Hepatitis E virus in domestic pigs and surface waters in Slovenia: Prevalence and molecular characterization of a novel genotype 3 lineage

Abstract

A novel hepatitis E virus (HEV) genotype 3 lineage was identified in Slovenian pig herds. Stool samples from six Slovenian pig farms were collected and tested for the presence of HEV RNA. Of 85 individual samples 15 (20.3%) were positive for HEV RNA of which 2/38 (5.3%), 6/21 (28.6%) and 7/26 (26.9%) were from suckling, weanling and fattening pigs, respectively. Additionally, 51 pooled porcine stool samples were tested in one of the biggest pig farm and the estimated infection rate of individual pig was calculated, resulting in 7.8%, 10.6% and 24.2% for suckling, weanling and fattening pigs, respectively. The majority of HEV positive porcine samples were from the same pig farm. Out of 17 Slovenian patients with confirmed recent hepatitis E in the period 1999–2011, the serum samples of 10 patients were tested and 3 samples turned out to be HEV RNA positive. Furthermore, 60 surface water samples were tested throughout the country, of which 2 (3.3%) were positive for HEV RNA, one of them in the near vicinity of a pig farm. All HEV strains were analysed at 5’ ORF1 and 5’ ORF2 regions and both genome regions confirmed that Slovenian HEV strains represent a distinct genotype 3 lineage, diverse from all other genotype 3 lineages available in GenBank and described in the literature to date. All but one HEV strains detected in pigs in Slovenia represent a monophyletic branch in phylogenetic trees, with a high degree of sequence identity. One human HEV strain belonged to genotype 1 and two to genotype 3 but did not match the new genotype 3 lineage detected in Slovenian pig herds.

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.