Molecular characterization of cetacean poxviruses along the coast of mainland Portugal.

Cetacean poxvirus (CePV) is the causative agent of tattoo skin disease (TSD) in dolphins, porpoises and whales, a condition characterized by pinhole, ring-like lesions or generalized tattoo-like skin lesions. This study genetically characterized cetacean poxviruses from stranded animals along mainland Portugal. Samples from skin lesions compatible with TSD were obtained from 4 odontocete species (Delphinus delphis, Stenella coeruleoalba, Phocoena phocoena, and Tursiops truncatus) and analyzed using a conventional PCR assay targeting the DNA polymerase gene partially. Among the positive samples (n = 29, 65.9%), a larger DNA polymerase gene fragment was obtained, allowing a robust phylogenetic analysis. Nineteen samples (43.2%) were successfully amplified and sequenced using Sanger sequencing. By combining 11 of these sequences with those from public databases, a maximum likelihood phylogenetic tree was constructed, revealing high heterogeneity within the group. These findings contribute to a better understanding of the genetic diversity, epidemiology, phylogenetics, and evolution of CePV.

Bagaza Virus in Wild Birds, Portugal, 2021.

Bagaza virus emerged in Spain in 2010 and was not reported in other countries in Europe until 2021, when the virus was detected by molecular methods in a corn bunting and several red-legged partridges in Portugal. Sequencing revealed high similarity between the 2021 strains from Portugal and the 2010 strains from Spain.

Emergence of rabbit haemorrhagic disease virus 2 in the archipelago of Madeira, Portugal (2016-2017).

We report the detection of rabbit haemorrhagic disease virus 2 (RHDV2) in the Madeira archipelago, Portugal. Viral circulation was confirmed by RT-qPCR and vp60 sequencing. Epidemiological data revealed the outbreak initiated in October 2016 in Porto Santo affecting wild and domestic rabbits. It was then detected three months later on the island of Madeira. Five haplotypes were identified and a genetic overall similarity of 99.54 to 99.89% was observed between the two viral populations. Unique single nucleotide polymorphisms were recognised in the Madeira archipelago strains, two of which resulting in amino acid substitutions at positions 480 and 570 in the VP60 protein. Phylogenetic investigation by Maximum Likelihood showed all the vp60 sequences from the Madeira archipelago group together with high bootstraps. The analysis also showed that the Madeira archipelago strains are closely related to the strains detected in the south of mainland Portugal in 2016, suggesting a possible introduction from the mainland. The epidemiological data and high genetic similarity indicate a common source for the Porto Santo and Madeira RHDV2 outbreaks. Human activity related to hunting was most probably at the origin of the Madeira outbreak.

AVIAN POXVIRUS INFECTION IN A FLAMINGO (PHOENICOPTERUS RUBER) OF THE LISBON ZOO.

Avian poxviruses (APV) are very large viruses spread worldwide in a variety of hosts. They are responsible for a disease usually referred to as pox, mainly characterized by nodular lesions on feather-free regions of the body. On May 2010, a young American flamingo (Phoenicopterus ruber) of the Lisbon Zoo (Portugal) developed a nodular lesion suggestive of poxvirus infection on its right foot. Avipoxvirus was isolated from the lesion and a fragment of the P4b-encoding gene was amplified by polymerase chain reaction. The nucleotide sequence of the amplicon was determined and analyzed. A close relationship (100% identity) was observed between the flamingo poxvirus and isolates from great bustard (Hungary 2005), house sparrow (Morocco 2009), MacQueen's bustard (Morocco 2011), and Houbara bustard (Morocco 2010 and 2011), suggesting interspecies transmission as a possible source of infection. To strengthen the investigation, the 5' and 3' ends of genes cnpv186 and cnpv 187, respectively, were also analyzed. The cnpv186-187 fragment exhibited 100% identity with MacQueen's bustard and Houbara bustard isolates, both from Morocco 2011. Phylogenetic analyses based in both fragments grouped the flamingo isolate consistently within clade B2 of canarypox. However, the phylogenetic relationships among the different representatives of avian poxviruses were more comprehensive in the tree based on the concatenated coding sequences of the cnpv186-187 fragment, rather than on the P4b-coding gene. The clearer displacement and distribution of the isolates regarding their host species in this last tree suggests the potential usefulness of this genomic region to refine avian poxvirus classification.

Depressive symptoms and pain complaints as predictors of later development of depression in Portuguese middle-aged women.

