Screening for hepatitis E virus genotype 3 in red deer (Cervus elaphus) and fallow deer (Dama dama), Portugal, 2018-2020.

Hepatitis E virus (HEV) genotype 3 is the most prevalent HEV genotype in Europe causing mostly asymptomatic infections in humans, but can also sporadically cause severe acute hepatitis, chronic liver disease, chronic hepatitis in immunocompromised patients and extra-hepatic manifestations. Although much is today known about the swine reservoir, no information is available on the occurrence of HEV from widely distributed deer species in Portugal. Here, we investigated the presence and characterized HEV in free-living deer in Portugal by screening stools from red deer (Cervus elaphus) (n = 95) and fallow deer (Dama dama) (n = 35) for HEV by a broad-spectrum nested RT-PCR, followed by sequencing and phylogenetic analysis. Two red deer females, sampled in central Portugal, showed to be shedding HEV (2.1%; 95% confidence interval: 0.58-7.35). Sequencing and genetic characterization showed that these two deer HEV sequences were 98.96% identical to each other, being both of HEV genotype 3 subgenotype 3e. The increasing numbers and distribution of deer in Portugal and the zoonotic features of the circulating HEV genotype 3 subgenotype 3e highlights the importance of continued surveillance directed to food-borne diseases, especially those involving wild animals and deer in particular.

Molecular dynamics and protein frustration analysis of human fused in Sarcoma protein variants in Amyotrophic Lateral Sclerosis type 6: An In Silico approach.

Amyotrophic lateral sclerosis (ALS) is the most frequent adult-onset motor neuron disorder. The disease is characterized by degeneration of upper and lower motor neurons, leading to death usually within five years after the onset of symptoms. While most cases are sporadic, 5%-10% of cases can be associated with familial inheritance, including ALS type 6, which is associated with mutations in the Fused in Sarcoma (FUS) gene. This work aimed to evaluate how the most frequent ALS-related mutations in FUS, R521C, R521H, and P525L affect the protein structure and function. We used prediction algorithms to analyze the effects of the non-synonymous single nucleotide polymorphisms and performed evolutionary conservation analysis, protein frustration analysis, and molecular dynamics simulations. Most of the prediction algorithms classified the three mutations as deleterious. All three mutations were predicted to reduce protein stability, especially the mutation R521C, which was also predicted to increase chaperone binding tendency. The protein frustration analysis showed an increase in frustration in the interactions involving the mutated residue 521C. Evolutionary conservation analysis showed that residues 521 and 525 of human FUS are highly conserved sites. The molecular dynamics results indicate that protein stability could be compromised in all three mutations. They also affected the exposed surface area and protein compactness. The analyzed mutations also displayed high flexibility in most residues in all variants, most notably in the interaction site with the nuclear import protein of FUS.

Norovirus gastroenteritis outbreaks in military units: a systematic review.

INTRODUCTION: Norovirus gastroenteritis is one of the most frequent causes of personnel unavailability in military units, being associated with significant morbidity and degradation of their operational effectiveness. The disease is usually mild but can be severe and life-threatening in young and healthy soldiers, who are prone to dehydration due to intensive daily activity. Despite its impact, the full extent of the norovirus gastroenteritis burden in military forces remains unclear. This systematic review aims to evaluate the impact and ascertain clinical and epidemiological features of norovirus outbreaks that have occurred in the military forces. METHODS: The systematic review followed the Preferred Reporting Items for Systemic Reviews and Meta-Analysis (PRISMA) guidelines and used three databases: PubMed, Scopus, and LILACs. Papers published up to 1 September 2019 were included without restrictions if they reported one or more outbreaks in the military forces on active duty, either on national territories or deployed overseas. RESULTS: A total of 343 papers were retrieved from the literature search. After inclusion/exclusion criteria a total of 39 eligible papers were considered. From 1988 (first reported outbreak in the military) to 2018 more than 101 norovirus outbreaks have been reported in the military, accounting for at least 24 332 cases. Secondary transmission was emphasised as the main route of norovirus transmission in the military forces, with eating outside the military setting an important route for the primary cases. CONCLUSIONS: The present review highlights that norovirus gastroenteritis has been a burden to military troops both in combat and on peacekeeping operations. Norovirus disease has been shown to exact a substantial toll on mission readiness and operational effectiveness. It is noteworthy that the impact of norovirus outbreaks among military units is underestimated because the literature review retrieved information from the armed forces from only nine countries.

Simultaneous norovirus outbreak in three Portuguese army bases in the Lisbon region, December 2017.

