Hepatitis E virus in wild and domestic rabbits from Portugal: a combined molecular and longitudinal serological study.

Hepatitis E virus (HEV), species Paslahepevirus balayani, poses a global public health threat, especially in developing countries, by causing acute enterically transmitted hepatitis. HEV infects various mammalian hosts and belongs to the genus Paslahepevirus in the family Hepeviridae. While swine are recognized as the main hosts of HEV, rabbits, which can also be affected by swine HEV-3 related strains, serve as the primary reservoir for the distinct emerging and zoonotic HEV-3ra subtype. In Portugal, where the European wild rabbit is abundant, their role in HEV epidemiology remains unclear. The primary aim of the present research was to evaluate the circulation and the potential for HEV infection within these species. This study employed a molecular and longitudinal serological approach to investigate HEV in Portuguese rabbits. Among the 205 wild rabbits tested, a seroprevalence of 2.44% (95% CI: 0.80-5.60) was found, with no significant associations with age, sex, localization, or sampling dates. Seropositive animals were found in the south and center regions of the country. HEV RNA was not detected in 120 fecal samples, suggesting a natural, low level, and widespread viral circulation. The study underscores the need for further research to comprehend HEV dynamics in these species, which is crucial for assessing potential transmission risks to humans.

Detection of hepatitis E virus genotype 3 in an Algerian mouse (Mus spretus) in Portugal.

Virus monitoring in small mammals is central to the design of epidemiological control strategies for rodent-borne zoonotic viruses. Synanthropic small mammals are versatile and may be potential carriers of several microbial agents. In the present work, a total of 330 fecal samples of small mammals were collected at two sites in the North of Portugal and screened for zoonotic hepatitis E virus (HEV, species Paslahepevirus balayani). Synanthropic small mammal samples (n = 40) were collected in a city park of Porto and belonged to the species Algerian mouse (Mus spretus) (n = 26) and to the greater white-toothed shrew (Crocidura russula) (n = 14). Furthermore, additional samples were collected in the Northeast region of Portugal and included Algerian mouse (n = 48), greater white-toothed shrew (n = 47), wood mouse (Apodemus sylvaticus) (n = 43), southwestern water vole (Arvicola sapidus) (n = 52), Cabrera's vole (Microtus cabrerae) (n = 49) and Lusitanian pine vole (Microtus lusitanicus) (n = 51). A nested RT-PCR targeting a part of open reading frame (ORF) 2 region of the HEV genome was used followed by sequencing and phylogenetic analysis. HEV RNA was detected in one fecal sample (0.3%; 95% confidence interval, CI: 0.01-1.68) from a synanthropic Algerian mouse that was genotyped as HEV-3, subgenotype 3e. This is the first study reporting the detection of HEV-3 in a synanthropic rodent, the Algerian mouse. The identified HEV isolate is probably the outcome of either a spill-over infection from domestic pigs or wild boars, or the result of passive viral transit through the intestinal tract. This finding reinforces the importance in the surveillance of novel potential hosts for HEV with a particular emphasis on synanthropic animals.

Increased risk of hepatitis E virus infection in workers occupationally exposed to sheep.

Hepatitis E virus (HEV) is an enteric RNA virus from the family Hepeviridae with five genotypes (genotypes 1-4 and 7) known to infect humans. HEV infection is known to have a zoonotic swine origin in industrialized countries. The role of pigs and wild boars as major reservoirs for human infection is today well-established; however, the list of new animal reservoirs is ever-expanding as new HEV strains are continuously being found in a broad host range. The recent detection of HEV in sheep stools brings concerns on the possibility of HEV transmission from these animals to humans, particularly in those occupationally exposed. The present work investigated the potential occupational risk of HEV infection in shepherds and sheep milk cheesemakers-workers occupationally exposed to ovine (WOEOs; N = 96)-from a region of the Centre of Portugal ('Serra da Estrela') based on the differences of anti-HEV IgG seroprevalence rates between these professionals and the general population (N = 192). The presence of HEV-specific antibodies in sheep (N = 90) from the same region was also evaluated. The HEV seroprevalence in WOEOs (29.3%) was found to be significantly higher (p = .0198) when compared with population controls (16.1%) which suggests an increased risk for HEV infection in these workers. HEV-specific antibodies were also found in 16.6% of the studied sheep showing that HEV circulates in these animals. Further studies are needed to confirm the zoonotic potential of sheep HEV.

