Research Article

Hepatitis E in Italy: A long-term prospective study

Luisa Romanò, Sara Paladini, Catia Tagliacarne, Marta Canuti, Silvia Bianchi,Alessandro Remo Zanetti⇑

Dipartimento di Sanità Pubblica-Microbiologia-Virologia, Università degli Studi di Milano, Via C. Pascal 36, 20133 Milan, Italy

See Editorial, pages 9–11

Background & Aims: In developed countries, hepatitis E is usu- and in large water-borne outbreaks causing a substantial rate of

ally associated with travelling to endemic areas, but a growingnumber of sporadic cases are also seen in patients with no travelhistory. The aim of this study was to assess the impact and themolecular epidemiology of hepatitis E in Italy.Methods: Between January 1994 and October 2009, we analyzed651 patients with acute non-A–C hepatitis. Diagnosis of hepatitisE was based on the presence of IgM anti-HEV and/or the detec-tion of HEV RNA by RT-PCR. Viral isolates were sequenced andphylogenetically characterized.Results: A total of 134 out of 651 (20.6%) patients tested hadacute hepatitis E. All were anti-HEV IgM and IgG positive and96 (71.6%) were also positive for HEV RNA. Moreover, 39 (6%)patients were anti-HEV IgG positive but negative for both IgManti-HEV and HEV RNA. A total of 109 (81.3%) patients developedhepatitis E travelling to endemic areas, 3 (2.3%) acquired intra-familial infection from relatives who developed travel-relateddisease, while 22 (16.4%) patients denied having travelled abroad.In all patients, acute disease had a self-limited course with ALTnormalization within 3–6 weeks. Phylogenetic analysis of 39 iso-lates from patients with a travel-related disease showed that theybelonged to genotype 1, while sequences from five patients withautochthonous hepatitis E belonged to genotype 3.Conclusions: In Italy, most cases of hepatitis E are travel related,caused by viral genotype 1, while autochthonous cases are causedby genotype 3. The prevalence of genotype 3 among pigs andboars suggests that HEV infection may have zoonotic origins innon-endemic countries.� 2010 European Association for the Study of the Liver. Publishedby Elsevier B.V. All rights reserved.

Introduction

Viral hepatitis type E is a public health problem of major concernin many developing countries where it occurs both sporadically

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Keywords: Hepatitis E; HEV; HEV RNA; IgM anti-HEV; IgG anti-HEV.Received 5 February 2010; received in revised form 7 June 2010; accepted 9 June2010; available online 20 August 2010q DOI of original article: 10.1016/j.jhep.2010.08.008.⇑ Corresponding author. Tel.: +39 02 50315126; fax: +39 02 50315120.E-mail address: alessandro.zanetti@unimi.it (A.R. Zanetti).Abbreviations: HEV, hepatitis E virus; IVDUs, intravenous drug users; HIV, humanimmunodeficiency virus; USA, United States of America; PCR, polymerase chainreaction; ORF, open reading frame; ALT, alanine amino transferase; HAV, hepatitisA virus.

morbidity and death, especially in pregnant women [1–3]. Inindustrialized countries, this disease is usually confined to travel-lers returning from endemic areas, even though a growing num-ber of sporadic cases have also been identified in patients with nohistory of recent travel to endemic countries [4–10]. Hepatitis E iscaused by a non-enveloped, single-stranded, positive-sense RNAvirus (HEV) of the genus Hepevirus, belonging to the Hepeviridaefamily [11]. A unique serotype and four major mammalian HEVgenotypes divided into several subtypes have been reported sofar [2,11,12]. Genotype 1 causes most human diseases in Asiaand Africa. Genotype 2 is quite rare and found to be geographi-cally limited to Mexico, Chad and Nigeria. Genotype 3 is spreadworldwide while genotype 4 is commonly found in Asia, and bothgenotypes 3 and 4 have been implicated in sporadic cases of hep-atitis E in humans and are present in swine herds and othermammals [2,10,13–16]. Genotypes 3 and 4 seem to be less viru-lent than genotypes 1 and 2 [2]. This may, at least in part, explainthe discrepancy between the relatively high anti-HEV antibodyprevalence versus the low incidence of autochthonous hepatitisE found in several developed countries, likely due to asymptom-atic infections by attenuated strains of HEV that rarely cause clin-ical disease. In fact, in contrast to the deep differences in thegeographical distribution of disease incidence, anti-HEV antibod-ies are detected worldwide [2,3,10].

Studies carried out in Italy showed that anti-HEV antibodiesare detectable in around 1–3% of individuals tested in the northernand central regions and in approximately 3–6% of those tested inthe south or in the main Islands [17,18]. Higher rates of anti-HEV have been found among intravenous drug users (IVDUs) espe-cially those infected with HIV, haemodialysis patients, homosexu-als, and in patients with chronic hepatitis C, suggesting that HEV aswell as being mainly transmittable through the faecal oral routecould also be transmitted parenterally [19–21]. Evidence showingHEV transmission through blood transfusion or blood componentsadds weight to this hypothesis [22].

