Genetically similar hepatitis E virus strains infect both humans and wild boars in the Barcelona area,Spain, and Sweden

Hepatitis E virus (HEV) is a hepatotropic virus, endemic in Europe where it infects humans and animals, with domestic pigs and wild boars as main reservoirs. The number of HEV‐infected cases with unknown source of infection increases in Europe. There are human HEV strains genetically similar to viruses from domestic pigs, and zoonotic transmission via consumption of uncooked pork meat has been shown. Due to continuous growth of the wild boar populations in Europe, another route may be through direct or indirect contacts with wild boars. In the Collserola Natural Park near Barcelona, Spain, the wild boars have spread into Barcelona city. In Sweden, they are entering into farmlands and villages. To investigate the prevalence of HEV and the risk for zoonotic transmissions, the presence of antibodies against HEV and HEV RNA were analysed in serum and faecal samples from 398 wild boars, 264 from Spain and 134 from Sweden and in sera from 48 Swedish patients with HEV infection without known source of infection. Anti‐HEV was more commonly found in Spanish wild boars (59% vs. 8%; p < 0.0001) while HEV RNA had similar prevalence (20% in Spanish vs. 15% in Swedish wild boars). Seven Swedish and three Spanish wild boars were infected with subtype 3f, and nine Spanish with subtype 3c/i. There were three clades in the phylogenetic tree formed by strains from wild boars and domestic pigs; another four clades were formed by strains from humans and wild boars. One strain from a Spanish wild boar was similar to strains from chronically infected humans. The high prevalence of HEV infections among wild boars and the similarity between wild boar HEV strains and those from humans and domestic pigs indicate that zoonotic transmission from wild boar may be more common than previously anticipated, which may develop into public health concern.     

Hepatitis E virus in sheep in Italy

Hepatitis E virus (HEV) is the leading cause of human enterically transmitted viral hepatitis occurring around the world both as outbreaks and as sporadic cases. The accumulating literature indicates that domestic pigs and wild boars are the main reservoirs of genotype 3 and genotype 4 for human infections in industrialized countries. However, the recent identification of HEV from various animal species poses additional potential concerns for HEV zoonotic infection. In this study, the role of sheep as potential host of hepatitis E virus (HEV) was investigated. By screening 192 sheep from seven farms located in Abruzzo Region (Southern Italy), HEV‐specific antibodies were detected in the sera of 41 animals (21.3%) whilst the RNA of HEV, genotype 3, was detected in 20 faecal (10.4%) and three serum samples (1.6%). Upon sequence analyses of a partial ORF2 gene region of eight HEV positive samples, the sheep sequences all grouped together within HEV genotype 3 subtype c, being most closely related to HEV strains identified in goat and wild boar from Abruzzo. This is the first study that demonstrates, serologically and molecularly, the presence of HEV in sheep population in a European country.     

Molecular detection of hepatitis E virus in wild boar population in eastern Romania

In industrialized countries, Hepatitis E is a recognized zoonosis, with wild boar and swine representing the main reservoirs for zoonotic genotype HEV ‐3 in Europe. Data related to HEV infection in wild boar population in Romania are restricted to serological surveys. Therefore, our main goal was to determine the HEV prevalence in wild boar population and to characterize HEV strains circulating in Romania. Using TaqMan real‐time RT ‐PCR assay, we analyzed the presence of RNA HEV in 45 liver samples and five spleen samples collected from 50 wild boars. Samples were collected during the 2013–2015 hunting seasons. Nine samples of 50 were tested positive for HEV RNA , resulting an overall prevalence of 18%. Phylogenetic analysis revealed that the isolates clustered in different HEV ‐3 monophyletic groups, depending on the sampling county. This is the first study signalling, based on molecular analysis, the presence of HEV in wild boar population from Romania. Also, in this study, we report the detection of HEV in splenic tissue from wild boar.     

