Short Communication Intervirology 2014;57:43–48 DOI: 10.1159/000354801
Received: February 28, 2013 Accepted after revision: July 25, 2013 Published online: October 22, 2013
Emergence of Autochthonous Infections with Hepatitis E Virus of Genotype 4 in Europe Yanis Bouamra a René Gérolami c Jean-Pierre Arzouni a Jean-Charles Grimaud d Pierre Lafforgue e Marjorie Nelli f Natacha Tivoli a Audrey Ferretti a Anne Motte a Philippe Colson a, b a
IHU Méditerranée Infection, Pôle des Maladies Infectieuses et Tropicales Clinique et Biologique, Fédération de Bactériologie-Hygiène-Virologie, Centre Hospitalo-Universitaire Timone, Assistance Publique – Hôpitaux Marseille (AP-HM), b Aix-Marseille Université, URMITE UM 63 CNRS 7278 IRD 198 Inserm U1095, Facultés de Médecine et de Pharmacie, c Service d’Hépato-Gastro-Entérologie, Centre Hospitalo-Universitaire Conception, AP-HM, d Service d’Hépato-Gastro-Enterologie, Centre Hospitalo-Universitaire Nord, AP-HM, e Service de Rhumatologie, Centre Hospitalo-Universitaire Sainte-Marguerite, AP-HM, Marseille, et f Service d’Hépato-Gastro-Entérologie, Centre Hospitalier de Salon, Salon-de-Provence, France
Key Words Hepatitis E virus · Genotype 4 · France · Laboratory-based surveillance · Zoonosis
Abstract In Europe, autochthonous hepatitis E is caused by genotype 3 hepatitis E virus (HEV) in almost all cases. A total of 15 infections with genotype 4 HEV were diagnosed in France from May 2009 to April 2012, and all but one of the HEV-4 strains implicated in these infections were genetically related and highly similar to HEV-4 sequences isolated from swine in Belgium. In addition, 5 autochthonous HEV-4 infections have been described in the region of Lazio, Italy, during March and April 2011, and these HEV sequences were 100% identical to one another but showed relatively low similarity (74– 85%) to HEV-4 RNA samples collected in France. We report 6 additional HEV-4 infections that were diagnosed from May to July 2012 which represented 50% of the HEV infections
© 2013 S. Karger AG, Basel 0300–5526/14/0571–0043$39.50/0 E-Mail [email protected] www.karger.com/int
diagnosed during this period in our clinical microbiology laboratory. Five of these HEV-4 strains were associated with autochthonous infections and were clustered together and with the majority of HEV-4 previously described in France, whereas the sixth strain was genetically divergent. Taken together with reports from other teams, these observations indicate that autochthonous infections with HEV-4 are emerging in Europe and have been transmitted by at least two distinct sources. © 2013 S. Karger AG, Basel
Over the last decade, hepatitis E virus (HEV) strains of genotype 3/4 have emerged as causative agents of autochthonous hepatitis E in developed countries, mostly Japan and Europe [1, 2]. A porcine reservoir has been identified for these two HEV genotypes, and food-borne zoonotic transmission is suspected, and demonstrated in a few cases [3–5]. Autochthonous hepatitis E in Japan has involved Philippe Colson Pôle des Maladies Infectieuses et Tropicales Clinique et Biologique Fédération de Bactériologie-Hygiène-Virologie, Centre Hospitalo-Universitaire Timone 264 rue Saint-Pierre, FR–13385 Marseille Cedex 05 (France) E-Mail philippe.colson @ univ-amu.fr
10
Fig. 1. HEV infections diagnosed in the
clinical microbiology laboratory of the Marseille university hospitals from January 2011 to October 2012, including with all HEV genotypes and genotype 4 viruses.
