Co-Circulation of Leishmania Parasites and Phleboviruses in a Population of Sand Flies Collected in the South of Portugal.

In the Old World, phlebotomine sand flies from the genus Phlebotomus are implicated in the transmission of Leishmania spp. parasites (Kinetoplastida: Trypanosomatidae) and viruses belonging to the genus Phlebovirus (Bunyavirales: Phenuiviridae). Two of the five sand fly species known to occur in Portugal, Phlebotomus perniciosus and Ph. ariasi, the former being the most ubiquitous, are recognized vectors of Leishmania infantum, which causes visceral leishmaniasis, the most prevalent form of leishmaniasis in the country. Phlebotomus perniciosus is also the vector of the neurotropic Toscana virus, which can cause aseptic meningitis. Entomological surveillance is essential to provide fundamental data about the presence of vectors and the pathogens they can carry. As such, and given the lack of data in Portugal, an entomological survey took place in the Algarve, the southernmost region of the country, from May to October 2018. Polymerase chain reaction assays were performed in order to detect the presence of the above-mentioned pathogens in sand fly pools. Not only were both Leishmania parasites and phleboviruses detected during this study, but more importantly, it was the first time their co-circulation was verified in the same sand fly population collected in Portugal.

Sandfly-Borne Phleboviruses in Portugal: Four and Still Counting.

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.

West Nile virus transmission potential in Portugal.

It is unclear whether West Nile virus (WNV) circulates endemically in Portugal. Despite the country's adequate climate for transmission, Portugal has only reported four human WNV infections so far. We performed a review of WNV-related data (1966-2020), explored mosquito (2016-2019) and land type distributions (1992-2019), and used climate data (1981-2019) to estimate WNV transmission suitability in Portugal. Serological and molecular evidence of WNV circulation from animals and vectors was largely restricted to the south. Land type and climate-driven transmission suitability distributions, but not the distribution of WNV-capable vectors, were compatible with the North-South divide present in serological and molecular evidence of WNV circulation. Our study offers a comprehensive, data-informed perspective and review on the past epidemiology, surveillance and climate-driven transmission suitability of WNV in Portugal, highlighting the south as a subregion of importance. Given the recent WNV outbreaks across Europe, our results support a timely change towards local, active surveillance.

Phylogenetic Analysis of Massilia phlebovirus in Portugal.

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.

Toscana Virus: Ten Years of Diagnostics in Portugal.

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 numbers 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.

