Search

[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. [PT] 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 tais 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. This work was partially supported by the FCT Project: Phleboviruses in Portugal: vectors pathogenesis and coinfections (PTDC/DTP-SAP/0859/2014). info:eu-repo/semantics/publishedVersion

This chapter summarizes the main features of the distribution of infected cells in ZIKV-infected mice and correlates it with pathological manifestations. The mouse innate immune system triggered by interferon IFN I is able to control the infection, unlike in humans where Zika virus proteins associate with IFN receptors causing their destruction and disabling the innate immune response. Thus, mouse models have to be modified to allow virus spreading and disease development. The spread of the virus through the mouse and its main consequences to the pathological mechanisms induced by infection of cells and tissues are summarized, with special emphasis on the major manifestations specific to the disease: congenital Zika syndrome, vertical transmission through the placenta, and sexual transmission through both male and female susceptibility to infection of the sexual organs.

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., Author summary In 2017, Aedes albopictus was reported for the first time in Portugal at two distinct locations, in the premises of a tire company in Penafiel, in the North, and nearby a golf course in Algarve, a tourism destination in the southernmost country region. The geographical spread of this species is promoted by egg and larvae transport in aquatic trade goods, as tires and aquatic plants, and adult anthropophilic behavior that favors passive land transportation. In Portugal, especially in the Southern region, temperate climate conditions are adequate for adult mosquitoes to survive most of the year. In a way to understand the genetic variability of Ae. albopictus populations introduced in Portugal, we analyzed 31 cytochrome C oxidase I gene (COI) partial sequences and 17 mitogenome sequences, integrating them in the context of the global Ae. albopictus phylogeographic diversity (i.e., 183 COI and 26 mitogenome sequences previously reported at worldwide level). Although COI haplotype 1 predominated, four additional haplotypes (2 to 5) were detected in Portugal. Subsequent in-depth mitogenome analysis revealed considerable genetic diversity, including not only sequences relating to mitogenomes reported mainly from Italy, Japan and China, but also two novel mitogenome mutational signatures. Our study indicates that Ae. albopictus is locally established in Portugal and intra-country dispersal may have already happened, highlighting the challenges for vector surveillance and control programs aiming at restraining arbovirus disease burden in the future.

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 establish, as its presence has been reported for three consecutive years, raising the public health awareness for future mosquito-borne diseases outbreaks.Author SummaryIn 2017, Aedes albopictus was reported for the first time in Portugal at two distinct locations, in the premises of a tyre company in Penafiel, in the North, and nearby a golf course in Algarve, a tourism destination in the southernmost country region. The geographical spread of this species is boosted by larvae and desiccation-resistant eggs transport in aquatic trade goods, as tires and aquatic plants, and adult anthropophilic behavior that favors passive land transportation. In Portugal, especially in the Southern region, temperate climate conditions are adequate for adult mosquitoes survival most of the year. In a way to understand the genetic variability of Ae. albopictus populations introduced in Portugal, we analyzed 31 cytochrome C oxidase I gene (COI) partial sequences and 17 mitogenome sequences, integrating them in the context of the global Ae. albopictus phylogeographic diversity (i.e., 183 COI and 26 mitogenome sequences previously reported at worldwide level). Although COI haplotype 1 predominated, four additional haplotypes (2 to 5) were detected in Portugal. Subsequent in-depth mitogenome analysis revealed considerable genetic diversity, including not only sequences relating to mitogenomes reported mainly from Italy, Japan and China, but also two novel mitogenome mutational signatures.Our study indicate that Ae. albopictus is locally established in Portugal and intra-country dispersal may have already happened, highlighting the challenges for vector surveillance and control programs aiming at restraining arbovirus disease burden in the future.

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 Loul&eacute, 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 &deg, C and an average maximum temperature of 26.2 &deg, 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 Loul&eacute, 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.