8 7 6 5 4 3 2 1 0
120 2- 11 20 3- 11 20 4- 11 20 5- 11 20 6- 11 20 7- 11 20 8- 11 20 9- 11 20 10 11 -2 11 011 -2 12 011 -2 0 1- 11 20 2- 12 20 3- 12 20 4- 12 20 5- 12 20 6- 12 20 7- 12 20 8- 12 20 9- 12 20 10 12 -2 01 2
Number of PCR-documented HEV infections
9
HEV-3/4 [2, 6], whereas until recently, HEV-3 had been found in nearly all European cases [1, 2, 4, 7–14]. Indeed, autochthonous infection with HEV-4f was first diagnosed in Germany in 2006–2007 from the report of a single case [15]. Six cases were then diagnosed in France from May 2009 to April 2012 of HEV-4 that were related to subtype 4b and genetically related to one another, although these sequences were found in both the south-east and north-east region and there was no apparent epidemiological link between the cases [10, 11, 16]. Moreover, HEV-4 RNA obtained in France were highly similar to HEV-4 sequences retrieved from swine in Belgium in 2010 [16, 17]. In addition, 5 autochthonous HEV-4 infections have been described in the region of Lazio, Italy, during March and April 2011 [12]. These HEV-4 sequences were retrieved from patients who did not report recent travel to HEV hyperendemic regions and were classified as 4d and 100% identical with one another, although neither an epidemiological link nor a common source was identified. Furthermore, these sequences showed relatively low similarity (74–85%) to HEV-4 RNA that had been described in France, suggesting multiple sources for these infections. Finally, 9 additional HEV-4 infections were identified in France in 2011, 6 of which in patients living in northern France [13]. Pork meat consumption and travel abroad in China were reported in 5 cases and in 1 case, respectively. Phylogenetic analyses revealed that HEV-4 RNA recovered from the 8 autochthonous cases were both clustered together and close to the other HEV-4 sequence described in France, whereas the HEV-4 from the imported case was clustered with sequences obtained in China. 44
Intervirology 2014;57:43–48 DOI: 10.1159/000354801
All genotypes
Genotype 4
In the Marseille university hospitals, 5 HEV-4 infections were identified from February 2011 to April 2012 (fig. 1) [11, 16]. We describe here 6 additional cases that were diagnosed from May 2012 to July 2012 based on positive testing of anti-HEV IgM and HEV RNA, as previously described [16]. During this 3-month period, these HEV-4 infections represented 50% of the HEV infections (6/12) diagnosed in our laboratory (fig. 1). It is worth noting that all 6 of these patients ate pig liver sausage uncooked; none ate shellfish nor reported any professional exposure to pigs or sewage. Two of the patients were married and reported several potential sources of HEV infection, including keeping pigs at home, manufacturing their own pig liver sausages, and drinking water from their own well. One patient had returned from China 5 weeks ago following a 10-day stay. Based on the analysis of a 424-bp fragment of HEV open reading frame 2 (ORF2), HEV-4 RNA recovered from the serum of this patient showed only 85% nucleotide identity with the HEV-4 RNA from the 5 other patients. This infection was likely acquired in China; the best BLAST matches (96.6–97.8% identity) against the NCBI GenBank nucleotide sequence database were sequences described in humans and swine in China (fig. 2). However, strikingly, similarity was concurrently 97.1% with a sequence from the outbreak of autochthonous HEV-4 infections recently reported in Italy [12]. The 5 other HEV-4 sequences were clustered together and with the 5 HEV-4 previously described in our laboratory [11, 16]. The nucleotide similarity between these 10 sequences was 99.2–100%, and their best hits in GenBank (88.9–94.5% identity) were sequences originating from swine or humans samBouamra et al.
JX102545 FRA-MAR Case no.5 FRA_MAR JN794589 FRA-MAR 60 JX102544 FRA-MAR
Case no.4 FRA_MAR 59 100
Case no.6 FRA_MAR JN794587 FRA-MAR Case no.3 FRA_MAR JX1025467 FRA-MAR Case no.1 FRA_MAR BBH JX997466 CHN (Jiangsu, Nanjing) Hu 2006
96 99
Ref 4b BBH DQ279091 CHN Sw Mar-2007
Fig. 2. Phylogenetic tree based on a partial
(424 nucleotides; nucleotides 6,553–6,974 of the HEV genome accession No. AB291961) nucleotide sequence of ORF2 of the HEV genome. The HEV sequences collected in the present study are indicated by black boxes and a black background. The HEV sequences of genotype 4 obtained earlier in our laboratory are indicated by black boxes and a white background. The HEV sequences with the highest BLAST scores using the sequences from this study as queries are indicated in boldface. Sequence E2105 ITA Hu 2011 was kindly provided by Drs. Anna Rosa Garbuglia and Maria R. Capobianchi [12]. The nucleotide alignments were performed using ClustalX v2.0 (http://www.clustal.org/ download/current/). The tree was constructed using the MEGA v5.0 software (http://www.megasoftware.net/) with the neighbor-joining method, as described previously [16]. The branches with bootstrap values, obtained from 1,000 resamplings of the data, >50% are labeled on the tree. The avian HEV sequence AY043166 was used as an outgroup. The scale bar indicates the number of nucleotide substitutions per site. The HEV sequences are labeled with the GenBank accession numbers, countries of origin, host species, and collection or submission dates. Av, Avian; BBH, best BLAST hit; CHN, China; FRA, France; Hu, human; JPN, Japan; MAR, Marseille; Sw, swine; Rab, rabbit; Ref, reference sequences.