Detection of Hepatitis E Virus in Shellfish Harvesting Areas from Galicia (Northwestern Spain).

The hepatitis E virus (HEV) affects almost 20 million individuals annually, causing approximately 3.3 million acute liver injuries, 56,600 deaths, and huge healthcare-associated economic losses. Shellfish produced close to urban and livestock areas can bioaccumulate this virus and transmit it to the human population. The aim of this study was to evaluate the presence of HEV in molluscan shellfish, in order to deepen the knowledge about HEV prevalence in Galicia (northwestern Spain), and to investigate this as a possible route of HEV transmission to humans. A total of 168 shellfish samples was obtained from two different Galician rías (Ría de Ares-Betanzos and Ría de Vigo). The samples were analyzed by reverse transcription-quantitative PCR (RT-qPCR). RT-nested PCR and sequencing were used for further genotyping and phylogenetic analysis of positive samples. HEV was detected in 41 (24.4%) samples, at quantification levels ranging from non-quantifiable (<102 copies of the RNA genome (RNAc)/g tissue) to 1.1 × 105 RNAc/g tissue. Phylogenetic analysis based on the open reading frame (ORF)2 region showed that all sequenced isolates belonged to genotype 3, and were closely related to strains of sub-genotype e, which is of swine origin. The obtained results demonstrate a significant prevalence of HEV in bivalve molluscs from Galician rías, reinforcing the hypothesis that shellfish may be a potential route for HEV transmission to humans.

Bulk-Tank Milk Longitudinal Serosurvey Reveals Decreasing Schmallenberg Virus Circulation in a Confined Population of Portuguese Sheep.

Introduction: In 2011, Schmallenberg virus (SBV) was first detected in dairy cattle herds in The Netherlands and Germany having since then spread across Europe. Today studies are starting to show a decrease in new SBV infections, a circumstance that raises alerts for possible re-emergence if ideal conditions for vector development occur. To assess the potential decrease in SBV circulation, we performed a 2-year longitudinal serological investigation for SBV infection at the herd level by using bulk-tank milk of a specific sheep breed from central Portugal. Materials and Methods: Bulk-tank milk samples from 68 flocks were collected in both 2015 and 2016, and lactosera were tested for IgG anti-SBV by EIA. Results and Discussion: Results show that in 2015, 92.6% (95% confidence interval [CI]: 83.9-96.8) of the bulk-tank milk samples were positive, whereas in 2016 only 77.9% (95% CI: 66.7-86.1 of the samples from the same flocks were positive. Differences in the 2015/2016 seroprevalences showed to be statistically significant (p = 0.027). This significant decrease at the herd level seems to be in agreement with reported data from other European countries and raise alerts, since increasingly favorable conditions (higher number of susceptible animals) are now present, potentially favoring SBV epidemics if improved conditions for midge replication occur in the future.

Short communication: detection and molecular characterization of hepatitis E virus in domestic animals of São Tomé and Príncipe.

As in most of the African continent, the status of hepatitis E virus (HEV) infection in domestic animals in São Tomé and Príncipe, an archipelago off the western equatorial coast of Central Africa, is also completely unknown. In the present study, we investigated the presence of HEV among domestic animals in São Tomé and Príncipe. A total of 93 stool samples from different animal species (goat, cow, pig, chicken, duck, and monkey) were tested for HEV RNA using two real-time RT-PCR assays, followed by a nested RT-PCR assay for sequencing and phylogenetic analysis. A total of six samples (1 cow stool and 5 pig stools) were found to be positive for HEV RNA of which one pig stool was positive by broad spectrum nested RT-PCR. Phylogenetic analysis showed that the retrieved sequence clustered within HEV subgenotype 3f, similar to zoonotic strains of European countries and posing interesting questions on past introduction of European HEV into São Tomé and Príncipe archipelago. This is the first report describing the presence and molecular characterization of HEV in São Tomé and Príncipe.

