Localisation of swine hepatitis E virus in experimentally infected pigs

The distribution of intravenously inoculated swine hepatitis E virus (HEV) was assessed by in situ hybridisation for a period of 50 days. Evidence of apparent clinical disease was found in only one pig in the HEV infected group. The only gross lesion observed was mildly enlarged mesenteric lymph nodes at 50 days post infection (dpi). Histopathologically, mild lymphoplasmacytic infiltration and focal hepatocellular necrotic lesions were found in HEV-infected pigs. Swine HEV nucleic acids were detected by RT-PCR in the faeces at 3 dpi in 100% of the 18 pigs infected with the virus. Thereafter, the number of positives declined.The most consistent and intense signal was found in the liver of infected animals using in situ hybridisation. The positive cells were hepatocytes, Kupffer cells, bile epithelial cells and interstitial lymphocytes. Swine HEV RNA was localised in the cytoplasm of the hepatocytes, with a slightly granular pattern of staining, but hybridisation signals were not observed in degenerative or vacuolated hepatocytes. HEV was much less frequently detected in extrahepatic tissues such as lymph nodes, tonsil, spleen and small and large intestine. It was concluded that swine HEV had replicated primarily in the hepatocytes and infection resulted in subclinical infection with minimal histopathological changes in the liver.     

Hepatitis E virus in Cuba: A cross-sectional serological and virological study in pigs and people occupationally exposed to pigs

Surveillance of hepatitis E virus (HEV) in risk groups is an important strategy to monitor its circulation pattern and to timely detect changes thereof. The aims of this cross-sectional study were to estimate the prevalence of HEV infections in pigs and humans from different regions of the country, to identify risk factors for increasing anti-HEV IgG prevalence and to characterize HEV strains. The presence of anti-HEV antibodies was assessed by commercial ELISA in serum samples from the general population, farm and slaughterhouse employees, as well as pigs sampled in the three regions of Cuba from February to September 2016. Overall, individuals with occupational exposure to swine or swine products (70/248, 28.2%) were 4 times more likely to be seropositive compared to the general population (25/285, 8.7%; OR: 4.18; p < .001). Within the risk group, risk factors included age, number of years working in a professional activity with direct exposure to swine, geographic region and distance between residence and closest professional swine setting, while wearing gloves had a protective effect. Prevalence of total anti-HEV antibodies in swine was 88.2% (165/187) and HEV RNA was detected by real-time RT-PCR in 9.2% (16/173) swine stools. All HEV strains sequenced clustered within genotype 3. Some strains clearly belonged to subtype 3a, while another group of strains was related with subtypes 3b and 3 k but partial HEV sequences did not allow unequivocal subtype assignment. These findings suggest that the high HEV exposure in Cuban individuals with swine-related occupations could be due to enzootic HEV in certain regions, direct contact with infectious animals or their products as well as environmental contamination.     

Evidence of increased Hepatitis E virus exposure in Lao villagers with contact to ruminants

Although pigs are the main reservoir, ruminants have also been shown to be susceptible to hepatitis E virus (HEV). We investigated zoonotic transmission of HEV in rural settings of Lao People's Democratic Republic (Lao PDR) where humans are in close contacts with ruminants and where pigs are rare. Villagers with (n = 171, risk group) and without (n = 155, control group) cattle were recruited in seven villages in Vientiane Capital. Owners of pigs were excluded. Blood, as well as information on socio‐demographics, animal contact, dietary habits and awareness of zoonoses were collected to assess risk factors. Blood and rectal swabs were collected from cattle (n = 173) and other ruminants (27 goat, 5 buffaloes) to measure anti‐HEV antibody and virus prevalence. A similar anti‐HEV antibody seroprevalence was found in cattle (6.8%) and other ruminants (8%). HEV RNA was detected in none of the animal rectal swabs and human sera. Anti‐HEV IgG seroprevalence was higher in cattle farmers than in the control group (59.1% vs. 43.9%, p = 0.008) and increased significantly with age. Other risk factors included male gender, close contact with cattle and consumption of undercooked meat. We find that HEV is highly endemic in rural Laos and provide first evidence that HEV circulates in free‐roaming ruminants with open access to village water sources. Despite some awareness about hygiene, villagers are likely constantly exposed to zoonotic diseases by dietary and lifestyle habits. Cattle farmers had a higher risk of HEV infection than other villagers. Our study highlights the need to raise the awareness of the rural population about water‐ and food‐borne pathogens, and about the role of cattle as a possible source of infection. The knowledge gained on local risk factors and husbandry conditions should guide future awareness raising campaigns and promote appropriate hygienic measures including handwashing and the consumption of safe food and water.     