BBH JX997618 CHN (Jiangsu, Nanjing) Hu 2010 BBH JX997512 CHN (Jiangsu, Nanjing) Hu 2007
74
69
57
BBH JX997570 CHN (Jiangsu, Nanjing) Hu 2009 BBH EU676172 CHN Sw Oct-2008
92
BBH JX997567 CHN (Jiangsu, Nanjing) Hu 2009
82
BBH JX855794 CHN Sw Oct-2011 BBH JX847413 CHN Hu Sep-2012 Ref 4a AF151962 CHN Hu May-1999 100 Ref 4a AF151963 CHN Hu May-1999
Ref 4e AY723745 IND Sw Aug-2004 Ref 4d AY594199 CHN Sw Apr-2004
66
BBH FJ641994 CHN Sw 2008 BBH Ref 4d AJ272108 CHN Hu Feb-2000 BBH HM152568 CHN Sw Feb-2008
84 72 87
BBH GU206559 CHN Sw Feb-200887 BBH JQ655736 CHN Sw Nov-2012 BBH GU361892 CHN Sw May-2008
66
JX997615 CHN (Jiangsu, Nanjing) Hu 2010
94 56
Case no. 2 FRA-MAR E2105 ITA Hu 2011 (ref. 12) JX997565 CHN (Jiangsu, Nanjing) Hu 2009 JX997470 CHN (Jiangsu, Nanjing) Hu 2006 JX997500 CHN (Jiangsu, Nanjing) Hu 2007
98 JX997488 CHN (Jiangsu, Nanjing) Hu 2007
Ref 4g AB108537 CHN Hu 2000 Ref 4c AB097811 JPN Sw Dec-2002 67
Ref 4c AB161718 JPN Hu Feb-2004 Ref 1 AF051830
83
Ref 2 M74506 54 69
Ref 3rab FJ906895 Ref 3f EU495148 Ref 3e AB248521 Ref 3a AY575859 AY043166
0.1
pled in China from 2006 through 2011 (fig. 2). In addition, another fragment (303 nucleotides) of HEV ORF2 obtained from 2 autochthonous cases identified in our laboratory from May 2012 to July 2012 were clustered with the 14 HEV-4 sequences previously retrieved from
autochthonous cases in France, and showed high levels of similarity (98.6–100%) to these sequences [10, 13]. Then, the best matches in GenBank (94% identity) were sequences originating from swine, Macaca mulata and humans sampled in China (fig. 3). No other cases of
Hepatitis E Virus Genotype 4 in Europe
Intervirology 2014;57:43–48 DOI: 10.1159/000354801
45
JX102542 FRA-MAR JX102543 FRA-MAR JX102544 FRA-MAR BBH KC437302 FRA Hu 2011 JX102546 FRA-MAR Case no.6 FRA_MAR 64 Case no.4 FRA_MAR JX102545 FRA-MAR 65 BBH KC437304 FRA Hu 2011 BBH KC437303 FRA Hu 2011 BBH KC437301 FRA Hu 2011 BBH KC437305 FRA Hu 2011 100 62 BBH KC437308 FRA Hu 2011 64 BBH KC437307 FRA Hu 2011 BBH KC437306 FRA Hu 2011 BBH GU982294 FRA Hu May-2009 BBH AJ344177 CHN Hu Aug-2001 57 BBH AJ344188 CHN Hu Aug-2001 98 BBH AJ344186 CHN Hu Aug-2001 BBH A J344192 CHN Hu Aug-2001 50 BBH EU375331 CHN Sw Jan-2008 95 Ref 4b HM439284 CHN HU 2008 BBH GQ242164 CHN Ma Dec-2008 BBH GQ242165 CHN Ma Jan-2009 83 BBH HQ292707 CHN Sw Jun-2009 BBH GQ242160 CHN Ma Aug-2008 BBH EU375332 CHN Sw Jan-2008 BBH GQ242166 CHN Ma Jan-2009 BBH GQ242162 CHN Ma Nov-2008 BBH GQ242163 CHN Ma Nov-2008 BBH EU375330 CHN Sw Jan-2008 64 BBH EU375329 CHN Sw Jan-2008 BBH DQ279091 CHN Sw Mar-2007 BBH AB683162 JPN Hu Dec-2005 Ref 4b AF117280 TWN Sw Dec-1998 BBH AB301700 JPN Hu 2003 100 BBH / Ref4b AB168096 VNM Hu Mar-2004 Ref 4d AJ344181 CHN Hu Aug-2001 JX401928 ITA Hu Mar-2011 88 Ref 4d AJ272108 CHN Hu Feb-2000 Ref 4d AY 596320 CHN Sw Apr-2004 100 Ref 4d AY 594199 CHN Sw Apr-2004 Ref 4a AJ344171 CHN Hu Aug-2001 99 Ref 4a AF117275 TWN Hu Dec-1998 KC437309 FRA Hu 2011 Ref 4g AB108537 CHN Hu 2000 Ref 4f AB082558 JPN Hu Mar-2002 Ref 4c AB105897 JPN Hu Mar-2003 Ref 4c AB082545 JPN Hu Mar-2002 Ref 4c AB094223 JPN Sw Oct-2002 100 Ref 4c AB097811 JPN Sw Dec-2002 79 99 Ref 4c AB099347 JPN Hu Jan-2003 100 Ref 1 AF051830 Ref 1 X99441 Ref 2 M74506 99 Ref 3rab FJ906895 Ref 3rab GU937805 98 Ref 3a AY575859 69 Ref 3a AF060668 Ref 3b AB091394 Ref 3i FJ998008 Ref 3g AF455784 88 Ref 3e AB248520 Ref 3e AB248522 58 Ref 3f EU360977 53 Ref 3f EU495148 Ref Av AM943646 65
