Introduction

Hepatitis E virus (HEV) is the sole member of the genus Hepevirus (family Hepeviridae). Four genotypes (G1 to G4) infect humans, with G1 and G2 restricted to human host, and G3 and G4 circulating also in different animals (swine, deer, wild boar, and rabbit) (Meng 2010; Pavio et al. 2010). Usually, the disease is self-limiting and resolves within 2–6 weeks, but a small number of cases experience more severe forms of the disease leading to hepatic failure. The major symptoms of hepatitis E are fever, nausea, abdominal pain, vomiting, and jaundice. Hepatitis E infection during pregnancy may result in fulminant hepatitis (Pérez-Gracia et al. 2017) and in patients with chronic liver disease, HEV may worsen rapidly to a syndrome called acute-on-chronic liver failure (Kumar and Saraswat 2013).

Foodborne transmission is a major pathway for HEV infection in Europe and is mainly associated to consumption of raw or undercooked meat and products derived from swine, wild boar, and deer (EFSA BIOHAZ Panel 2017). Shellfish can also be a potential transmission vehicle, since they can concentrate viral particles from the surrounding marine environment during their filter feeding process. Indeed, HEV RNA has been detected in shellfish worldwide (Li et al. 2007; Song et al. 2010; Crossan et al. 2012; Diez-Valcarce et al. 2012; Pol-Hofstad et al. 2013; Gao et al. 2015; Mesquita et al. 2016).

In Italy, few studies have assessed the occurrence of HEV in shellfish for human consumption and HEV has never been detected in shellfish from approved harvesting areas (La Rosa et al. 2012; Iaconelli et al. 2015; Fusco et al. 2017). Furthermore, no studies have been conducted to investigate the occurrence of HEV in seawater from shellfish production areas, even if there is evidence of HEV presence in other water environments, including sewage and river water (La Rosa et al. 2010; Iaconelli et al. 2017; La Rosa et al. 2017c).

In the present study, shellfish samples collected in growing areas in Southern Italy were analyzed for the presence of HEV, along with marine waters collected in production areas and nearby sewage discharge points.

Materials and Methods

During 2015, 384 shellfish samples (298 mussels, 41 razor clams, 33 striped venus clams, and 12 wedge clams) were collected in 27 production areas of Campania Region, Southern Italy, distributed along 90 km of the coastal area. Sample preparation was performed according to ISO/TS 15216-1:2013 (ISO 2013). In the same period, 68 water samples (39 seawater samples from shellfish production areas and 29 water samples from discharge points) were collected in 10 selected production areas. Twenty liters of water samples was concentrated as previously described (La Rosa et al. 2017b). Viral nucleic acids from shellfish and water samples were extracted using NucliSense easyMAG (bioMérieux).

For shellfish samples, analysis of HEV was performed by real-time RT-(q)PCR assay according to Jothikumar et al. (2006) with slight modifications. PCR inhibition was ruled out by an external amplification control. A linearized plasmid containing the target sequence was used to generate the standard curve. Positive samples were subjected to genotyping through amplification of a portion of ORF1 using a broad range assay targeting the methyltransferase (MTase) gene of the ORF1 region of HEV (Fogeda et al. 2009). All positive PCR products were subjected to automated sequencing (Bio-Fab Research, Italy). Water samples were directly subjected to nested PCR as described for shellfish samples.

All positive samples were further analyzed using two different assays targeting the ORF2 region, to allow characterization at subtype level. Primers used in the present study are listed in Table 1.

Table 1 Primers used in the present study
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The phylogenetic tree was constructed using the neighbor-joining method by MEGA software version 6.0 and the reliability of the clusters was assessed by bootstrapping using 1000 replicates.

Results and Discussion

The presence of HEV was detected by real-time PCR in ten (eight mussels, two razor clams) of the 384 shellfish samples (2.6%), with positive samples collected from seven different production areas of the 27 included in the study (26%). Concentrations of HEV were below the quantification limit of the method and ranged between 30 and 90 genome copies/g of digestive tissue. Conventional amplification and characterization by sequencing was achieved only for two shellfish samples, characterized as G3.

