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.

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.

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.

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.

The enterovirus protease inhibitor rupintrivir exerts cross-genotypic anti-norovirus activity and clears cells from the norovirus replicon.

Potent and safe inhibitors of norovirus replication are needed for the treatment and prophylaxis of norovirus infections. We here report that the in vitro anti-norovirus activity of the protease inhibitor rupintrivir is extended to murine noroviruses and that rupintrivir clears human cells from their Norwalk replicon after only two passages of antiviral pressure. In addition, we demonstrate that rupintrivir inhibits the human norovirus (genogroup II [GII]) protease and further explain the inhibitory effect of the molecule by means of molecular modeling on the basis of the crystal structure of the Norwalk virus protease. The combination of rupintrivir with the RNA-dependent RNA polymerase inhibitors 2'-C-methylcytidine and favipiravir (T-705) resulted in a merely additive antiviral effect. The fact that rupintrivir is active against noroviruses belonging to genogroup I (Norwalk virus), genogroup V (murine norovirus), and the recombinant 3C-like protease of a GII norovirus suggests that the drug exerts cross-genotypic anti-norovirus activity and will thus most likely be effective against the clinically relevant human norovirus strains. The design of antiviral molecules targeting the norovirus protease could be a valuable approach for the treatment and/or prophylaxis of norovirus infections.

Pet veterinarians have no increased risk of hepatitis E compared to the general population.

The discovery of autochthonous hepatitis E in industrialized countries associated to genotype 3 has changed substantially the understanding about hepatitis E virus (HEV) circulation. In these countries transmission has been associated to the consumption and direct contact to swine, a well-known reservoir for HEV. More animal reservoirs for HEV might exist since antibodies to HEV have been detected in other animal species, such as dogs and cats. Given the intimate contact between these pets and humans, the potential zoonotic HEV transmission from dogs and cats deserves to be explored. To address this issue, 493 sera from pet veterinarians (373) and matched general population (120) were tested for the presence of anti-HEV IgG using a commercial ELISA. Antibodies to HEV were found in 9.9% veterinarians and 13.3% general population. No statistically significant difference was found between these two groups showing that pet veterinarians have no increased risk to hepatitis E compared to the general population.

Antibodies to hepatitis E in Portuguese mothers and their newborns.

The high mortality of pregnant women is one of the main features of hepatitis E virus (HEV) infection in endemic countries. With the emergence of genotype 3 HEV in industrialized countries, attention should be given to pregnant women from these regions since the effect of genotype 3 HEV in pregnancy is not yet known. In this study, archived sera dating from 1993 from mothers with no history of traveling to HEV endemic countries and their newborns were tested for anti-HEV IgG antibodies. Four of the 12 maternal sera were positive for IgG anti-HEV, indicating exposure to this virus. Their newborns were also positive, showing higher anti-HEV IgG levels. Overall, these findings demonstrate not only an efficient transplacental transport of anti-HEV IgG, but also circulation of autochthonous HEV in Portugal in the early 1990s.

Favipiravir (T-705) inhibits in vitro norovirus replication.

Human noroviruses are the primary cause of foodborne gastroenteritis. Potent and safe inhibitors are needed for the treatment/prophylaxis of norovirus infections. We demonstrate that Favipiravir [T-705, a drug in advanced clinical development for the treatment of infections with the influenza virus] inhibits in vitro murine norovirus replication. Time-of-drug addition studies reveal that T-705 exerts its activity at a time-point that coincides with onset of viral RNA synthesis, which is in line with the viral polymerase as the presumed target.

Inhibition of norovirus replication by the nucleoside analogue 2'-C-methylcytidine.

We here report on the activity of 2'-C-methylcytidine (2CMC) [a nucleoside polymerase inhibitor of the hepatitis C virus (HCV)] on the in vitro replication of (murine) norovirus (MNV). 2CMC inhibits (i) virus-induced CPE formation, (ii) viral RNA synthesis and (iii) infectious progeny formation with EC(50) values of ∼2µM. 2CMC acts at a time-point that coincides with the onset of viral RNA synthesis. Even following 30 passages of selective pressure no MNV-resistant virus was selected, which is in line with the high barrier to resistance of the nucleoside analogue for HCV. When combined with the broad-spectrum RNA virus inhibitor ribavirin, a marked antagonistic activity was observed indicating that these molecules should not be combined for the treatment of norovirus infections. Our results suggest that 2'-C-methyl nucleoside analogues should be further explored for the treatment and prophylaxis of norovirus infections.

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.

A foodborne outbreak of norovirus gastroenteritis associated with a Christmas dinner in Porto, Portugal, December 2008.