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution(CC BY) license (http://creativecommons.org/licenses/by/4.0/), Background: Mosquito-borne viruses, such as Zika, dengue, yellow fever, and chikungunya, are important causes of human diseases nearly worldwide. The greatest health risk for arboviral disease outbreaks is the presence of the most competent and highly invasive domestic mosquito, Aedes aegypti. In Cabo Verde, two recent arbovirus outbreaks were reported, a dengue outbreak in 2009, followed by a Zika outbreak in 2015. This study is the first entomological survey for Ae. aegypti that includes all islands of Cabo Verde archipelago, in which we aim to evaluate the actual risk of vector-borne arboviruses as a continuous update of the geographical distribution of this species. Methods: In order to assess its current distribution and abundance, we undertook a mosquito larval survey in the nine inhabited islands of Cabo Verde from November 2018 to May 2019. Entomological larval survey indices were calculated, and the abundance analyzed. We collected and identified 4045 Ae. aegypti mosquitoes from 264 positive breeding sites in 22 municipalities and confirmed the presence of Ae. aegypti in every inhabited island. Results: Water drums were found to be the most prevalent containers (n = 3843; 62.9%), but puddles (n = 27; 0.4%) were the most productive habitats found. The overall average of the House, Container, and Breteau larval indices were 8.4%, 4.4%, and 10.9, respectively. However, 15 out of the 22 municipalities showed that the Breteau Index was above the epidemic risk threshold. Conclusion: These results suggest that if no vector control measures are considered to be in place, the risk of new arboviral outbreaks in Cabo Verde is high. The vector control strategy adopted must include measures of public health directed to domestic water storage and management., This research as also the APC were funded by the National Institute of Public Health of Cabo Verde grant number 65.06.01.04.29.01.-Reforço Do Laboratório Nacional De Saúde Pública.

© 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/)., 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., This research was supported in part by the Portuguese Foundation for Science and Technology (FCT) through the project “Phleboviruses in Portugal: vectors, pathogenesis and co-infections” (PTDC/DTP-SAP/0859/2014). J.L. was supported by a research lectureship by the Zoology Department of the University of Oxford. M.G. was supported by Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ). This manuscript received the support of Fundação para a Ciência e a Tecnologia, grant number UIDB/04295/2020 and UIDP/04295/2020.

Introduction: Phlebovirus genus (family Phenuiviridae), consists of 10 species of viruses composed by different serologically related viruses. Toscana virus (which belongs to Sandfly Fever Naples Virus- SFNV- species complex) is neurotropic and its presence was reported for the first time in Portugal in 1983. An entomological survey conducted in 2007–2008, in Arrabida region, in south Portugal detected two new phleboviruses of unknown pathogenic potential, provisionally named Arrabida and Alcube ascribed to SFNV and Salehabad Virus (SALV) species, respectively. Apart from a previous characterization of the Arrabida virus [1] these viruses, including the Toscana virus, have not been previously studied at the ultrastructural level. We conducted a comparative ultrastructural study to evaluate their morphology and cytopathic effects in cell cultures. Materials and methods: Cells infected and mock-infected with virus suspensions of Toscana, Arrabida and Alcube viruses were collected at 4 days after infection and sequentially fixed in 3% glutaraldehyde, 1% osmium tetroxide and 0.5% uranyl acetate. Thin sections of Epon-Araldite embedded samples were studied in a JEOL 1200EX electron microscope. Results: Toscana virus displays numerous extracellular virus aggregates adhering to the cell membranes and occasional particles inside the Golgi cisternae. Arrabida virus infected cells showed extensive proliferation of membranes of the endoplasmic reticulum. Alcube virus induced marked degenerative changes in infected cells. Extracellular particles from all virus infections had diameter of 76 to 80 nm and could not be distinguished by size or morphology. Discussion and conclusions: Our results show that Toscana produces much more virus particles than Arrabida but no intracelular proliferation of membranes compared to the Arrabida virus. The differences may at least in part be related to the longer adaptation of the Toscana virus to the cultured cells. In contrast Alcube virus belongs to a different species and shows strong cytopathic effects with extensive alterations of cellular organelles, suggesting that it may be more pathogenic than the other viruses.