Belgian Wildlife as Potential Zoonotic Reservoir of Hepatitis E Virus

Hepatitis E is an acute human liver disease in healthy individuals but may become chronic in immunocompromised patients. It is caused by the hepatitis E virus (HEV) and can have a zoonotic origin, particularly in high‐income countries. In this study, 383 sera from wild boars were selected for serology; for virological analyses, 69 sera and 61 livers from young wild boars were used. A total of 189 and 235 sera of, respectively, red deer and roe deer were collected for serological analysis. For virological analyses, 84 and 68 sera and 29 and 27 livers from, respectively, red and roe deer were sampled. An apparent seroprevalence of 34% (95% CI 29.71–39.46) was found in wild boars, of 1% (95% CI 0–2.4) in red deer and 3% (95% CI 0.8–4.2) in roe deer. To assess the ELISA screening prevalence, Western blot (WB) analyses were carried out, a receiver operating characteristic curve analysis was performed and different scenarios with varying ELISA specificities relative to WB were analysed. Seroprevalence remained high whatever the scenario in the wild boar population. In wild boar, 4 of 69 sera and 4 of 61 livers were detected as positive for HEV RNA. All sequences obtained from sera belonged to genotype HEV‐3. HEV RNA, belonging to genotype HEV‐3, was detected in one of 29 red deer livers. Wild boar can be considered as a host reservoir of the virus in Belgium. However, in contrast to the epidemiological role played by them in other countries, the low prevalence in deer makes these species an unlikely reservoir. This evidence needs further investigation to determine in which situation deer can serve as reservoir. These results also raise the question of the dynamics of HEV infection between wild fauna, domestic pigs and humans.     

Hepatitis E virus strains infecting Swedish domestic pigs are unique for each pig farm and remain in the farm for at least 2 years

Hepatitis E virus (HEV) genotype 3 (HEV3) is distributed globally and infects both humans and animals, mainly domestic pigs and wild boars, which are the major reservoirs. In this study, the prevalence of HEV among Swedish pigs was investigated by HEV RNA analysis in 363 faecal samples from 3‐month‐old piglets sampled twice (2013 and 2014) in 30 Swedish pig farms. Four different types of farms were investigated; organic, conventional closed (keeping the sow), satellites in a sow pool (conventional farms sharing sows) and conventional non‐closed farms (purchasing gilts). More than two‐thirds (77%) of the farms had HEV‐infected pigs. HEV RNA was found in faeces from 79 pigs (22%). Partial ORF1 could be sequenced in 46 strains. Phylogenetic analysis revealed a unique HEV3 strain for each farm. Strains sampled more than a year apart from the same farm were closely related, indicating that the same HEV strain is present for several years on the farm. Despite that only 4% of the Swedish pig farms were investigated, two farms had strains similar to those from humans, another had strains similar to wild boar HEV. The uniqueness of strains from each farm indicates a possibility to identify a source of infection down to farm level. This knowledge may be used by the farms to investigate the effectiveness of good hygiene routines to reduce the amount of HEV and thus the infection risk in the farm, and for Swedish public health authorities to identify cases of HEV transmissions from consumption of uncooked pork.     

Susceptibility of Pigs to Zoonotic Hepatitis E Virus Genotype 3 Isolated from a Wild Boar

In Europe, zoonotic hepatitis E virus (HEV) genotype 3 strains mainly circulate in humans, swine and wild boar. The aim of this study was to investigate the potential transmission of a wild boar originating HEV strain (WbHEV) to swine by intravenous or oral inoculation and to study the consequences of infection of a WbHEV strain, a WbHEV strain previously passaged in a pig and a swine HEV strain after oral inoculation. Firstly, an intravenous infection was performed for which five piglets were divided into two groups with three pigs inoculated with a WbHEV field strain and two pigs inoculated with a HEV‐negative swine liver homogenate. All pigs were necropsied 8, 9 and 10 days post‐inoculation. Secondly, an oral infection of 56 days was performed on 12 piglets divided into four groups inoculated with a WbHEV strain, a WbHEV strain previously passaged in swine, a swine HEV strain or a HEV‐negative swine liver homogenate. After intravenous inoculation, HEV RNA was detected in serum, bile, liver, spleen, duodenum, jejunum, colon, lung, gastro‐hepatic lymph nodes and faeces in all infected piglets. After oral inoculation, HEV RNA was detected in serum, bile, liver, gastro‐hepatic lymph nodes and faeces. Most of HEV‐inoculated pigs became seropositive at day 15. This study provides experimental evidence of early viral spread throughout the organism after intravenous infection with a WbHEV strain and supports the notion that such a zoonotic strain could be transmitted via the natural faecal–oral route of infection between wild boar and pigs but also between pigs.     

