Hepatitis E virus in domestic pigs and surface waters in Slovenia: Prevalence and molecular characterization of a novel genotype 3 lineage
Highlights
► Hepatitis E virus was described for the first time in pigs in Slovenia. ► A total of 85 individual and 51 pooled samples were tested with the detection rate of up to 28.6% in weanling pigs. ► Partial ORF 1 and ORF 2 genome regions were analysed confirming the emergence of a new genotype 3 lineage. ► In addition, hepatitis E viral RNA was detected in surface water samples in the near vicinity of a pig farm. ► Infection of pigs and humans through the environment may occur.
Introduction
Hepatitis E virus (HEV) has been recognized as an important cause of non-A, B, C hepatitis in humans throughout the world (Meng, 2010). In Europe, there are variable reports on acute HEV infection, ranging from 5–15% in patients with non-A, B, C hepatitis (Lewis et al., 2010). From the epidemiological point of view, HEV displays two distinct patterns, with genotypes 1 and 2 being responsible for major outbreaks in endemic regions in developing countries and genotypes 3 and 4 as zoonotic HEV responsible for sporadic cases of hepatitis in developed countries (Teshale et al., 2010). In humans, the clinical presentation of HEV infection is indistinguishable from hepatitis A and is presented mainly as acute hepatitis with jaundice, abdominal pain, hepatomegaly, nausea, vomiting and fever. The incubation period is up to 40 days, so it is difficult to recognize the source of infection, especially in sporadic cases (Pavio et al., 2010). However, contaminated water sources have been reported to be the major cause of HEV outbreaks, especially for genotypes 1 and 2 in endemic regions (Sailaja et al., 2009). In developed countries with sporadic HEV 3 or 4 genotype infections, contaminated water has not been implicated in human HEV infection, although HEV RNA has been detected in surface water and should be considered as a potential infection source (Rutjes et al., 2009). The mortality rate seems to be higher in developing countries and among pregnant women (Navaneethan et al., 2008, Pavio and Mansuy, 2010). The transmission route is mainly faecal-oral but, in addition to waterborne outbreaks in developing countries, some cases of foodborne infections have also been reported throughout the world, linked with the consumption of raw meat and porcine liver products (Lewis et al., 2010, Teo, 2010).
Four HEV genotypes (genotypes 1–4) have been described in the literature to date, all of them belonging to a single serotype (Mushahwar, 2008). In addition to the four known genotypes, distinct avian HEV strains have been described (Haqshenas et al., 2001) and a study group from Germany recently found HEV in rats, not closely related to other described HEV strains (Johne et al., 2010). The determination of HEV genotype is usually made by sequence analysis of the appropriate genome segment. A systematic review of different HEV genotypes with detailed sequence analysis of whole genome or partial genome segments was done by Lu et al. (2006). The most appropriate genome fragment best reflecting the whole genome sequence was found to be the 5’ end of the ORF2 region (nt position 5994–6294), partial coding region of a viral capsid protein. On the basis of nucleotide diversity, genotype 3, as the most frequently detected genotype in Europe, was divided into 10 subtypes, representing lineages 3a–3j. The authors’ explanation was that the high diversity within genotypes 3 and 4 might be due to the zoonotic potential of those genotypes (Lu et al., 2006). Genotype 3 is the most common genotype found in various animals in Europe, mostly domestic pigs and wild boars (Pavio et al., 2010). There are many reports on HEV prevalence in domestic pigs in Europe. In recent studies, it was shown that more than 50% of porcine samples collected at pig farms contained HEV RNA (Rutjes et al., 2007, Vasickova et al., 2009). In individual pig stool samples, the prevalence of HEV RNA was reported to be between 6–46.9%, the highest being in the age group of 10–17 weeks (Forgách et al., 2010, Martelli et al., 2010, McCreary et al., 2008). Although infected pigs usually remain asymptomatic, some degree of underlying hepatitis may occur (Teo, 2010).
In this study, we report for the first time the detection of HEV in Slovenian domestic pigs, environmental samples and humans and provide an insight into the molecular diversity of the detected strains. HEV strains representing a distinct genotype 3 lineage, diverse from all other genotype 3 lineages available in GenBank and described in the literature to date were identified.
Section snippets
Sample collection
Animal stool samples were collected in 2004–2005 for molecular characterization of enteric viruses present in Slovenia, as described previously (Steyer et al., 2008, Zimšek Mijovski et al., 2010). Briefly, stool samples from pigs were sampled by age and were grouped into three age-groups: suckling (0–3 weeks), weanling (3–10 weeks), and fattening (more than 10 weeks). Porcine stool samples were collected at six pig farms, A–F, distributed throughout the country (Fig. 1). In total, 38 samples
Results
Of 85 unpooled pig stool samples, 15 (20.3%) were positive when tested with primers amplifying highly conserved ORF2/3 junction region (Table 1). As shown in Table 1, prevalence of HEV RNA in individual samples ranged from 5.3% in suckling pigs to 28.6% in weanling pigs. HEV RNA was detected only in samples collected from farms D and E. Majority of the positive samples were detected at farm D, which is one of the biggest pig farms located in the central region of Slovenia; and 4 individual
Discussion
There are many endemic regions of HEV in developing countries but in Europe, mainly sporadic cases of acute HEV have been described in the literature (Pavio and Mansuy, 2010). In previous studies, HEV genotype 3 was most commonly detected in animal reservoirs, mainly pigs and wild boars, also in Europe (Pavio et al., 2010). In this manuscript, we report for the first time the prevalence and molecular characterization of HEV in domestic pigs from Slovenian pig farms. It should be mentioned that
Acknowledgements
This work was partially supported by the Slovenian Research Agency, Ministry of Agriculture, Forestry and Food (Post-doc Project Z3-0037 and Project V4-0512) and EU 6th Framework Program “EVENT” (SP22-CT-2004-502571). The authors express their gratitude to the veterinarians at the pig farms, Uroš Jamnikar, BSc and Damjan Rajapakse Jerina, BSc for sample collection and Irena Šest for excellent laboratory technical assistance.
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