Direct contact and environmental contaminations are responsible for HEV transmission in pigs

Hepatitis E virus (HEV) can cause enterically-transmitted hepatitis in humans. The zoonotic nature of Hepatitis E infections has been established in industrialized areas and domestic pigs are considered as the main reservoir. The dynamics of transmission in pig herds therefore needs to be understood to reduce the prevalence of viremic pigs at slaughter and prevent contaminated pig products from entering the food chain. An experimental trial was carried out to study the main characteristics of HEV transmission between orally inoculated pigs and naïve animals. A mathematical model was used to investigate three transmission routes, namely direct contact between pigs and two environmental components to represent within-and between-group oro-fecal transmission. A large inter-individual variability was observed in response to infection with an average latent period lasting 6.9 days (5.8; 7.9) in inoculated animals and an average infectious period of 9.7 days (8.2; 11.2). Our results show that direct transmission alone, with a partial reproduction number of 1.41 (0.21; 3.02), can be considered as a factor of persistence of infection within a population. However, the quantity of virus present in the environment was found to play an essential role in the transmission process strongly influencing the probability of infection with a within pen transmission rate estimated to 2 ⋅ 10^− 6g ge^− 1d^− 1(1 ⋅ 10^− 7; 7 ⋅ 10^− 6). Between-pen environmental transmission occurred to a lesser extent (transmission rate: 7 ⋅ 10^− 8g ge^− 1d^− 1(5 ⋅ 10^− 9; 3 ⋅ 10^− 7) but could further generate a within-group process. The combination of these transmission routes could explain the persistence and high prevalence of HEV in pig populations.     

Natural and experimental hepatitis E virus genotype 3 - infection in European wild boar is transmissible to domestic pigs

Hepatitis E virus (HEV) is the causative agent of acute hepatitis E in humans in developing countries, but sporadic and autochthonous cases do also occur in industrialised countries. In Europe, food-borne zoonotic transmission of genotype 3 (gt3) has been associated with domestic pig and wild boar. However, little is known about the course of HEV infection in European wild boar and their role in HEV transmission to domestic pigs. To investigate the transmissibility and pathogenesis of wild boar-derived HEVgt3, we inoculated four wild boar and four miniature pigs intravenously. Using quantitative real-time RT-PCR viral RNA was detected in serum, faeces and in liver, spleen and lymph nodes. The antibody response evolved after fourteen days post inoculation. Histopathological findings included mild to moderate lymphoplasmacytic hepatitis which was more prominent in wild boar than in miniature pigs. By immunohistochemical methods, viral antigens were detected mainly in Kupffer cells and liver sinusoidal endothelial cells, partially associated with hepatic lesions, but also in spleen and lymph nodes. While clinical symptoms were subtle and gross pathology was inconspicuous, increased liver enzyme levels in serum indicated hepatocellular injury. As the faecal-oral route is supposed to be the most likely transmission route, we included four contact animals to prove horizontal transmission. Interestingly, HEVgt3-infection was also detected in wild boar and miniature pigs kept in contact to intravenously inoculated wild boar. Given the high virus loads and long duration of viral shedding, wild boar has to be considered as an important HEV reservoir and transmission host in Europe.

Electronic supplementary material

The online version of this article (doi:10.1186/s13567-014-0121-8) contains supplementary material, which is available to authorized users.     