In 1999, three novel hepatitis E isolates, phylogenetically dis-tinct from the Burmese (genotype 1) and Mexican (genotype 2)strains, were identified in Greece and Italy in patients with acutehepatitis E who had not travelled to areas typically consideredendemic for HEV [23,24]. These findings, together with the iden-tification of an additional HEV strain in a patient from the USAwith no recent foreign travel history, suggested that HEV may

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be native to Europe and the USA [25,26]. In addition, the closehomology found at that time between an HEV-like sequence fromswine herds in the USA and the novel HEV isolate from the USpatient suggested a possibility of a zoonosis [26,27]. Untilrecently, hepatitis E was believed to be associated only with aself-limited course. However, recent studies showed that HEVmay be the cause of chronic hepatitis occasionally leading to cir-rhosis in some immunocompromised patients, especially thoseundergoing organ transplantation [28–31].

To better understand the epidemiological and clinical impactof hepatitis E in Italy, a prospective study was carried out on con-secutive patients hospitalized with acute non-A–C hepatitis dur-ing the last 15 years.

n = 651

Patients with acute non-A-C hepatitis

n = 134 (20.6%)

anti-HEV IgM and IgG positive

n = 39 (6%)

anti-HEV IgG positive

n = 96 (71.6%)

HEV RNA positive in sera

n = 22/26 (84.6%)

HEV RNA positive in stools

n = 478 (73.4%)

HEV negative

Fig. 1. Cases of acute hepatitis type E identified among 651 patientshospitalized with acute non-A–C hepatitis.

Materials and methods

Between January 1994 and October 2009, 651 consecutive patients were hospi-talized with a diagnosis of acute viral non-A–C hepatitis (negativity for IgManti-HAV, HBsAg, IgM anti-HBc, anti-HCV, HCV RNA, and exclusion of autoimmu-nity, alcohol or hepatotoxic drugs). For each patient enrolled in the study, a serumsample collected during the early acute phase of illness and stools collected from89 patients were available for testing. The case definition for acute hepatitis E wasbased on presence of IgM anti-HEV and/or presence of HEV RNA in sera or stools.

Additional serum samples (mean 2.8, range 2–5) were collected from patientsidentified as having hepatitis E, during the course of the disease. Patients withoutmarkers of HEV infection were defined as having acute non-A–E hepatitis.

To identify possible risk factors for viral hepatitis during the 6 months pre-ceding the onset of illness, each patient was interviewed using a pre-coded setof questions on their lifestyle and travel history.

Total or IgG anti-HEV antibodies were detected during the study-time by dif-ferent commercially available immunoassays (HEV EIA, Abbott Labs, Germany;HEV ELISA, Genelabs Diagnostics, Singapore; HEV IgG, Dia.Pro, Diagnostic BioP-robes, Italy); for the detection of IgM anti-HEV antibodies, we employed an in-house assay at the beginning of the study [23]. Later on, different commerciallyavailable assays (HEV IgM ELISA, Genelabs Diagnostics, Singapore; HEV IgM, Dia.-Pro, Diagnostic BioProbes, Italy) were used.

Viral RNA was extracted from 200 ll of serum or from 200 ll of 10% faecalsuspension using the QIAamp MinElute Virus Spin kit (QIAGEN, Hilden, Ger-many). The presence of HEV RNA was detected by two different nested RT-PCRs,employing primers derived, respectively, from the ORF1 and ORF2 of the viralgenome of HEV, as previously described [23,32].