Hepatitis E Virus Genotype 1 Cases Imported to Portugal from India, 2016.

Hepatitis E in industrialized countries is mainly associated with genotype 3 hepatitis E virus (HEV) and normally causes a sporadic self-limiting disease in immunocompetent individuals. Unlike genotype 3, genotypes 1 and 2 circulate in developing countries, produce severe disease and occur in the epidemic form. Hepatitis E occurring in travellers returning from endemic areas in developing countries is not a novel epidemiological occurrence, however the vast majority of cases remain to be genetically studied. The present study describes two cases of severe acute hepatitis E that required hospitalization for 6 and 9 days in two individuals of Indian nationality that had recently migrated to Portugal to work. The retrieved HEV sequences both belonged to genotype 1 and had a high degree of nucleotide sequence identity, clustering with strains isolated in India and Nepal, in 2013 and 2014. Confirmed HEV genotypes of increased pathogenicity like genotype 1 are being introduced into otherwise naïve populations of industrialized countries such as European countries with consequences difficult to predict. As far as we know the present study is the first in Portugal to describe and genetically characterize imported cases of hepatitis E infection caused by HEV genotype 1.

A nationwide serosurvey of hepatitis E virus antibodies in the general population of Portugal.

Background: Evidence has shown that Hepatitis E virus (HEV) genotype 3 is autochthonous in industrialized countries due to zoonotic transmission through direct contact or consumption of raw or undercooked meat from domestic swine or wild boar. As there is lack of data on seroprevalence of HEV in the general Portuguese population, a wide survey was conducted as part of the HEPeCONTROL project (60DT2), under EEA grants funding. Methods: Sera from a representative sample of the Portuguese population (n = 1656) at different geographic locations (30 territorial units), and age (0-99 years) were collected between July 2015 and February 2016. The sera were tested for the presence of anti-HEV IgG and IgM by EIA using one of the two most commonly used commercial immunoassays in Europe. Results: The overall HEV IgG seroprevalence was found to be 16.3% increasing with age (P < 0.05) from 0.6% in the 0-9 years group to 30.1% in people older than 70 years. The seroprevalence also varied geographically with generally higher seropositivities (25-30%) in the most rural areas of Portugal. However, the geographical differences were not statistically significant (P > 0.05). Out of 1656 samples, 8 were positive for anti-HEV IgM indicating current of recent HEV infection but no significant differences were found concerning age groups, regions and sex. Conclusions: The present nation-wide survey provides insight in the epidemiology of HEV in Portugal and confirms that HEV is endemic in the Portuguese population.

Hepatitis E virus genotype 3 in mussels (Mytilus galloprovinciallis), Spain.

Coastal waters can become contaminated with both human waste from sewage treatment plants and runoff following manure application. Thus, shellfish produced close to land can bioaccumulate enteric viruses of human and animal origin, including zoonotic hepatitis E virus that infect both human and swine. The goal of this study was to evaluate the presence of HEV in shellfish from Galicia (NW Spain), a densely populated region with a strong tradition of swine farming, and one of the most important regions in the world for mussel production. We tested 81 mussel batches by RT-qPCR followed by conventional broad-spectrum nested RT-PCR and phylogenetic analysis. We have obtained 12 positive samples by RT-qPCR (14.81%) with HEV contamination levels ranging from 6.7 × 10(1) to 8.6 × 10(4) RNA copies/g digestive tissue. Phylogenetic analysis based on a 330 nt region of the ORF 1 showed that all sequenced isolates belonged to the zoonotic genotype 3 subgenotype e, being closely related to strains of human and swine origin. Results show that shellfish may be a potential route for HEV transmission to humans.

A Public Health initiative on hepatitis E virus epidemiology, safety and control in Portugal--study protocol.

