Accepted Manuscript Hepatitis E virus genotype 3 in mussels (Mytilus galloprovinciallis), Spain João R. Mesquita, Danielle Oliveira, Enrique Rivadulla, Joana Abreu-Silva, Miguel F. Varela, Jesús L. Romalde, Maria S.J. Nascimento PII:
S0740-0020(15)30104-0
DOI:
10.1016/j.fm.2016.03.009
Reference:
YFMIC 2540
To appear in:
Food Microbiology
Received Date: 27 October 2015 Revised Date:
11 March 2016
Accepted Date: 11 March 2016
Please cite this article as: Mesquita, J.R., Oliveira, D., Rivadulla, E., Abreu-Silva, J., Varela, M.F., Romalde, J.L., Nascimento, M.S.J., Hepatitis E virus genotype 3 in mussels (Mytilus galloprovinciallis), Spain, Food Microbiology (2016), doi: 10.1016/j.fm.2016.03.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Hepatitis E virus genotype 3 in mussels (Mytilus galloprovinciallis), Spain
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João R. Mesquita1,2, Danielle Oliveira3, Enrique Rivadulla4, Joana Abreu-
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Silva3, Miguel F. Varela4, Jesús L. Romalde4*, Maria S.J. Nascimento2,3
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Genéticos/Universidade do Porto, Campus Agrário de Vairão, Vairão, Portugal.
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Escola Superior Agrária, Instituto Politécnico de Viseu, Portugal.
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CIBIO/UP, Centro de Investigação em Biodiversidade e Recursos
Laboratório de Microbiologia, Departamento de Ciências Biológicas,
Faculdade de Farmácia da Universidade do Porto, Portugal
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Universidade de Santiago de Compostela, 15782 Santiago de Compostela,
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Spain.
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Departamento de Microbiología y Parasitología, CIBUS-Facultad de Biología,
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Submitted to: Food Microbiology, March 2015
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*Corresponding author
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Phone: +34 881816908
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Fax: +34 881816966
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email:
[email protected].
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Abstract
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Coastal waters can become contaminated with both human waste from sewage
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treatment plants and runoff following manure application. Thus, shellfish
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produced close to land can bioaccumulate enteric viruses of human and animal
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origin, including zoonotic hepatitis E virus that infect both human and swine.
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The goal of this study was to evaluate the presence of HEV in shellfish from
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Galicia (NW Spain), a densely populated region with a strong tradition of swine
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farming, and one of the most important regions in the world for mussel
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production. We tested 81 mussel batches by RT-qPCR followed by
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conventional broad-spectrum nested RT-PCR and phylogenetic analysis. We
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have obtained 12 positive samples by RT-qPCR (14.81%) with HEV
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contamination levels ranging from 6.7 X 101 to 8.6 X 104 RNA copies/g
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digestive tissue. Phylogenetic analysis based on a 330 nt region of the ORF 1
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showed that all sequenced isolates belonged to the zoonotic genotype 3
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subgenotype e, being closely related to strains of human and swine origin.
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Results show that shellfish may be a potential route for HEV transmission to
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humans.
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Keywords: Hepatitis E virus, Shellfish, detection
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1. Introduction
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Hepatitis E virus (HEV) (genus Hepevirus, family Hepeviridae) is an
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hepatotropic
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containing three open reading frames (ORF) (Meng et al., 2012). ORF1
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encodes a 1693 amino acid protein containing functional motifs and domains
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present in the non-structural proteins of other positive-stranded RNA viruses,
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ORF2 encodes the capsid protein, and ORF3 encodes a protein essential for
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virus egress (Kamar et al., 2015). Comparative analyses of the nucleotide
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sequences of HEV strains led to the identification of at least four recognized
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genotypes that cause disease in humans mainly through the fecal-oral route
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(Meng et al., 2012).