In the absence of viremia, the diagnostics of Zika virus (ZIKV) infections must rely on serologicaltechniques. InordertoimprovetheserologicaldiagnosisofZIKV,ZIKV-IgAandZIKV-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. This work has been partially funded by the ISCIII Project “PI16CIII/00037”. info:eu-repo/semantics/publishedVersion

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. This work was partially funded by the FCT project “New arboviruses isolated in Portugal. Risk assessment and public health application (PTDC/SAU-SAP/119199/2010)” and EU grant FP7-613996 Vmerge. info:eu-repo/semantics/publishedVersion

Zika virus is an emerging arbovirus transmitted by Aedes sp. mosquitoes like the Dengue and Chikungunya viruses. Zika virus was until recently considered a mild pathogenic mosquito-borne flavivirus with very few reported benign human infections. In 2007, an epidemic in Micronesia initiated the turnover in the epidemiological history of Zika virus and more recently, the potential association with congenital microcephaly cases in Brazil 2015, still under investigation, led the World Health Organization (WHO) to declare a Public Health Emergency of International Concern on February 1, 2016. Here, we present the clinical and laboratory aspects related to the first four imported human cases of Zika virus in Portugal from Brazil, and alert, regarding the high level of traveling between Portugal and Brazil, and the ongoing expansion of this virus in the Americas, for the threat for Zika virus introduction in Europe and the possible introduction to Madeira Island where Aedes aegypti is present. info:eu-repo/semantics/publishedVersion

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/)., 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.

Zika virus is a flavivirus transmitted by mosquitoes from the Aedes genus, sexually, or by vertical transmission. It was discovered in 1947 in Africa and it is thought to produce, in some cases, microcephaly in newborns and the Guilan–Barre syndrome in adults. The otherwise low pathogenicity of the virus for humans precluded its detailed study. Recently, outbreaks in Pacific islands, with a virus related to Asian strains, were followed by a large outbreak in Brazil, which easily spread to all Latin-American countries and Cape Verde in África. Ultrastructural information on Zika’s effects on cells is very limited. Also an early histopathological study signaled alterations on other organs, which have not been further explored. (...) info:eu-repo/semantics/publishedVersion

Free PMC Article: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5685210/ Members of the ZIKV reference laboratory group: Portugal: Maria João Alves, National Institute of Health Dr. Ricardo Jorge, Lisbon With international travel, Zika virus (ZIKV) is introduced to Europe regularly. A country's ability to robustly detect ZIKV introduction and local transmission is important to minimise the risk for a ZIKV outbreak. Therefore, sufficient expertise and diagnostic capacity and capability are required in European laboratories. To assess the capacity, quality, operational specifics (guidelines and algorithms), technical and interpretation issues and other possible difficulties that were related to ZIKV diagnostics in European countries, a questionnaire was conducted among national reference laboratories in 30 countries in the European Union/European Economic Area (EU/EEA) in May 2016. While the coverage and capacity of ZIKV diagnostics in the EU/EEA national reference laboratories were found to be adequate, the assessment of the quality and needs indicated several crucial points of improvement that will need support at national and EU/EEA level to improve ZIKV preparedness, response and EU/EEA ZIKV surveillance activities. This study was conducted with ECDC-funding SRS-003-2016-RS. info:eu-repo/semantics/publishedVersion

Culex (Culex) pipiens (Diptera: Culicidae) has two recognized biotypes, pipiens and molestus, which differ in physiology and behaviour; this difference may influence vectorial capacity for West Nile virus (WNV). Our goal was first to determine the presence of Cx. pipiens populations in 31 locations in Portugal and to subsequently analyse their host-feeding preferences and habitat determinants. Molecular identification of Cx. pipiens forms and their hybrids was performed in 97 females; bloodmeal sources were identified in 59 engorged specimens. Overall, 61.9% of specimens were identified as Cx. pipiens f. pipiens, 20.6% as Cx. pipiens f. molestus, and 17.5% as hybrid forms. Culex pipiens f. pipiens fed preferentially on birds, and Cx. pipiens f. molestus on humans. Hybrid forms fed mostly on birds, but human bloodmeals were common. With reference to habitat, Cx. pipiens f. pipiens and hybrid forms were positively correlated with peri-urban habitats. Our results confirm the sympatric presence of different Cx. pipiens biotypes in 14 of the 31 locations studied. Peri-urban areas were a common habitat of all biotypes and may represent zones of hybridization. The feeding preferences and sympatric distribution of the Cx. pipiens biotypes observed in Portugal favour the epizootic circulation of WNV and the occurrence of disease outbreaks of WNV.