To molecularly characterize the identified strains, amplicons were purifiedwith NucleoSpin Extract II (Macherey–Nagel GmbH & Co. KG, Düren, Germany)and then directly sequenced by automated DNA sequencing on the genetic ana-lyzer ABI PRISM 3100 (Applied Biosystem, Foster City, CA). Phylogenetic treeswere constructed by means of the Neighbor-Joining method and Kimura 2-parameter model using the MEGA package, version 4.0 [33]. A bootstrap re-sam-pling analysis was performed (1000 replicates) to test the robustness of the tree.The HEV sequences obtained in this study were deposited into GenBank underaccession numbers (from HM446588 to HM446626 and from HM446627 toHM446631). HEV ORF2 sequences downloaded from the GenBank databasesequences were included for comparison in the phylogenetic analysis. Referencesequences of ORF2 corresponded to the following strains derived from Burma: B1(M73218), B2 (D10330); from India: I1 (X98292), I2 (X99441), I3 (AF076239),IakI90 (AF124407), Yam-67 (AF459438); from Pakistan: P1 (M80581), P2(AF185822); from China: C1 (D11092), C2 (L25595), C3 (L08816), C4 (D11093),T1 (AJ272108), Hebei (M94177), swCH25 (AY594199), CCC220 (AB108537); fromNepal: TK15/92 (AF051830); from Japan: wbJSG1 (AB222182), HE-JF2(AB079763), JSO-Hyo03L (AB189073), HE-JA04-1911 (AB248520), swJ8-5(AB248521), swJ12-4 (AB248522), HE-JF1 (AB079762), JAK-Sai (AB074915), HE-JTB94-1 (AB292649), HE-JF3 (AB079764), HE-JI4 (AB080575), swJ570(AB073909); from Egypt: 94-Egypt (AF051352); from Morocco: Morocco(AY230202); from Mexico: Mx1 (M74506); from Chad: Chad T3 (U62121), T3(AY204877); from Kyrgyzstan: Osh205 (AF455784); from USA: US1 (AF060668),US2 (AF060669), swUS1 (AF082843); from France: MaR7300083 (EU116337),TLS39 (EU495230), TLS41 (EU495231), Fr-33 (EU543566), Fr-2 (EF053274),TLS13 (EU495212), HEVMars6318189 (EF028801), 6100709 (EF061399); fromSpain: C12 (FJ464744), SWP8 (EU723516), SW627 (EU723513), SWP7(EU723515); from United Kingdom: swUK (AF503511); from Canada: Arkell(AY115488); from Mongolia: swMN06 (AB290312); from The Netherlands:

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NLSW28 (AF336292), NLSW15 (AF332620), NLSW68 (AY032756), NLSW85(AF336295), NLSW91 (AY032759); from Taiwan: TW12SW (AF296165),NLSW105 (AF336298); TW13SW (AF296166).

Phylogenetic analysis of the HEV strains was performed according to Lu et al.[12].

Statistical analysis

Statistical analysis was conducted using Epi Info software (version 3.5). Differ-ences in frequency were compared by v2 test. Mean values were compared byStudent’s t test. A p value of <0.05 was considered significant.

Results

The mean age of the 651 patients enrolled in the study was37 years, ranging from 1 to 93 years; 61.7% of patients weremales and 38.3% females, and the mean ALT peak value duringhospitalization was 1596 IU/L, ranging from 101 to 12,520 IU/L.

As shown in Fig. 1, 134 out of 651 (20.6%) patients tested werediagnosed with acute hepatitis E. No significant difference wasfound in the frequency of acute hepatitis E as regards the yearsunder scrutiny. All sera collected from such patients were posi-tive for both IgM and IgG anti-HEV antibodies, and 96 (71.6%)patients were also positive for HEV RNA in at least one of theserum samples collected. In addition, serial stool samples (mean3, range 2–4) collected from 22 of 26 viremic patients were alsofound positive for viral RNA. Finally, 39 out of 651 (6%) patientswere anti-HEV IgG positive but negative for both IgM anti-HEVand HEV RNA.

As reported in Table 1, 82.3% of patients with acute hepatitis Ewere males, the mean age was 31.3 years, ranging from 3 to68 years. During hospitalization, ALT mean peak was 2539.6 IU/L with a wide range of values (122–12,290 IU/L). Compared withpatients with non-A–E acute hepatitis, the 134 patients whosecondition fully met the definition of HEV acute hepatitis weremore frequently male, younger and suffered from more severedisease with higher ALT levels.

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Table 1. Demographic and clinical characteristics of patients with acutehepatitis non-A–E or with acute hepatitis E.

Acute hepatitis type

non-A-E n = 478

E n = 134

p value

Gender56.8% %3.28

50.0<

Age (years): naeM 38.1 31.3 <0.05 egnaR 1 — 93 3 — 68

ALT (IU/L): naeM 1339.9 2539.6 <0.05egnaR 101 — 12,520 122 — 12,290

43.2% 17.7%MF

Table 2. Demographic and clinical characteristics of patients with travel andnon-travel related acute hepatitis type E.

p value

Gender

Age (years): naeMegnaR

ALT (IU/L): naeMegnaR

MF

Travel relatedn = 109

Nonrelated

-travel

n = 22

79.1% 20.9%

94.4% 5.6% n.s.

30 43 <0.001 3 — 68 17 — 65

2558.9 2397.3 n.s.122 — 12,290 589 — 6000

Acute hepatitis type E

Research Article

As for risk factors (Fig. 2), 109 (81.3%) HEV positive patientsdeveloped acute hepatitis after their return from travel to ende-mic areas such as the Indian sub-continent, Morocco, and others.The mean time elapsing from their return home and the onset ofthe disease was 1.8 weeks (range < 1–4 weeks). Three patients(2.3%) acquired intra-familial infection (secondary cases) fromtheir households who developed hepatitis E after visiting ende-mic areas. Such patients (two girls aged 3 and 5 years and oneboy aged 13 years) had never travelled abroad and developedthe disease 2.5, 4, and 5 weeks after the onset of hepatitis E intheir households. Crucially, it must be added, 98 (89.9%) of theinfected ‘travelling’ patients had been visiting their father-land;while the remaining 11 (10.1%) patients were native Italians.Finally, 22 patients (16.4%) did not travel abroad, had no contactwith people coming from endemic areas, and did not have otherpossible risk factors such as drug addiction, blood transfusion,consumption of raw or undercooked shellfish or occupational risk(swine handlers, pig farmers).

