Hepatitis E virus (HEV) is a causative agent of enterically transmitted acute hepatitis in humans (1). It is a major public health issue in developing countries, where it causes large waterborne epidemics (2). In industrialized countries, it is an emerging problem, as an increasing number of sporadic cases for which the origins are still unclear (3) have been reported for patients who have not traveled to HEV-endemic areas.

HEV is a nonenveloped virus with a single-stranded positive RNA genome of 7.2 kb composed of 3 open reading frames (ORFs). HEV is the sole member of the family Hepeviridae (4) and has been classified into 4 major genotypes and 24 subtypes. Genotype 1 is divided into 5 subtypes (1a to 1e), genotype 2 into 2 subtypes (2a and 2b), genotype 3 into 10 subtypes (3a to 3j), and genotype 4 into 7 subtypes (4a to 4g) (5). Although genotypes 1 and 2 are endemic to developing countries, genotypes 3 and 4 are the cause of sporadic cases. HEV is the only hepatitis virus that is also found in a wide variety of animals (6). Genotype 3 can infect humans as well as swine, wild boar, deer, and mongoose (7–10). It is generally agreed that swine are widely infected all over the world (6). HEV seroprevalence varies greatly depending on countries; 22.7% to 88.4% of pigs are seropositive at 6 months of age (11,12). Among pigs slaughtered at ≈25 weeks of age, the prevalence of HEV fecal excretion ranges from 4% to 41% (13,14). Viral RNA sequences from pigs and humans can be closely related (15,16), and cross-species infection of genotypes 3 and 4 from human to pig and pig to nonhuman primate has been demonstrated experimentally (17). To date, only 2 cases of zoonotic transmission from consumption of raw or undercooked sika deer and wild boar meat have been clearly identified in Japan with near or 100% homology between the sequences from the patient and the consumed meat (7,8).

A few reports have shown close phylogenetic relationships between sequences identified in swine and in humans. However, these studies were based on limited numbers of sequences with little geographic or temporal data (18–21).

In France, HEV seroprevalence in the human population ranges from 3.2% to 16.6%, depending on the geographic regions studied (22,23). The number of reported viral hepatitis E cases is increasing. Although only 38 cases were reported in 2006, a total of 340 cases were diagnosed in 2010, of which 70% were declared autochthonous with no recent history of patients traveling abroad (French National Reference Laboratory, unpub. data). In the swine reservoir, a recent nationwide survey performed at slaughterhouses showed high prevalence of HEV. HEV seroprevalence in swine ranges from 31% at the individual level to 65% at the farm level. In that study, HEV prevalence in pig liver was estimated at 4%, meaning that HEV-infected pig livers can enter the food chain (24). Moreover, it has been shown that regional products made from raw pig liver may contain HEV (25). In France, pork is the most widely eaten type of meat (26) and could represent an HEV reservoir with a high risk for zoonotic transmission.

To assess the zoonotic risk for transmission from swine to humans in France, we studied HEV sequences in both hosts. HEV sequences collected from every human autochthonous case of hepatitis E infection and HEV-positive pig livers collected at slaughterhouses, both within 18 months, were analyzed. Epidemiologic and spatial–temporal data corresponding to phylogenetic analyses of partial ORF2 sequences were used to investigate whether swine are a major source of HEV contamination in France.

Although zoonotic transmission of hepatitis E virus from swine to human has been well accepted, little data are available on HEV sequences circulating in human and swine populations within a country during a restricted period. We investigated a large number of HEV sequences, collected from 106 patients and 43 swine over an 18-month period. The patients were mostly male (72%) and >55 years of age. This finding is in agreement with a previous report on acute HEV infection in France, which found that men accounted for 68% (36/53) of the cases (31). The situation in industrialized countries contrasts with that in regions where attack rates for waterborne outbreaks of HEV genotype 1 are higher among young adults (15–40 years of age) (1). This observation suggests that the 2 epidemiologic profiles may involve different contamination routes. There are differences in hygiene and meat consumption habits in these regions. Moreover, no animal reservoirs have been yet described for the genotypes involved in waterborne outbreaks (genotypes 1 or 2) (6), suggesting that zoonotic transmission might be limited in any HEV-endemic areas.

All 149 HEV sequences belonged to genotype 3 and were divided into at least 3 subtypes according to the classification elaborated by Lu et al. (5). Sequences sharing a minimum of 90% similarity were considered as belonging to the same subtype. Among these sequences, 137 belonged to subtypes 3f, 3c, and 3e. For 12 sequences, 8 human HEV and 4 swine HEV, there was some classification uncertainty because they were close to 7 different subtypes but shared <90% homology. The difficulty in classifying this undefined subtype might be because of partial sequencing of the strains identified, although Lu et al. showed that the 5′ end of the ORF2 region matches the complete genomic sequence for HEV classification better than other regions of the HEV genome (5). Using the nucleotide BLAST database (http://blast.ncbi.nlm.nih.gov/Blast), sequences from this undefined subtype are close but share <90% homology to 3a and 3c sequences detected in the Netherlands or 3h and 3i sequences detected in Germany. This undefined subtype also clusters on its own (>90% homology) and could be a new subtype that is specific to France. Comparison of autochthonous HEV from France with HEV from neighboring countries shows that the same main subtypes are found: 3f is found all over Europe; 3c in the Netherlands, Italy, and Hungary; and 3e in the UK, the Netherlands, Germany, and Hungary (15,18,32–35). This finding suggests that some subtypes may have emerged and evolved locally through animal trading.