Serological evidence of hepatitis E virus infection in pigs and jaundice among pig handlers in Bangladesh

Hepatitis E virus (HEV) is the most common cause of viral hepatitis in humans. Pigs may act as a reservoir of HEV, and pig handlers were frequently identified with a higher prevalence of antibodies to HEV. The objectives of this study were to identify evidence of HEV infection in pigs and compare the history of jaundice between pig handlers and people not exposed to pigs and pork. Blood and faecal samples were collected from 100 pigs derived from three slaughterhouses in the Gazipur district of Bangladesh from January to June, 2011. We also interviewed 200 pig handlers and 250 non‐exposed people who did not eat pork or handled pigs in the past 2 years. We tested the pig sera for HEV‐specific antibodies using a competitive ELISA and pig faecal samples for HEV RNA using real‐time RT‐PCR. Of 100 pig sera, 82% (n = 82) had detectable antibody against HEV. Of the 200 pig handlers, 28% (56/200) demonstrated jaundice within the past 2 years, whereas only 17% (43/250) of controls had a history of jaundice (p < .05). Compared to non‐exposed people, those who slaughtered pigs (31% versus 15%, p < .001), reared pigs (37% versus 20%, p < .001), butchered pigs (35% versus 19%, p < .001) or involved in pork transportation (28% versus 13%, p < .001) were more likely to be affected with jaundice in the preceding 2 years. In multivariate logistic regression analysis, exposure to pigs (odds ratio [OR]: 2.2, 95% CI: 1.2–3.9) and age (OR: 0.97, 95% CI: 0.95–0.99) was significantly associated with jaundice in the past 2 years. Pigs in Bangladesh demonstrated evidence of HEV infection, and a history of jaundice was significantly more frequent in pig handlers. Identifying and genotyping HEV in pigs and pig handlers may provide further evidence of the pig's role in zoonotic HEV transmission in Bangladesh.     

The reduction of CSFV transmission to untreated pigs by the pestivirus inhibitor BPIP: A proof of concept

5-[(4-Bromophenyl)methyl]-2-phenyl-5H-imidazo[4,5-c]pyridine (BPIP) is a representative molecule of a novel class of highly active in vitro inhibitors of the replication of Classical swine fever virus (CSFV). We recently demonstrated in a proof of concept study that the molecule has a marked effect on viral replication in CSFV-infected pigs. Here, the effect of antiviral treatment on virus transmission to untreated sentinel pigs was studied. Therefore, BPIP-treated pigs (n = 4), intra-muscularly infected with CSFV, were placed into contact with untreated sentinel pigs (n = 4). Efficient transmission of CSFV from four untreated seeder pigs to four untreated sentinels was observed. In contrast, only two out of four sentinel animals in contact with BPIP-treated seeder animals developed a short transient infection, of which one was likely the result of sentinel to sentinel transmission. A significant lower viral genome load was measured in tonsils of sentinels in contact with BPIP-treated seeder animals compared to the positive control group (p = 0.015). Although no significant difference (p = 0.126) in the time of onset of viraemia could be detected between the groups of contact animals, a tendency towards the reduction of virus transmission was observed. Since sentinel animals were left untreated in this exploratory trial, the study can be regarded as a worst case scenario and gives therefore an underestimation of the potential efficacy of the activity of BPIP on virus transmission.     

Seroprevalence of hepatitis E virus in dogs in Switzerland

Hepatitis E virus (HEV) is the causative agent of an acute and in most cases self‐limiting hepatitis. Of the four major HEV genotypes that infect humans, genotype 3 and 4 are zoonotic and have been identified in humans but predominantly in pigs and wild boar, which are considered the main reservoirs. However, the known host range of zoonotic HEV may be increasing to comprise additional species, including companion animals. Several studies have identified contact with dogs as a risk factor for HEV infection in humans, yet information on the occurrence of HEV in Swiss dogs is lacking. To examine a possible risk of exposure, this study was designed to assess the seroprevalence of HEV in 84 Swiss dogs. Serum and plasma samples collected from four veterinary clinics were screened for HEV‐specific antibodies by HEV‐antibody ELISA test kit. In addition, information of 22 dogs regarding the country of origin, the type of dog feed and any history of hunting was recorded. Samples from seropositive animals were also screened for the presence of HEV RNA by quantitative real‐time RT‐PCR (qRT‐PCR). Overall, 38% (32 of 84) of the dogs tested seropositive for anti‐HEV, indicating exposure to HEV. Among the 22 dogs for which information was available, HEV‐specific antibodies were detected in three of five dogs that were born abroad, in one of two dogs that were fed a raw meat‐based diet, and in one hunting dog. No viral RNA could be detected in any of the serum and plasma samples; thus, the genotype of the strains remained undetermined. This study provides further evidence for canine exposure and susceptibility to HEV and highlights the need to further assess the risks of HEV transmission to humans with contact to dogs.     