No differences in ALT levels were detected among patientswho acquired HEV abroad compared to those who never visitedendemic areas (Table 2). Patients belonging to the latter groupwere older (mean age 43 years, range 17–65 versus 30 years,range 3–68; p <0.001) and somewhat more likely males thanfemales (94.4% versus 79.1%), even though gender difference

Travel abroad 81.3%

(109/134)Autochthonous 16.4% (22/134)

Secondary cases 2.3% (3/134)

2 M

1 So

1 An

1 Sr

1 Ca

0 5

Fig. 2. Acute hepatitis E among 134 patien

36 Journal of Hepatology 20

was not significant (p = 0.2). Among HEV positive patients, acutedisease had a self-limited course with normalization of ALT levelswithin 3–6 weeks in most patients. One patient (male, 50 years)acquired HAV and HEV co-infection travelling to India. Thispatient who was found positive for HEV RNA and for anti-HEVof both the IgM and IgG classes, and negative for IgM anti-HAVat hospitalization, became IgM anti-HAV positive 1 week later.In this case, the disease had a more severe course (ALT peak12,990 IU/L, total bilirubin 14.09 mg/dl, alkaline phosphatase1153 U/L, c-glutamyl transpeptidase 1160 U/L) than thatobserved in patients infected with HEV alone, and showed nor-malization of liver function tests after 8 weeks. The mean dura-tion of HEV RNA presence, calculated in serial samples collectedfrom 22 patients with acute HEV, was 8 days (range 3–19 days)in sera and 11 days (range 8–21 days) in stools. IgM anti-HEVtended to disappear in 3–4 weeks, while anti-HEV of the IgG classcould be detected at decreasing levels throughout follow up (upto 25 months post-infection).

Sequencing and phylogenetic analysis of ORF2 PCR productsof 39 viral isolates (named from MI_TR1 to MI_TR39), derivedfrom patients with acute HEV who travelled abroad, showed thatthey all were infected with a virus of genotype 1. Out of the 39viral isolates, 37 were of subtype 1a and 2 of subtype 1c

N° of cases

22 Pakistan

43 Bangladesh

orocco

malia

gola

i Lanka

pe Verde

38 India

10 15 20 25 30 35 40 45

ts according to their travel history.

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Fig. 3. Phylogenetic tree based on partial ORF2 gene nucleotide sequences of 39 HEV sequences (in bold and named from MI_TR1 to MI_TR39) derived from patientswho travelled to endemic areas and 5 HEV sequences (in bold and named from Hu_It2 to Hu_It6) derived from patients with no history of travelling abroad. Thepercentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches.

JOURNAL OF HEPATOLOGY

(Fig. 3). On the contrary, ORF2 sequences (named from Hu It2 toHu It6) obtained from samples of five patients (all males, meanage 45 years, range 34–65 years) with no history of travel abroad,belonged to genotype 3.

In particular, as reported in Fig. 4, two of these sequencesbelonged to subtype 3f, two to subtype 3e and one to subtype3h, together with the Italian sequence previously published byour group in 1999 (It1, subtype 3h) and other human, swine,and wild boar strains identified elsewhere.

Discussion

Viral hepatitis type E is highly endemic in tropical and subtropi-cal countries with poor sanitation where large outbreaks, usually

Journal of Hepatology 20

associated with faecal contamination of water supply, occur fre-quently [1,3,10]. Industrialized countries like North-America,Europe, Australia, New Zealand and part of Asia (Japan, Taiwan,Hong Kong) were traditionally considered non-endemic areaswith only sporadic cases largely confined to travellers returningfrom endemic areas. However, in recent years, a growing numberof sporadic cases of hepatitis E in patients who were never linkedto areas of HEV endemicity were reported in developed regions[4–10].

From our data it emerged that 134 out of 651 (20.6%) patientsconsecutively hospitalized from the mid-nineties to date with adiagnosis of acute viral non-A–C hepatitis indeed had hepatitisE. In addition, 6% (39 out of 651) patients were found to beanti-HEV IgG positive but negative for both IgM anti-HEV and

11 vol. 54 j 34–40 37

Fig. 4. Phylogenetic tree of five HEV sequences (in bold and named from Hu_It2 to Hu_It6) derived from patients who never travelled abroad, based on partial ORF2gene nucleotide sequences. Sequence of It1 was manually added (broken line) in the tree according to data from a previous phylogenetic analysis [12].