INTRODUCTION: Norovirus outbreaks frequently occur in communities and institutional settings acquiring a particular significance in armed forces where prompt reporting is critical. Here we describe the epidemiological, clinical and laboratorial investigation of a multicentre gastroenteritis outbreak that was detected simultaneously in three Portuguese army units with a common food supplier, Lisbon region, between 5 and 6 December 2017. METHODS: Questionnaires were distributed to all soldiers stationed in the three affected army units, and stool specimens were collected from soldiers with acute gastrointestinal illness. Stool specimens were tested for common enteropathogenic bacteria by standard methods and screened for a panel of enteric viruses using a multiplex real-time PCR assay. Food samples were also collected for microbiological analysis. Positive stool specimens for norovirus were further genotyped. RESULTS: The three simultaneous acute gastroenteritis outbreaks affected a 31 (3.5%) soldiers from a total of 874 stationed at the three units and lasted for 2 days. No secondary cases were reported. Stool specimens (N=11) were negative for all studied enteropathogenic agents but tested positive for norovirus. The recombinant norovirus GII.P16-GII.4 Sydney was identified in all positive samples with 100% identity. CONCLUSIONS: The results are suggestive of a common source of infection plausibly related to the food supplying chain. Although centralisation of food supplying in the army has economic advantages, it may contribute to the multifocal occurrence of outbreaks. A rapid intervention is key in the mitigation of outbreak consequences and in reducing secondary transmission.

Crenosoma Striatum in Lungs of European Hedgehogs (Erinaceus Europeus) from Portugal.

Crenosoma striatum is a host-specifi c metastrongiloid nematode causing respiratory tract disease in hedgehogs (Erinaceus europaeus). Since few studies have reported C. striatum in hedgehogs and little genetic data is available concerning this lungworm, this study aimed to determine the occurrence of C. striatum in a population sample of hedgehogs from Portugal, additionally providing morphological, histological and molecular data. From 2017 to 2018 a survey of infection was carried out in 11 necropsied hedgehogs. Worms were extracted from fresh lung tissues and microscopically evaluated. Molecular characterization of partial mitochondrial (12S rRNA) and nuclear (18S rRNA) genes was performed. The presence of lungworms in pulmonary tissues of five hedgehogs (45.5%) was detected. Morphological and histopathological analyses evidenced adult forms of nematodes consistent with C. striatum. Molecular characterization of 18S rRNA genes confirmed the classifi cation as C. striatum. Also, novel genetic data characterizing the mitochondrial (12S rRNA) gene of C. striatum is presented. This is the first report of C. striatum infection in hedgehogs of Portugal. The findings here reported provide new insights regarding the geographic distribution and the molecular identification of this lungworm species.

Helminths associated with Norops fuscoauratus (Squamata, Dactyloidae) in highland marshes of the Brazilian semi-arid.

Helminthological studies may contribute with valuable information on host biology and conservation. Herein, we provide new data on helminths infecting the lizard Norops fuscoauratus, testing one of the factors considered most important in parasitic ecology: host size. We analysed 25 specimens of N. fuscoauratus from three highland marshes in the Brazilian semi-arid. Eight taxa of helminths belonging to Nematoda, Trematoda and Acanthocephala were found. Physaloptera sp. showed the higher prevalence (40%), with a mean intensity of infection of 3.3 ± 1.46 (1-16) and mean abundance 1.32 ± 0.65 (0-16). Norops fuscoauratus represents four new host records for the helminths Cyrtosomum sp., Pharyngodon travassosi, Strongyloides sp. and Centrorhynchus sp. There is no relationship of host body size (P = 0.79) and mass (P = 0.50) with parasite richness. In addition, the present study contributes to the knowledge of the parasitic fauna of N. fuscoauratus and the Neotropical region.

[The future of psychiatry and the psychiatrist of the future]. / De toekomst van de psychiatrie en de psychiater van de toekomst.