Pain complaints and depressive symptoms seem to be risk factors for future depressive episodes. We present a one-year follow-up study on Portuguese women and primary health care (initial sample: n = 503). We used the Center for Epidemiologic Studies Depression Scale (CES-D), a short questionnaire on pain, and clinical interviews. We used forward stepwise logistic regression analysis to define models that enable the prediction of developing a major depressive episode in the one-year follow-up. We concluded that nondepressed women with (a) high scores (≥28) at the CES-D, (b) a high severity of pain index, and (c) fatigue complaints at the baseline were particularly at risk.

Thermostability study of virulent Newcastle disease viruses isolated in Southern Angola.

Newcastle disease (ND) is endemic in Angola. Several outbreaks of ND occurred in small backyard flocks and village chickens with high mortality in the southern provinces of the country, Cunene, Namibe and Huíla, in 2016 and 2018. In those years, 15 virulent ND virus (NDV) strains were isolated and grouped within subgenotype 2 of genotype VII (subgenotype VII.2). We now present a study on the thermostability of the isolates, aiming at the selection of the most thermostable strains that, after being genetically modified to reduce their virulence, can be adapted to the production of vaccines less dependent on cold chain and more adequate to protect native chickens against ND. Heat-inactivation kinetics of haemagglutinin (Ha) activity and infectivity (I) of the isolates were determined by incubating aliquots of virus at 56 °C for different time intervals. The two isolates from Namibe province showed a decrease in infectivity of 2 log10 in ≤ 10 min, therefore belonging to the I-phenotype, but while the NB1 isolate from 2016 maintained the Ha activity up to 30 min and was classified as thermostable virus (I-Ha+), the Ha activity of the 2018 NB2 isolate decreased by 2 log2 in 30 min, being classified as a thermolabile virus (I-Ha-). Of the 13 NDV isolates from Huíla province, 10 isolates were classified as thermostable, eight with phenotype I+Ha+ and 2 with phenotype I-Ha+. The other three isolates from this province were classified as thermolabile viruses (I-Ha-).Contribution: This study will contribute to the control and/or eradication of Newcastle disease virus in Angola. The thermostable viral strains isolated from chickens in the country can be genetically manipulated by reverse genetic technology in order to reduce their virulence and use them as a vaccine in the remote areas of Angola.

First detection of Bagaza virus in Common magpies (Pica pica), Portugal 2023.

Bagaza virus (BAGV) is a mosquito-borne flavivirus of the family Flaviviridae, genus Orthoflavivirus, Ntaya serocomplex. Like other viruses of the Ntaya and Japanese encephalitis serocomplexes, it is maintained in nature in transmission cycles involving viremic wild bird reservoirs and Culex spp. mosquitoes. The susceptibility of red-legged partridge, ring-necked pheasant, Himalayan monal and common wood pigeon is well known. Determining whether other species are susceptible to BAGV infection is fundamental to understanding the dynamics of disease transmission and maintenance. In September 2023, seven Eurasian magpies were found dead in a rural area in the Mértola district (southern Portugal) where a BAGV-positive cachectic red-legged partridge had been found two weeks earlier. BAGV had also been detected in several red-legged partridges in the same area in September 2021. Three of the magpies were tested for Bagaza virus, Usutu virus, West Nile virus, Avian influenza virus and Avian paramyxovirus serotype 1, and were positive for BAGV only. Sequencing data confirmed the specificity of the molecular detection. Our results indicate that BAGV is circulating in southern Portugal and confirm that Eurasian magpie is potential susceptible to BAGV infection. The inclusion of the abundant Eurasian magpie in the list of BAGV hosts raises awareness of the potential role of this species as as an amplifying host.

High pathogenic avian influenza A(H5) viruses of clade 2.3.4.4b in Europe-Why trends of virus evolution are more difficult to predict.