Research Article

HEV RNA. Since this finding does not allow us to distinguishwhether such a pattern should be interpreted as a sign of pastHEV infections in patients with ongoing acute hepatitis ofunknown cause or, vice versa, an acute hepatitis E infection withan early disappearance of viral RNA and IgM antibodies, datafrom these patients were excluded from further elaboration.

A total of 81.3% of patients with hepatitis E developed the dis-ease after their return from endemic areas or acquired intra-familial HEV (2.3%, secondary cases) from such infected house-holds. Thus travelling to disease-endemic areas directly or indi-

38 Journal of Hepatology 20

rectly accounted for 83.6% of cases diagnosed with hepatitis E.However, it must be noted that most (89.9%) of such patientswere immigrants who had visited their countries of origin andhad then returned to Italy. Thus, we can infer that the risk ofHEV exposure may depend not only on travelling arrangementsand destinations, but also on an albeit momentary return to lessprotected local habits and customs. The remaining 22 patients(16.4%) were classified as autochthonous acquired hepatitis E,since all denied travel abroad. Patients belonging to the lattergroup were older and somewhat more likely males than females

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than the former group. The course and outcome of clinical illnesswere similar in both groups. All 134 patients developed self-lim-ited disease with ALT level normalization within 3–6 weeks, andHEV RNA disappearance within 8 days in serum samples andwithin 11 days in stools.

Sequencing and phylogenetic analysis of PCR products derivedfrom 39 patients with travel-related hepatitis E showed a fullmatch between the genotype’s geography (in our cases all hadgenotype 1, subtypes 1a and 1c) and the area where the travellerhad acquired infection (mostly in the Indian sub-continent). Sucha finding further supports the epidemiological link between tra-vel history to endemic areas and the onset of the disease withbiological plausibility. On the other hand, all five sequencesderived from patients with autochthonous hepatitis E belongedto genotype 3; two of which were of subtype 3f, two of subtype3e, and one of subtype 3h. This finding is in agreement with whatwas reported more than 10 years ago suggesting that HEV may benative in Europe and in the USA and, more recently, with severalpublished studies showing that genotype 3 is the major cause ofhuman hepatitis E in non-endemic countries and is also com-monly present among swine herds and other mammals[2,10,16,25,26,34–37]. In Italy, HEV genotype 3 is widespreadamong farm pigs and wild boars. Recently, HEV RNA could bedetected in stools collected from 42% of randomly selectedhealthy pigs from six different swine farms and from bile samplesof 25% of wild boars [38,39]. Phylogenetic analysis of the nucleo-tide sequences obtained from 16 PCR products derived from suchinfected pigs indicated that all strains belonged to genotype 3[38]. In particular, one group of seven Italian strains clusteredclose to human and to swine European HEV strains (92–96% sim-ilarity) [38]. A recent study carried out on HEV derived from 15Italian pigs, showed that all identified strains belonged to geno-type 3 and were similar to human and swine subtypes 3e, 3f,and 3c circulating in Europe [39]. Moreover, phylogenetic analy-sis of 10 PCR products from sequences derived from wild boarsshows that only one HEV strain was circulating in the populationof animals examined, and that this strain was close to human andswine European strains [40]. In the same region, findings from asurvey carried out to determine anti-HEV in sera collected from1422 pigs of 39 different pig farms showed that 50.2% of the ani-mals hosted in 38 out of the 39 farms examined (97.5%) wereantibody positive. This confirms that HEV is widespread in pigsin Italy and is likely to be endemic in many farms [41]. Similarfindings were found in the USA and in other European countries,including Spain, France, Southwest England, Germany, Swedenand The Netherlands, strengthening the fact that swine and wildboars may represent a huge reservoir for HEV genotype 3 in suchcountries [34–37,42–47]. In particular, the high degree of similar-ity found between human and swine HEV strains (in some caseshuman/swine strains isolated are even identical) suggests possi-ble zoonotic transmission from domestic swine to humans[2,10,16,25,26]. In this context, viral RNA was detected in com-mercial pig liver in Japan as well as in the USA and in The Neth-erlands, and transmission of HEV through consumption of raw orundercooked deer or wild boar meat has been reported [48–54].

HEV genotypes 1 and 2 appear to be more pathogenic forhumans than genotypes 3 and 4 that are responsible for the occa-sional cases of acute viral hepatitis E in these settings. This mayexplain, at least in part, the difference between the relatively highprevalence of anti-HEV antibody found in several populationgroups compared to the relatively small number of patients with

Journal of Hepatology 20

locally acquired hepatitis E. In other words, this discrepancy maybe the result of subclinical, inapparent infections caused by theless virulent HEV genotype 3 derived from swine or from otherdomestic or wild animals that only rarely causes clinical diseasein immunocompetent hosts.

In conclusion, in Italy most cases of acute hepatitis E are travelrelated and caused by viral genotype 1, while sporadic autoch-thonous cases are caused by genotype 3 which is commonlyfound in domestic pigs and wild boars; a finding supporting thehypothesis that infection may have a zoonotic origin.