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Globally, according to the World Health Organization (WHO), HEV genotypes 1-
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4 are cause for substantial public health concerns afflicting almost 20 million
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individuals annually and causing acute liver injury in approximately 3.3 million,
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with circa 56 600 deaths (WHO, 2015). Moreover, increasing reports of
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morbidity in the form of chronic hepatitis E in the immunosuppressed and the
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recognition of hepatitis E extra-hepatic manifestations have been raising alert in
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a Public Health perspective (Breda et al., 2014; Kamar et al., 2015; Santos et
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al., 2013). Genotypes 1 and 2 are prevalent in developing countries in Asia,
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Africa, and Central America, where hepatitis E is highly endemic, being
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transmitted through fecal contaminated water supplies or food products. In
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industrialized countries HEV has until recently been exclusively considered in
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travelers returning from HEV endemic regions. However, the discovery of
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autochthonous HEV in industrialized countries has changed the comprehension
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of HEV infection in these regions, now known to be mainly due to genotype 3
positive-sense,
single-stranded
RNA
virus
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non-enveloped,
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present in swine to such an extent that they are now considered important
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reservoirs for human disease (Berto et al., 2012a; Berto et al, 2012b; Mesquita
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et al., 2014). Consequently, it is expected that spillover of swine and human
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waste may contaminate the environment with HEV enabling routes for human
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exposure and subsequent hepatitis E infection.
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Coastal waters are often contaminated not only with human waste originated
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from sewage treatment plants, but also due to runoff following manure
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application, especially in countries where a high density of farming is present.
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As such, shellfish produced close to land are known to bioaccumulate not only
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human but also animal enteric viruses (Grodzki et al., 2014; Manso and
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Romalde, 2013). The role of bivalve molluscs as vehicles for enteric viruses is
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for long established since these agents are likely to survive for a long period of
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time in the water column, especially if associated with particulate matter and
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sediments, and are not inactivated during food preparation since bivalves are
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also often consumed raw or slightly cooked (Mesquita et al., 2011; Grodzki et
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al., 2014).
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The role of shellfish as vehicles for HEV infection has been gathering the
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attention of the scientific community however it is yet unclear. HEV has been
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widely detected in shellfish from the United Kingdom (Crossan et al., 2012),
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however several recent reports indicate the complete lack of HEV detection on
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shellfish, namely in Italy (La Rosa et al., 2012), France (Grodzki et al., 2014)
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and Denmark (Krog et al., 2014). Nevertheless, anecdotal data have linked the
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consumption of shellfish to hepatitis E both in Vietnam and Japan (Koizumi et
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al., 2004; Inagaki et al., 2015), and also on a cruise ship (Said et al., 2009).
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The goal of the present study was to evaluate the presence of HEV in shellfish
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from Galicia (NW Spain), one of the most important regions in the world for
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mussel production.
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2. Materials and methods
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2.1. Origin and processing of shellfish samples
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A total of 81 mussel (Mytilus galloprovincialis) batches corresponding to 7
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production beds collected over a 18-month period (October 2010–March 2012)
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for a previous study (Manso and Romalde, 2013) were used. After collection,
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batches (10 individuals) of mussel hepatopancreas were processed according
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to the developed standard method ISO/TS 15216-1:2013 with slight
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modifications. RNA extraction was carried out with NucleoSpin RNA Virus kit
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(Macherey-Nagel, Germany) following manufacturer’ specifications after adding
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an extraction control, in accordance with ISO/TS 15216-1:2013.
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2.2. RT-qPCR probe assay for HEV screening and quantification
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A RT-qPCR targeting the ORF3 region of HEV (Jothikumar et al., 2006) was
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applied to undiluted and diluted (1/10) RNA extracts. RT-qPCR was performed
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using the KAPA SYBR® FAST One-Step qRT-PCR Kit (Boston, USA) with
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primers JVHEVF/JVHEVR and probe JVHEVP, a TaqMan® probe containing a
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5′ 6-carboxy fluorescein fluorophore and 3′ black hole quencher. The thermal
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profile was comprised of 42 °C for 5 min and 95 °C for 5 min, followed by 40
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cycles of 95 °C for 3 sec and 60 °C for 20 sec.
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Quantification was estimated, in accordance with ISO/TS 15216-1:2013, by
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standard curves constructed with serial dilutions of purified viral RNA, plotting
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number of RNA viral genome copies per gram of digestive tissue.
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2.3. Nested broad-spectrum RT-PCR assay
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Nucleic acids from RT-qPCR positive samples were tested for the presence of
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HEV RNA by using a nested broad-spectrum RT-PCR with amplification within
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the RNA-dependent RNA-polymerase (RdRP) gene of the open reading frame
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(ORF) 1 region of HEV genome (Johne et al, 2010). The first round RT-PCR
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was performed using the Qiagen One-Step RT-PCR Kit (Hilden, Germany) with
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primers HEV-cs and HEV-cas that amplify a 472-bp fragment. The thermal
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profile was comprised of 42 °C for 60 min and 95 °C for 15 min, followed by 40
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cycles of 94 °C for 1 min, 50 °C for 1 min and 74 ° C for 1 min, with a final
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extension at 72 °C for 10 min. In the second round PCR, 5 µl of the first round
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products were amplified with KAPA HiFi Hotstart ReadyMix, KAPA Biosystems
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(Woburn, MA, USA) using the primers HEV-csn and HEV-casn that amplify a
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334-bp fragment. The thermal profile consisted of 95 °C for 5 min and 35 cycles
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of 94 °C for 30 sec, 50 °C for 30 sec and 72 °C for 1 min, with a final extension
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at 72 °C for 10 min.