Since 2016, A(H5Nx) high pathogenic avian influenza (HPAI) virus of clade 2.3.4.4b has become one of the most serious global threats not only to wild and domestic birds, but also to public health. In recent years, important changes in the ecology, epidemiology, and evolution of this virus have been reported, with an unprecedented global diffusion and variety of affected birds and mammalian species. After the two consecutive and devastating epidemic waves in Europe in 2020-2021 and 2021-2022, with the second one recognized as one of the largest epidemics recorded so far, this clade has begun to circulate endemically in European wild bird populations. This study used the complete genomes of 1,956 European HPAI A(H5Nx) viruses to investigate the virus evolution during this varying epidemiological outline. We investigated the spatiotemporal patterns of A(H5Nx) virus diffusion to/from and within Europe during the 2020-2021 and 2021-2022 epidemic waves, providing evidence of ongoing changes in transmission dynamics and disease epidemiology. We demonstrated the high genetic diversity of the circulating viruses, which have undergone frequent reassortment events, providing for the first time a complete overview and a proposed nomenclature of the multiple genotypes circulating in Europe in 2020-2022. We described the emergence of a new genotype with gull adapted genes, which offered the virus the opportunity to occupy new ecological niches, driving the disease endemicity in the European wild bird population. The high propensity of the virus for reassortment, its jumps to a progressively wider number of host species, including mammals, and the rapid acquisition of adaptive mutations make the trend of virus evolution and spread difficult to predict in this unfailing evolving scenario.

A population-based assessment of the relationship between menopausal and depressive symptoms in Portuguese women.

Empirical research on the relationship between menopause and depression has found contradictory results. Some authors suggest that depression is related to menopausal symptoms and not to menopausal status per se. Our aim is to study the relationship between menopausal status and depressive symptoms in a sample (n = 728) of middle-aged Portuguese women, taking into account the presence of menopausal symptoms. Depressive symptoms were measured with the Centre for Epidemiological Studies Depression Scale (CES-D). There is a relatively high significant correlation between depressive and menopausal symptomatology (rs = .47). The increase of depressive symptomatology in perimenopause is still significant, even when adjusting for the presence of menopausal symptomatology.

Molecular characterization and phylogenetic analysis of Newcastle disease viruses isolated in southern Angola, 2016-2018.

Newcastle disease (ND) is a highly contagious viral disease that affects many bird species worldwide. This study presents the results of the molecular characterization and phylogenetic analysis of 15 virulent ND viruses (NDV) isolated from chickens during outbreaks reported in 2016 and 2018, in the provinces of Namibe and Huíla, in southern Angola. A 561-nucleotide fragment of the F gene was amplified by RT-PCR and sequenced for molecular characterization. Results showed that in all isolates the amino acid sequence comprising the cleavage site of fusion protein is characteristic of virulent viruses (RRQKR/F). Blast analysis revealed high similarity (99.2%) between two isolates from Huíla province, HLA4 and HLA6, and strain 5620 (GenBank accession number KY747479) isolated from chickens in the neighboring country Namibia, in 2016. The other isolates investigated are more related (97.0%) with strain 6195 (GenBank accession number KY747480), also isolated in Namibia in 2016. Phylogenetic analysis performed by Maximum Likelihood, Neighbor-joining and Bayesian methods revealed that like the strains isolated in Namibia, the isolates from southern Angola also belong to subgenotype 2 of genotype VII (VII.2). The network analysis revealed that NBA1 isolate from Angola is closer to a common ancestor than the isolates from Namibia, suggesting that transmission of ND viruses occurred from Angola to Namibia.

Snapshot of the Phylogenetic Relationships among Avian Poxviruses Circulating in Portugal between 2017 and 2023.

Avipoxvirus (APV), a linear dsDNA virus belonging to the subfamily Chordopoxvirinae of the family Poxviridae, infects more than 278 species of domestic and wild birds. It is responsible for causing avian pox disease, characterized by its cutaneous and diphtheric forms. With a high transmission capacity, it can cause high economic losses and damage to the ecosystem. Several diagnostic methods are available, and bird vaccination can be an effective preventive measure. Ten APV-positive samples were analyzed to update the molecular characterization and phylogenetic analysis of viruses isolated in Portugal between 2017 and 2023. A P4b gene fragment was amplified using a PCR, and the nucleotide sequence of the amplicons was determined using Sanger sequencing. The sequences obtained were aligned using ClustalW, and a maximum likelihood phylogenetic tree was constructed. With this study, it was possible to verify that the analyzed sequences are distributed in subclades A1, A2, B1, and B3. Since some of them are quite similar to others from different countries and obtained in different years, it is possible to conclude that there have been several viral introductions in Portugal. Finally, it was possible to successfully update the data on Avipoxviruses in Portugal.

Genome Characterization and Spaciotemporal Dispersal Analysis of Bagaza Virus Detected in Portugal, 2021.

