www.elsevier.com/locate/vetmic

Veterinary Microbiology 122 (2007) 16–24

Association of hepatitis E virus (HEV) and postweaning

multisystemic wasting syndrome (PMWS) with lesions

of hepatitis in pigs

M. Martın a,b,*, J. Segales a, F.F. Huang b, D.K. Guenette b,E. Mateu a, N. de Deus a, X.J. Meng b

a Departament de Sanitat i d’Anatomia Animals, Centre de Recerca en Sanitat Animal (CReSA),

Universitat Autonoma de Barcelona, Bellaterra, Barcelona, Spainb Center for Molecular Medicine and Infectious Diseases, College of Veterinary Medicine,

Virginia Polytechnic Institute and State University, Blacksburg, VA, USA

Received 18 April 2006; received in revised form 18 December 2006; accepted 20 December 2006

Abstract

The aim of the study was to determine the presence of swine hepatitis E virus (HEV) RNA and antibodies in postweaning

multisystemic wasting syndrome-affected (n = 114) and non-affected (n = 46) pigs and the possible association with hepatitis

lesions. Forty-four pigs were RT-PCR positive (28.2%); 25 of them were PMWS cases, while 19 were non-PMWS pigs. In both

groups, HEV RT-PCR results were associated with hepatitis (OR = 5.61 for PMWS-affected pigs and OR = 5.17 for non-PMWS

affected pigs; p = 0.01). No interaction was detected in a logistic regression between PMWS occurrence and HEV infection for

the development of hepatitis lesions. Seropositivity to HEV was more likely to occur in pigs with hepatitis (51.9%) compared to

pigs without hepatitis (36.1%; p = 0.03). Significant differences in optical densities were notices comparing the lesional stage of

pigs ( p = 0.009). While pigs with slight or moderate hepatitis were seropositive, pigs with more severe lesions were seronegative

to HEV. These results indicate that swine HEV infection can be a significant contributor to the development of moderate hepatitis

in pigs regardless of the PMWS status.

# 2007 Elsevier B.V. All rights reserved.

Keywords: HEV; Hepatitis E; Porcine circovirus type 2; Postweaning multisystemic wasting syndrome; Swine

* Corresponding author at: Departament de Sanitat i d’Anatomia

Animals, Facultat de Veterinaria, Universitat Autonoma de Barce-

lona, 08193 Bellaterra, Barcelona, Spain. Tel.: +34 935811046;

fax: +34 935813297.

E-mail address: marga.martin@uab.es (M. Martın).

0378-1135/$ – see front matter # 2007 Elsevier B.V. All rights reserved

doi:10.1016/j.vetmic.2006.12.020

1. Introduction

Hepatitis E virus (HEV) is a small non-enveloped

RNA virus of approximately 7.2 kb which has recently

been classified within the family Hepeviridae, genus

Hepevirus (Emerson et al., 2004). HEV is the

.

M. Martın et al. / Veterinary Microbiology 122 (2007) 16–24 17

causative agent of hepatitis E, an important cause of

acute hepatitis in humans in many developing

countries although sporadic cases are increasingly

reported in industrialized countries (Emerson and

Purcell, 2003). The disease appears often as a self-

limiting acute hepatitis with a low case-fatality rate;

however, in pregnant women it can cause up to 25%

mortality because of the development of fulminant

acute hepatitis (Purcell and Emerson, 2001).

In 1997, a novel strain of HEV in pigs, designated

swine HEV, was identified in swine herds in the United

States and was shown to be genetically and

serologically related to human HEV strains (Meng

et al., 1997). Swine HEV can infect non-human

primates, and conversely a US-2 strain of human HEV

infected pigs (Meng et al., 1998; Halbur et al., 2001).

These findings raised a concern about human

infections with swine HEV. In fact, pig handlers in

the United States and other countries have been found

to be at increased risk of zoonotic infection by HEV

(Drobeniuc et al., 2001; Meng et al., 2002; Withers

et al., 2002). It is now recognized that swine are a

reservoir for HEV and swine HEV can infect humans

(Garkavenko et al., 2001; van der Poel et al., 2001;

Yoo et al., 2001; Choi et al., 2003; Takahashi et al.,

2003; Banks et al., 2004). In Spain, HEV RNA has

been detected in human cases of hepatitis, urban

sewage and swine faecal samples, confirming thus that

autochthonous HEV strains circulate in both human

and animal populations in Spain (Pina et al., 2000;

Clemente-Casares et al., 2003).

