISF radiation.
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Parasitologia médica Entomologia médica Vírus Mosquitos
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Instituto de Higiene e Medicina Tropical