83
75
Fig. 3. Phylogenetic tree based on a partial
(204 nucleotides; nucleotides 6,025–6,326 of the HEV genome accession No. AB291961) nucleotide sequence of ORF2 of the HEV genome. The phylogenetic analysis is as described in figure 2, except that the avian HEV sequence AM943646 was used as an outgroup. The sequences are labeled as described above with the following exceptions: the sequences from this study are indicated by a black box with a black background and the abbreviations are the same, with the addition of ITA, Italy; TWN, Taiwan; and VNM, Vietnam.
82
69
89
57 73
0.05
HEV-4 infection were detected in our center from August 2012 to June 2013. Worldwide, HEV-4 strains have been mostly described in Japan and China in humans and swine [18–20]. Several HEV-4 subtypes have been described in Japan, including 46
Intervirology 2014;57:43–48 DOI: 10.1159/000354801
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subtypes b and f, in addition to HEV-4 transmission through pig meat consumption [3, 21]. In China, HEV-4 has emerged over the last decade and overtaken HEV-1 as the predominant strain in humans and swine [19, 20, 22]. The HEV molecular epidemiology has been found to be Bouamra et al.
complex in this country, as several HEV-4 subtypes, including subtypes b and d, have been detected, and sequences recovered from humans were found (although inconsistently) to share a high level of nucleotide similarity with those found from swine in the same geographical area [19, 20, 22]. Otherwise, HEV-4 have been collected in India, Indonesia, South Korea, Taiwan and Vietnam [18, 20, 23–27], including in autochthonous hepatitis E, and an epidemiological link with pigs or wild boars has been observed in cases diagnosed in Bali and South Korea [14, 23, 25]. In addition, HEV-4 infection was reported in 2010 in England in a patient who returned from a 2-month stay in India 1 month before presenting jaundice [28]. HEV-4 collected in Japan and France have been associated with higher liver cytolysis and a lower prothrombin index relative to HEV-3, and more severe illness was described in Japan for HEV-4 compared to HEV-3 [29, 30]. However, further studies are needed to determine if differences in the hepatitis E presentation and outcome might be linked to the HEV genotypic patterns or the host or both.
Overall, previous data indicate the emergence of autochthonous HEV-4 infections in Europe, originating from different sources, and these findings create an incentive to implement a surveillance of HEV-4 infections on that continent to aid in documenting the presence of this genotype in European pigs or wild boars and identify the possible importation of HEV-4 from Asia.
Acknowledgements We are thankful to Drs. Anna Rosa Garbuglia and Maria R. Capobianchi, Laboratory of Virology, L. Spallanzani National Institute for Infectious Diseases, Rome, Italy, for providing the sequence of HEV genotype 4 from Italy.
