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Sandfly-Borne Phleboviruses in Portugal: Four and Still Counting

Publication . Amaro, Fátima; Zé-Zé, Líbia; Alves, Maria João

According to ICTV, there are currently 66 known phlebovirus species. More than 40 of these viruses were isolated or detected in phlebotomine sandflies and some of them are known pathogens. In Portugal, information about sandfly-borne phleboviruses is scarce and scattered sandfly-borne diseases are neglected and often not considered in differential diagnoses. The main objective of this work was to gather the existing information and to raise awareness about the circulating phleboviruses in this country. To date, Massilia and Alcube phleboviruses have been isolated from sandflies in southern Portugal. Human infections with Toscana and Sicilian phleboviruses have been reported, as well as seroprevalence in cats and dogs. More studies are needed in order to understand if the viruses isolated during the entomological surveys have an impact on human health and to fully understand the real importance of the already recognized pathogens in our country.

2022-08-13Journal article

Open access

Molecular Identification and Ecology of Portuguese Wild-Caught Phlebotomine Sandfly Specimens

Publication . Zé-Zé, L.; Amaro, F.; Osório, H.; Giovanetti, M.; Lourenço, J.; Alves, M.J.

Phlebotomine sandflies are important vectors of Leishmania spp. and phleboviruses causing disease in animals and humans. Morphological identification of phlebotomine sandflies to the species level is challenging, requiring microscopical examination of the genitalia, which is demanding and time consuming. Molecular sandfly species identification can be a practical solution to save resources since it enables further molecular studies capable of generating data, such as biting preferences by blood meal analysis. In this study, resorting to a sandfly dataset collected between 2014 and 2018 across Portuguese territory under active mosquito surveillance and sandfly specific surveys, we used molecular methods to explore the genetic diversity and spatial distribution, further exploring ecological co-variants of four sandfly species—Phlebotomus ariasi, P. perniciosus, P. sergenti, and Sergentomyia minuta—all of which are of public health importance. Sandflies were collected from Spring to Autumn (May–November) following local temperature patterns. P. perniciosus was the most widespread detected species, with a nationwide distribution. All studied species clustered together with known samples from the Iberian Peninsula. Further monitoring studies of sandfly species diversity, distribution, and seasonality are essential for surveillance and control of sandfly-borne pathogens both nationally and globally

2022-02-11Journal article

Open access

Toscana Virus: Ten Years of Diagnostics in Portugal

Publication . Amaro, Fátima; Zé-Zé, Líbia; Luz, Maria Teresa; Alves, Maria João

[EN] Introduction: Toscana virus (TOSV) is an emerging sandfly-borne virus within the Phlebovirus genus. Although most infections caused by this virus present as asymptomatic or with minimal symptomatology, TOSV may emerge as a febrile disease or sporadic cases of neurological disease such as meningitis or meningoencephalitis. This pathogen is distributed throughout the Mediterranean basin, along with the spatial distribution of its recognized sandfly vector, Phlebotomus perniciosus. Portugal, after Italy, was the second country considered endemic for this virus, with the first case of acquired infection published in 1985. Although little is known about the circulation of this virus in Portugal, the laboratory diagnosis of TOSV is available at the Centre for Vectors and Infectious Diseases Research of the National Institute of Health Dr. Ricardo Jorge (CEVDI/INSA), since 2007. The aim of this study is to report the results of the diagnosis of TOSV at the CEVDI/INSA, between 2009 and 2018. Material and Methods: The diagnosis of TOSV in the CEVDI/INSA is included in the arboviruses and vector-borne neurotropic viruses panels or can be performed, when specified, for TOSV only. Direct detection is made in cerebrospinal fluid samples and is available for TOSV by specific real-time reverse transcription polymerase chain reaction followed by conventional real-time reverse transcription polymerase chain reaction for sequencing purposes, if positive. For indirect diagnosis, performed in serum samples, an in-house immunofluorescence assay for the detection of IgM and IgG antibodies against TOSV is used. A commercial immunofluorescence assay consisting in a mosaic of four phleboviruses is also available, in which, in addition to TOSV, antibody detection for sandfly fever Naples virus, sandfly fever Sicilian virus and sandfly fever Cyprus virus can be done. All diagnostic tests requested by clinicians to the CEVDI/ INSA for arboviruses, neurotropic viruses and/or TOSV between January 2009 and December 2018, were included in this study. Results: During the study period, the CEVDI/INSA received samples from 608 patients with diagnostic requests for TOSV. Five acute TOSV infections and one acute sandfly fever Sicilian virus infection were confirmed in serum samples. Three other patients had serological evidence of previous contact with the virus. Two of the six patients with acute infection developed febrile syndrome, and the other four presented with neurological disease: meningitis (n = 2), meningoencephalitis (n = 1) and severe depression of consciousness (n = 1). These infections were most likely acquired in the districts of Faro (3), Lisbon (2) and Setúbal (1). Discussion: In Portugal, the number of laboratory diagnostic requests for TOSV is low when compared to the number of requests for other less prevalent vector-borne viruses. The Faro district presented the highest number of TOSV-specific diagnostic requests which seems to indicate a higher level of recognition by clinicians in that region. Febrile syndrome and neurological disease were the clinical manifestations that were present in acute cases. In this study, in addition to the Faro district, recent infections were also detected in the districts of Lisbon and Setúbal. It is probable that TOSV may be distributed throughout the mainland territory since its main vector is present from north to south. In 2017, the sandfly fever Sicilian virus was associated for the first time with human disease in our country, thus alerting to the circulation of this phlebovirus. Conclusion: Even though the number of identified cases in Portugal is low, TOSV circulates and causes disease in our country. The diagnosis of this and other phleboviruses should not be neglected in the differential diagnosis of febrile syndrome and viral meningitis and meningoencephalitis, especially during the warmer months, when the vector’s activity is higher.

2021-05-28Journal article

Open access

Phylogenetic Analysis of Massilia phlebovirus in Portugal

Publication . Amaro, F.; Zé-Zé, L.; Lourenço, J.; Giovanetti, M.; Becker, S.C.; Alves, M.J.

In the last two decades, molecular surveys of arboviruses have enabled the identification of several new viruses, contributing to the knowledge of viral diversity and providing important epidemiological data regarding possible new emerging viruses. A combination of diagnostic assays, Illumina sequencing and phylogenetic inference are here used to characterize two new Massilia phlebovirus strains isolated from sandflies collected in the Arrábida region, Portugal. Whole genome sequence analysis enabled their identification as reassortants and the recognition of genomic variants co-circulating in Portugal. Much is still unknown about the life cycle, geographic range, evolutionary forces and public health importance of these viruses in Portugal and elsewhere, and more studies are needed.

2021-07-20Journal article

Open access