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.

Mutation rate of SARS-CoV-2 and emergence of mutators during experimental evolution.

Background and objectives: To understand how organisms evolve, it is fundamental to study how mutations emerge and establish. Here, we estimated the rate of mutation accumulation of SARS-CoV-2 in vitro and investigated the repeatability of its evolution when facing a new cell type but no immune or drug pressures. Methodology: We performed experimental evolution with two strains of SARS-CoV-2, one carrying the originally described spike protein (CoV-2-D) and another carrying the D614G mutation that has spread worldwide (CoV-2-G). After 15 passages in Vero cells and whole genome sequencing, we characterized the spectrum and rate of the emerging mutations and looked for evidences of selection across the genomes of both strains. Results: From the frequencies of the mutations accumulated, and excluding the genes with signals of selection, we estimate a spontaneous mutation rate of 1.3 × 10 -6 ± 0.2 × 10-6 per-base per-infection cycle (mean across both lineages of SARS-CoV-2 ± 2SEM). We further show that mutation accumulation is larger in the CoV-2-D lineage and heterogeneous along the genome, consistent with the action of positive selection on the spike protein, which accumulated five times more mutations than the corresponding genomic average. We also observe the emergence of mutators in the CoV-2-G background, likely linked to mutations in the RNA-dependent RNA polymerase and/or in the error-correcting exonuclease protein. Conclusions and implications: These results provide valuable information on how spontaneous mutations emerge in SARS-CoV-2 and on how selection can shape its genome toward adaptation to new environments. Lay Summary: Each time a virus replicates inside a cell, errors (mutations) occur. Here, via laboratory propagation in cells originally isolated from the kidney epithelium of African green monkeys, we estimated the rate at which the SARS-CoV-2 virus mutates-an important parameter for understanding how it can evolve within and across humans. We also confirm the potential of its Spike protein to adapt to a new environment and report the emergence of mutators-viral populations where mutations occur at a significantly faster rate.

Combined detection of molecular and serological signatures of viral infections: The dual assay concept.

The recent worldwide spread of viral infections has highlighted the need for accurate, fast, and inexpensive disease diagnosis and monitorization methods. Current diagnostics tend to focus either on molecular or serological testing. In this work we propose a dual detection assay approach for viral diseases, where both serological and molecular assays are combined in a single analysis performed on a magnetoresistive system. This type of assay guarantees an accurate assessment of the infection phase, saving time and costs associated with multiple independent tests. Zika and dengue viruses were used as model diseases for the validation of the system. Human IgG anti-zika and anti-dengue antibodies were successfully detected in infected patients' serum, using a novel approach combining competitive and sandwich strategies in a magnetoresistive portable platform. Specificity and sensitivity values of 100% were obtained. Calibration curves with dynamic ranges between 10 ng/mL and 1 µg/mL were established achieving LODs of 1.26 and 1.38 nM for IgG anti-ZIKV and anti-DENV antibodies, respectively. Viral RNA detection down to a few hundreds of pM was also successfully carried out after the design of specific oligo probes and primers for RT-PCR amplification. Dual assays were performed for both viruses, where viral RNA and anti-virus antibodies in serum samples were simultaneously detected. The results obtained for the detection of the molecular and serological targets in the dual assay format show no significant difference between the ones obtained individually, proving the feasibility and accuracy of the dual detection assay. This assay format represents a new paradigm in viral infections diagnostics.

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.

Mitogenome diversity of Aedes (Stegomyia) albopictus: Detection of multiple introduction events in Portugal.

Aedes albopictus, along with Ae. aegypti, are key arbovirus vectors that have been expanding their geographic range over the last decades. In 2017, Ae. albopictus was detected for the first time at two distinct locations in Portugal. In order to understand how the Ae. albopictus populations recently introduced in Portugal are genetically related and which is their likely route of invasion, we performed an integrative cytochrome C oxidase I gene (COI)- and mitogenome-based phylogeographic analysis of mosquitoes samples collected in Portugal in 2017 and 2018 in the context of the global Ae. albopictus diversity. COI-based analysis (31 partial sequences obtained from 83 mosquitoes) revealed five haplotypes (1 to 5), with haplotype 1 (which is widely distributed in temperate areas worldwide) being detected in both locations. Haplotypes 2 and 3 were exclusively found in Southern region (Algarve), while haplotype 4 and 5 were only detected in the North of Portugal (Penafiel, Oporto region). Subsequent high discriminatory analyses based on Ae. albopictus mitogenome (17 novel sequences) not only confirmed a high degree of genetic variability within and between populations at both geographic locations (compatible with the Ae. albopictus mosquito populations circulating in Europe), but also revealed two mitogenome mutational signatures not previously reported at worldwide level. While our results generally sustain the occurrence of multiple introduction events, fine mitogenome sequence inspection further indicates a possible Ae. albopictus migration within the country, from the Northern introduction locality to the Southern region. In summary, the observed scenario of high Ae. albopictus genetic diversity in Portugal, together with the detection of mosquitoes in successive years since 2017 in Algarve and Penafiel, points that both Ae. albopictus populations seem to be already locally established, as its presence has been reported for three consecutive years, raising the public health awareness for future mosquito-borne diseases outbreaks.

