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Co-circulation of a novel phlebovirus and Massilia virus in sandflies, Portugal

Publication . Amaro, Fátima; Zé-Zé, Líbia; Alves, M.J.; Börstler, J.; Clos, J.; Lorenzen, S.; Becker, S. C.; Schmidt-Chanasit, J.; Cadar, D.

Background: In Portugal, entomological surveys to detect phleboviruses in their natural vectors have not been performed so far. Thus, the aims of the present study were to detect, isolate and characterize phleboviruses in sandfly populations of Portugal. Findings: From May to October 2007–2008, 896 female sandflies were trapped in Arrábida region, located on the southwest coast of Portugal. Phlebovirus RNA was detected by using a pan-phlebovirus RT-PCR in 4 out of 34 Phlebotomus perniciosus pools. Direct sequencing of the amplicons showed that 2 samples exhibited 72 % nucleotide identity with Arbia virus, and two showed 96 % nucleotide identity with Massilia virus. The Arbia-like virus (named Alcube virus) was isolated in cell culture and complete genomic sequences of one Alcube and two Massila viruses were determined using next-generation sequencing technology. Phylogenetic analysis demonstrated that Alcube virus clustered with members of the Salehabad virus species complex. Within this clade, Alcube virus forms a monophyletic lineage with the Arbia, Salehabad and Adana viruses sharing a common ancestor. Arbia virus has been identified as the most closely related virus with 20-28 % nucleotide and 10-27 % amino acid divergences depending on the analysed segment. Conclusions: We have provided genetic evidence for the circulation of a novel phlebovirus species named Alcube virus in Ph. perniciosus and co-circulation of Massilia virus, in Arrábida region, southwest of Portugal. Further epidemiological investigations and surveillance for sandfly-borne phleboviruses in Portugal are needed to elucidate their medical importance.

2015-10-24Journal article

Open access

Insect-specific flaviviruses, a worldwide widespread group of viruses only detected in insects

Publication . Calzolari, M.; Zé-Zé, Líbia; Vásquez, A.; Seco, M.P.S.; Amaro, Fátima; Dottori, M.

Several flaviviruses are important pathogens for humans and animals (Dengue viruses, Japanese encephalitis virus, Yellow-fever virus, Tick-borne encephalitis virus, West Nile virus). In recent years, numerous novel and related flaviviruses without known pathogenic capacity have been isolated worldwide in the natural mosquito population. However, phylogenetic studies have shown that genomic sequences of these viruses diverge from other flaviviruses. Moreover, these viruses seem to be exclusive of insects (they do not seem to grow on vertebrate cell lines), and were already defined as mosquito-only flaviviruses or insect-specific flaviviruses. At least eleven of these viruses were isolated worldwide, and sequences ascribable to other eleven putative viruses were detected in several mosquito species. A large part of the cycle of these viruses is not well known, and their persistence in the environment is poorly understood. These viruses are detected in a wide variety of distinct mosquito species and also in sandflies and chironomids worldwide; a single virus, or the genetic material ascribable to a virus, was detected in several mosquito species in different countries, often in different continents. Furthermore, some of these viruses are carried by invasive mosquitoes, and do not seem to have a depressive action on their fitness. The global distribution and the continuous detection of new viruses in this group point out the likely underestimation of their number, and raise interesting issues about their possible interactions with the pathogenic flaviviruses, and their influence on the bionomics of arthropod hosts. Some enigmatic features, as their integration in the mosquito genome, the recognition of their genetic material in DNA forms in field-collected mosquitoes, or the detection of the same virus in both mosquitoes and sandflies, indicate that the cycle of these viruses has unknown characteristics that could be of use to reach a deeper understanding of the cycle of related pathogenic flaviviruses.

2015-07-31Journal article

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Electron-microscopy characterization of cells infected with a new phlebovirus isolated in sandflies from south Portugal

Publication . Amaro, Fátima; Zé-Zé, Líbia; Alves, M.J.; Alves de Matos, A.P.

Phlebovirus genus is comprised by arthropod-borne viruses and is one of the five genera that compose Bunyaviridae family [1]. Viruses within this family mature intracellularly and their RNA replicates in the cytoplasm, while the viral proteins transit through the Endoplasmic Reticulum and Golgi apparatus [2]. Serological studies in human Portuguese populations have proven that Toscana virus, a neurotropic phlebovirus, is present in Portugal but there were no data about phleboviral activity in arthropod vectors [3]. In this context, and in order to detect sandflies naturally infected by phleboviruses, an entomological surveillance was conducted in Arrabida region, in the south of Portugal.

2015-08Journal article

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Genetic characterization of Arrabida virus, a novel phlebovirus isolated in South Portugal

Publication . Amaro, F.; Hanke, D.; Zé-Zé, L.; Alves, M.J.; Becker, S.C.; Höper, D.

In order to detect phleboviruses’ natural infection in sandflies, an entomological survey was carried out, from May to October in 2007 and 2008, in Arrábida region in the south of Portugal. The isolation of a new phlebovirus was achieved after inoculation of a sandfly pool homogenate in Vero E6 cells. Based on the phylogenetic analysis of the complete genome sequences from the Small, Medium and Large, segments obtained with Next Generation sequencing, we can assume that the new phlebovirus, provisionally named Arrabida virus, is closely related to Massilia, Granada and Punique viruses. This is the first isolation of a sandfly-borne phlebovirus from the Sandfly Naples Fever Virus group in Portugal. Further investigation is needed in order to assess the importance of this phlebovirus for Public Health.

2016-03-02Journal article

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Climatic and Physiographic Variables to Evaluate Culex pipiens s.l. Risk and Habitat

Publication . Proença, M. da Conceição; Alves, Maria João; Rebelo, M.T.

Using a geographic information system (GIS), the relations between a georeferenced data set of Culex pipiens s.l . collected in Portugal mainland during seven years (2006-2012) and meteorological and physiographic parameters are evaluated. This work is one of the results of a long-term surveillance program of pernicious insects that act as vectors of various diseases; its focus is on the possibility of prevention that can be achieved with abundance data. The focus on Culex pipiens is justified by its abundance and its competence as a vector for numerous health issues. The cumulative distribution of monthly captures by each meteorological parameter allows to compute thresholds corresponding to mosquito massive presence related to 90% of the captures. Using the weather parameters measured in the network of weather stations across the country, a monthly average of each parameter of interest (temperature, humidity, etc.) is computed and an interpolation of the results is made to produce raster maps corresponding to each month. The previously obtained thresholds are applied to each map, producing spatial masks with the relevant zones for each parameter. The intersection of the various masks for each month shows the most densely populated area of Culex, and the ensemble allows us to observe the evolution of mosquito presence through the critical season, which is from May to October at these latitudes. In parallel, mosquito abundance data are related to physiographic parameters. The relative distribution of female mosquitoes across land cover types in each month allows identifying which classes and seasons are most relevant. Orthometric altitude related to the presence of 90% of the catches shows the limits reached by mosquitoes in each month. The results are applied to the previously obtained climate envelopes, delimiting critical areas where the level of risk of transmission of the pathogens for which Culex pipiens is a competent vector is high and countermeasures should be concentrated, allowing its planning, and targeting on a monthly basis. The described procedure can be used with other relevant vectors in any region of the world, whenever abundance data is available.

2022-08-04Journal article

Open access