An outbreak of acute norovirus gastroenteritis was detected and epidemiologically linked to a Christmas dinner reunion of 22 recent graduate students in a restaurant in Porto, Portugal, in December 2008. A retrospective cohort study was carried out using online standardised questionnaires. Sixteen primary and three secondary cases were identified and the risk ratios with 95% confidence intervals for each food item were calculated. The response rate to the online questionnaires was 96%. The outbreak met all four Kaplan s criteria and the attack rate was 73%. Norovirus GII.4 2006b was detected in stools and emesis samples of two primary cases. The ingestion of soup and lettuce salad was considered a risk factor for this norovirus outbreak, as determined by statistical analysis. Our investigation demonstrated two routes of transmission of norovirus starting with foodborne exposure followed by secondary person-to-person spread. To our knowledge this is the first study identifying norovirus as the causative agent of a foodborne outbreak in Portugal.

The natural prenylated flavone artelastin is an inhibitor of ROS and NO production.

Artelastin, a prenylated flavone previously isolated from Artocarpus elasticus, was evaluated for its effect on the production of reactive oxygen species (ROS) by human polymorphonuclear neutrophils (PMNs) and nitric oxide (NO) by J774 murine macrophage cell line. Artelastin showed to be an inhibitor of ROS production due to a strong O2- scavenging activity. No effect was observed on the activity of myeloperoxidase (MPO). Artelastin showed also to be an inhibitor of NO production without NO scavenging activity. This flavone seems to interfere with the expression of the inducible nitric oxide synthase (iNOS) immediately after LPS-IFNgamma-macrophage stimulation.

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.

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.

Xanthone derivatives: new insights in biological activities.

Xanthones or xanthen-9H-ones (dibenzo-gamma-pirone) comprise an important class of oxygenated heterocycles whose role is well-known in Medicinal Chemistry. The biological activities of this class of compounds are associated with their tricyclic scaffold but vary depending on the nature and/or position of the different substituents. In this review, an array of biological/pharmacological effects is presented for both natural and synthetic xanthone derivatives, with an emphasis on some significant studies on structure-activity relationships. The antitumor activity of some xanthones as well as the related targets, particularly PKC modulation studies, is also discussed in detail. Examples of the "hit" compounds involved in cancer therapy, namely DMXAA, psorospermin, mangiferin, norathyriol, mangostins, and AH6809, a prostanoid receptor antagonist, are also mentioned. Finally, a historical perspective of these xanthonic derivatives, their relevance as therapeutic agents and/or their uses as pharmacological tools and as extract components in folk medicine are also highlighted.

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.

Inhibition of lymphocyte proliferation by prenylated flavones: artelastin as a potent inhibitor.

Eight natural prenylated flavones, previously isolated from Artocarpus elasticus, were evaluated for their effect on the mitogenic response of human lymphocytes to PHA. They all exhibited a dose-dependent suppression effect. An interesting relationship was observed between their antiproliferative activity and their chemical structure. Indeed, the most potent flavones possessed a 3,3-dymethylallyl group (prenyl) at C-8, such as artelastin, which exhibited the highest antiproliferative activity. Studies of the mechanism underlying its effect revealed that artelastin had an irreversible inhibitory effect on the PHA-induced lymphocyte proliferation and could affect the course of the ongoing mitogenic response either at the initial induction phase or at the late phase of proliferation. This prenylated flavone was also shown to be a potent inhibitor of both T- and B-lymphocyte mitogen induced proliferation although B-mitogenic response was the more sensitive one. Artelastin did not affect either the basal levels of the early marker of activation CD69 on non-stimulated splenocytes or its expression on ConA- or LPS-stimulated splenocytes. However, it decreased the production of IFN-gamma, IL-2, IL-4 and IL-10 in ConA-stimulated splenocytes. Furthermore, artelastin had no effect on apoptosis of splenocytes.

Gastroenteritis outbreak associated with faecal shedding of canine norovirus in a Portuguese kennel following introduction of imported dogs from Russia.

We describe an outbreak of acute gastroenteritis (AGE) in dogs in March 2011 in a Portuguese kennel after the introduction of imported Russian dogs. Canine norovirus was detected in faecal samples of all dogs with AGE symptoms. Partial sequence analysis of the RT-PCR products confirmed that all canine norovirus strains were identical. The canine norovirus infection disseminated rapidly in 2 days to all dogs in the kennel demonstrating the highly contagious nature of this virus. The incubation period was <48 h, the diarrhoeal disease was self-limiting and the viral shedding lasted <7 days. Overall, the epidemiological features of this outbreak resembled those of human norovirus infections.

Detection and characterization of hepatitis E virus in domestic pigs of different ages in Portugal.

This study represents the primary hepatitis E virus (HEV) surveillance in domestic pigs in Portugal, five pig farms were investigated in 5 different Portuguese regions, ten faecal samples were collected at four different stages of the production. All faecal samples were tested for hepatitis E virus by real-time RT-PCR. At least one sample from each farms of all age groups tested positive for HEV. The prevalence in the pig herds varied from 10% to 30% and the mean prevalence was 32% in weaners, 20% in growers, 32% in fatteners and 4% in adult dry sows. Phylogenetic analysis of the detected HEV sequences indicated that the circulating virus strains belong under the genotype 3.