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2.4. Sequencing and phylogenetic analysis
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RT-PCR products of the second round PCR (334-bp fragment) were separated
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by electrophoresis in a 1.5% agarose gel and appropriately sized bands were
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excised and purified with the GRS PCR & Gel Band Purification Kit (GRISP,
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Porto, Portugal) and sequenced in both directions using the BigDye Terminator
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v1.1 Cycle Sequencing kit (PE Applied Biosystems, Foster City, CA, USA).
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Sequence editing and multiple alignments were performed with the BioEdit
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software package, version 2.1 (Ibis Biosciences, Carlsbad, CA, USA).
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Phylogenetic analysis was performed using MEGA version 6.0 software.
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3. Results and Discussion
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From the 81 mussel batches initially studied by RT-qPCR, HEV RNA was
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detected in 12 (14.81%). Contamination levels ranged from 6.7 X 101 to 8.6 X
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104 RNA copies/g digestive tissue and all positive samples yielded high
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extraction efficiencies (>10 %; as determined by ISO/TS 15216-1:2013). After
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retesting by nested broad-spectrum RT-PCR it was possible to obtain 6
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amplified products. These amplified products were subjected to sequencing and
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phylogenetic analysis in order to obtain information about their genetic
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relatedness with HEV reference strains of both animal and human origin.
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Phylogenetic analysis based on a 330 nt region of the ORF 1 showed that all
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sequenced isolates belonged to genotype 3 subgenotype e, being closely
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related to strains of human and animal (swine and wild boar) origin (Figure 1).
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Results here presented are intriguing since the high majority of studies in
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Europe generally points to the absence of HEV on shellfish (Grodzki et al.,
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2014; La Rosa et al., 2012; Kroget al., 2014). On the opposite, a single study
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from the UK detected HEV in 97% of the sampled shellfish (Crossan et al.,
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2012). However attention should be paid to the collection site of the majority of
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samples from the UK study. This site was near a slaughterhouse and a meat
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preparation purification plant that processes pigs, in a 10 m2 area around an
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outfall (drain/sewage pipe) directly in line with the processing plant. We
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consider that this is not the typical ecosystem selected for shellfish beds hence
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beds that, although close to a high population density area, are approved for
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use in the production of shellfish for human consumption (Manso and Romalde,
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2013), after a mandatory depuration period (as determined by the European
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Commission regulation (EC) No 853/2004). Nonetheless, although current
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depuration procedures may reduce HEV loads to some extent, they are
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ineffective for the total elimination of the viruses from shellfish (Manso and
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Romalde, 2013).
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4. Conclusion
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HEV infections are cause for serious health concerns affecting almost 20 million
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individuals every year worldwide. This study confirms that HEV genotype 3 is
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present in shellfish causing apprehension on a new potential route for HEV
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transmission to humans. The complete spectrum of animal reservoirs and
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routes for infection are believed to be yet fully understood and warrant further
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research in this topic.
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Acknowledgements
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This work was supported in part by Grant 2014–PG110 from the Xunta de
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Galicia (Spain).
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Figure legend
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Figure 1. Phylogenetic reconstruction based on the ORF1 region using the
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Neighbor-Joining algorithm. Bootstrap values (1000 re-samplings) are shown at
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each branch point. The evolutionary distances were computed using the
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Maximum Composite Likelihood method. Bar, substitutions per nucleotide
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position. Sequences are defined in tree as Strain|Origin|Genotype (accession
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number). Sequences obtained in this work were deposited in have been
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deposited in the GenBank with accession numbers LN887195-LN887200.
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ACCEPTED MANUSCRIPT Highlights 1. Detection of HEV in shellfish harvested areas is described 2. HEV levels ranged from 6.7 X 101 to 8.6 X 104 RNA copies/g digestive tissue 3. All sequenced isolates belonged to the zoonotic genotype 3e
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4. Shellfish constitute a potential route for HEV transmission to humans