Disclosure Statement The authors have no conflicts of interest to disclose.
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Bouamra et al.
Copyright: S. Karger AG, Basel 2013. Reproduced with the permission of S. Karger AG, Basel. Further reproduction or distribution (electronic or otherwise) is prohibited without permission from the copyright holder.
Received: February 28, 2013 Accepted after revision: July 25, 2013 Published online: October 22, 2013
Emergence of Autochthonous Infections with Hepatitis E Virus of Genotype 4 in Europe Yanis Bouamra a René Gérolami c Jean-Pierre Arzouni a Jean-Charles Grimaud d Pierre Lafforgue e Marjorie Nelli f Natacha Tivoli a Audrey Ferretti a Anne Motte a Philippe Colson a, b a
IHU Méditerranée Infection, Pôle des Maladies Infectieuses et Tropicales Clinique et Biologique, Fédération de Bactériologie-Hygiène-Virologie, Centre Hospitalo-Universitaire Timone, Assistance Publique – Hôpitaux Marseille (AP-HM), b Aix-Marseille Université, URMITE UM 63 CNRS 7278 IRD 198 Inserm U1095, Facultés de Médecine et de Pharmacie, c Service d’Hépato-Gastro-Entérologie, Centre Hospitalo-Universitaire Conception, AP-HM, d Service d’Hépato-Gastro-Enterologie, Centre Hospitalo-Universitaire Nord, AP-HM, e Service de Rhumatologie, Centre Hospitalo-Universitaire Sainte-Marguerite, AP-HM, Marseille, et f Service d’Hépato-Gastro-Entérologie, Centre Hospitalier de Salon, Salon-de-Provence, France
Key Words Hepatitis E virus · Genotype 4 · France · Laboratory-based surveillance · Zoonosis
Abstract In Europe, autochthonous hepatitis E is caused by genotype 3 hepatitis E virus (HEV) in almost all cases. A total of 15 infections with genotype 4 HEV were diagnosed in France from May 2009 to April 2012, and all but one of the HEV-4 strains implicated in these infections were genetically related and highly similar to HEV-4 sequences isolated from swine in Belgium. In addition, 5 autochthonous HEV-4 infections have been described in the region of Lazio, Italy, during March and April 2011, and these HEV sequences were 100% identical to one another but showed relatively low similarity (74– 85%) to HEV-4 RNA samples collected in France. We report 6 additional HEV-4 infections that were diagnosed from May to July 2012 which represented 50% of the HEV infections
© 2013 S. Karger AG, Basel 0300–5526/14/0571–0043$39.50/0 E-Mail [email protected] www.karger.com/int
diagnosed during this period in our clinical microbiology laboratory. Five of these HEV-4 strains were associated with autochthonous infections and were clustered together and with the majority of HEV-4 previously described in France, whereas the sixth strain was genetically divergent. Taken together with reports from other teams, these observations indicate that autochthonous infections with HEV-4 are emerging in Europe and have been transmitted by at least two distinct sources. © 2013 S. Karger AG, Basel
Over the last decade, hepatitis E virus (HEV) strains of genotype 3/4 have emerged as causative agents of autochthonous hepatitis E in developed countries, mostly Japan and Europe [1, 2]. A porcine reservoir has been identified for these two HEV genotypes, and food-borne zoonotic transmission is suspected, and demonstrated in a few cases [3–5]. Autochthonous hepatitis E in Japan has involved Philippe Colson Pôle des Maladies Infectieuses et Tropicales Clinique et Biologique Fédération de Bactériologie-Hygiène-Virologie, Centre Hospitalo-Universitaire Timone 264 rue Saint-Pierre, FR–13385 Marseille Cedex 05 (France) E-Mail philippe.colson @ univ-amu.fr
10
Fig. 1. HEV infections diagnosed in the
clinical microbiology laboratory of the Marseille university hospitals from January 2011 to October 2012, including with all HEV genotypes and genotype 4 viruses.