BACKGROUND: The training curriculum of the psychiatrist needs to be reviewed. To determine the model of the future psychiatrist, the question of how the future psychiatry will look needs to be answered first.
AIM: Assessment of relevant developments in psychiatry and the organisation of psychiatric care with the aim to propose a profile of the future psychiatrist.
METHOD: The recent history of psychiatry as a starting point for a vision of the future.
RESULTS: 1. Psychiatry must use an integrative anthropological theory, in order to be able to understand psychopathology in its essence. 2. Content-driven moral leadership is necessary to prevent psychiatry being tempted by hypes. 3. The tacit social contract between the medical profession and society is under pressure; both parties distrust each other and psychiatrists must regain trust and renew the contract. 4. Psychiatric care must remain affordable, which means a review of the current organisation of care; psychiatry will become network psychiatry. 5. Psychiatrists will work in a network of care providers and the patient will be in control. The psychiatrist will have a flexible role, ranging from managerial to supportive and advisory. Keeping psychotherapeutic skills up-to-date is an essential requirement. 6. Future training should focus on the sustainable employability of the psychiatrist as a human being, on knowledge of history of psychiatry and its socio-economic context, and policy and engagement; and on the skill of conceptual thinking (philosophy).
CONCLUSION: The profession of psychiatry needs a new job profile. The process of development and elaboration should contain the following core elements: broad education, conceptual skills, sensitivity for activism, social involvement, and expertise in treatment of patients with complex problems in diverse settings.

Antimicrobial alumina nanobiostructures of disulfide- and triazole-linked peptides: Synthesis, characterization, membrane interactions and biological activity.

Due to the its physical-chemical properties, alumina nanoparticles have potential applications in several areas, such as nanobiomaterials for medicinal or orthodontic implants, although the introduction of these devices poses a serious risk of microbial infection. One convenient strategy to circumvent this problem is to associate the nanomaterials to antimicrobial peptides with broad-spectrum of activities. In this study we present two novel synthesis approaches to obtain fibrous type alumina nanoparticles covalently bound to antimicrobial peptides. In the first strategy, thiol functionalized alumina nanoparticles were linked via disulfide bond formation to a cysteine residue of an analog of the peptide BP100 containing a four amino acid spacer (Cys-Ala-Ala-Ala). In the second strategy, alumina nanoparticles were functionalized with azide groups and then bound to alkyne-decorated analogs of the peptides BP100 and DD K through a triazole linkage obtained via a copper(I)-catalyzed cycloaddition reaction. The complete physical-chemical characterization of the intermediates and final materials is presented along with in vitro biological assays and membrane interaction studies, which confirmed the activity of the obtained nanobiostructures against both bacteria and fungi. To our knowledge, this is the first report of aluminum nanoparticles covalently bound to triazole-peptides and to a disulfide bound antimicrobial peptide with high potential for biotechnological applications.

In silico analysis and molecular dynamics simulation of human superoxide dismutase 3 (SOD3) genetic variants.

Oxidative stress is a major factor in aging processes. Superoxide dismutase 3 (SOD3) plays a key role in the protection of extracellular oxidative stress. Missense mutations in SOD3 have been described to be associated with the occurrence of pulmonary, cardiovascular, and neoplastic diseases. This study aims to analyze the effects of missense mutations on the SOD3 structure and function by modeling a complete SOD3 structure as well as analyzing the differences between the wild-types and mutants using computational simulations. Here, ten algorithms were used to predict the structural and functional effects of missense mutations. A complete model of SOD3 protein was made by ab initio and comparative modeling using the Rosetta algorithm and validated by PROCHECK, Verify 3D, QMEAN, and ProSa. Molecular dynamics (MD) simulations were performed and analyzed using the GROMACS package. The deleterious potential of the A58T and R231G mutants was not predicted by the majority of the used algorithms. The analyzed mutations were predicted as destabilizing by at least one algorithm. The MD analyses indicated that protein flexibility may be increased by all of the analyzed mutations, while the protein-ligand stability may be decreased. They also suggested that the variants A91T and R231G increase the overall dimensions of SOD3 and decrease its accessible surface area. Our findings, therefore, indicated that the analyzed mutations could affect the protein structure and its ability to interact with other molecules, which may be related to the functional impairment of SOD3 upon A58T and R231G mutations, as well as their involvement in pathologies.

Country-wide surveillance of norovirus outbreaks in the Portuguese Army, 2015-2017.

INTRODUCTION: Gastrointestinal infections are among the most common foodborne and waterborne diseases in military populations, with direct implications in operational efficiency and force readiness. Through the surveillance system of reportable acute gastrointestinal illness in the Portuguese Army, four norovirus outbreaks were identified between October 2015 and October 2017 in mainland Portugal and the Azores archipelago. The present study documents the epidemiological, clinical and laboratory investigations of these norovirus outbreaks. METHODS: Cases were investigated and epidemiological questionnaires were distributed to all soldiers in each military setting where the outbreaks occurred. Stool samples from soldiers with acute gastroenteritis illness were collected and screened for common enteropathogenic agents. Food and water samples served on the settings were also collected for microbiological investigation. Norovirus-positive samples were further characterised by sequence analysis using a public automated genotyping tool. RESULTS: The four outbreaks affected a total of 99 soldiers among the 618 stationed on base units and in a military exercise. A total of 27 soldiers provided a stool sample, of which 20 were positive for norovirus by real-time PCR. Phylogenetic analysis showed that the noroviruses involved were all genogroup II, namely GII.17, GII.Pe-GII.4 Sydney 2012, GII.P2-GII.2 and GII.P16-GII.2. Of note, 30 soldiers had to receive treatment at the military hospital due to severity of symptoms. CONCLUSION: In this short, two-year surveillance period, a total of four norovirus gastroenteritis outbreaks were detected in the Portuguese Army which caused a considerable morbidity, showing once again the impact of norovirus on Army effectiveness and force readiness.

