Hepatitis E virus genotype 3 in mussels (Mytilus galloprovinciallis), Spain
Introduction
Hepatitis E virus (HEV) (genus Hepevirus, family Hepeviridae) is an hepatotropic non-enveloped, positive-sense, single-stranded RNA virus containing three open reading frames (ORF) (Meng et al., 2012). ORF1 encodes a 1693 amino acid protein containing functional motifs and domains present in the non-structural proteins of other positive-stranded RNA viruses, ORF2 encodes the capsid protein, and ORF3 encodes a protein essential for virus egress (Kamar et al., 2015). Comparative analyses of the nucleotide sequences of HEV strains led to the identification of at least four recognized genotypes that cause disease in humans mainly through the fecal-oral route (Meng et al., 2012).
Globally, according to the World Health Organization (WHO), HEV genotypes 1–4 are cause for substantial public health concerns afflicting almost 20 million individuals annually and causing acute liver injury in approximately 3.3 million, with circa 56 600 deaths (WHO, 2015). Moreover, increasing reports of morbidity in the form of chronic hepatitis E in the immunosuppressed and the recognition of hepatitis E extra-hepatic manifestations have been raising alert in a Public Health perspective (Breda et al., 2014, Kamar et al., 2015, Santos et al., 2013). Genotypes 1 and 2 are prevalent in developing countries in Asia, Africa, and Central America, where hepatitis E is highly endemic, being transmitted through fecal contaminated water supplies or food products. In industrialized countries HEV has until recently been exclusively considered in travelers returning from HEV endemic regions. However, the discovery of autochthonous HEV in industrialized countries has changed the comprehension of HEV infection in these regions, now known to be mainly due to genotype 3 and the result of zoonotic transmission. In fact, HEV genotype 3 are widely present in swine to such an extent that they are now considered important reservoirs for human disease (Berto et al., 2012a, Berto et al., 2012b, Mesquita et al., 2014). Consequently, it is expected that spillover of swine and human waste may contaminate the environment with HEV enabling routes for human exposure and subsequent hepatitis E infection.
Coastal waters are often contaminated not only with human waste originated from sewage treatment plants, but also due to runoff following manure application, especially in countries where a high density of farming is present. As such, shellfish produced close to land are known to bioaccumulate not only human but also animal enteric viruses (Grodzki et al., 2014, Manso and Romalde, 2013). The role of bivalve molluscs as vehicles for enteric viruses is for long established since these agents are likely to survive for a long period of time in the water column, especially if associated with particulate matter and sediments, and are not inactivated during food preparation since bivalves are also often consumed raw or slightly cooked (Mesquita et al., 2011, Grodzki et al., 2014).
The role of shellfish as vehicles for HEV infection has been gathering the attention of the scientific community however it is yet unclear. HEV has been widely detected in shellfish from the United Kingdom (Crossan et al., 2012), however several recent reports indicate the complete lack of HEV detection on shellfish, namely in Italy (La Rosa et al., 2012), France (Grodzki et al., 2014) and Denmark (Krog et al., 2014). Nevertheless, anecdotal data have linked the consumption of shellfish to hepatitis E both in Vietnam and Japan (Koizumi et al., 2004, Inagaki et al., 2015), and also on a cruise ship (Said et al., 2009).
The goal of the present study was to evaluate the presence of HEV in shellfish from Galicia (NW Spain), one of the most important regions in the world for mussel production.
Section snippets
Origin and processing of shellfish samples
A total of 81 mussel (Mytilus galloprovincialis) batches corresponding to 7 production beds collected over a 18-month period (October 2010–March 2012) for a previous study (Manso and Romalde, 2013) were used. After collection, batches (10 individuals) of mussel hepatopancreas were processed according to the developed standard method ISO/TS 15216-1:2013 with slight modifications. RNA extraction was carried out with NucleoSpin RNA Virus kit (Macherey–Nagel, Germany) following manufacturer’
Results and discussion
From the 81 mussel batches initially studied by RT-qPCR, HEV RNA was detected in 12 (14.81%). Contamination levels ranged from 6.7 × 101 to 8.6 × 104 RNA copies/g digestive tissue and all positive samples yielded high extraction efficiencies (>10%; as determined by ISO/TS 15216-1:2013). After retesting by nested broad-spectrum RT-PCR it was possible to obtain 6 amplified products. These amplified products were subjected to sequencing and phylogenetic analysis in order to obtain information
Conclusion
HEV infections are cause for serious health concerns affecting almost 20 million individuals every year worldwide. This study confirms that HEV genotype 3 is present in shellfish causing apprehension on a new potential route for HEV transmission to humans. The complete spectrum of animal reservoirs and routes for infection are believed to be yet fully understood and warrant further research in this topic.
Acknowledgments
This work was supported in part by Grant 2014–PG110 from the Xunta de Galicia (Spain).
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