In September 2021, Bagaza virus (BAGV), a member of the Ntaya group from the Flavivirus genus, was detected for the first time in Portugal, in the heart and the brain of a red-legged partridge found dead in a hunting ground in Serpa (Alentejo region; southern Portugal). Here we report the genomic characterization of the full-length sequence of the BAGV detected (BAGV/PT/2021), including phylogenetic reconstructions and spaciotemporal analyses. Phylogenies inferred from nucleotide sequence alignments, complemented with the analysis of amino acid alignments, indicated that the BAGV strain from Portugal is closely related to BAGV strains previously detected in Spain, suggesting a common ancestor that seems to have arrived in the Iberia Peninsula in the late 1990s to early 2000s. In addition, our findings support previous observations that BAGV and Israel turkey meningoencephalitis virus (ITV) belong to the same viral species.

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.

Multiyear surveillance of influenza A virus in wild birds in Portugal.

This report presents the results of a multiyear (2005 to 2009) study of avian influenza virus (AIV) occurrence in wild birds in Portugal. A total of 5691 samples from wild birds belonging to 13 different orders were examined. Ninety-three samples tested positive for AIV by matrix reverse transcriptase-polymerase chain reaction, giving a total prevalence of 1.63%. Twenty-one viruses were successfully cultured in embryonated chicken eggs, which represent a rate of viral infectivity of 22.6% in the samples. Nine subtypes of haemagglutinin (H1, H3 to H7, H9 to H11) and eight subtypes of neuraminidase (N1 to N4, N6 to N9) were identified in 20 different combinations. The most prevalent subtypes of haemagglutinin detected were H5, H1 and H4, while for neuraminidase subtypes N2 and N6 were the most common. The subtype combinations H4N6 and H1N1 were predominant (15.1%). All H5 and H7 viruses detected in the present study were low pathogenic for poultry as determined by the sequence of amino acids at the cleavage site of haemagglutinin. The full-length nucleotide sequences of five H5, one H7 and five N3 genes were analysed phylogenetically. The Bayesian analysis revealed that all but one of the strains analysed were closely related to isolates detected in the same period in North and Central European countries. Three H5N3 isolates, all from 2007, formed a separate cluster in both H5 and N3 phylogenetic trees. This study provides evidence that various subtypes of AIV, including subtypes H5 and H7, circulate in Portugal, which may pose a risk to industrial poultry.

A population-based assessment of midlife Portuguese women's experience of perimenopause and menopause.

The frequency of changes experienced by menopausal women is variable in different cultures. It is our purpose to identify the symptoms that Portuguese women consider to be related to menopause. We also wish to evaluate the relations between these symptoms and menopausal status, sociodemographic and lifestyle factors, and medication-menopausal hormone therapy (MHT) and psychotropics. In women with a low educational level, the frequency of symptoms and the consumption of psychotropic medication is higher, and the practice of exercise is lower, as is the use of MHT. Our results may be useful in providing guidance to health care professionals.

Simple Method for Establishing Primary Leporidae Skin Fibroblast Cultures.

Commercial hare and rabbit immortalized cell lines are extremely limited regarding the many species within the lagomorpha order. To overcome this limitation, researchers and technicians must establish primary cell cultures derived from biopsies or embryos. Among all cell types, fibroblasts are plastic and resilient cells, highly convenient for clinical and fundamental research but also for diagnosis, particularly for viral isolation. Here, we describe a fast and cheap method to produce primary fibroblast cell cultures from leporid species, using dispase II, a protease that allows dermal-epidermal separation, followed by a simple enzymatic digestion with trypsin. This method allows for the establishment of an in vitro cell culture system with an excellent viability yield and purity level higher than 85% and enables the maintenance and even immortalization of leporid fibroblastic cells derived from tissues already differentiated.

Development and validation of a real-time PCR for the detection and quantification of porcine circovirus type 2.

Porcine circovirus type 2 (PCV2) is a spherical and non-enveloped virus belonging to the genus Circovirus of the Circoviridae family with a single stranded circular DNA genome. This virus, already detected worldwide, has been associated to several diseases and was implicated as the etiological agent of a disease named postweaning multisystemic wasting syndrome. Several methods have been described for the detection of PCV2, being real-time PCR the most simple and reliable. As far as we know, all the real-time PCR systems described until now are based on ORF2 gene, that exhibit the highest variability. This paper reports the development and validation of a real-time PCR targeted to ORF1 and based on a TaqMan probe for the detection of porcine circovirus type 2 DNA in swine samples. Due to the lack of PCV1 samples, the ability of the test to discriminate between PCV1 and PCV2 positive samples was evaluated in silico. Estimations of 100% specificity and 100% sensitivity were obtained based on the qPCR results with panel of 81 swine samples (known PCV2-positive (n = 50); known PCV2-negative (n = 17); samples positive to other common swine viral pathogens (n = 13) and one sample from a BFDV-positive parrot (n = 1)). Intra- and inter-assay coefficients of variation obtained with three positive samples of different viral charges in five replicates or in five independent assays were below the acceptance threshold. The limit of detection determined with a recombinant plasmid containing the amplicon, led to conclude that this assay can detect at least three plasmid copies.

