Hepatitis E virus genotype 3 in mussels (Mytilus galloprovinciallis), Spain

doi:10.1016/j.fm.2016.03.009

Food Microbiology

Volume 58, September 2016, Pages 13-15

https://doi.org/10.1016/j.fm.2016.03.009 Get rights and content

Highlights

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Detection of HEV in shellfish harvested areas is described.
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HEV levels ranged from 6.7 × 10¹ to 8.6 × 10⁴ RNA copies/g digestive tissue.
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All sequenced isolates belonged to the zoonotic genotype 3e.
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Shellfish constitute a potential route for HEV transmission to humans.

Abstract

Coastal waters can become contaminated with both human waste from sewage treatment plants and runoff following manure application. Thus, shellfish produced close to land can bioaccumulate enteric viruses of human and animal origin, including zoonotic hepatitis E virus that infect both human and swine. The goal of this study was to evaluate the presence of HEV in shellfish from Galicia (NW Spain), a densely populated region with a strong tradition of swine farming, and one of the most important regions in the world for mussel production. We tested 81 mussel batches by RT-qPCR followed by conventional broad-spectrum nested RT-PCR and phylogenetic analysis. We have obtained 12 positive samples by RT-qPCR (14.81%) with HEV contamination levels ranging from 6.7 × 10¹ to 8.6 × 10⁴ RNA copies/g digestive tissue. Phylogenetic analysis based on a 330 nt region of the ORF 1 showed that all sequenced isolates belonged to the zoonotic genotype 3 subgenotype e, being closely related to strains of human and swine origin. Results show that shellfish may be a potential route for HEV transmission to humans.

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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Hepatitis E virus genotype 3 in shellfish, United Kingdom
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A systematic review and a meta-analysis of case-control and cohort studies (including transversal studies) were performed to determine the main risk factors associated with sporadic hepatitis E infection. Suitable scientific articles were identified through a systematic literature search and subjected to a methodological quality assessment. From each study, odds-ratio (OR) measures were extracted/calculated, as well as study characteristics such as population type, design, and risk factor hierarchy. Mixed-effects meta-analyses models were adjusted by population type to appropriate data partitions.
Seventy-seven cohort and case-control studies conducted between 1986 and 2016 and investigating risk factors in mixed population, susceptible population, and pregnant women, were included in this meta-analysis. Hepatitis E cases were defined with serological exams and differentiated whenever the serological exam is associated or not with symptoms.
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Citation Excerpt :
HAV and HEV have rarely been detected in raw vegetables and fruits worldwide, which could probably be due to that these foods would not be the main route of transmission, or to the low number of research studies carried out on them up to date (Brassard et al., 2012; Khan et al., 2014; Marti et al., 2017; Maunula et al., 2013; Parada-Fabián et al., 2016; Purpari et al., 2019; Randazzo et al., 2018c; Shin et al., 2019; Terio et al., 2017). Fig. 2 shows the main matrices in which viruses have been detected worldwide (Abad et al., 1997; Amri et al., 2011; Banks et al., 2010; Bazzardi et al., 2014; Benabbes et al., 2013; Berto et al., 2012; Bigoraj et al., 2014; Bouwknegt et al., 2007; Boxman et al., 2019; Brassard et al., 2012; Chan et al., 2017; Chironna et al., 2002; Cioffi et al., 2018; Coelho et al., 2003; Colson et al., 2010; Cossaboom et al., 2016; Croci et al., 2007; Croci et al., 2000; Crossan et al., 2012; De Medici et al., 2001; Demirci et al., 2019; DePaola et al., 2010; Di Bartolo et al., 2015; Di Bartolo et al., 2012; Di Pasquale et al., 2019; Diez-Valcarce et al., 2012; Elamri et al., 2006; Fang et al., 2016; Feagins et al., 2007; Formiga-Cruz et al., 2002; Fusco et al., 2019; Fusco et al., 2017; Geng et al., 2019; Gharbi-Khelifi et al., 2007; Giannini et al., 2018; Gutiérrez-Vergara et al., 2015; Hansman et al., 2008; Heldt et al., 2016; Iaconelli et al., 2015; Intharasongkroh et al., 2017; Khan et al., 2014; Kingsley et al., 2002; Kitahashi et al., 1999; Kittigul et al., 2010; Korsman et al., 2019; La Rosa et al., 2018; La Rosa et al., 2012; Le Guyader et al., 2000; Le Guyader et al., 1994; Li et al., 2007; Macaluso et al., 2006; Manso et al., 2010; Manso and Romalde, 2013; Marti et al., 2017; Martin-Latil et al., 2014; Maunula et al., 2013; Mesquita et al., 2016; Monge and Arias, 1996; Montone et al., 2019; Moor et al., 2018; Muniain-Mujika et al., 2003; Mykytczuk et al., 2017; Namsai et al., 2011; Nenonen et al., 2006; O'Hara et al., 2018; Parada-Fabián et al., 2016; Pavio et al., 2017; Pereira et al., 2018; Polo et al., 2015; Purpari et al., 2019; Randazzo et al., 2018c; Roldán et al., 2013; Romalde et al., 2002; Shin et al., 2019; Sincero et al., 2006; Suffredini et al., 2017; Szabo et al., 2015; Terio et al., 2017; Wang et al., 2008; Wilhelm et al., 2014; Wong et al., 2019; Yazaki et al., 2003). Transmission of HAV by contaminated food or water is a well-recognized phenomenon, and the first records of outbreaks involving food date from the 1950s decade (Roos, 1956).
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Hepatitis E virus genotype 3 in mussels (Mytilus galloprovinciallis), Spain

Highlights

Abstract

Introduction

Section snippets

Origin and processing of shellfish samples

Results and discussion

Conclusion

Acknowledgments

J. Virol. Methods

Clin. Res. Hepatol. Gastroenterol.

Int. J. Food Microbiol.

Food Microbiol.

Prevalence and transmission of hepatitis E virus in domestic swine populations in different European countries

BMC Res. Notes

Detection and characterization of hepatitis E virus in domestic pigs of different ages in Portugal

Zoonoses Public Health

First report of chronic hepatitis E in renal transplant recipients in Portugal

J. Infect. Dev. Ctries.

Hepatitis E virus genotype 3 in shellfish, United Kingdom

Emerg. Infect. Dis.

Bioaccumulation efficiency, tissue distribution, and environmental occurrence of hepatitis E virus in bivalve shellfish from France

Appl. Environ. Microbiol.