BACKGROUND: The discovery of autochthonous hepatitis E in industrialized countries has changed the understanding of hepatitis E virus (HEV) infection in these regions, now known to be mainly due to zoonotic transmission of genotype 3. The foodborne route of transmission via consumption of contaminated meat from HEV infected pigs is well documented as well as the direct occupational exposure to animal reservoirs. Accumulating evidence also points to an emerging potential threat to blood safety after the identification of viremic blood donors and the documentation of HEV-contaminated blood or blood products. Moreover, the origin of several iatrogenic cases remains unclear and porcine-derived pharmaceutic products have been suspected as a cause. Severe morbidity following HEV infection in patients receiving immunosuppressive therapy and in those with severe immunodeficiency from other causes has been recently recognized as a serious consequence of this infection in industrialized countries. In Portugal no large-scale HEV seroprevalence study has been undertaken, no professional risk groups have been identified, and the risk of blood donation from HEV silent infected donors is unknown. The present paper describes seroepidemiological and molecular approaches to answer these questions. METHODS/DESIGN: To address these issues a study protocol was designed that will approach: i) the seroprevalence of HEV among the Portuguese general population; ii) HEV infection among butchers and slaughterhouse workers (occupational risk); iii) the silent HEV infection in Portuguese blood donors (HEV transfusion-associated risk); iv) the potential HEV contamination of porcine-derived pharmaceutical products. Commercial enzyme immunoassays and real-time/conventional RT-PCR assays will be used. DISCUSSION: This study is the first evaluation of the seroepidemiological status to HEV infection of the Portuguese population, the first to potentially identify professional risk groups, and to evaluate the safety of blood and blood products and porcine-derived pharmaceutics in Portugal. It will generate valuable data applicable for preventive and control measures against HEV infection (e.g., introduction of systematic screening of blood donors, control of blood products or porcine derived pharmaceutical products), thus helping to manage the burden of this viral disease.

Serological Evidence for Schmallenberg Virus Infection in Sheep of Portugal, 2014.

Between November and December of 2014, a serosurvey was set up to evaluate the presence of Schmallenberg virus (SBV) antibodies in sheep of Portugal. Sera (n = 1068) were tested using an indirect enzyme linked immunosorbent assay (ID Screen(®) Schmallenberg virus indirect, IDvet Innovative Diagnostics, Montpellier, France). The estimated occurrence of immunogobulin G (IgG) antibodies against SBV in sheep of Portugal was 12.8% (95% confidence interval 11.0-15.0%). This is the first study reporting the presence of SBV antibodies in sheep of Portugal.

Multiple enteropathogenic viruses in a gastroenteritis outbreak in a military exercise of the Portuguese Army.

BACKGROUND: Gastroenteritis is one of the most common infectious diseases in the military populations and can diminish operational effectiveness and impede force readiness. OBJECTIVES: The present study investigates the cause and the source of an acute gastroenteritis outbreak that occurred during a military exercise of the Portuguese Army, in February 2013. STUDY DESIGN: A retrospective investigation was performed and stool samples, food items and water were screened for common foodborne bacteria and viruses, namely Norovirus GI, Norovirus GII, Astrovirus, Rotavirus, Adenovirus and Sapovirus. RESULTS: From the total of 160 soldiers that participated in the military exercise 20 developed gastroenteritis (attack rate of 12.5%). Symptoms were predominantly vomiting (n=17, 85%) and diarrhoea (n=9, 45%). The first cases occurred 24-48h after drinking water from the creek, the plausible origin of the outbreak. The epidemic peak was registered 2 days after and the last cases 6 days after, upon returning to base. No pathogenic bacteria were found in stools however virological analysis revealed the presence of multiple enteropathogenic viruses, namely Norovirus GI (GI.3), Norovirus GII (GII.4 New Orleans 2009), Astrovirus and Sapovirus, as single or co-infections. Food and water samples were not tested for the presence of viruses due to exhaustion of samples on bacteriological analysis. CONCLUSIONS: To the best of our knowledge this is the first report of a viral gastroenteritis outbreak among military personnel in the Portuguese Army.