West Nile virus in horses during the summer and autumn seasons of 2015 and 2016, Portugal.

West Nile fever (WNF) is an emergent disease in Europe, under surveillance in the European Union. Following a 5-year period of apparent silence (autumn 2010 to summer 2015), West Nile virus (WNV) reemerged in the South of Portugal, in July 2015. Here we present data from the onset, geographic location within mainland Portugal, and outcome of clinical cases of WNV infection in horses in 2015 and 2016. During the transmission seasons of 2015 and 2016, twenty-seven horses, most symptomatic (n=20) were found positive to IgM, pr-E immunoglobulins and VNT, leading to the subsequent report to Animal Disease Notification System of the European Commission (ADNS) by the Portuguese National Authority for Animal Health. Outbreaks occurred in the middle summer (August) and early/mid autumn (October/November) of 2015 and 2016, in the southern regions of the country (Alentejo and Algarve). Compared with the previous WNV transmission seasons of 2004 and 2010, a higher number of cases were reported in 2015 and 2016. The results of our study contribute to increase information concerning the geographic areas affected and time period for WNV transmission risk in Portugal.

Development and validation of a blocking ELISA test for the detection of avian influenza antibodies in poultry species.

Avian influenza (AI) is an infectious viral disease usually asymptomatic in wild birds that can spread to domestic poultry and cause large-scale outbreaks. Some of the AI viruses (AIV) can cross the species barrier and induce fatal disease to humans, a matter of great concern worldwide. Early detection of AIV is of major importance for disease control, since prompt implementation of adequate measures can prevent spread of the virus and therefore further outbreaks. This paper reports the development and validation of a blocking ELISA using a monoclonal antibody against a conserved structural protein for the serologic diagnosis of all AI virus subtypes from domestic bird species, allowing the quick, easily automated and low-cost screening of a high number of farms. The test will be of great value not only for surveillance, but also for monitoring the efficiency of vaccination programs. Cut-off values were established in 20% of inhibition for turkey sera and in 50% of inhibition for chicken and duck sera. Estimations of 100% specificity and 100% sensitivity were obtained based on the results of known AI positive (n=130) and negative (n=208) sera, including serum samples from birds infected with other common avian viral pathogens (n=7). ROC analysis showed an area under curve (AUC) of 1.0 for chicken, duck and turkey sera, indicating that the blocking ELISA was able to perfectly discriminate between negative and positive samples of any of the poultry species tested. Inter- and intra-assay coefficients of variation were above the acceptance threshold. Furthermore, the ELISA titers were similar to the known hemagglutination inhibition titers of three positive reference sera indicating sensitivity comparable with the golden standard HI method. The method here described is an economically attractive alternative to the commercial ELISAs currently available.

A real time Taqman RT-PCR for the detection of rabbit hemorrhagic disease virus 2 (RHDV2).

A specific real time RT-PCR for the detection of RHDV2 was developed and validated using RHDV and RHDV2 RNA preparations from positive field samples. The system was designed to amplify a 127 nucleotide-long RNA region located within the vp60 gene, based on the alignment of six sequences originated in Portugal, obtained in our laboratory, and 11 sequences from France and Italy. The primers and probe target sequences are highly conserved in the vast majority of the RHDV2 sequences presently known. In the sequences showing variability, only one mismatch is found per strain, usually outlying the 3' end of the primer or probe hybridization sequences. The specificity of the method was demonstrated in vitro with a panel of common rabbit pathogens. Standardization was performed with RNA transcripts obtained from a recombinant plasmid harboring the target sequence. The method was able to detected nine RNA molecules with an efficiency of 99.4% and a R(2) value of 1. Repeatability and reproducibility of the method were very high, with coefficients of variation lower than 2.40%. The assay was proven a valuable tool to diagnose most of RDVH2 circulating strains, and may be also useful to monitor viral loads, and consequently, disease progression and vaccination efficacy.