Serologic evidence for Schmallenberg virus circulation at high altitude, Central Portugal, 2015-2016.

A few years after the initial 2011 large scale Schmallenberg virus (SBV) epidemic that affected Europe, a subsequent decrease in new SBV infections was observed presumably associated with natural substitution of previously exposed animals. In the present work, a 2-year prospective serosurvey was performed to evaluate SBV circulation in a population of sheep living at high altitude in the central region of Portugal and with restricted movement. Blood from a representative sample of 168 autochthonous sheep was collected in 2015 and again in 2016, and tested for the presence of anti-SBV IgG by ELISA. Of the 2015 sample collection, seven animals tested positive for anti-SBV IgG, corresponding to a seroprevalence of 4.2% while of the 2016 sample collection, 10 presented SBV antibodies, showing a seroprevalence of 6.0% (p = 0.619). Results show that SBV is endemic in sheep of central Portugal, even in herds at high altitude locations. When comparing anti-SBV seroprevalences of 2015/2016 found in this study, to one detected in 2014 in the same region, a steep decrease could be observed (p < 0.001). This is in accordance with what has been documented in Western European countries, where a decrease in the number of SBV-infected sheep has been found, a fact which may pose a new threat for SBV re-emergence.

Rubella antibodies in cord blood sera in Portugal: association with maternal age and vaccination status.

This study evaluated the impact of maternal vaccination against rubella on the levels of specific rubella IgG (rIgG) in 198 newborn cord sera samples. Detailed maternal vaccination data were available. Specific rIgG was measured using a commercial enzyme immunoassay. Most mothers (78.8%) had been vaccinated against rubella at least once in their lives. In 15 (7.6%) cord sera samples, the concentration of specific rIgG was below 11 IU/ml, which was classified as seronegative. Statistical analysis using multiple logistic regression (n = 198) showed that newborns of mothers born between 1986 and 1995, and those born to unvaccinated mothers, were more likely to be seronegative (odds ratio (ORs) 5.2 and 4.9, respectively, adjusted for sex and gestational age). For vaccinated mothers (n = 156), those born between 1986 and 1995 were more likely to have seronegative newborns (OR 11.5 adjusting for sex, gestational age and time since last vaccination). Mothers of the 15 (7.6%) seronegative newborns might have been susceptible to rubella during pregnancy. Checking the vaccination status therefore recommended.

Nanobiostructure of fibrous-like alumina functionalized with an analog of the BP100 peptide: Synthesis, characterization and biological applications.

The functionalization of alumina nanoparticles of specific morphology with antimicrobial peptides (AMP) can be a promising strategy for modeling medical devices and packaging materials for cosmetics, medicines or food, since the contamination by pathogens could be reduced. In this paper, we show the synthesis of a fibrous-like alumina nanobiostructure, as well as its functionalization with the peptide EAAA-BP100, an analog of the antimicrobial peptide BP100. The antibacterial activity of the obtained material against some bacterial strains is also investigated. The covalent binding of the peptide to the nanoparticles was promoted by a reaction between the carboxyl group of the glutamate side chain (E1) of the peptide and the amino groups of the alumina nanoparticles, previously modified by reaction with 3-aminopropyltrietoxysilane (APTES). The functionalized nanoparticles were characterized by zeta potential measurements, Fourier transform infrared spectroscopy, and other physicochemical techniques. Although the obtained alumina nanobiostructure shows a relatively low degree of substitution with EAAA-BP100, antibacterial activities against Escherichia coli and Salmonella typhimurium strains are appreciably higher than the activities of the free peptide. The obtained results can affect the design of new hybrid nanobiomaterials based on nanoparticles functionalized with AMP.

Prevalence of hepatitis E virus antibodies in workers occupationally exposed to swine in Portugal.

