Widespread diffusion of genotype 3 hepatitis E virus among farming swine in Northern Italy

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Abstract

Hepatitis E virus (HEV) causes acute hepatitis in humans, and infects several animal species, mostly asymptomatically. Swine and human HEV strains are genetically related suggesting both a zoonotic and a possible foodborne transmission. The prevalence of swine HEV was investigated in 274 randomly selected pigs from six different swine farms of Northern Italy, testing viral RNA in stools by nested reverse-transcription-polymerase chain reaction. HEV genome was detected in 115 stools (42%). All farms resulted positive for HEV, with a prevalence ranging between 12.8% and 72.5%. HEV-positive pigs were detected in all age groups and production stages tested, although infection was more prevalent in weaners than in the older fatteners (42.2% vs. 27.0%). Genetic characterization of swine strains identified was performed by sequencing and database alignment. Phylogenetic analysis on the nucleotide sequences from 16 positive PCR products indicated that all strains belonged to genotype 3. In particular, one group of seven Italian strains clustered close (91.6–96.2% identity) to human and swine European HEV strains.

Introduction

Hepatitis E virus (HEV; genus Hepevirus, family Hepeviridae) is a small non-enveloped virus with a single-stranded positive sense RNA genome. The virus genome is approximately 7.2 kb, and contains three open reading frames (ORF). ORF1 encodes non-structural proteins including an RNA-dependent RNA polymerase, ORF2 the capsid protein, and ORF3 a small protein with unclear function (Li et al., 2006, Lu et al., 2006).

HEV causes human hepatitis E (Huang et al., 2002), generally associated to a low (<1%) mortality rate, except for pregnant women (20–25% mortality) (Emerson and Purcell, 2003). The disease is endemic in many developing countries of Asia and Africa, and in Mexico, where it usually occurs as epidemics (Okamoto, 2007). The natural transmission of HEV is fecal–oral, and contaminated drinking water following heavy rains or flooding is a major source of infection (Emerson and Purcell, 2003, Okamoto, 2007). Sporadic cases of disease have also been reported in US, Japan and Europe (Meng et al., 1998b, Zanetti et al., 1999, Clemente-Casares et al., 2003, Li et al., 2006, Reuter et al., 2006, Renou et al., 2007). Many of these are associated with traveling through endemic countries (Clemente-Casares et al., 2003, Renou et al., 2007), although an increasing number of cases have been reported among patients without history of traveling abroad (Emerson and Purcell, 2003). In industrialized countries, anti-HEV antibodies have also been detected in a significant proportion of healthy individuals (Mast et al., 1997, Meng et al., 2002).

There is increasing evidence that HEV may represent a zoonotic agent (Meng et al., 1997, Tei et al., 2003, Yazaki et al., 2003, Sonoda et al., 2004, Banks et al., 2007). Different species of domestic and wild mammals have been reported to have anti-HEV antibodies (Lu et al., 2006, Okamoto, 2007), and/or to be infected with viruses closely related to human HEV (Tei et al., 2003, Banks et al., 2004). Conversely, avian hepatitis E viruses are genetically distant from human viruses (Huang et al., 2004). HEV encompasses at least four genotypes, which differ with respect to geographic distribution, host range and pattern of infection, although they are similar serologically (Tei et al., 2003, Banks et al., 2004, Lu et al., 2006, Okamoto, 2007).

Genotype 1, associated with large waterborne outbreaks, and genotype 2 are prevalent in Asia and Africa, and in Chad, Mexico and Nigeria, respectively, infecting mostly humans (Zheng et al., 2006, Caron et al., 2006). In addition to infecting humans, genotype 3 is also widespread in swine herds in industrialized countries including North America, Europe, and Japan. Genotype 4 is mainly distributed in Asia, and is associated with sporadic cases and foodborne outbreaks (Lu et al., 2006).

Swine HEV (Meng et al., 1997) usually infect pigs sub-clinically, and the swine may be an important reservoir of the virus (Li et al., 2006, Lu et al., 2006). In fact, increasing evidence of autochthonous cases of hepatitis E in industrialized regions suggests a zoonotic transmission of HEV from pigs (Drobeniuc et al., 2001, Lu et al., 2006). HEV strains identified in swine show a close genetic relatedness to human genotypes 3 and 4 strains (Emerson and Purcell, 2003, Clemente-Casares et al., 2003, Banks et al., 2004, Lu et al., 2006). Experimental interspecies transmission of HEV between non-human primates and pigs has been demonstrated (Meng et al., 1998a), and seroepidemiological studies have shown that pig handlers have an increased risk of HEV infection (Meng et al., 2002). Finally, studies in Japan have linked human HEV cases to consumption of raw or undercooked meat from deer, wild boars and pigs (Tei et al., 2003, Yazaki et al., 2003, Masuda et al., 2005). In France a direct zoonotic transmission of genotype 3 HEV from a pet pig to its owner was also described (Renou et al., 2007).

In the Mediterranean Area, hepatitis E cases in humans have been reported sporadically, including Italy (Zanetti et al., 1999), and seroprevalence appears to be around 5%. To date very little information exists on the prevalence, epidemiology and genetic characteristics of swine HEV in Italy (Caprioli et al., 2007).

In this study we evaluated the prevalence of swine HEV in asymptomatic pigs from six farms of Northern Italy, and compared the genome sequences identified with HEV sequences of different origins reported worldwide.

Section snippets

Samples

From January 2006 to June 2006, 274 fecal samples were collected from randomly selected pigs of 3–4 (weaners) or 8–9 months of age (fatteners), gilts (0 parities), young sows (1–2 parities) and old sows (>2 parities) belonging to six different farms of Northern Italy. The farm size ranged from 500 to 1100 sows. All pigs sampled appeared to be clinically healthy. From each farm, at least 10 animals were sampled for each production stage, except for the two farrow-to-weaning farms where fatteners

HEV detection in swine fecal samples

HEV RNA was detected in 115 of 274 pigs tested (42%). All six farms resulted positive for HEV, with a prevalence ranging between 12.8% and 72.5% (Table 1). HEV-positive pigs were detected and in all age groups and production stages tested (Table 2). In the fattening stage, a higher HEV prevalence was observed between weaners (90–120-day-old), with 27 positives out of 64 tested (42.2%), while in fatteners (>120-day-old) the prevalence was 27%. Among breeding animals, 43.1% of gilts tested

Discussion

This study represents the first extended survey on swine HEV prevalence conducted in Italy, and demonstrates that HEV infection is largely widespread in Italian pig farms. The overall rate of virus shedding observed (42% of 276 fecal samples tested) is markedly higher than we detected (5.9%) previously in farms of the same area (Caprioli et al., 2007). At least partially, this may be due to the individual stools examined in the present study vs. fecal pools in our earlier investigation, where

Acknowledgements

This study was partially supported by the European Commission, DG Research Quality of Life Program 6th Framework (EVENT, SP22-CT-2004-502571) “EVENT” (FP6-2002-SSP-1).

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