Constant Hepatitis E Virus (HEV) Circulation in Wild Boar and Red Deer in Spain: An Increasing Concern Source of HEV Zoonotic Transmission

Hepatitis E is a viral zoonosis that affects multiple hosts. The complete dynamics of infection in wildlife are still unknown, but the previous fact facilitates the maintenance and circulation of the virus, posing a risk to human health in the case of meat consumption from susceptible animals. In Spain, it has been shown how domestic pigs, cattle and wildlife (i.e. wild boar and red deer) clearly interact in hunting farms, generating a complex epidemiological situation in terms of interspecies pathogen transmission. Therefore, in this study, we aimed to (i) evaluate the circulation of the virus in geographically close domestic (Iberian pigs) and wild animals (wild boar and deer) living in hunting areas from central Spain over an 8‐year period (2003–2010) and (ii) to determine whether HEV could be used as a marker of domestic–wildlife contact. For these purposes, a longitudinal analysis of Iberian pig, wild boar and red deer samples (n = 287) through virological and serological tests was conducted to shed light upon the circulation events of HEV. Regarding HEV RNA detection by real‐time RT‐PCR, 10.12% samples (95% CI: 5.44–14.8) from wild boar and 16.05% samples (95% CI: 8.06–24.04) from red deer were positive. As for the Iberian pigs, none of the 48 samples was positive for HEV RNA detection. In the serological analysis, 43.75% (95% CI: 29.75–57.75) from Iberian pig, 57.40% (95% CI: 48.10–66.70) from wild boar and 12.85% (95% CI: 5.01–20.69) samples from red deer presented anti‐HEV antibodies. Positive samples were distributed among all study years (2003–2010). These results depict the urgent need to improve the inspection and surveillance of these species and their products. In the case of HEV, it is clear that the stable and constant presence of the virus in wildlife and its contact with Iberian pigs pose a risk for human health as they are all destined for human consumption.     

Emergent subtype of hepatitis E virus genotype 3 in wild boar in Spain

Wild boar (Sus scrofa) is considered as the main wildlife reservoir of zoonotic hepatitis E virus (HEV) genotypes. The aim of this study was to evaluate the circulation of HEV in free‐ranging wild boar in the Doñana National Park (DNP), Spain. Blood samples were collected from 99 wild boar in the DNP during 2015. Sera were analysed in parallel using indirect ELISA and real‐time RT‐PCR. A total of 57 of the 99 tested animals (57.6%; 95%CI: 47.8%–67.3%) had anti‐HEV antibodies, indicating that this virus is widespread in wild boar in the DNP. HEV RNA was detected in one animal and phylogenetic analysis showed that the sequence isolated belonged to subtype 3r. The results suggest a potential risk of zoonotic transmission of this novel HEV‐3 subtype, which could be of public health concern. Further studies are required to assess the role of wild boar in the epidemiology of HEV‐3r and to determine the infectivity of this emergent HEV subtype in other species, including humans.     

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.     

Presence of Hepatitis E Virus in a RED Deer (Cervus elaphus) Population in Central Italy

Hepatitis E is an acute human disease caused by the hepatitis E virus (HEV). In addition to humans, HEV has been detected in several animal species and is recognized as a zoonotic pathogen. Pigs, wild boar and deer can be reservoir. In this study, we evaluated HEV prevalence in a free‐living red deer (Cervus elaphus) population in central Italy by detecting virus‐specific antibodies and RNA in sera. A total of 35 of 251 red deer sera were positive for anti‐HEV IgG. HEV RNA was detected in 10 of 91 sera examined. Two genomic fragments targeted by diagnostic PCRs in the capsid region were sequenced, both matching with genotype 3 HEV. Overall results confirmed the occurrence of HEV infection in deer also in Italy.     