The concept of zoonotic hepatitis E in industrialized countries has emerged with the discovery of swine strains of hepatitis E virus (HEV) genotype 3, closely related to human HEV. Different routes of zoonotic HEV transmission have been recognized, including contact with infected pigs. Workers occupationally exposed to swine (WOES) have been considered a risk group for HEV infection, but contradictory results have been reported. In the present study, we searched for anti-HEV IgG in WOES (butchers, slaughterhouse workers, veterinarians and pig farmers; n = 114) and in the general population (n = 804) in order to investigate the potential occupational risk of zoonotic HEV infection in this work group. A significantly higher (p = 0.008) anti-HEV IgG seroprevalence was found in WOES (30.7 %) when compared with the general population (19.9 %). Multivariate analysis showed that having professions with exposure to pigs for more than 16.5 years was a risk factor for being positive for anti-HEV IgG (aOR of 5.4, 95 % CI 1.9-15.6, p = 0.002). To our knowledge, this is the first study on the prevalence of anti-HEV IgG in WOES in Portugal, also showing increased probability for infection in this group.

Persistence of rubella and mumps antibodies, following changes in the recommended age for the second dose of MMR vaccine in Portugal.

In Portugal, the recommended age for the second dose of MMR (MMR2) was changed from 10-13 years to 5-6 years for those born in 1994 and afterwards. This study aimed to assess if MMR schedule and time elapsed from the last dose are associated with the concentration of rubella and mumps IgG antibodies. Three Portuguese birth cohorts (convenience samples) were selected for this study (66, 59 and 41 participants born respectively in 1990-1993, 1994-1995 and 2001-2003). Geometric mean concentrations (GMC) for mumps IgG were respectively 36, 30 and 38 RU/ml (P = 0·236) and for rubella IgG were 18, 20 and 17 IU/ml (P = 0·641). For both specific antibodies, no differences were observed with time since MMR2. Receiving MMR2 at 5-6 or 10-13 years was not associated with concentration of both antibodies. The GMC of rubella IgG was lower in males (P = 0·029). Taking into account previous evidence and the logistics needed to change vaccination schedules, it seems reasonable that sustaining very high coverage with two doses of MMR is currently the most pragmatic way to control mumps and rubella rather than any changes to the schedule.

Hepatitis E Virus in Sylvatic and Captive Wild Boar from Portugal.

Hepatitis E virus (HEV) is a zoonotic agent today considered a major Public Health issue in industrialized countries. HEV strains belonging to zoonotic genotype 3 are widely present in swine, being today considered important reservoirs for human disease. Unlike in swine, only scarce data are available on the circulation of HEV in wild boar. This study describes the detection and molecular characterization of HEV in livers from sylvatic wild boar hunted in Portugal and destined for consumption. Additionally, the detection of HEV in stools of a confined wild boar population also destined for consumption is also described. A total of 80 liver samples collected during the hunting season of 2011/2012 and 40 stools collected in February 2012 from a wild boar breeding farm in Portugal were tested by a nested broad-spectrum RT-PCR assay targeting open reading frame (ORF) 1. Twenty livers (25.0%) and 4 stools (10%) were positive for HEV. Phylogenetic analysis showed that all strains clustered with sequences classified as HEV genotype 3 subgenotype e. To our knowledge, this is the first report documenting the occurrence and molecular analysis of HEV in sylvatic and captive wild boar destined for human consumption in Portugal. This report demonstrates for the first time the circulation of HEV in wildlife reservoirs of Portugal adding knowledge to the epidemiology of HEV in wild boar populations.

Evidence of autochthonous hepatitis E in a Portuguese pediatric cohort, 1992-1995.

Despite the growing importance of hepatitis E virus (HEV) in industrialized countries, minimal attention has been given to autochthonous HEV infection in children. The present study screened archived sera (N = 71) from a Portuguese pediatric cohort collected in 1992-1995 for the presence of antibodies against HEV, using enzyme immunoassays and immunodot. Anti-HEV IgG was detected in two children and anti-HEV IgM in one, suggesting an acute HEV infection. This shows that HEV was circulating in the paediatric population of Portugal in the early 1990s.

Outbreak of Porcine Epidemic Diarrhea Virus in Portugal, 2015.

An outbreak of porcine epidemic diarrhea virus (PEDV) in the South of Portugal in January 2015 and the spread of PEDV northwards in the territory are described. Comparative analysis of the amplified sequences showed a very high (99.0%) identity with the PEDV variant most recently reported in the United States and also show complete (100%) identity to the strains recently reported in Germany, supporting the hypothesis that a unique strain is currently circulating in Europe. The origin of this PEDV variant still needs to be elucidated and further studies in the remaining European countries may contribute to the knowledge.