High HEV presence in four different wild boar populations in East and West Germany

Swine Hepatitis E virus (HEV) can be transmitted from pigs to humans causing hepatitis. A high prevalence of HEV in wild boar populations is reported for several European countries, but actual data for Germany are missing. During the hunting season from October to December 2007 liver, bile and blood samples were collected from wild boars in four different German regions. The samples were tested for HEV RNA by quantitative PCR (qPCR) and anti-HEV IgG antibodies by two different ELISAs and a Line immunoassay. A seroprevalence of 29.9% using ELISA and 26.2% in the Line immunoassay was determined. The seroprevalence rate varied greatly within the analyzed regions. However, qPCR analysis revealed a higher prevalence of 68.2% positive animals with regional differences. Surprisingly, also adult wild sows and wild boars were highly HEV positive by qPCR. Compared to liver and serum samples, bile samples showed a higher rate of positive qPCR results. Sequencing and phylogenetic analysis of a 969 nt fragment within ORF 2 revealed that all isolates clustered within genotype 3 but differed in the subtype depending on the hunting spot. Isolates clustered within genotypes 3i, 3h, 3f and 3e. Within one population HEV isolates were closely related, but social groups of animals in close proximity might be infected with different subtypes. Two full-length genomes of subtypes 3i and 3e from two different geographic regions were generated. The wild boar is discussed as one of the main sources of human autochthonous infections in Germany.     

A Study of the Ability of a TK‐Negative and gI/gE‐Negative Pseudorabies Virus (PRV) Mutant Inoculated by Different Routes to Protect Pigs Against PRV Infection

The capacity of a TK‐negative (TK ^‐) and gI/gE‐negative (gI/gE ^‐) pseudorabies virus (PRV) mutant to protect pigs against Aujeszky's disease carried out by experimental infection with a virulent PRV strain, was tested. There were three groups, each of four susceptible pigs which were inoculated twice by two different schedules. Group 1 received the modified virus by the intradermal (first inoculation)‐intramuscular (second inoculation) routes; group 2 was treated by the intranasal (first inoculation)‐intramuscular (second inoculation) routes. The third group was left untreated as the control. All of the pigs were challenged intranasally with a virulent PRV strain and they were subsequently injected with dexamethasone. Two pigs in each group were necropsied on days 5 and 15 after dexamethasone inoculation. The challenge exposure resulted in mild clinical signs, increase in growth and a shorter period of virus shedding in vaccinated pigs, whereas the control group showed severe signs of Aujeszky's disease. No difference in the titre of the virulent virus which was excreted by pigs of all three groups, was observed and all animals seroconverted. Both the mutant strain and the wild‐type virus established a latent infection although only the latter was reactivated and shed. Slight lesions were observed in target tissues of the vaccinated animals and no significant differences were detected between the two inoculation schedules.     

Hepatitis E virus: Animal reservoirs and zoonotic risk

Hepatitis E virus (HEV) is a small, non-enveloped, single-strand, positive-sense RNA virus of approximately 7.2 kb in size. HEV is classified in the family Hepeviridae consisting of four recognized major genotypes that infect humans and other animals. Genotypes 1 and 2 HEV are restricted to humans and often associated with large outbreaks and epidemics in developing countries with poor sanitation conditions, whereas genotypes 3 and 4 HEV infect humans, pigs and other animal species and are responsible for sporadic cases of hepatitis E in both developing and industrialized countries. The avian HEV associated with Hepatitis-Splenomegaly syndrome in chickens is genetically and antigenically related to mammalian HEV, and likely represents a new genus in the family. There exist three open reading frames in HEV genome: ORF1 encodes non-structural proteins, ORF2 encodes the capsid protein, and the ORF3 encodes a small phosphoprotein. ORF2 and ORF3 are translated from a single bicistronic mRNA, and overlap each other but neither overlaps ORF1. Due to the lack of an efficient cell culture system and a practical animal model for HEV, the mechanisms of HEV replication and pathogenesis are poorly understood. The recent identification and characterization of animal strains of HEV from pigs and chickens and the demonstrated ability of cross-species infection by these animal strains raise potential public health concerns for zoonotic HEV transmission. It has been shown that the genotypes 3 and 4 HEV strains from pigs can infect humans, and vice versa. Accumulating evidence indicated that hepatitis E is a zoonotic disease, and swine and perhaps other animal species are reservoirs for HEV. A vaccine against HEV is not yet available.     