Introdução: O vírus Toscana (TOSV) é um vírus emergente, transmitido por flebótomos, que pertence ao género Phlebovirus. Apesar de a maioria das infeções causadas por este vírus serem assintomáticas ou apresentarem uma sintomatologia ligeira, o TOSV pode causar síndrome febril ou casos esporádicos de doença neurológica tal como meningite ou meningoencefalite. Este agente patogénico encontra-se distribuído por toda a bacia do Mediterrâneo, de acordo com as áreas de distribuição do seu vetor reconhecido, Phlebotomus perniciosus. Depois de Itália, Portugal foi o segundo país considerado endémico para este vírus após a publicação, em 1985, do primeiro caso de infeção adquirida no nosso território. Apesar do pouco conhecimento acerca da circulação deste vírus, no nosso país, o diagnóstico laboratorial de TOSV está disponível em Portugal, desde 2007, no Centro de Estudos de Vetores e Doenças Infeciosas do Instituto Nacional de Saúde Dr. Ricardo Jorge (CEVDI/INSA). O objetivo deste trabalho é dar a conhecer os resultados do diagnóstico de TOSV em Portugal, de 2009 a 2018, no CEVDI/INSA. Material e Métodos: O diagnóstico de TOSV no CEVDI/INSA está inserido nos painéis 'arbovírus' e 'vírus neurotrópicos transmitidos por vetores' ou pode ser realizado, quando especificado, só para TOSV. O diagnóstico direto é realizado em amostras de líquido cefalorraquidiano e encontra-se disponível no CEVDI/INSA por RT-PCR em tempo real, específico para TOSV, seguido de RT-PCR convencional, no caso de a amostra ser positiva na primeira técnica, para confirmação por sequenciação. Para o diagnóstico indireto, realizado em amostras de soro, é utilizado uma técnica de imunofluorescência in-house, para a deteção de anticorpos IgM e IgG anti-TOSV. Também está disponível uma imunofluorescência comercial, com um mosaico de quatro flebovírus, onde para além do TOSV, são testados anticorpos contra três vírus da febre por flebótomos, nomeadamente Nápoles, Sicília e Chipre. Neste trabalho foram considerados os pedidos de diagnóstico ao CEVDI/INSA para arbovírus, vírus neurotrópicos e/ou TOSV, de janeiro de 2009 a dezembro de 2018. Resultados: No período em estudo, foram enviadas ao CEVDI/INSA, amostras de 608 indivíduos com pedido de diagnóstico de TOSV. Foram confirmadas cinco infeções agudas por TOSV e uma infeção aguda por vírus Sicília em amostras de soro. Três outros doentes apresentaram prova serológica de contacto prévio com o TOSV. Dois dos doentes com infeção aguda apresentaram síndrome febril, mas quatro evidenciaram quadros neurológicos: meningite (n = 2), meningoencefalite (n = 1) e alterações graves do estado de consciência (n = 1). Estas infeções foram, muito provavelmente, adquiridas nos distritos de Faro (3), Lisboa (2) e Setúbal (1). Discussão: Em Portugal, o número de pedidos de diagnóstico laboratorial para TOSV é baixo quando comparado com o número de pedidos para outros vírus transmitidos por vetores. O distrito de Faro foi o que apresentou o número mais alto de pedidos de diagnóstico específicos para TOSV, o que parece demonstrar que existe um maior reconhecimento pelos clínicos daquela região. Síndrome febril e doença neurológica foram as manifestações clínicas nos casos agudos. Neste estudo, além do distrito de Faro, foram também detetadas infeções recentes nos distritos de Lisboa e Setúbal. É provável que o TOSV se encontre distribuído por todo o território continental, uma vez que o seu principal vetor está presente de norte a sul. Em 2017, o vírus Sicília foi associado, pela primeira vez, a doença humana no nosso país, alertando para a circulação deste flebovírus. Conclusão: Apesar do número de casos identificados em Portugal ser baixo, o TOSV circula e causa doença no nosso país. Este e outros flebovírus não deveriam ser negligenciados no diagnóstico diferencial de síndrome febril e de meningites e meningoencefalites virais, em especial nos meses mais quentes, quando é maior a atividade dos seus vetores.

Seasonal Dynamics and Spatial Distribution of Aedes albopictus (Diptera: Culicidae) in a Temperate Region in Europe, Southern Portugal.

Aedes albopictus is an invasive mosquito that has colonized several European countries as well as Portugal, where it was detected for the first time in 2017. To increase the knowledge of Ae. albopictus population dynamics, a survey was carried out in the municipality of Loulé, Algarve, a Southern temperate region of Portugal, throughout 2019, with Biogents Sentinel traps (BGS traps) and ovitraps. More than 19,000 eggs and 400 adults were identified from May 9 (week 19) and December 16 (week 50). A positive correlation between the number of females captured in the BGS traps and the number of eggs collected in ovitraps was found. The start of activity of A. albopictus in May corresponded to an average minimum temperature above 13.0 °C and an average maximum temperature of 26.2 °C. The abundance peak of this A. albopictus population was identified from September to November. The positive effect of temperature on the seasonal activity of the adult population observed highlight the importance of climate change in affecting the occurrence, abundance, and distribution patterns of this species. The continuously monitoring activities currently ongoing point to an established population of A. albopictus in Loulé, Algarve, in a dispersion process to other regions of Portugal and raises concern for future outbreaks of mosquito-borne diseases associated with this invasive mosquito species.