There is growing evidence that Zika virus (ZIKV) can cause devastating infant brain defects and other neurological disorders in humans. However, no specific antiviral therapy is available at present. We tested a series of 2'-C- or 2'-O-methyl-substituted nucleosides, 2'-C-fluoro-2'-C-methyl-substituted nucleosides, 3'-O-methyl-substituted nucleosides, 3'-deoxynucleosides, derivatives with 4'-C-azido substitution, heterobase-modified nucleosides, and neplanocins for their ability to inhibit ZIKV replication in cell culture. Antiviral activity was identified when 2'-C-methylated nucleosides were tested, suggesting that these compounds might represent promising lead candidates for further development of specific antivirals against ZIKV. This work was supported by the Czech Science Foundation (grantGA14–29256S); the Ministry of Education, Youth, and Sports of the Czech Republic, under the NPU I program (grant LO1218); the National Subvention for the Development of Research Organizations (grant RVO:61388963); the European Virus Archive Goes Global project, which has received funding from the European Union’s Horizon 2020 research and innovation program (grant 653316). info:eu-repo/semantics/publishedVersion

The recent detection of Anaplasma phagocytophilum in Portugal stimulated further research on the agent's enzootic cycle, which usually involves rodents. Thus a total 322 rodents belonging to five species, including 30 Apodemus sylvaticus (wood mouse), 65 Mus musculus (house mouse), 194 M. spretus (algerian mouse), 5 Rattus norvegicus (brown rat) and 28 R. rattus (black rat), were studied by indirect immunofluorescent assay (IFA) and/or polymerase chain reaction (PCR) for A. phagocytophilum exposure in four sampling areas of mainland and two areas of Madeira Island, Portugal. Overall, 3.6% (7/194) of M. spretus presented with IFA-positive results. Seropositive mice were detected in all three mainland sampling areas where this species was captured, with prevalence of 5.2% (5/96) and 5.0% (1/20) for the Ixodes-areas of Arrábida and Mafra, and 1.3% (1/78) for Mértola, a difference that was not statistically significant (p0.05). The majority of IFA-positive mice were detected in spring when considering either Arrábida alone (p = 0.026) or all M. spretus sampling areas together (p = 0.021), although the significance of this association was not evident after Bonferroni correction. Nevertheless, neither the seropositive M. spretus, nor additional samples of 10% seronegative rodents from mainland, and 16% of rodents collected in Madeira Island showed evidence of A. phagocytophilum active infections when spleen and/or lung samples were tested by PCR. Either the M. spretus results represents residual antibodies from past A. phagocytophilum infections, present infections with limited bacteremia, or cross-reactions with closely related agents deserves more investigation.

Free PMC Article: www.ncbi.nlm.nih.gov/pmc/articles/pmid/26497645/ 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. This work was partially funded by the FCT project “New arboviruses isolated in Portugal. Risk assessment and public health application" (PTDC/SAU-SAP/119199/2010).