8 7 6 5 4 3 2 1 0
120 2- 11 20 3- 11 20 4- 11 20 5- 11 20 6- 11 20 7- 11 20 8- 11 20 9- 11 20 10 11 -2 11 011 -2 12 011 -2 0 1- 11 20 2- 12 20 3- 12 20 4- 12 20 5- 12 20 6- 12 20 7- 12 20 8- 12 20 9- 12 20 10 12 -2 01 2
Number of PCR-documented HEV infections
9
HEV-3/4 [2, 6], whereas until recently, HEV-3 had been found in nearly all European cases [1, 2, 4, 7–14]. Indeed, autochthonous infection with HEV-4f was first diagnosed in Germany in 2006–2007 from the report of a single case [15]. Six cases were then diagnosed in France from May 2009 to April 2012 of HEV-4 that were related to subtype 4b and genetically related to one another, although these sequences were found in both the south-east and north-east region and there was no apparent epidemiological link between the cases [10, 11, 16]. Moreover, HEV-4 RNA obtained in France were highly similar to HEV-4 sequences retrieved from swine in Belgium in 2010 [16, 17]. In addition, 5 autochthonous HEV-4 infections have been described in the region of Lazio, Italy, during March and April 2011 [12]. These HEV-4 sequences were retrieved from patients who did not report recent travel to HEV hyperendemic regions and were classified as 4d and 100% identical with one another, although neither an epidemiological link nor a common source was identified. Furthermore, these sequences showed relatively low similarity (74–85%) to HEV-4 RNA that had been described in France, suggesting multiple sources for these infections. Finally, 9 additional HEV-4 infections were identified in France in 2011, 6 of which in patients living in northern France [13]. Pork meat consumption and travel abroad in China were reported in 5 cases and in 1 case, respectively. Phylogenetic analyses revealed that HEV-4 RNA recovered from the 8 autochthonous cases were both clustered together and close to the other HEV-4 sequence described in France, whereas the HEV-4 from the imported case was clustered with sequences obtained in China. 44
Intervirology 2014;57:43–48 DOI: 10.1159/000354801
All genotypes
Genotype 4
In the Marseille university hospitals, 5 HEV-4 infections were identified from February 2011 to April 2012 (fig. 1) [11, 16]. We describe here 6 additional cases that were diagnosed from May 2012 to July 2012 based on positive testing of anti-HEV IgM and HEV RNA, as previously described [16]. During this 3-month period, these HEV-4 infections represented 50% of the HEV infections (6/12) diagnosed in our laboratory (fig. 1). It is worth noting that all 6 of these patients ate pig liver sausage uncooked; none ate shellfish nor reported any professional exposure to pigs or sewage. Two of the patients were married and reported several potential sources of HEV infection, including keeping pigs at home, manufacturing their own pig liver sausages, and drinking water from their own well. One patient had returned from China 5 weeks ago following a 10-day stay. Based on the analysis of a 424-bp fragment of HEV open reading frame 2 (ORF2), HEV-4 RNA recovered from the serum of this patient showed only 85% nucleotide identity with the HEV-4 RNA from the 5 other patients. This infection was likely acquired in China; the best BLAST matches (96.6–97.8% identity) against the NCBI GenBank nucleotide sequence database were sequences described in humans and swine in China (fig. 2). However, strikingly, similarity was concurrently 97.1% with a sequence from the outbreak of autochthonous HEV-4 infections recently reported in Italy [12]. The 5 other HEV-4 sequences were clustered together and with the 5 HEV-4 previously described in our laboratory [11, 16]. The nucleotide similarity between these 10 sequences was 99.2–100%, and their best hits in GenBank (88.9–94.5% identity) were sequences originating from swine or humans samBouamra et al.
JX102545 FRA-MAR Case no.5 FRA_MAR JN794589 FRA-MAR 60 JX102544 FRA-MAR
Case no.4 FRA_MAR 59 100
Case no.6 FRA_MAR JN794587 FRA-MAR Case no.3 FRA_MAR JX1025467 FRA-MAR Case no.1 FRA_MAR BBH JX997466 CHN (Jiangsu, Nanjing) Hu 2006
96 99
Ref 4b BBH DQ279091 CHN Sw Mar-2007
Fig. 2. Phylogenetic tree based on a partial
(424 nucleotides; nucleotides 6,553–6,974 of the HEV genome accession No. AB291961) nucleotide sequence of ORF2 of the HEV genome. The HEV sequences collected in the present study are indicated by black boxes and a black background. The HEV sequences of genotype 4 obtained earlier in our laboratory are indicated by black boxes and a white background. The HEV sequences with the highest BLAST scores using the sequences from this study as queries are indicated in boldface. Sequence E2105 ITA Hu 2011 was kindly provided by Drs. Anna Rosa Garbuglia and Maria R. Capobianchi [12]. The nucleotide alignments were performed using ClustalX v2.0 (http://www.clustal.org/ download/current/). The tree was constructed using the MEGA v5.0 software (http://www.megasoftware.net/) with the neighbor-joining method, as described previously [16]. The branches with bootstrap values, obtained from 1,000 resamplings of the data, >50% are labeled on the tree. The avian HEV sequence AY043166 was used as an outgroup. The scale bar indicates the number of nucleotide substitutions per site. The HEV sequences are labeled with the GenBank accession numbers, countries of origin, host species, and collection or submission dates. Av, Avian; BBH, best BLAST hit; CHN, China; FRA, France; Hu, human; JPN, Japan; MAR, Marseille; Sw, swine; Rab, rabbit; Ref, reference sequences.