First report of wild boar susceptibility to Porcine circovirus type 3: High prevalence in the Colli Euganei Regional Park (Italy) in the absence of clinical signs

The genus Circovirus includes one of the most relevant infectious agents affecting domestic pigs, Porcine circovirus type 2 (PCV ‐2). The wild boar susceptibility to this pathogen has also been demonstrated although the actual epidemiological role of wild populations is still debated. In recent times, a new circovirus, Porcine circovirus type 3 (PCV ‐3), has been discovered and reported in the presence of several clinical conditions. However, no information is currently available about PCV ‐3 circulation and prevalence in wild boar. To fill this gap, 187 wild boar serum samples were collected in the Colli Euganei Regional Park (Northern Italy) and screened for PCV ‐3, demonstrating a high viral prevalence (approximately 30%). No gender differences were demonstrated while a lower infection prevalence was observed in animals younger than 12 months compared to older ones, differently from what described in commercial pigs. Almost all sampled animals were in good health conditions and no association was proven between PCV ‐3 status and clinical syndromes in wild animals. The genetic characterization of selected strains enlightened a relevant variability and the absence of closely related strains originating from domestic pigs. Therefore, the observed scenario is suggestive of multiple introductions from other wild or domestic swine populations followed by prolonged circulation and independent evolution. Worldwide, this study reports for the first time the high susceptibility of the wild boar to PCV ‐3 infection. The high prevalence and the absence of association with clinical signs support the marginal role of this virus in the wild boar population ecology. However, its epidemiological role as reservoir endangering commercial swine cannot be excluded and will require further investigations.     

Experimental Evaluation of Faecal Escherichia coli and Hepatitis E Virus as Biological Indicators of Contacts Between Domestic Pigs and Eurasian Wild Boar

Domestic pigs and Eurasian wild boar (Sus scrofa) share several important viral and bacterial pathogens. Therefore, direct and indirect contacts between domestic pigs and wild boar present a risk of pathogen spillover and can lead to long‐term perpetuation of infection. Biological indicators could be a powerful tool to understand and characterize contacts between wild boar and domestic pigs. Here, faecal Escherichia coli and Hepatitis E virus (HEV) were explored as potential biological indicators under experimental conditions. The data gained in our pilot study suggest that faecal E. coli can be used as biological indicator of contact between wild boar and domestic pig. For HEV, faecal transmission was also confirmed. However, molecular studies on full‐genome basis did not reveal markers that would allow tracing of transmission direction. Based on these promising results, future field studies will especially target the practicability of E. coli microbiome molecular typing as surrogate of contacts at the wildlife–livestock interface.     

Spatio‐temporal Analysis of African Swine Fever in Sardinia (2012–2014): Trends in Domestic Pigs and Wild Boar

African swine fever (ASF) is a notifiable viral disease affecting domestic pigs and wild boars that has been endemic in Sardinia since 1978. Several risk factors complicate the control of ASF in Sardinia: generally poor level of biosecurity, traditional breeding practices, illegal behaviour in movements and feeding of pigs, and sporadic occurrence of long‐term carriers. A previous study describes the disease in Sardinia during 1978–2013. The aim of this study was to gain more in‐depth knowledge of the spatio‐temporal pattern of ASF in Sardinia during 2012 to May 2014, comparing patterns of occurrence in domestic pigs and wild boar and identifying areas of local transmission. African swine fever notifications were studied considering seasonality, spatial autocorrelation, spatial point pattern and spatio‐temporal clusters. Results showed differences in temporal and spatial pattern of wild boar and domestic pig notifications. The peak in wild boar notifications (October 2013 to February 2014) occurred six months after than in domestic pig (May to early summer 2013). Notifications of cases in both host species tended to be clustered, with a maximum significant distance of spatial association of 15 and 25 km in domestic pigs and wild boars, respectively. Five clusters for local ASF transmission were identified for domestic pigs, with a mean radius and duration of 4 km (3–9 km) and 38 days (6–55 days), respectively. Any wild boar clusters were found. The apparently secondary role of wild boar in ASF spread in Sardinia could be explained by certain socio‐economic factors (illegal free‐range pig breeding or the mingling of herds. The lack of effectiveness of previous surveillance and control programmes reveals the necessity of employing a new approach). Results present here provide better knowledge of the dynamics of ASF in Sardinia, which could be used in a more comprehensive risk analysis necessary to introduce a new approach in the eradication strategy.     