Experimental Infection with Mycobacterium Avium,Serotype 2, in Pigs: 4. Contact Infection from Orally Inoculated Pigs*

In 3 experiments, 13 pigs were inoculated orally with 0.5 mg Mycobacterium avium daily for 5 days (1 mg = 32–68×10⁶ viable units). Five to 8 days after inoculation, 16 non-inoculated pigs were added to the inoculated pigs.Cultures from faeces showed excretion of M. avium from all the inoculated pigs for some time within the period 16 to 65 days after the last inoculation; the greatest numbers of organisms (62000 per 100 g faeces) were found at about the middle of the excretion period (Table 2). Two of the contact pigs were found to excrete M. avium in small numbers, 1 at 15, the other at 37 and 44 days after contact.All the inoculated pigs and 13 of the contact pigs showed positive intradermal tuberculin reactions. Post-mortem examination showed tuberculous lesions in all the inoculated pigs (Table 3). M. avium was transmitted to 15 of 16 pigs by contact. Five of these pigs showed gross lesions, 10 of them microscopic lesions only; in 9 of them the infection was proved by culture.     

Hepatitis E virus in swine and effluent samples from slaughterhouses in Brazil

Hepatitis E is an infectious disease which virus (HEV) is highly disseminated in swine herd populations. Sporadic acute human hepatitis E cases have been associated to genotype 3 and 4 strains of HEV also reported in swine populations of endemic and non-endemic areas. With the aim to evaluate the incidence of animals with current infection of HEV, 115 bile samples were collected from three slaughterhouses under inspection by Animal Sanitary Protection Agency of Rio de Janeiro, Brazil. In parallel, effluent samples were collected from six sewage pipe exit sites of two slaughterhouses. HEV RNA was detected in 11 out of 115 (9.6%) bile samples collected and three waste samples from one slaughterhouse. Viral loads observed for bile samples varied from 10(1)-10(5) genome copies/mL and for effluent samples mean load was 10(2) genome copies/mL. Sequencing and phylogenetic analysis classified samples within genotype 3 subtype 3b closely related to the sample obtained from the first reported autochthonous human case and samples from swine of commercial herds in Brazil. Our data demonstrates that although most animals achieve slaughter age (around 20 weeks old) already immune to HEV, a significant number of animals are with current infection at commercial age. Further studies should be addressed to consider risk analysis and possible evaluation of inspection regulations considering food safety measures regarding hepatitis E zoonotic aspect in Brazil.     

Experimental Dual Infection of Specific Pathogen‐Free Pigs with Porcine Reproductive and Respiratory Syndrome Virus and Pseudorabies Virus

Twenty 6‐week‐old specific pathogen‐free pigs were divided into four groups. On day 0 of the experiment, PRRSV–PRV (n = 6) and PRRSV (n = 4) groups were intranasally inoculated with porcine reproductive and respiratory syndrome virus (PRRSV) (10^5.6 TCID₅₀). On day 7, the PRRSV–PRV and PRV (n = 6) groups were intranasally inoculated with pseudorabies virus (PRV) (10^3.6 TCID₅₀). Control pigs (n = 4) were kept as uninoculated negative controls. Half of the pigs in each group were euthanized and necropsied on day 14 or 21. Clinical signs such as depression and anorexia were observed in the PRRSV–PRV and PRV groups after inoculation with PRV. Although febrile response was observed after virus inoculations, the duration of that response was prolonged in the PRRSV–PRV group compared with the other groups. The lungs in the PRRSV–PRV group failed to collapse and were mottled or diffusely tan and red, whereas the lungs of the pigs in the other groups were grossly normal. Histopathologically, interstitial pneumonia was present in all PRRSV‐inoculated pigs, but the pneumonic lesions were more severe in the PRRSV–PRV group. Mean PRRSV titres of tonsil and lung in the PRRSV–PRV group were significantly (P < 0.05) higher than that in the PRRSV group on day 21. These results indicate that dual infection with PRRSV and PRV increased clinical signs and pneumonic lesions in pigs infected with both viruses, as compared to pigs infected with PRRSV or PRV only, at least in the present experimental conditions.     