BBH JX997618 CHN (Jiangsu, Nanjing) Hu 2010 BBH JX997512 CHN (Jiangsu, Nanjing) Hu 2007
74
69
57
BBH JX997570 CHN (Jiangsu, Nanjing) Hu 2009 BBH EU676172 CHN Sw Oct-2008
92
BBH JX997567 CHN (Jiangsu, Nanjing) Hu 2009
82
BBH JX855794 CHN Sw Oct-2011 BBH JX847413 CHN Hu Sep-2012 Ref 4a AF151962 CHN Hu May-1999 100 Ref 4a AF151963 CHN Hu May-1999
Ref 4e AY723745 IND Sw Aug-2004 Ref 4d AY594199 CHN Sw Apr-2004
66
BBH FJ641994 CHN Sw 2008 BBH Ref 4d AJ272108 CHN Hu Feb-2000 BBH HM152568 CHN Sw Feb-2008
84 72 87
BBH GU206559 CHN Sw Feb-200887 BBH JQ655736 CHN Sw Nov-2012 BBH GU361892 CHN Sw May-2008
66
JX997615 CHN (Jiangsu, Nanjing) Hu 2010
94 56
Case no. 2 FRA-MAR E2105 ITA Hu 2011 (ref. 12) JX997565 CHN (Jiangsu, Nanjing) Hu 2009 JX997470 CHN (Jiangsu, Nanjing) Hu 2006 JX997500 CHN (Jiangsu, Nanjing) Hu 2007
98 JX997488 CHN (Jiangsu, Nanjing) Hu 2007
Ref 4g AB108537 CHN Hu 2000 Ref 4c AB097811 JPN Sw Dec-2002 67
Ref 4c AB161718 JPN Hu Feb-2004 Ref 1 AF051830
83
Ref 2 M74506 54 69
Ref 3rab FJ906895 Ref 3f EU495148 Ref 3e AB248521 Ref 3a AY575859 AY043166
0.1
pled in China from 2006 through 2011 (fig. 2). In addition, another fragment (303 nucleotides) of HEV ORF2 obtained from 2 autochthonous cases identified in our laboratory from May 2012 to July 2012 were clustered with the 14 HEV-4 sequences previously retrieved from
autochthonous cases in France, and showed high levels of similarity (98.6–100%) to these sequences [10, 13]. Then, the best matches in GenBank (94% identity) were sequences originating from swine, Macaca mulata and humans sampled in China (fig. 3). No other cases of
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JX102542 FRA-MAR JX102543 FRA-MAR JX102544 FRA-MAR BBH KC437302 FRA Hu 2011 JX102546 FRA-MAR Case no.6 FRA_MAR 64 Case no.4 FRA_MAR JX102545 FRA-MAR 65 BBH KC437304 FRA Hu 2011 BBH KC437303 FRA Hu 2011 BBH KC437301 FRA Hu 2011 BBH KC437305 FRA Hu 2011 100 62 BBH KC437308 FRA Hu 2011 64 BBH KC437307 FRA Hu 2011 BBH KC437306 FRA Hu 2011 BBH GU982294 FRA Hu May-2009 BBH AJ344177 CHN Hu Aug-2001 57 BBH AJ344188 CHN Hu Aug-2001 98 BBH AJ344186 CHN Hu Aug-2001 BBH A J344192 CHN Hu Aug-2001 50 BBH EU375331 CHN Sw Jan-2008 95 Ref 4b HM439284 CHN HU 2008 BBH GQ242164 CHN Ma Dec-2008 BBH GQ242165 CHN Ma Jan-2009 83 BBH HQ292707 CHN Sw Jun-2009 BBH GQ242160 CHN Ma Aug-2008 BBH EU375332 CHN Sw Jan-2008 BBH GQ242166 CHN Ma Jan-2009 BBH GQ242162 CHN Ma Nov-2008 BBH GQ242163 CHN Ma Nov-2008 BBH EU375330 CHN Sw Jan-2008 64 BBH EU375329 CHN Sw Jan-2008 BBH DQ279091 CHN Sw Mar-2007 BBH AB683162 JPN Hu Dec-2005 Ref 4b AF117280 TWN Sw Dec-1998 BBH AB301700 JPN Hu 2003 100 BBH / Ref4b AB168096 VNM Hu Mar-2004 Ref 4d AJ344181 CHN Hu Aug-2001 JX401928 ITA Hu Mar-2011 88 Ref 4d AJ272108 CHN Hu Feb-2000 Ref 4d AY 596320 CHN Sw Apr-2004 100 Ref 4d AY 594199 CHN Sw Apr-2004 Ref 4a AJ344171 CHN Hu Aug-2001 99 Ref 4a AF117275 TWN Hu Dec-1998 KC437309 FRA Hu 2011 Ref 4g AB108537 CHN Hu 2000 Ref 4f AB082558 JPN Hu Mar-2002 Ref 4c AB105897 JPN Hu Mar-2003 Ref 4c AB082545 JPN Hu Mar-2002 Ref 4c AB094223 JPN Sw Oct-2002 100 Ref 4c AB097811 JPN Sw Dec-2002 79 99 Ref 4c AB099347 JPN Hu Jan-2003 100 Ref 1 AF051830 Ref 1 X99441 Ref 2 M74506 99 Ref 3rab FJ906895 Ref 3rab GU937805 98 Ref 3a AY575859 69 Ref 3a AF060668 Ref 3b AB091394 Ref 3i FJ998008 Ref 3g AF455784 88 Ref 3e AB248520 Ref 3e AB248522 58 Ref 3f EU360977 53 Ref 3f EU495148 Ref Av AM943646 65