Emergence of the Asian lineage of Zika virus in Angola: an outbreak investigation.

BACKGROUND: Zika virus infections and suspected microcephaly cases have been reported in Angola since late 2016, but no data are available about the origins, epidemiology, and diversity of the virus. We aimed to investigate the emergence and circulation of Zika virus in Angola. METHODS: Diagnostic samples collected by the Angolan Ministry of Health as part of routine arboviral surveillance were tested by real-time reverse transcription PCR by the Instituto Nacional de Investigação em Saúde (Ministry of Health, Luanda, Angola). To identify further samples positive for Zika virus and appropriate for genomic sequencing, we also tested samples from a 2017 study of people with HIV in Luanda. Portable sequencing was used to generate Angolan Zika virus genome sequences from three people positive for Zika virus infection by real-time reverse transcription PCR, including one neonate with microcephaly. Genetic and mobility data were analysed to investigate the date of introduction and geographical origin of Zika virus in Angola. Brain CT and MRI, and serological assays were done on a child with microcephaly to confirm microcephaly and assess previous Zika virus infection. FINDINGS: Serum samples from 54 people with suspected acute Zika virus infection, 76 infants with suspected microcephaly, 24 mothers of infants with suspected microcephaly, 336 patients with suspected dengue virus or chikungunya virus infection, and 349 samples from the HIV study were tested by real-time reverse transcription PCR. Four cases identified between December, 2016, and June, 2017, tested positive for Zika virus. Analyses of viral genomic and human mobility data suggest that Zika virus was probably introduced to Angola from Brazil between July, 2015, and June, 2016. This introduction probably initiated local circulation of Zika virus in Angola that continued until at least June, 2017. The infant with microcephaly in whom CT and MRI were done had brain abnormalities consistent with congenital Zika syndrome and serological evidence for Zika virus infection. INTERPRETATION: Our analyses show that autochthonous transmission of the Asian lineage of Zika virus has taken place in Africa. Zika virus surveillance and surveillance of associated cases of microcephaly throughout the continent is crucial. FUNDING: Royal Society, Wellcome Trust, Global Challenges Research Fund (UK Research and Innovation), Africa Oxford, John Fell Fund, Oxford Martin School, European Research Council, Departamento de Ciência e Tecnologia/Ministério da Saúde/National Council for Scientific and Technological Development, and Ministério da Educação/Coordenação de Aperfeicoamento de Pessoal de Nível Superior.

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.

First case of confirmed congenital Zika syndrome in continental Africa.

Background: Zika virus has been responsible for recent outbreaks in the western hemisphere with known neurological complications such as microcephaly. This complication has not been previously documented in continental Africa. Methods: Neurological evaluation of the newborn was performed after birth, at one and two months of age. The mother and the newborn sera samples were tested by immunofluorescent assay (IFA; immunoglobulin G [IgG] and IgM) for Zika virus and the presence of Zika virus ribonucleic acid (RNA) was checked by qualitative real-time reverse transcription polymerase chain reaction (RT-PCR) in placenta, blood and urine samples. Results: We report on a newborn, born in Portugal, with microcephaly with confirmed congenital Zika virus infection (Asian lineage) imported from Angola with typical clinical and imaging findings. Conclusions: To our knowledge this is the first report that shows the circulation of the Asian lineage in Angola and the first report of a congenital Zika syndrome in continental Africa.

Detection of the Invasive Mosquito Species Aedes (Stegomyia) albopictus (Diptera: Culicidae) in Portugal.