Pigs infected with HEV develop a subclinical

infection; however microscopic lesions of hepatitis are

observed in both experimentally (Halbur et al., 2001)

and naturally (Meng et al., 1997) infected pigs. Swine

HEV has been shown to replicate in liver as well as in

gastrointestinal tract and other extrahepatic tissues

(Williams et al., 2001; Choi and Chae, 2003; Meng,

2003).

Porcine circovirus type 2 (PCV2), the primary

causative agent of postweaning multisystemic wasting

syndrome (PMWS), is also considered a hepatitis-

inducing virus in pigs (Rosell et al., 2000). In fact,

approximately 45% of PMWS-affected pigs have a

mild to severe hepatitis (Segales et al., 2004)

consisting of lympho-histiocytic infiltrations in portal

areas or irregularly distributed throughout the hepatic

parenchyma (Rosell et al., 2000). These hepatic

lesions resemble those described in pigs naturally and

experimentally infected with swine HEV (Meng et al.,

1997; Halbur et al., 2001).

The objective of the study was to explore the

possible participation of swine HEV and PMWS with

the presence of hepatitis lesions in pigs.

2. Materials and methods

2.1. Case selection and sampling

A total of 160 pigs of 1–5 months were included in

this study. All animals came from conventional

Spanish pig farms and were submitted alive to the

Pathology Diagnostic Laboratory at the Veterinary

Faculty of Barcelona (Spain). One hundred and

fourteen had been submitted under suspicion of

PMWS that was later confirmed by fulfilling the case

definition criteria for this disease as reported else-

where (Segales and Domingo, 2002). At arrival,

animals were bled via the cranial cava vein and

euthanized with an overdose of sodium pentobarbital.

Serum samples were kept frozen at �80 8C until used

for analyses. During necropsy, samples of tissues

including liver were taken, fixed by immersion in 10%

buffered formalin and processed for routine histo-

pathologic examination. Additionally, 46 pigs sub-

mitted to the laboratory for conditions other than

PMWS were also studied. For these pigs blood was

taken as well as samples of liver, bile, lymph nodes

and faeces. Fresh and fixed material was available for

these later animals.

In the 160 studied pigs, other conditions diagnosed

were bacterial pneumonia, porcine reproductive and

respiratory syndrome (PRRS), enteric salmonellosis,

colibacillosis, swine influenza, coccidiosis, Glasser’s

disease and streptococcal meningitis.

2.2. Histopathology

Within the PMWS group, 64 (56.1%) animals had

hepatitis and 50 (43.9%) had no liver lesions as

determined by the microscopic examination. Thirteen

out of 46 (28.3%) non-PMWS animals had hepatitis.

Hepatic lesions were scored from I to IVaccording to a

previously described classification for PMWS-affected

pigs (Rosell et al., 2000). With this criteria, 54 animals

M. Martın et al. / Veterinary Microbiology 122 (2007) 16–2418

(50 PMWS and four non-PMWS) had stage I lesions

(slight multifocal lymphohistiocytic infiltrates in the

portal tracts and/or irregularly distributed in the liver

parenchyma), 19 pigs (10 PMWS and 9 non-PMWS)

had stage II lesions (intermediate to intense lympho-

histiocytic inflammation of the portal tracts often

accompanied by clusters of mononuclear inflammatory

cells in the liver parenchyma), three pigs with PMWS

had stage III lesions (multifocal clusters of mono-

nuclear inflammatory infiltrates within the hepatic

parenchyma, a variable hepatic plate disorganization,

and frequent presence of apoptotic bodies), and one

PMWS-affected pig was classified as having stage IV

lesions (generalized perilobular fibrosis, with disorga-

nization of liver plates and massive loss of hepatocytes,

and remaining stroma of the lobules diffusely infiltrated

by mononuclear cells).

2.3. RT-PCR to detect swine HEV

For both groups, a semi-nested RT-PCR was used to

detect swine HEV RNA in serum (n = 156). Addition-

ally, in the other non-PMWS pigs, the same RT-PCR

was performed on fresh samples of liver, bile, faeces

and mesenteric lymph nodes. The primers were

designed based upon the alignment of the available

partial ORF2 sequences of Spanish HEV strains (VH2

[AF195062], BCN [AF058684] and E11 [AF195063]),

the prototype swine HEV strain [AF082843], and eight

other swine HEV strains identified from pigs in USA

and Japan (Huang et al., 2002; Takahashi et al., 2003).

