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Short Communication
Detection of hepatitis E virus in wild boar (Sus scrofa) livers
Mamadou Kaba a,b, Bernard Davoust a,c, Jean-Lou Marié c, Philippe Colson a,b,*
a
URMITE CNRS UMR 6236 IRD 198, Facultés de Médecine et de Pharmacie, Université de la
Méditerranée (Aix-Marseille-II), 27 boulevard Jean Moulin, 13385 Marseille cedex 05, France
b
Pôle des Maladies Infectieuses et Tropicales Clinique et Biologique, Fédération de
Bactériologie-Hygiène-Virologie, Centre Hospitalo-Universitaire Timone, 264 rue SaintPierre 13385, Marseille CEDEX 05, France
c
Direction Régionale du Service de Santé des Armées de Toulon, BP 80, 83800 Toulon Armées,
France
* Corresponding author. Tel.: +33 4 91387969
E-mail address: philippe.colson@ap-hm.fr (P. Colson).
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Abstract
The routes of transmission of hepatitis E virus (HEV) in industrialised countries are
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largely unknown, but several studies suggest that HEV can be a zoonosis derived from pigs.
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The aim of the present study was to determine the prevalence of HEV in the wild boar (Sus
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scrofa) and to determine the genetic relationships between HEV sequences recovered from
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wild boars and from domestic pigs and humans. HEV RNA was detected by real time reverse
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transcriptase PCR in 7/285 (2.5%) liver samples from wild boars hunted in South-Eastern
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France. HEV sequences were recovered from five wild boars and belonged to genotype 3f.
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These sequences shared 89-100% nucleotide identity with each other and were genetically
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close to HEV sequences recovered from humans in Southern France. Wild boars in South-
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Eastern France may be a source of HEV transmission to humans.
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Keywords: Hepatitis E virus; Wild boars; Zoonosis; Autochthonous transmission; France
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Hepatitis E virus (HEV) is a leading cause of acute viral hepatitis in tropical and
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subtropical countries (Dalton et al., 2008). In Western Europe, in the USA and in Japan, an
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increasing number of autochthonous cases of hepatitis E have been described recently that
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involve HEV genotypes 3 and 4. The overall case fatality in humans with hepatitis E is 14%,
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but may reach 2030% in pregnant women and higher levels in patients with chronic liver
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disease.
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HEV transmission routes are poorly documented in industrialised countries, although a
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growing body of data indicates that pigs are a major reservoir for HEV genotypes 3 and 4
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(Dalton et al., 2008). A limited amount of data is available on HEV prevalence in wild boars in
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Europe (de Deus et al., 2008a; Kaci et al., 2008) and no data are available from France, where
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many autochthonous hepatitis E cases have been reported recently (Mansuy et al., 2008; Renou
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et al., 2008). The aim of this study was to assess the prevalence of HEV in wild boars in South-
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Eastern France and to determine the genetic relationship between HEV sequences from wild
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boars and those previously recovered from pigs and from human hepatitis E cases in this region.
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Samples of liver were collected from 285 wild boars (Sus scrofa) hunted in South-
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Eastern France from September 2007 to January 2008 (Table 1). Samples were homogenised in
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10% (W/V) sterile phosphate-buffered saline, then clarified by centrifugation at low speed for
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5 min. Total RNA was extracted from 200 µL supernatant using the MagNA Pure LC RNA
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Isolation Kit (Roche Diagnostics). HEV RNA was detected by real-time PCR and sequenced
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(Colson et al., 2007).
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HEV RNA was detected in the livers of 7/285 (2.5%) wild boars (Table 1). HEV
sequences were obtained in 5/7 HEV RNA-positive samples and shared 89-100% nucleotide
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identity with each other, and 80-98% identity with genotype 3 HEV sequences obtained from
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human hepatitis E cases (n = 39) diagnosed in our laboratory (GenBank accession numbers are
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listed in Table 1 of Supplementary material, Appendix A). Phylogenetic analysis showed that
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all wild boar HEV sequences belonged to genotype 3f, but did not cluster together (Fig. 1).
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Three HEV sequences were 100% identical and shared 94% and 97% nucleotide identity,
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respectively, with HEV sequences recovered from a human in France (EU543567) and from
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swine manure in Spain (DQ141120). Another sequence (FJ718694) shared 97-98% nucleotide
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identity with sequences recovered from human hepatitis E cases diagnosed in our laboratory
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(EF028801) and in South-Western France (EU495208-EU495209). The fifth sequence
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recovered from a wild boar (FJ718695) was close to sequences recovered from humans in
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Southern France (EU495225, EF061402).
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The present study is the first reporting HEV detection in wild boars in France. Although
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HEV has been found in wild boars in other European countries and in Japan (de Deus et al.,
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2008a; Kaci et al., 2008; Martelli et al., 2008), the substantial number of cases of human
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hepatitis E reported in France, mostly in its Southern part, including seven fatal cases, have
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prompted and extended the search for sources of HEV infection and routes of transmission in
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this country (Renou et al., 2008; Mansuy et al., 2009).