The epidemiology of blood-contaminated needlestick injuries among veterinarians in Portugal.

Needlestick injuries (NIs) are considered a substantial occupational health and safety hazard in contemporary health care practice. Unlike human medicine where much effort has been devoted to reduce the incidence of these events, the same aggressive approach has not been used in veterinary medicine. This study investigated the occurrence of blood-contaminated NIs in Portuguese veterinarians. Participants of a veterinary meeting were asked to complete a questionnaire-based survey. Univariate and multivariate logistic regression analyses were performed to produce predicted probabilities for NI episodes in veterinarians. From the total of 373 enrolled veterinarians, 293 (78.5%) reported having had at least one NI during their professional life. Veterinarians working with dogs were more likely to have experienced a NI (adjusted odds ratio [aOR]: 145.74, P < .001). The high level of NIs observed in these professionals shows that NIs are a potential occupational health problem in Portuguese veterinarians, with the possibility for transmission of haematogenous zoonosis.

First report of a norovirus outbreak associated with the variant Sydney 2012 in Portugal.

INTRODUCTION: This study describes the investigation of a gastroenteritis outbreak in a group of students, associated with a dinner reunion in February 2013 in Porto, Portugal. METHODOLOGY: An anonymous structured questionnaire was developed and sent to 34 students who attended the dinner reunion. Eighteen students completed the questionnaire and thirteen met the case definition (attack rate of 72%). Stools from two students were screened for norovirus by RT-PCR using primer pairs that target the highly conserved polymerase gene and the capsid gene. RESULTS: Norovirus genotyping confirmed the variant Sydney 2012 as the probable cause of the outbreak. CONCLUSION: This is the first report of an outbreak associated with the new variant Sydney 2012 in Portugal.

Seroprevalence of canine norovirus in 14 European countries.

To investigate the prevalence of the recently described genogroup VI canine noroviruses (CNVs) in dogs in Europe, we tested 510 serum samples from dogs in 14 European countries for anti-IgG CNV antibodies. Seropositive dogs were found throughout Europe. Dogs with antibodies against human noroviruses were also found.

The viral polymerase inhibitor 2'-C-methylcytidine inhibits Norwalk virus replication and protects against norovirus-induced diarrhea and mortality in a mouse model.

Human noroviruses are a major cause of food-borne illness, accountable for 50% of all-etiologies outbreaks of acute gastroenteritis (in both developing and developed countries). There is no vaccine or antiviral drug for the prophylaxis or treatment of norovirus-induced gastroenteritis. We recently reported the inhibitory effect of 2'-C-methylcytidine (2CMC), a hepatitis C virus polymerase inhibitor, on the in vitro replication of murine norovirus (MNV). Here we evaluated the inhibitory effect of 2CMC on in vitro human norovirus replication through a Norwalk virus replicon model and in a mouse model by using AG129 mice orally infected with MNV. Survival, weight, and fecal consistency were monitored, and viral loads in stool samples and organs were quantified. Intestines were examined histologically. 2CMC reduced Norwalk virus replicon replication in a dose-dependent manner and was able to clear cells of the replicon. Treatment of MNV-infected AG129 mice with 2CMC (i) prevented norovirus-induced diarrhea; (ii) markedly delayed the appearance of viral RNA and reduced viral RNA titers in the intestine, mesenteric lymph nodes, spleen, lungs, and stool; (iii) completely prevented virus-induced mortality; and (iv) resulted in protective immunity against a rechallenge. We demonstrate for the first time that a small-molecule inhibitor of norovirus replication protects from virus-induced disease and mortality in a relevant animal model. These findings pave the way for the development of potent and safe antivirals as prophylaxis and therapy of norovirus infection.

Natural compounds with cell growth inhibitory activity in human tumor cell lines.