[Background] The evolution of species boundaries and the relative impact of selection and gene flow on genomic divergence are best studied in populations and species pairs exhibiting various levels of divergence along the speciation continuum. We studied species boundaries in Iberian barbels, Barbus and Luciobarbus, a system of populations and species spanning a wide degree of genetic relatedness, as well as geographic distribution and range overlap. We jointly analyze multiple types of molecular markers and morphological traits to gain a comprehensive perspective on the nature of species boundaries in these cyprinid fishes., [Results] Intraspecific molecular and morphological differentiation is visible among many populations. Genomes of all sympatric species studied are porous to gene flow, even if they are not sister species. Compared to their allopatric counterparts, sympatric representatives of different species share alleles and show an increase in all measures of nucleotide polymorphism (S, H d, K, π and θ). High molecular diversity is particularly striking in L. steindachneri from the Tejo and Guadiana rivers, which co-varies with other sympatric species. Interestingly, different nuclear markers introgress across species boundaries at various levels, with distinct impacts on population trees. As such, some loci exhibit limited introgression and population trees resemble the presumed species tree, while alleles at other loci introgress more freely and population trees reflect geographic affinities and interspecific gene flow. Additionally, extent of introgression decreases with increasing genetic divergence in hybridizing species pairs., [Conclusions] We show that reproductive isolation in Iberian Barbus and Luciobarbus is not complete and species boundaries are semi-permeable to (some) gene flow, as different species (including non-sister) are exchanging genes in areas of sympatry. Our results support a speciation-with-gene-flow scenario with heterogeneous barriers to gene flow across the genome, strengthening with genetic divergence. This is consistent with observations coming from other systems and supports the notion that speciation is not instantaneous but a gradual process, during which different species are still able to exchange some genes, while selection prevents gene flow at other loci. We also provide evidence for a hybrid origin of a barbel ecotype, L. steindachneri, suggesting that ecology plays a key role in species coexistence and hybridization in Iberian barbels. This ecotype with intermediate, yet variable, molecular, morphological, trophic and ecological characteristics is the local product of introgressive hybridization of L. comizo with up to three different species (with L. bocagei in the Tejo, with L. microcephalus and L. sclateri in the Guadiana). In spite of the homogenizing effects of ongoing gene flow, species can still be discriminated using a combination of morphological and molecular markers. Iberian barbels are thus an ideal system for the study of species boundaries, since they span a wide range of genetic divergences, with diverse ecologies and degrees of sympatry., This work was supported financially by Fundação para a Ciência e a Tecnologia (FCT) project grant POCTI/BSE/35121/2000 to MJA, and PhD fellowships SFRH/BD/13067/2003 from FCT (co-participated by FEDER, POCI 2010 and FSE), Proc.85547 from Fundação Calouste Gulbenkian and from the Arizona State University School of Life Sciences to HFG. This research received support from the SYNTHESYS Project ES-TAF-1605 to HFG, http://www.synthesys.info/, which is financed by European Community Research Infrastructure Action under the FP7 “Capacities” Programme, at the Museo Nacional de Ciencias Naturales (CSIC).

West Nile virus (WNV) and Japanese encephalitis virus (JEV) are important mosquito-borne viruses of the Flaviviridae family, associated with encephalitis, mainly in humans and horses. WNV is also pathogen for many bird species. The incidence of human and animal WNV infections in Europe has risen, mostly in recent years, and JEV was detected in 2011 in mosquitoes collected in Italy and may emerge in Europe in the same way as other flaviviruses had emerged recently (USUTU and Bagaza virus) and should be regarded as a potential threat to public health. Prompt identification and discrimination between WNV and JEV provides critical epidemiological data for prevalence studies and public and animal health management policies. Here we describe a quantitative one-step duplex TaqMan RT-PCR, targeting non-structural protein 2A gene (NS2A-qRT-PCR), based on only one primer pair and two probes for differential diagnosis of WNV and JEV. Also this assay enables the detection of both WNV lineages (WNV-1 and WNV-2). To access the specificity of NS2A-qRT-PCR a panel of different arboviruses were used. The assay was shown to be specific for both WNV lineages (WNV-1 and WNV-2), WNV related Kunjin virus and JEV, since no cross-reactions were observed with other tested arboviruses. Sensitivity of the assay was determined using serial dilutions of in vitro-transcribed RNA from WNV and JEV. The duplex NS2A-qRT-PCR assay was shown to be very sensitive, being able to detect 10 copies of WNV and JEV RNA. This assay is a suitable tool for the diagnosis of WNV and JEV, and provides a valuable addition to the methods currently available for routine diagnosis of these zoonoses and for surveillance studies.

Presentation at ESCV 2016: Oral 39 Zika virus (ZIKV) belongs to the genus Flavivirus and was first isolated from the blood of a febrile sentinel rhesus monkey during a study of yellow fever in 1947, in Zika Forest, Uganda. ZIKV is transmitted by Aedes sp. Mosquitoes, as Dengue, Yellow fever and Chikungunya viruses, and was until recently considered a mild pathogenic mosquito-borne flavivirus with very few reported human cases of self-limiting acute febrile illnesses most often with maculopapular rash, headache, malaise and conjunctivitis, all followed by full recovery without sequel. During 60 years, cases of ZIKV infections were detected only sporadically in Africa, and South and Southeast Asia. (...) info:eu-repo/semantics/publishedVersion