83
75
Fig. 3. Phylogenetic tree based on a partial
(204 nucleotides; nucleotides 6,025–6,326 of the HEV genome accession No. AB291961) nucleotide sequence of ORF2 of the HEV genome. The phylogenetic analysis is as described in figure 2, except that the avian HEV sequence AM943646 was used as an outgroup. The sequences are labeled as described above with the following exceptions: the sequences from this study are indicated by a black box with a black background and the abbreviations are the same, with the addition of ITA, Italy; TWN, Taiwan; and VNM, Vietnam.
82
69
89
57 73
0.05
HEV-4 infection were detected in our center from August 2012 to June 2013. Worldwide, HEV-4 strains have been mostly described in Japan and China in humans and swine [18–20]. Several HEV-4 subtypes have been described in Japan, including 46
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subtypes b and f, in addition to HEV-4 transmission through pig meat consumption [3, 21]. In China, HEV-4 has emerged over the last decade and overtaken HEV-1 as the predominant strain in humans and swine [19, 20, 22]. The HEV molecular epidemiology has been found to be Bouamra et al.
complex in this country, as several HEV-4 subtypes, including subtypes b and d, have been detected, and sequences recovered from humans were found (although inconsistently) to share a high level of nucleotide similarity with those found from swine in the same geographical area [19, 20, 22]. Otherwise, HEV-4 have been collected in India, Indonesia, South Korea, Taiwan and Vietnam [18, 20, 23–27], including in autochthonous hepatitis E, and an epidemiological link with pigs or wild boars has been observed in cases diagnosed in Bali and South Korea [14, 23, 25]. In addition, HEV-4 infection was reported in 2010 in England in a patient who returned from a 2-month stay in India 1 month before presenting jaundice [28]. HEV-4 collected in Japan and France have been associated with higher liver cytolysis and a lower prothrombin index relative to HEV-3, and more severe illness was described in Japan for HEV-4 compared to HEV-3 [29, 30]. However, further studies are needed to determine if differences in the hepatitis E presentation and outcome might be linked to the HEV genotypic patterns or the host or both.
Overall, previous data indicate the emergence of autochthonous HEV-4 infections in Europe, originating from different sources, and these findings create an incentive to implement a surveillance of HEV-4 infections on that continent to aid in documenting the presence of this genotype in European pigs or wild boars and identify the possible importation of HEV-4 from Asia.
Acknowledgements We are thankful to Drs. Anna Rosa Garbuglia and Maria R. Capobianchi, Laboratory of Virology, L. Spallanzani National Institute for Infectious Diseases, Rome, Italy, for providing the sequence of HEV genotype 4 from Italy.
Disclosure Statement The authors have no conflicts of interest to disclose.
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