The Application and Interpretation of IgG Avidity and IgA ELISA Tests to Characterize Zika Virus Infections.

In the absence of viremia, the diagnostics of Zika virus (ZIKV) infections must rely on serological techniques. In order to improve the serological diagnosis of ZIKV, ZIKV-IgA and ZIKV-IgG avidity assays were evaluated. Forty patients returning from ZIKV endemic areas, with confirmed or suspected ZIKV infections were studied. Samples were classified as early acute, acute and late acute according to the number of days post illness onset. Low avidity IgG was only detected at acute and late acute stages and IgA mostly at the early acute and acute stages. The date of sampling provides useful information and can help to choose the best technique to use at a determined moment in time and to interpret low avidity IgG and IgA results, improving the serological diagnosis of ZIKV.

Practical Guidelines for Studies on Sandfly-Borne Phleboviruses: Part II: Important Points to Consider for Fieldwork and Subsequent Virological Screening.

In this series of review articles entitled "Practical guidelines for studies on sandfly-borne phleboviruses," the important points to be considered at the prefieldwork stage were addressed in part I, including parameters to be taken into account to define the geographic area for sand fly trapping and how to organize field collections. Here in part II, the following points have been addressed: (1) factors influencing the efficacy of trapping and the different types of traps with their respective advantages and drawbacks, (2) how to process the trapped sand flies in the field, and (3) how to process the sand flies in the virology laboratory. These chapters provide the necessary information for adopting the most appropriate procedures depending on the requirements of the study. In addition, practical information gathered through years of experience of translational projects is included to help newcomers to fieldwork studies.

Practical Guidelines for Studies on Sandfly-Borne Phleboviruses: Part I: Important Points to Consider Ante Field Work.

The purpose of this review is to provide practical information to help researchers intending to perform "from field to laboratory" studies on phleboviruses transmitted by sandflies. This guideline addresses the different steps to be considered starting from the field collection of sandflies to the laboratory techniques aiming at the detection, isolation, and characterization of sandfly-borne phleboviruses. In this guideline article, we address the impact of various types of data for an optimal organization of the field work intending to collect wildlife sandflies for subsequent virology studies. Analysis of different data sets should result in the geographic positioning of the trapping stations. The overall planning, the equipment and tools needed, the manpower to be deployed, and the logistics to be anticipated and set up should be organized according to the objectives of the field study for optimal efficiency.

Seroprevalence of vector-borne pathogens and molecular detection of Borrelia afzelii in military dogs from Portugal.

BACKGROUND: Canine vector-borne diseases (CVBDs) are increasingly being reported worldwide and represent a serious threat to both animal and public health. Military dogs may constitute a risk group for the agents causing these diseases, as they frequently work outdoors in different areas and are thus exposed to vector arthropods. In order to assess the risk of exposure of this type of dogs, a serological and molecular survey was conducted in military working dogs in Portugal. One hundred apparently healthy dogs were surveyed. Serum samples were tested for antigens of Angiostrongylus vasorum and Dirofilaria immitis; and for antibodies to A. vasorum, Anaplasma spp., Babesia spp., Ehrlichia canis, Leishmania infantum, Rickettsia spp. and Toscana virus. Serum was tested by polymerase chain reaction for Borrelia burgdorferi (sensu lato), with sequencing of the DNA products. RESULTS: Forty-nine per cent of the dogs were seropositive for antibodies against Rickettsia spp., 16 % for Anaplasma spp., 13 % for L. infantum, 7 % for E. canis, 5 % for A. vasorum (including 1 % positive for both antibodies and circulating antigens), 3 % for Babesia spp. and 1 % positive for Toscana virus. B. burgdorferi (s.l.) was detected in eight out of 94 dogs tested (8.5 %) and in three cases (3.2 %) nucleotide sequence analysis showed identity with the genospecies Borrelia afzelii. No positive cases were recorded for D. immitis. Overall, 66 % of the dogs were positive for at least one out of the eight tested CVBD agents, six of which are zoonotic (i.e. Anaplasma spp., Borrelia spp., E. canis, L. infantum, Rickettsia spp. and Toscana virus). Serological specific antibody detection against more than one CVBD agent (including molecular detection of Borrelia spp.) was recorded in 25 % of the dogs, comprising 19 % with positive reaction to two agents, 5 % to three agents and 1 % to four agents. CONCLUSIONS: These results reveal a high occurrence of CVBD agents in military working dogs in Portugal and highlight the need to maintain a comprehensive and regular prophylaxis to reduce the contact between working dogs and those pathogens. For the first time in Portugal, B. afzelii DNA was identified in dogs and a dog was found seropositive for antibodies against Toscana virus.