Twenty six compounds (coumarins, flavonoids and alkaloids) were evaluated for their ability to inhibit the growth of three human tumor cell lines: breast adenocarcinoma (MCF-7), non-small cell lung cancer (NCI-H460) and melanoma (A375-C5). Three of them [3-hydroxyflavone (6), 2'-hydroxy-3,4,4',5,6'-pentamethoxychalcone (11), Siderin (20)] were very potent in inhibiting all human tumor cell lines tested. The structure / activity relationship is discussed.

Sulfated small molecules targeting eBV in Burkitt lymphoma: from in silico screening to the evidence of in vitro effect on viral episomal DNA.

Epstein-Barr virus (EBV) infects more than 90% of the world population. Following primary infection, Epstein-Barr virus persists in an asymptomatic latent state. Occasionally, it may switch to lytic infection. Latent EBV infection has been associated with several diseases, such as Burkitt lymphoma (BL). To date, there are no available drugs to target latent EBV, and the existing broad-spectrum antiviral drugs are mainly active against lytic viral infection. Thus, using computational molecular docking, a virtual screen of a library of small molecules, including xanthones and flavonoids (described with potential for antiviral activity against EBV), was carried out targeting EBV proteins. The more interesting molecules were selected for further computational analysis, and subsequently, the compounds were tested in the Raji (BL) cell line, to evaluate their activity against latent EBV. This work identified three novel sulfated small molecules capable of decreasing EBV levels in a BL. Therefore, the in silico screening presents a good approach for the development of new anti-EBV agents.

Prevalence and transmission of hepatitis E virus in domestic swine populations in different European countries.

BACKGROUND: Hepatitis E virus (HEV) genotype 3 and 4 can cause liver disease in human and has its main reservoir in pigs. HEV investigations in pigs worldwide have been performed but there is still a lack of information on the infection dynamics in pig populations. FINDINGS: The HEV transmission dynamics in commercial pig farms in six different European countries was studied. The data collected show prevalence in weaners ranging from 8% to 30%. The average HEV prevalence in growers was between 20% and 44%. The fatteners prevalence ranged between 8% and 73%. Sows prevalence was similar in all countries. Boar faeces were tested for HEV only in Spain and Czech Republic, and the prevalence was 4.3% and 3.5% respectively. The collected data sets were analyzed using a recently developed model to estimate the transmission dynamics of HEV in the different countries confirming that HEV is endemic in pig farms. CONCLUSIONS: This study has been performed using similar detection methods (real time RT-PCR) for all samples and the same model (SIR model) to analyse the data. Furthermore, it describes HEV prevalence and within-herd transmission dynamics in European Countries (EU): Czech Republic, Italy, Portugal, Spain, The Netherlands and United Kingdom, confirming that HEV is circulating in pig farms from weaners to fatteners and that the reproductive number mathematical defined as R0 is in the same range for all countries studied.

Treatment of Akata EBV-positive cells with doxorubicin causes more EBV reactivation than treatment with etoposide.

BACKGROUND/AIMS: EBV has been associated with Burkitt lymphoma (BL). It establishes a latent infection but its reactivation has been observed in patients receiving long-term chemotherapy. The effect of doxorubicin on virus reactivation has been described previously, but the effect of etoposide or cytarabine on EBV reactivation has not been reported in the literature. The aim of this work was to carry out such a study. METHODS: Akata EBV-positive cell lines were treated with etoposide, doxorubicin or cytarabine. Viable cells were analyzed by trypan blue, programmed cell death by TUNEL assay, mRNA levels by RT-PCR and cellular or viral proteins by Western blot. Viruses were visualized by electron microscopy. RESULTS: All of the studied drugs caused cell death by apoptosis. Comparing the effect of etoposide and doxorubicin (at their IC(50)) in the EBV-positive cells, etoposide caused less EBV reactivation than doxorubicin. Cytarabine apparently did not reactivate EBV. CONCLUSION: When treating Akata EBV-positive cells with the respective IC(50) of the following drugs, etoposide induced less EBV reactivation than doxorubicin, and cytarabine apparently did not induce EBV reactivation.