Hepatitis E Virus in Wild Boar in the Central Northern Part of Italy

Hepatitis E virus (HEV) is responsible for sporadic acute hepatitis in developed countries, where the infection is acquired probably through ingestion of contaminated food, in addition to travel‐related cases. In this study, the circulation of HEV in wild boar from nine Italian provinces was evaluated. An overall seroprevalence of 10.2% was found, although there were differences among the provinces, while no samples were positive for HEV RNA detection. This study indicates an active circulation of HEV in the Italian wild boar populations and suggests to consider the zoonotic risk in handling and eating meat from this animal.     

Assessing the Risk of African Swine Fever Introduction into the European Union by Wild Boar

The presence of African swine fever (ASF) in the Caucasus region and Russian Federation has increased concerns that wild boars may introduce the ASF virus into the European Union (EU). This study describes a semi‐quantitative approach for evaluating the risk of ASF introduction into the EU by wild boar movements based on the following risk estimators: the susceptible population of (1) wild boars and (2) domestic pigs in the country of origin; the outbreak density in (3) wild boars and (4) domestic pigs in the countries of origin, the (5) suitable habitat for wild boars along the EU border; and the distance between the EU border and the nearest ASF outbreak in (6) wild boars or (7) domestic pigs. Sensitivity analysis was performed to identify the most influential risk estimators. The highest risk was found to be concentrated in Finland, Romania, Latvia and Poland, and wild boar habitat and outbreak density were the two most important risk estimators. Animal health authorities in at‐risk countries should be aware of these risk estimators and should communicate closely with wild boar hunters and pig farmers to rapidly detect and control ASF.     

The African swine fever virus isolate Belgium 2018/1 shows high virulence in European wild boar

African swine fever (ASF) is one of the most important and complex viral diseases in domestic pigs and wild boar. Over the last decade, the disease has spread to several European and Asian countries and is now one of the major threats to profitable pig production worldwide. One of the more recently affected western countries is Belgium. To date, only wild boar are affected in a rather defined area in the Luxembourg region close to France, Luxembourg and Germany. While detailed sequence analyses were recently performed, biological characterization was still pending. Here, we report on the experimental inoculation of four sub‐adult wild boar to further characterize the virus and its distribution in different tissues. After oronasal inoculation with the virus strain ‘Belgium 2018/1’, all animals developed an acute and severe disease course with typical pathomorphological and histopathological lesions. Organs and blood samples were positive in qPCR, haemadsorption test and antigen lateral flow devices (LFD). Virus and viral genome were also detected in genitals and accessory sex glands of two boars. There were no antibodies detectable in commercial antibody ELISAs, antibody LFDs and indirect immunoperoxidase tests. Thus, the genotype II ASF virus isolate ‘Belgium 2018/1’ showed a highly virulent phenotype in European wild boar similar to parental viruses like Armenia 2007 and other previously characterized ASFV strains. The study also provided a large set of well‐characterized sample materials for test validation and assay harmonization.     