Contact transmission of vesicular stomatitis virus New Jersey in pigs

OBJECTIVE: To determine how viral shedding and development or lack of clinical disease relate to contact transmission of vesicular stomatitis virus New Jersey (VSV-NJ) in pigs and determine whether pigs infected by contact could infect other pigs by contact. ANIMALS: 63 pigs. PROCEDURE: Serologically naive pigs were housed in direct contact with pigs that were experimentally inoculated with VSV-NJ via ID inoculation of the apex of the snout, application to a scarified area of the oral mucosa, application to intact oral mucosa, or ID inoculation of the ear. In a second experiment, pigs infected with VSV-NJ by contact were moved and housed with additional naive pigs. Pigs were monitored and sampled daily for clinical disease and virus isolation and were serologically tested before and after infection or contact. RESULTS: Contact transmission developed only when vesicular lesions were evident. Transmission developed rapidly; contact pigs shed virus as early as 1 day after contact. In pens in which contact transmission was detected, 2 of 3 or 3 of 3 contact pigs were infected. CONCLUSIONS AND CLINICAL RELEVANCE: Transmission was lesion-dependent; however, vesicular lesions often were subtle with few or no clinical signs of infection. Contact transmission was efficient, with resulting infections ranging from subclinical (detected only by seroconversion) to clinical (development of vesicular lesions). Long-term maintenance of VSV-NJ via contact transmission alone appears unlikely. Pigs represent an efficient large-animal system for further study of VSV-NJ pathogenesis and transmission.     

Experimental infection of pigs with different doses of the African swine fever virus Armenia 07 strain by intramuscular injection and direct contact

We experimentally infected pigs with the African swine fever virus (ASFV) Armenia 07 strain (genotype II) to analyze the effect of different dose injections on clinical manifestations, virus-shedding patterns, histopathology, and transmission dynamics by direct contact. Each three pigs and four pigs were injected intramuscularly with 0.1 fifty percent hemadsorbing doses (HAD₅₀)/ml, 10¹ HAD₅₀/ml and 10⁶ HAD₅₀/ml of ASFV Armenia 07 strain, respectively. Each two of three pigs injected with 0.1 HAD₅₀/ml and 10¹ HAD₅₀/ml died by 10 days post inoculation. All pigs had a gross lesion of splenomegaly. Perigastric and renal lymph nodes were enlarged and resembled blood clots in nine of ten pigs. It was revealed that 0.1 HAD₅₀/ml of this ASFV was sufficient to infect healthy pigs by intramuscular injection and caused sub-acute lethal disease. For the transmission study, two 8-week-old pigs were injected intramuscularly with 10³ HAD₅₀/ml of the same virus. Each of the experimentally inoculated pigs was co-housed with two 8-week-old naive pigs. All contact pigs exhibited clinical manifestations at 6 or 7 days after the experimentally inoculated pigs developed pyrexia. These findings suggest that this strain may spread slowly within a herd. Histologically, lymph nodes resembled blood clots were formed by severe blood absorption and followed hemorrhage result of disruption of the lymphoid sinus filling with absorbed red blood cells. The severity of the gross and histological lesions depended on duration after infection, regardless of the difference of injection doses in this study.     

Seroconversion of pigs in contact with dogs exposed to canine coronavirus.

In order to determine if canine coronavirus (CCV) could be transmitted to pigs, two dogs were inoculated orally with virulent CCV. After 24 h, the dogs were moved to an isolation room that contained three three-day-old pigs. A wire mesh fence, allowing close contact between the animals, separated the dogs from the pigs. The dogs and pigs were observed for 14 days for clinical signs of disease. Samples of blood were obtained from dogs and pigs immediately before the dogs were inoculated with virus and 14 and 28 days later. The dogs developed mild clinical signs of an infection, but the pigs remained normal throughout the observation period. The dogs shed CCV for eight days after exposure. All three pigs developed neutralizing antibodies against CCV and transmissible gastroenteritis virus by 14 days after they were exposed to the dogs.