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The HEV prevalence in wild boars in the present study is similar to that reported in
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Japan (2.3%), but is lower than in Italy, Spain, Hungary and Germany, where HEV RNA was
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detected in 5.3%25% of wild boars (de Deus et al., 2008a; Kaci et al., 2008; Martelli et al.,
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2008). Although we were unable to estimate the age of wild boars in the present study, they are
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usually hunted when older than 12 months. In previous studies, HEV RNA was detected in
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wild boars of various ages (Martelli et al., 2008). These data contrast with those observed in
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domestic pigs, which are mostly HEV RNA-positive from 24 months of age (de Deus et al.,
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2008b). Most importantly, our findings clearly indicate that wild boars represent a source of
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HEV transmission at the age at which they are hunted. The high nucleotide identity between
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HEV sequences recovered in the present study and from humans in Southern France suggests
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that wild boars might represent a source of autochthonous HEV transmission to humans in this
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geographical area.
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Although several studies have implicated zoonotic transmission of HEV from wild
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boars to humans, only one study has confirmed food-borne transmission of HEV by ingestion
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of wild boar meat on the basis of HEV sequence comparison (Li et al., 2005). In a case-control
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study in Germany, Wichmann et al. (2008) recently showed that consumption of wild boar
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meat was independently associated with autochthonous HEV infection. Direct exposure to wild
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boar blood might also represent a source of HEV transmission for hunters or butchers. In blood
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donors from South-Western France and Japan, hunting was associated with a higher prevalence
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of antibodies against HEV (Mansuy et al., 2008). Of note, nearly half a million wild boars are
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killed in France each year and there has been an 8-fold increase in this number globally from
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1974, the largest increase being in the Mediterranean region.1
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In summary, the present study indicates that wild boars represent an HEV reservoir in
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South-Eastern France and that wild boars should be considered a possible source of HEV
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transmission to humans through direct or indirect exposure in this geographical area. Hunters
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should consider wearing gloves when they are exposed to wild boar blood and wild boar meat
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should be adequately cooked before its consumption.
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http://www.oncfs.gouv.fr/events/point_faune/mammifere/2004/sanglier_brochure_extrait.pdf
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Conflict of interest statement
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None of the authors of this paper has a financial or personal relationship with other
people or organisations that could inappropriately influence or bias the content of the paper.
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Acknowledgements
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We thank Professor Hervé Richet for reviewing this manuscript.
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Appendix A. Supplementary material.
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Supplementary data associated with this article can be found in the online version at
doi:….
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References
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Colson, P., Coze, C., Gallian, P., Henry, M., De Micco, P., Tamalet, C., 2007. Transfusionassociated hepatitis E, France. Emerging Infectious Diseases 13, 648-649.
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Gortazar, C., Segales, J., 2008a. Epidemiological study of hepatitis E virus infection in
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N., Rostaing, L., Alric, L., Moreau, J., Peron, J. M., Izopet, J., 2009. Acute hepatitis E
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Delogu, M., Ostanello, F., 2008. Detection of hepatitis E virus (HEV) in a demographic
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Table 1
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Hepatitis E virus (HEV) RNA detection in wild boar liver samples related to the place of
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hunting.
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Origins of wild boars tested
Var Department
Bouches-du-Rhône Department
Total
Number
Number of samples
Number of HEV sequences
Genotype
of liver
positive for HEV
obtained from wild boar
(subtype)
samples
RNA (% positive) 1
liver samples
278
7 (2.5%)
5
3 (f)
7
0 (0.0%)
ND 2
ND
285
7 (2.5%)
5

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1
HEV RNA-positive by in house real time reverse transcriptase PCR.
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2
ND, Not done.
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Figure legend
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Fig. 1. Phylogenetic tree constructed by the neighbour-joining method based on partial
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nucleotide sequences of the 5’ open reading frame 2 region of the HEV genome (337 bp). The
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phylogenetic analysis includes (1) HEV sequences recovered from wild boar liver samples in
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the present study (in boldface on a black font); (2) sequences recovered from human hepatitis E
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cases diagnosed in our laboratory (in boldface and framed by a black line); (3) GenBank
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sequences corresponding to the best BLAST2 hits on the wild boar HEV sequences recovered
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in the present study (in boldface on a grey font); and (4) sequences with previously identified
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genotypes and subtypes. Avian HEV sequence (AY043166) was used as an outgroup. HEV
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sequence names are labelled as follows: Host: Hu, Human; Sw, Pig; and Wb, Wild boar;
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GenBank accession number, country of origin, genotype and subtype (in parenthesis).
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Bootstrap values are indicated when 50% as a percentage obtained from 1,000 resamplings of
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the data. The scale bar represents a genetic distance of 0.05 substitutions per site.
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Abbreviations: Av, Avian; BBH, Best BLAST hit; CH, China; EG, Egypt; FR, France; G3f,
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Genotype 3f; Hu, Human; HUN, Hungary; IN, India; JA, Japan; Mrs, Marseille (France); MX,
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Mexico; NL, Netherlands; NP, Nepal; SP, Spain; Sw, Pig; TH, Thailand; TW, Taiwan; UK,
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United Kingdom; US, United States of America; Wb, Wild boar. Wild boar HEV sequences
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recovered in the present study are deposited in the GenBank database under accession
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numbers: FJ718691FJ718695.
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See: http://www.ncbi.nlm.nih.gov/BLAST/