The proportion of each subtype in both species was then estimated, and the proportions of subtypes 3f, 3c, and 3e were found to be almost the same. Such a similar distribution of subtypes suggests an active circulation of the virus between the 2 host species in France. In the Netherlands, proportions of subtypes in human compared with animals or environmental strains were found to differ markedly, 6% versus 43% for 3f and 75% versus 35% for 3c (18), suggesting a limited number of contamination events through these 2 possible contamination pathways in this country.

Although HEV is widely distributed across France, some geographic regions showed higher rates of infection in humans. Most (67%) cases of autochthonous hepatitis E were found in southern France and particularly in the Provence-Alpes-Côte-d’Azur region (30%). These results are consistent with HEV seroprevalence in blood donors being higher in southern (16.6%) than in northern France (3.2%) (22,23). Furthermore, this observation correlates with results of a previous national survey in France showing an increasing north-to-south gradient of acute hepatitis E (31). In contrast, in the animal reservoir, most HEV sequences were detected in the main pig-producing area located in northwestern France. Nevertheless, the low number (only 2) of human cases observed in this region with a high density of pig farms suggests that the number of contamination events through the environmental pathway is limited. In the Ile de France region (Paris area), a high number (11%) of cases of hepatitis E was also reported. This finding could be partially explained by the high population density (18%) in this area; a few cases were reported after traveling and eating uncooked pork products in southern France.

To further analyze sequence similarities between human and swine HEV strains, we determined the similarities in nucleotides between human and swine sequences. HEV has a high mutation rate because of its error-prone RNA-dependent RNA polymerase and is probably present as a quasispecies in an infected host (36). Thus, low (<1%) variability in nucleotides may correspond to a unique strain. Analyzing human HEV sequences, 100% nt similarity was found in 4 groups of 2–3 patients. These patients were not related, but they may have been exposed to an unknown common source of contamination.

Swine sequences amplified from livers of animals within the same herd were found to be homogeneous, with a maximum difference of 3 nt along the 306 nt sequenced. Except for 3 groups of 2 herds, all the sequences were different (<99%). These 3 groups included herds that were sampled at the same time or 5 months apart and that were geographically close (a few kilometers). This high similarity of partial sequences might be explained by a possible exchange of animals between nearby herds, which is a common practice. However, it cannot be excluded that movements of farm workers and veterinarians or spreading of infected slurry might contribute to HEV transmission between herds. Swine HEV infection spreads easily within a herd through the fecal–oral route (37). This geographic clustering of HEV strains detected in animals was also observed in Sweden (19).

Because animals from the same herd can have a difference of 3 nt over the same amplified sequence, human and porcine HEV sequences with >99% similarities may be considered as coming from related strains. A comparison of human and swine sequences showed that 2 pairs of sequences were similar (99.3%). In both cases, swine and human sequences were detected in different geographic areas. The swine sequences were identified first and later in humans. Pork meat is the most widely eaten meat in France (34.7 kg/inhabitant/year), and it is distributed and consumed throughout France (26). In our study, HEV sequences were amplified from liver, but other meat might be a vector for HEV infection because it has been shown that other organs such as muscles can be HEV positive (38). Considering the geographic distances and the detection of these HEV sequences in animals first, it seems reasonable to assume that foodborne infection may play a major role in autochthonous cases of hepatitis E. The high similarity observed suggests that these 2 cases could be the result of zoonotic transmission. Furthermore, because since these sequences are not geographically linked, contamination through environmental exposure can be ruled out.

In addition to the high degree of similarity observed between human and swine sequences and the identical proportion of each subtype in both hosts, no specific nucleotide substitutions have been identified when sequences from different host species were compared. These results are in line with the possible absence of a species barrier for HEV strains of genotype 3. However, before concluding that there are no host restriction determinants, further analysis of longer sequences is required.

This unique large-scale study on human and swine sequences with spatial–temporal data suggests that zoonotic transmission of HEV is involved in autochthonous cases. The swine reservoir is widely infected with HEV, and infected livers enter the food chain. Living in southern France seems to be associated with more frequent exposure to HEV (67% of cases). This observation might be linked to cultural food habits specific to southern France and frequent consumption of products made from raw swine liver (25).

Slurry from swine is often spread onto local fields, but there are few (only 2) cases reported in Brittany compared with other regions. The spread of HEV into the environment may not have major consequences but cannot be ignored. Contact with animals; consumption of contaminated water, vegetables, or shellfish; or unknown routes of transmission need to be investigated. In conclusion, taken together, these results confirm the major role played by the swine reservoir of HEV in autochthonous cases of hepatitis E. This study underlines the need for a surveillance and control plan, either at the level of pig production or at the level of food processing, to limit human exposure to HEV through consumption of pork products.