The growing number of people travelling to exotic countriesin addition to the impressive migratory flux of people fromHEV endemic areas to Europe may cause changes in the epidemi-ological picture of hepatitis E on our continent. All this requiresconstant surveillance and the implementation of appropriatepublic health measures to control and prevent this disease. In thisregard, it should be mentioned that safe and effective recombi-nant hepatitis E vaccines have already been successfully adminis-tered [55,56].

Financial disclosure

This study was funded in part by the Ministero dell’Istruzione,dell’Università e della Ricerca (COFIN, Contract No.2003060911_004) and in part by the Università degli Studi diMilano (FIRST 2006, Fondo Interno Ricerca Scientifica eTecnologica).

Conflict of interest

The authors who have taken part in this study declared that theydo not have anything to disclose regarding funding or conflict ofinterest with respect to this manuscript.

Acknowledgments

We are grateful to many clinicians who supplied samples for test-ing. In particular, we are indebted with Dr Paolo Fabris (OspedaleS. Bortolo, Vicenza) for his skilful collaboration.

References

[1] Jilani N, Das BC, Husain SA, Baweda UK, Chattopadhya D, Gupta RK, et al.Hepatitis E and fulminant hepatic failure during pregnancy. J GastroenterolHepatol 2007;22:676–682.

[2] Purcell RH, Emerson SU. Hepatitis E: an emerging awareness of an olddisease. J Hepatol 2008;48:494–503.

[3] Aggarwal R, Naiks S. Epidemiology of hepatitis E: current status. J Gastro-enterol Hepatol 2009;24:1484–1493.

[4] Schwartz E, Jenks NP, Van Damme P, Galun E. Hepatitis E virus infection intravellers. Clin Infect Dis 1999;42:1312–1314.

[5] Pina S, Buti M, Cotrina M, Piella J, Girones R. HEV identified in serum fromhuman with acute hepatitis and in sewage of animal origin in Spain. JHepatol 2000;33:826–833.

[6] Tsang TH, Denison EK, Williams HV, Venczel LV, Ginsberg MM, Vugia DJ.Acute hepatitis E infection acquired in California. Clin Infect Dis2000;30:618–619.

[7] Widdowson MA, Jaspers WJ, van der Poel WH, Verschoor F, de Roda HusmanAM, Winter HL, et al. Cluster of cases of acute hepatitis associated withhepatitis E virus infection acquired in The Netherlands. Clin Infect Dis2003;36:29–33.

[8] Mansuy JM, Peron JM, Abravanel F, Poirson H, Dubois M, Miedouge M, et al.Hepatitis E in the south west of France in individuals who have never visitedan endemic area. J Med Virol 2004;74:419–424.

11 vol. 54 j 34–40 39

Research Article

[9] Ijaz S, Arnold E, Banks M, Bendall RP, Cramp ME, Cunningham R, et al. Non-

travel-associated hepatitis E in England and Wales: demographic, clinical,and molecular epidemiological characteristics. J Infect Dis 2005;192:1166–1172.

[10] Dalton HR, Bendal L, Ijaz S, Banks M. Hepatitis E: an emerging infection indeveloped countries. Lancet Infect Dis 2008;8:698–709.

[11] Emerson SU, Purcell RH. Hepatitis E virus. In: Knipe DM, Howley PM, editors.Fields Virology. Philadelphia: Lippincott Williams & Wilkins; 2007. p.3047–3058.

[12] Lu L, Li L, Hagedorn CH. Phylogenetic analysis of global hepatitis E virussequences: genetic diversity, subtypes and zoonosis. Rev Med Virol2006;16:5–36.

[13] Arankalle VA, Chobe LP, Joshi MV, Chadha MS, Kundu B, Walimbe AM.Human and swine hepatitis E virus from Western India belong to differentgenotypes. J Hepatol 2002;36:417–425.

[14] Cooper K, Huang FF, Batista L, Rayo CD, Bezanilla JC, Toth TE, et al.Identification of genotype 3 hepatitis E virus (HEV) in serum and fecalsamples pigs in Thailand and Mexico, where genotype 1 and 2 HEV strainsare prevalent in the respective human populations. J Clin Microbiol2005;43:1684–1688.

[15] Mushahwar IK. Hepatitis E virus: molecular virology, clinical features,diagnosis, transmission, epidemiology, and prevention. J Med Virol2008;80:646–658.

[16] Hsieh S-Y, Meng X-J, Wu Y-H, Liu S-T, Tam AW, Lin D-Y, et al. Identity of anovel swine hepatitis E virus in Taiwan forming a monophyletic group withTaiwan isolates of human hepatitis E virus. J Clin Microbiol1999;37:3828–3834.