In water samples, HEV RNA was detected in five of 39 seawater samples (12.8%) distributed in three production areas, all characterized as belonging to G3 HEV. Water samples from discharge points were all negative for HEV RNA. In one production area, HEV detection was achieved both in shellfish and in seawater samples (samples 46502B and MI-NA12) and sequence analysis showed an identity of 99.2–100% among seawater and shellfish samples.

Unfortunately, no amplification was obtained using the additional assays targeting the ORF2 regions and therefore subtyping of the strains was not feasible.

Figure 1 shows the phylogenetic tree constructed in the ORF1 region, including the study sequences (in bold), along with G3 human and swine sequences detected previously in Italy, and G1, G2, G4 sequences retrieved from GenBank. Sequences obtained from shellfish and water samples (GenBank accession numbers: MF796385–MF796391) grouped with G3 human and swine sequences previously detected in Italy.

Fig. 1
figure 1

Phylogenetic tree of hepatitis E virus. The evolutionary history was inferred by using the Maximum Likelihood method based on the Kimura 2-parameter model. The percentage of trees in which the associated taxa clustered together is shown next to the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 23 nucleotide sequences. Codon positions included were 1st + 2nd + 3rd + noncoding. There were a total of 132 positions in the final dataset. Evolutionary analyses were conducted in MEGA6

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The present study highlights the presence of HEV in shellfish from commercial harvesting areas in Southern Italy at low prevalence (2.6%) and with concentrations always below 102 genome copies/g. Viral infections are frequently associated to shellfish consumption, often as a consequence of sewage contamination of harvesting areas and undercooking of the product (Bellou et al. 2013). Few episodes of hepatitis E following shellfish consumption have been described (Koizumi et al. 2004; Said et al. 2009) even if shellfish consumption has been reported as a risk factor for HEV infection in several sporadic cases (Chau et al. 2006; Sadler et al. 2006; Renou et al. 2008; La Rosa et al. 2011; Inagaki et al. 2015). Furthermore, studies conducted in European countries have detected HEV RNA in shellfish with prevalence values ranging from 3 to 14.8% (Diez-Valcarce et al. 2012; Pol-Hofstad et al. 2013; Mesquita et al. 2016), though several studies reported no detection (La Rosa et al. 2010; Fusco et al. 2013; Grodzki et al. 2014; Krog et al. 2014; Prpic et al. 2015). In Spain, concentrations ranging from 67 to 8.6 × 104 genome copies/g were found (Mesquita et al. 2016). In Italy, HEV has never been detected in commercial harvesting areas (La Rosa et al. 2012; Iaconelli et al. 2015; Fusco et al. 2017), and only one study documented HEV contamination in bivalves used for biomonitoring and collected in areas not approved for harvesting (Donia et al. 2012).

Seawater samples collected in production areas tested also positive for HEV in this study. To our knowledge, no evidence of HEV contamination of coastal waters has been reported in Italy before. However, a few studies have documented HEV’s presence in sewage and river waters in Italy (La Fauci et al. 2010; La Rosa et al. 2010; Idolo et al. 2013; Iaconelli et al. 2015; Marcheggiani et al. 2015), demonstrating environmental circulation of this virus. Moreover, slurry samples from farms located in Northern Italy were found to be HEV-positive, pointing out the possibility of surface water contamination with animal strains, as a consequence of run-off waters, percolation from pig farms or agronomic use of pig slurry (La Rosa et al. 2017a).

The present study is the first to demonstrate the occurrence of HEV both in shellfish from commercial harvesting areas and in marine waters collected in the same production areas in Italy, with strains showing a high degree of nucleotide identity. These data could help to refine risk assessment of foodborne transmission of HEV, and will support the establishing of an integrated surveillance to reduce the risk of shellfish-associated illnesses.