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

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.

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

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.

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

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.

Mosquito surveillance for prevention and control of emerging mosquito-borne diseases in Portugal - 2008-2014.

Mosquito surveillance in Europe is essential for early detection of invasive species with public health importance and prevention and control of emerging pathogens. In Portugal, a vector surveillance national program-REVIVE (REde de VIgilância de VEctores)-has been operating since 2008 under the custody of Portuguese Ministry of Health. The REVIVE is responsible for the nationwide surveillance of hematophagous arthropods. Surveillance for West Nile virus (WNV) and other flaviviruses in adult mosquitoes is continuously performed. Adult mosquitoes-collected mainly with Centre for Disease Control light traps baited with CO2-and larvae were systematically collected from a wide range of habitats in 20 subregions (NUTS III). Around 500,000 mosquitoes were trapped in more than 3,000 trap nights and 3,500 positive larvae surveys, in which 24 species were recorded. The viral activity detected in mosquito populations in these years has been limited to insect specific flaviviruses (ISFs) non-pathogenic to humans. Rather than emergency response, REVIVE allows timely detection of changes in abundance and species diversity providing valuable knowledge to health authorities, which may take control measures of vector populations reducing its impact on public health. This work aims to present the REVIVE operation and to expose data regarding mosquito species composition and detected ISFs.

Detection of mosquito-only flaviviruses in Europe.

The genus Flavivirus, family Flaviviridae, includes a number of important arthropod-transmitted human pathogens such as dengue viruses, West Nile virus, Japanese encephalitis virus and yellow fever virus. In addition, the genus includes flaviviruses without a known vertebrate reservoir, which have been detected only in insects, particularly in mosquitoes, such as cell fusing agent virus, Kamiti River virus, Culex flavivirus, Aedes flavivirus, Quang Binh virus, Nakiwogo virus and Calbertado virus. Reports of the detection of these viruses with no recognized pathogenic role in humans are increasing in mosquitoes collected around the world, particularly in those sampled in entomological surveys targeting pathogenic flaviviruses. The presence of six potential flaviviruses, detected from independent European arbovirus surveys undertaken in the Czech Republic, Italy, Portugal, Spain and the UK between 2007 and 2010, is reported in this work. Whilst the Aedes flaviviruses, detected in Italy from Aedes albopictus mosquitoes, had already been isolated in Japan, the remaining five viruses have not been reported previously: one was detected in Italy, Portugal and Spain from Aedes mosquitoes (particularly from Aedes caspius), one in Portugal and Spain from Culex theileri mosquitoes, one in the Czech Republic and Italy from Aedes vexans, one in the Czech Republic from Aedes vexans and the last in the UK from Aedes cinereus. Phylogenetic analysis confirmed the close relationship of these putative viruses to other insect-only flaviviruses.

[Toscana virus in the Portuguese population: serosurvey and clinical cases]. / Vírus Toscana na população Portuguesa: vigilância sero-epidemiológica e casos clínicos.