The Asian tiger mosquito Aedes albopictus is an invasive mosquito originating from the Asia-Pacific region. This species is of major concern to public and veterinary health because of its vector role in the transmission of several pathogens, such as chikungunya, dengue, and Zika viruses. In Portugal, a National Vector Surveillance Network (REde de VIgilância de VEctores—REVIVE) is responsible for the surveillance of autochthonous, but also invasive, mosquito species at points of entry, such as airports, ports, storage areas, and specific border regions with Spain. At these locations, networks of mosquito traps are set and maintained under surveillance throughout the year. In September 2017, Ae. albopictus was detected for the first time in a tyre company located in the North of Portugal. Molecular typing was performed, and a preliminary phylogenetic analysis indicated a high similarity with sequences of Ae. albopictus collected in Europe. A prompt surveillance response was locally implemented to determine its dispersal and abundance, and adult mosquitoes were screened for the presence of arboviral RNA. A total of 103 specimens, 52 immatures and 51 adults, were collected. No pathogenic viruses were detected. Despite the obtained results suggest low abundance of the population locally introduced, the risk of dispersal and potential establishment of Ae. albopictus in Portugal has raised concern for autochthonous mosquito-borne disease outbreaks.

Dengue virus serotype 3 and Chikungunya virus co-infection in a traveller returning from India to Portugal, November 2016.

We report a case of a laboratory-confirmed Dengue and Chikungunya viruses co-infection imported from India to Portugal in early November 2016. The patient developed fever, retro-orbital pain and generalized myalgia after returning from Delhi, Jaipur, Agra, Rishikesh, Goa and Mumbai. This case highlights the importance of these arboviruses to public health in India where high rates of co-infection have been reported in the last few years, and demonstrates how challenging the laboratory diagnosis of imported co-infection cases can be in non-endemic areas.

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.

Characterization Through Multilocus Sequence Analysis of Borrelia turdi Isolates from Portugal.

Borrelia turdi is a spirochete from the Borrelia burgdorferi complex, first reported in Japan, that has been increasingly detected in Europe. This genospecies is mostly associated with avian hosts and their ornithophilic ticks such as Ixodes frontalis. In this study, we isolated B. turdi from five I. frontalis feeding on Turdus merula, Turdus philomelos, Parus major and Troglodytes troglodytes, and one Ixodes ricinus feeding on a T. merula in Portugal. These isolates were genetically characterised according to their 5S-23S rRNA intergenic spacer, 16S rRNA and through typing of seven housekeeping genes (multilocus sequence typing). Multilocus sequence analyses revealed that the strains isolated in our study, although belonging to B. turdi genospecies, are not identical to the B. turdi reference strain Ya501. Instead, our strains are separated into a clear defined group, suggesting that the European samples diverged genetically from the strain originally detected in Japan. Population analysis of 5S-23S rRNA sequences can further resolve subpopulations within B. turdi, but more samples from a large geographical scale and host range would be needed to assess potential phylogeographical patterns within this genospecies.

Human case of West Nile neuroinvasive disease in Portugal, 2015.

A case of West Nile virus (WNV) infection was reported in the Algarve region, Portugal, in the first week of September 2015. WNV is known to circulate in Portugal, with occasional reports in horses and birds (2004 to 2011) and very sporadically human cases (in 2004 and in 2010). Here we present the clinical and laboratory aspects related to the first human case of West Nile neuroinvasive disease reported in Portugal.

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.

Portuguese hosts for Ornithodoros erraticus ticks.

The hematophagous soft tick Ornithodoros erraticus feeds nocturnally on multiple warm-blooded vertebrate hosts. This tick is often found living buried in the soil of traditional pigpens. O. erraticus is an important infectious disease vector both for humans and animals. In the Iberian Peninsula, this tick serves as the vector of human tick-borne relapsing fever caused by the spirochete Borrelia hispanica. The natural ecosystems maintaining this spirochete are not well understood, with details of competent vertebrate reservoirs and tick-host interactions poorly understood. Investigation of arthropod blood meal composition provides evidence linking the vector to specific hosts, providing insights into possible disease reservoirs. Ticks collected from two pigpens located in southern Portugal were subjected to blood meal analysis. PCR amplification of vertebrate cytochrome b was used to disclose the original host from which 349 ticks had derived their previous blood meal. Host origins for blood meal analysis from 79 of 349 ticks revealed that 46.8% had previously fed from pigs, 35.4% human, 13.9% bovine, 5.1% sheep, 1.3% rodent, and 1.3% from birds. Three samples revealed mixed blood meals, namely, human-pig (1.3%), sheep-pig (1.3%), and bovine-pig (1.3%). The major role of pigs as hosts is consistent with fieldwork observations and underlines the importance of pigs for maintaining O. erraticus tick populations. Humans serve as accidental hosts, frequently confirmed by reports from both producers and veterinarians. Other livestock species and wildlife prevalent in the region appear only to have a minor role in maintaining this tick. The results demonstrate the importance of blood meal analysis to determine tick hosts providing a tool for investigation of sylvatic cycle for Borrelia hispanica.