[17] Zanetti AR, Dawson GJ and the Study Group of Hepatitis E. Hepatitis E inItaly: a seroepidemiological survey. J Med Virol 1994;42:318–320.

[18] Coppola RC, Masia G. Seroprevalence and epidemiological features of hepatitisE virus infection in Italy. In: Rizzetto M, Purcell RH, Jerin J, Verme G, editors.Viral hepatitis and liver disease. Torino: Minerva; 1997. p. 609–611.

[19] Montella F, Rezza G, Di Sora F, Pezzotti P, Recchia O. Association betweenhepatitis E virus and HIV infection in homosexual men. Lancet1994;344:1433.

[20] Pisanti FA, Coppola A, Galli C. Association between hepatitis C and hepatitisE viruses in southern Italy. Lancet 1994;344:746–747.

[21] Romanò L, Tanzi E, Brancatelli S, Raimondo G, Galli C, Zanetti AR. Prevalenceof anti-HEV among patients with chronic viral hepatitis and healthyindividuals in Southern Italy. In: Buisson Y, Coursaget Y, Kane M, editors.Enterically-transmitted hepatitis viruses. Tours, France: La Simarre; 1996. p.237–239.

[22] Matsubayashi K, Kang JH, Sakata H, Takahashi K, Shindo M, Kato M, et al. Acase of transfusion-transmitted hepatitis E caused by blood from a donorinfected with hepatitis E virus via zoonotic food-borne route. Transfusion2008;48:1368–1375.

[23] Schlauder GG, Dasi SM, Zanetti AR, Tassopoulos NC, Mushahwar IK. Novelhepatitis E virus (HEV) isolates from Europe: evidence for additionalgenotypes of HEV. J Med Virol 1999;57:243–251.

[24] Zanetti AR, Schlauder GG, Romanò L, Tanzi E, Fabris P, Dawson GJ, et al.Identification of a novel variant of hepatitis E virus in Italy. J Med Virol1999;57:356–360.

[25] Kwo PY, Schlauder GG, Carpenter HA, Murphy PJ, Rosemblatt JE, Dawson GJ,et al. Acute hepatitis E by a new isolate acquired in the United States. MayoClin Proc 1997;72:1133–1136.

[26] Schlauder GG, Dawson GJ, Erker JC, Kwo PY, Knigge MF, Smalley DL, et al. Thesequence and phylogenetic analysis of a novel hepatitis E virus isolated froma patient with acute hepatitis reported in the United States. J Gen Virol1998;79:447–456.

[27] Meng XJ, Purcell RH, Halbur PG, Lehman JR, Webb DM, Tsareva TS, et al. Anovel virus in swine is closely related to the human hepatitis E virus. ProcNatl Acad Sci USA 1997;94:9860–9865.

[28] Gerolami R, Moal V, Colon P. Chronic hepatitis E with cirrhosis in a kidney-transplant recipient. N Engl J Med 2008;258:859–860.

[29] Haagsma EB, van den Berg AP, Porte RJ, Benne CA, Vennema H, Reimerink JH,et al. Chronic hepatitis E virus infection in liver transplant recipients. LiverTranspl 2008;14:547–553.

[30] Kamar N, Selves J, Mansuy JM, Ouezzani L, Peron JM, Guitard J, et al.Hepatitis E virus and chronic hepatitis in organ-transplant recipients. N EnglJ Med 2008;358:811–817.

[31] Bihl F, Negro F. Chronic hepatitis E in the immunosuppressed: a new sourceof trouble? J Hepatol 2009;50:435–437.

[32] Shrestha SM, Shreshta S, Tsuda F, Nishizawa T, Gotanda Y, Takeda N, et al.Molecular investigation of hepatitis E virus infection in patients with acutehepatitis in Kathmandu, Nepal. J Med Virol 2003;69:207–214.

40 Journal of Hepatology 20

[33] Kumar S, Tamura K, Jakobsen IB, Nei M. Molecular evolutionary geneticsanalysis software. Bioinformatics 2001;17:1244–1245. Available from:http://www.megasoftware.net/.

[34] Garkavenko O, Obriadina A, Meng J, Anderson DA, Bernard HJ, Schroeder BA,et al. Detection and characterisation of swine hepatitis E virus in NewZealand. J Med Virol 2001;62:525–529.

[35] Banks M, Heath GS, Grierson SS, King DP, Gresham A, Girones R, et al.Evidence for the presence of hepatitis E virus in pigs in the United Kingdom.Vet Rec 2004;154:223–227.

[36] Borgen K, Herremans T, Duizer E, Vennema H, Rutjes S, Bosman A, et al. Non-travel related hepatitis E virus genotype 3 infections in The Netherlands; acase series 2004–2006. BMC Infect Dis 2008;8:61.

[37] Rutjes SA, Lodder WJ, Lodder-Vershoor F, van der Berg HHJL, Vennema H,Duizer E, et al. Sources of hepatitis E virus genotype 3 in The Netherlands.Emerg Infect Dis 2009;15:381–387.