The host blood-feeding patterns of mosquito vectors affects the likelihood of human exposure to zoonotic pathogens, including West Nile Virus (family Flaviviridae, genus Flavivirus, WNV). In Portugal, data are unavailable regarding the blood-feeding habits of common mosquito species, including Culex pipiens L., considered the primary vector of WNV to humans. The sources of bloodmeals in 203 blood-fed mosquitoes of nine species collected from June 2007 to November 2010 in 34 Portuguese counties were analyzed by sequencing cytochrome-b partial fragments. Cx. pipiens was the most common species collected and successfully analyzed (n = 135/78). In addition, blood-fed females of the following species were analyzed: Ochlerotatus caspius Pallas (n = 20), Culex theileri Theobald (n = 16), Anopheles maculipennis s.l. Meigen (n = 10), Culiseta longiareolata Macquart (n = 7), Aedes aegypti L. (n = 6), Culex perexiguus Theobald (n = 3), Culiseta annulata Schrank (n = 3), and Ochlerotatus detritus Haliday (n = 3). The Cx. pipiens mosquitoes fed predominantly on birds (n = 55/78, 70.5%), with a high diversity of avian species used as hosts, although human blood was identified in 18 specimens (18/78, 23.1%). No significant differences were found between the host-feeding patterns of blood-fed Cx. pipiens collected in residential and nonresidential habitats. The occurrence of human derived blood meals and the presence of a mix avian-human bloodmeal accordingly suggest this species as a potential vector of WNV. Therefore, in Portugal, Cx. pipiens may play a role both in the avian-to-avian enzootic WNV cycle and in the avian-to-mammal transmission. In this context, the identity of Cx. pipiens (considering the forms molestus and pipiens) and the potential consequence on feeding behavior and WNV transmission are discussed.

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. © 2012 Crown.

SUMMARYToscana virus (TOSV) is an emergingPhlebovirusof growing interest as a human pathogen in the Mediterranean Basin. In Portugal, however, little is known about the prevalence of TOSV infection. The aim of this work was to perform a seroprevalence study in patients with requests for laboratory diagnosis of vector-borne viruses. A total of 538 patients with and without neurological signs from 2004 to 2008 were studied by in-house indirect immunofluorescence assay and commercial enzyme-linked immunosorbent assays. A prevalence of 4·2% for IgG antibodies was found in the group of patients with neurological signs. Five (3%) of these had recent infections. In the group with no neurological signs, the IgG prevalence was 1·3%. Two samples, belonging to two patients, were also confirmed with plaque reduction neutralization tests with the TOSV ISS. Phl.3 Italian strain. This work showed that TOSV is present and causing disease from north to south in Portugal. The probable circulation of different phlebovirus serotypes in Portugal emphasizes the need for further studies.

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.

Data relating to hantavirus infection in Spain are scarce and limited to rural areas. The aim of this work was to study the seroprevalence of hantavirus infection in the Autonomous Community of Madrid (ACM), a region containing both rural and urban populations in different ecological settings. Sera from 3852 individuals (1849 male, 2003 female) were screened by indirect inmunofluorescence, with Vero E6 cells infected with Puumala, Hantaan and Seoul viruses as antigens. Screen-positive results were confirmed by Western blot with recombinant Seoul virus nucleocapsid protein as antigen. Antibodies against hantavirus were detected in 12 sera (0.31%). No statistical differences were found according to sex and age. The highest prevalence was found in the southeastern area, significantly higher than the central and north-western areas. The most frequent serological pattern was reactivity against all three viruses used (33.3% of all positive sera). Therefore, this study confirms the presence of hantavirus infection in the ACM, including for the first time an urban area of Spain, but with the highest prevalence in a rural area. Serological evidence suggests that there is more than one circulating serotype.

An agent pathogenic for laboratory albino Swiss mice was isolated from a pool of Haemaphysalis punctata ticks collected from cattle on a farm located in Alcácer do Sal county, southern Portugal. The isolated virus was shown to be distinct from but serologically related to virus members of the Bhanja antigenic group. This new virus in the family Bunyaviridae was named Palma for the farm where ticks have been collected for several studies.