Monitoring of African Swine Fever in the Wild Boar Population of the Most Recent Endemic Area of Spain

Wild boars are natural hosts for African swine fever (ASF). The ASF virus (ASFV) can persist for long periods in the environment, such as in ticks and contaminated products, which may be sources of infection for wild boar populations. African swine fever was eradicated in domestic pig populations in Spain in 1995, after 35 years of significant effort. To determine whether ASFV can persist in wild boar hosts after it has been eradicated from domestic pigs and to study the role of wild boar in helping ASFV persist in the environment, we checked for the presence of ASFV in wild boars in Doñana National Park, one of the largest natural habitats of wild boar in Spain and one of the last areas where ASF was endemic prior its eradication. Samples from 158 animals collected between 2006 and 2010 were analysed using serological and nucleic acid‐based diagnostic techniques recommended by the World Organization for Animal Health (OIE). None of the samples was found to be positive. These results confirm the absence of disease in wildlife in what was once one of the areas most affected by ASF in Spain, and they suggest that wild boars play a limited role in ASFV persistence. These results confirm that ASFV cannot persist in isolated wild boar populations for long periods of time without the interaction of other factors such as re‐infection by contact with domestic pigs or by feeding on contaminated swill.     

Porcine circovirus 3 is highly prevalent in serum and tissues and may persistently infect wild boar (Sus scrofa scrofa)

Porcine circovirus 3 (PCV‐3) prevalence has been minimally investigated in wild boar; dynamics of infection and viral tissue distribution are currently unknown. In this study, serum samples from 518 wild boar (from years 2004 to 2018) were used to study frequency of infection. Also, serum samples from 19 boar captured and recaptured at least two times for a period of time from 1 month to 1 year were collected to determine PCV‐3 infection dynamics. Finally, to elucidate PCV‐3 DNA organic distribution, sera, different tissues and faeces were obtained from 35 additional wild boar. PCV‐3 DNA was extracted and amplified with a conventional PCR. For the PCV‐3 PCR‐positive sera from the longitudinally sampled and different tissue types, a quantitative PCR was performed. Genome sequence was obtained from a number of PCV‐3 PCR‐positive samples from different years, different time‐points of infection and tissues. Obtained results confirmed the susceptibility of wild boar to the virus, showing high frequency of PCV‐3 detection (221 out of 518, 42.66%) and demonstrating circulation at least since 2004. Compiled data indicate the possibility of long‐term infections, since 5 out of 10 PCV‐3 PCR‐positive boars longitudinally sampled showed positivity in samplings separated for more than 5 months. All tested tissue types’ harboured PCV‐3 genome, with the highest percentage of PCR positivity in submandibular lymph node, tonsil, lung, liver, spleen and kidney. The amount of DNA in all tested PCV‐3 PCR‐positive samples was moderate to low. All partial and complete PCV‐3 sequences obtained from wild boar displayed high nucleotide identity, higher than 98%. In conclusion, this study further confirms that wild boar is susceptible to PCV‐3 infection, showing high frequency of detection in this animal species. Furthermore, PCV‐3 can be found in different tissues of wild boar and is apparently able to cause persistent infection.     

Statistical Exploration of Local Transmission Routes for African Swine Fever in Pigs in the Russian Federation, 2007–2014

African swine fever (ASF) is a devastating viral disease of swine that is present in both pigs and wild boar in the western part of the Russian Federation and the eastern part of the European Union. It represents a significant threat for the European pig production industry as neither treatment nor vaccine is available. This study analysed the spatial and spatio‐temporal distributions of ASF cases that were reported in domestic pigs and wild boar for assessing the likelihood of wild boar‐to‐domestic pig and farm‐to‐farm transmission routes in the epidemic that occurred from 2007 to 2014 in the Krasnodar and the Tver regions, two of the most affected areas of the Russian Federation. Results suggest that in both regions, the spatial proximity to an infectious farm was a strong risk factor for infection of a susceptible farm. In the Krasnodar region, the results of the statistical analysis suggest that the epidemics in wild boar and in domestic pigs were independent from each other. In contrast, there seemed to be a dependence between the two epidemics in the Tver region. But because outbreaks in domestic pigs were not statistically significantly clustered around wild boar cases, the joint spatial distribution of wild boar cases and of outbreaks in domestic pigs in the Tver region may be explained by regular spillovers from the domestic pig to the wild boar population. These findings confirm the need to maintain high biosecurity standards on pig farms and justify strict control measures targeted at domestic pig production such as culling of infected herds and local movement restrictions.