[38] Di Bartolo I, Martelli F, Inglese N, Pourshaban M, Caprioli A, Ostanello F, et al.Widespread diffusion of genotype 3 hepatitis E virus among farming swinein northern Italy. Vet Microbiol 2008;132:47–55.

[39] Martelli F, Toma S, Di Bartolo I, Caprioli A, Ruggeri FM, Lelli D, et al.Detection of Hepatitis E Virus (HEV) in Italian pigs displaying differentpathological lesions. Res Vet Sci 2010;88:492–496.

[40] Martelli F, Caprioli A, Zengarini M, Marata A, Fiegna C, Di Bartolo I, et al.Detection of hepatitis E virus (HEV) in a demographic managed wild boar(Sus scrofa scrofa) population in Italy. Vet Microbiol 2008;126:74–81.

[41] Martinelli N, Luppi A, Lelli D, Sozzi E, Canelli E, Moreno Martin AM, et al.Prevalence of hepatitis E virus antibodies among pigs in northern Italy.XXXV Meeting Annuale Società Italiana di Patologia ed Allevamento deiSuini, 12–13 marzo 2009, Modena. p. 431–437.

[42] Dalton HR, Stableforth W, Thurairajah P, Hazeldine S, Remnarace R, UsamaW, et al. Autochthonous hepatitis E in Southwest England: natural history,complications and seasonal variation, and hepatitis E virus IgG seropreva-lence in blood donors, the elderly and patients with chronic liver disease.Eur J Gastroenterol Hepatol 2008;20:784–790.

[43] Wichmann O, Achimanski S, Koch J, Kohler M, Rothe C, Plentz A, et al.Phylogenetic and case-control study on hepatitis E virus infection inGermany. J Infect Dis 2008;198:1732–1741.

[44] Legrand-Abravanel F, Mansuy JM, Dubois M, Kamar N, Peron JM, Rostaing L,et al. Hepatitis E virus genotype 3 diversity, France. Emerg Infect Dis2009;15:110–114.

[45] Peralta B, Mateu E, Casas M, de Deus N, Martin M, Pina S. Geneticcharacterization of the complete coding regions of genotype 3 hepatitis Evirus isolated from Spanish swine herds. Virus Res 2009;139:111–116.

[46] Mansuy JM, Abravanel F, Miedouge M, Mengelle C, Merviel C, Dubois M,et al. Acute hepatitis E in south-west France over a 5-years period. J ClinVirol 2009;44:74–77.

[47] Norder H, Sundqvist L, Magnusson L, Ostergaard Breum S, Lofdahl M, BreumS, et al. Endemic hepatitis E in two Nordic countries. Euro Surveill 2009;14(19).

[48] Yazaki Y, Mizuo H, Takahashi M, Nishizawa T, Sasaki N, Gotanda Y, et al.Sporadic acute or fulminant hepatitis E in Hokkaido, Japan, may be food-borne, as suggested by the presence of hepatitis E virus in pig liver as food. JGen Virol 2003;84:2351–2357.

[49] Matsuda H, Okada K, Takahashi K, Mishiro S. Severe hepatitis E virusinfection after ingestion of uncooked liver from a wild boar. J Infect Dis2003;188:944.

[50] Tei S, Kitajima N, Takahashi K, Mishiro S. Zoonotic transmission of hepatitis Evirus from deer to human beings. Lancet 2003;362:371–373.

[51] Takahashi K, Kitajima N, Abe N, Mishiro S. Complete or near-completenucleotide sequences of hepatitis E virus genome recovered from a wild boar, adeer, and four patients who ate the deer. Virology 2004;330:501–505.

[52] Tamada Y, Yano K, Yatsuhashi H, Inoue O, Mawatari F, Ishibashi H.Consumption of wild boar linked to cases of hepatitis E. J Hepatol2004;40:869–870.

[53] Bouwknegt M, Lodder-Verschoor F, van der Poel WH, Rutjes SA, RodaHusman AM. Hepatitis E virus RNA in commercial porcine livers in TheNetherlands. J Food Prot 2007;70:2889–2895.

[54] Feagins AR, Opriessnig T, Guenette DK, Halbur PG, Meng XJ. Detection andcharacterization of infectious hepatitis E virus from commercial pig liversold in local grocery stores in the USA. J Gen Virol 2007;88:912–917.

[55] Shrestha MP, Scott RM, Joshi DM, Mammen MP, Thapa N, Myint KS, et al.Safety and efficacy of a recombinant hepatitis E vaccine. N Engl J Med2007;356:895–903.

[56] Zhang J, Liu CB, Li RC, Li YM, Zheng YJ, Li YP, et al. Randomized-controlledphase II clinical trial of a bacterically expressed recombinant hepatitis Evaccine. Vaccine 2009;27:1869–1874.

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