Toscana virus (Phlebovirus genus, Bunyaviridae family) is a neurotropic virus which circulates in the Mediterranean Basin. Although Portugal has been the second country where its presence was reported, the existence of this virus in our country has been referred only sporadically, and there is a lack of knowledge regarding the prevalence of antibodies in the population. Thus, the objective of this study was to analyse the prevalence of antibodies anti-Toscana virus in the human population in our country. Sero-epidemiological investigations were performed with indirect immunofluorescence assay (IFA) and enzyme linked immunosorbent assay (ELISA) tests. The study population consisted of a control population (blood donors, n=150), a population considered at risk (n=236) and a population of individuals with symptoms and laboratory diagnostic request for vector-borne viruses. The latter population was divided into two groups: those individuals with neurological symptoms (n=165) and those without neurological symptoms (n=373). We tested sera from a total of 924 individuals. The seroprevalence of IgG antibodies in the control population was 2%. In the population considered at risk, the prevalence was 3.4%. In the population with central nervous system disease, we detected a seroprevalence of 4.2%. For the same type of antibodies and in subjects without central nervous system disease, the prevalence was 1.3%. Five cases of recent infection (3%) were detected in the population with neurological signs. Those infections have been acquired in the districts of Faro, Coimbra, Aveiro and Lisbon. The associated clinical diagnoses were meningitis, meningoencephalitis and rash. The observed seroprevalences were, in general, lower than reported in other endemic countries. Only 5 of the 29 sera which gave positive results by IFA and ELISA were confirmed by plaque reduction neutralization tests with the Italian strain ISS.Phl.3. This can indicate the presence of more than one Toscana virus serotype circulating in Portugal and emphasizes the need for more research about this etiological agent in our country.

Molecular characterization of a new isolate of Borrelia lusitaniae derived from Apodemus sylvaticus in Portugal.

A total of 196 small mammals were collected in Portugal and tested for Borrelia burgdorferi sensu lato. Tissue samples were taken from each animal and cultured in Barbour-Stoenner-Kelly (BSK)-II medium. The single strain of spirochete isolated was confirmed as Borrelia lusitaniae by genetic analyses. This is the first report of B. lusitaniae isolated from Apodemus sylvaticus.

[Phleboviruses laboratory diagnosis (Toscana virus)]. / Diagnóstico laboratorial de flebovírus (Vírus Toscana).

Viruses from the Phlebovirus genus (family Bunyaviridae) are arboviruses that can be transmitted to humans by mosquitoes (Rift Valey virus) or by sandflies. In phleboviruses group we can find the etiological agents for Sandfly Fever (SF) that is spread in the Mediterranean basin, Middle East, Pakistan and India. In Portugal, the importance of phleboviruses to the Public Health is recognized because there have been detected cases of Toscana virus infection wich is responsible for severe cases of SF that involve encephalitis/meningitis. On the other hand, Phlebotomus perniciosus, one of Toscana virus vectors, is widespread in Portugal.

Molecular detection of Rickettsia felis, Rickettsia typhi and two genotypes closely related to Bartonella elizabethae.

A total of 56 fleas were collected from mice, rats, and one hedgehog in national parks of mainland Portugal and the Madeira Island. All fleas were tested for the presence of bacteria of the genera Rickettsia and Bartonella using PCR assays. In fleas from mainland Portugal, we detected Rickettsia felis in one Archaeopsylla erinacei maura flea and in one Ctenophtalmus sp. In five Leptopsylla segnis fleas taken from rats in the Madeira Island, we identified Rickettsia typhi. In addition, in four fleas from the genera Ornithophaga and Stenoponia collect from mice and a rat in mainland Portugal, we detected the presence of two new Bartonella genotypes closely related to Bartonella elizabethae. Our findings emphasize the potential risk of flea-transmitted infections in mainland Portugal and the Madeira archipelago, and extend our knowledge of the potential flea vectors of human pathogens.