RNA was extracted with TriZol (Life Tech, USA) from

100 ml of serum or with the RNeasy mini kit (Qiagen,

Hilden, Germany) for tissue samples. Reverse tran-

scription was performed at 42 8C for 1 h with Super-

Script II (Invitrogen, USA). The semi-nested PCR was

performed using degenerate primers which can detect

potential genetically diversified field strains of swine

HEV: forward primer for both rounds (F1) 50-TTVGGGCTYCTYGACTTTGC-30, external reverse

(R1) 50-CCRCCRAGAAGYGTATCAGC-30, and inter-

nal reverse (R2) 50-CCRCGRCCCACCTCACCAAC-

30 (V = A or C or G; Y = C or T; R = A or G). PCR

parameters for both rounds of PCR included an initial

denaturation step at 94 8C for 6 min, followed by 35

cycles of denaturation for 50 s at 94 8C, annealing for

30 s at 48 8C, extension for 60 s at 72 8C, and a final

incubation at 72 8C for 7 min. The expected product of

the semi-nested RT-PCR was 212 bp. Negative (water)

and positive (prototype strain of swine HEV, GenBank

accession number AF082843) controls were included in

each set of RT-PCRs. The amplified PCR products were

examined by 1% agarose gel electrophoresis. A pig was

considered to be RT-PCR HEV positive when at least

one of the examined samples was positive.

2.4. Swine HEV sequencing

Twelve PCR products were sequenced (10 corre-

sponding to pigs with hepatitis and two from pigs

without hepatitis), and the resulting sequences were

compared genetically and phylogenetically to other

known HEV strains available in GenBank including

representatives of genotypes 1–4. Sequence align-

ments were generated by CLUSTALX (1.83) (Thomp-

son et al., 1997). Phylogenetic tree was constructed by

the neighbour-joining method (Saitou and Nei, 1987)

based on the partial nucleotide sequence of the ORF2

region (212 nucleotides). Bootstrap values were

determined on 1000 iterations of the data sets and

the final tree was displayed using the TreeView

program (1.6.6) (http://taxonomy.zoology.gla.ac.uk/

rod/treeview.html). The HEV sequences reported in

this paper were designated as UAB1–UAB12 and have

been deposited in GenBank database with accession

numbers: DQ383734–DQ383745.

2.5. Serology to swine HEV

IgG antibodies to HEV were detected using a

previously standardized in-house ELISA (Meng et al.,

1997). Briefly, 96-well plates were coated with a

purified 55 kDa truncated recombinant capsid protein

of the Sar-55 strain of human HEV (kindly provided by

Drs. Robert Purcell and Suzanne Emerson of the

National Institutes of Health, Bethesda, MD) in 0.1 M

sodium carbonate buffer (pH 9.6). After 2 h incubation

at 37 8C, the solution was replaced with 100 ml/well of

blocking buffer (10% FBS + 0.5% gelatine in washing

buffer). Serum samples in duplicate were added to each

well and incubated at 37 8C for 30 min. Plates were

washed four times with washing buffer before adding

HRP-conjugated goat anti-swine IgG as the secondary

antibody (Kirkegaard and Perry Laboratories, Inc.,

USA). After incubation at 37 8C for 30 min, the plates

were washed four times with washing buffer. ABTS

M. Martın et al. / Veterinary Microbiology 122 (2007) 16–24 19

solution was added to each well for colour develop-

ment. The optical density (OD) of each sample was read

with an ELISA plate reader with a 405 nm filter. Test

samples with OD values equal to or greater than the cut-

off value (0.300) were considered positive for anti-HEV

IgG (Meng et al., 1997). Preimmune and hyperimmune

anti-HEV swine sera were used as negative and positive

controls in each plate.

2.6. Statistical analyses

The x2-test (Fischer’s exact test) was used to

analyze the relationship between histopathology and

either RT-PCR or ELISA results (positive or negative).

The Mann–Whitney test was used to correlate the

ELISA OD values with liver histopathological results.

In order to compare the possible interaction between

PMWS and the HEV status for the presence of

hepatitis lesions, a stratified analysis was done and the

Mantel–Haenszel x2 was calculated. Additionally, a

backward stepwise logistic regression was performed

where the hepatitis status was considered to be the

outcome variable. All analyses were done using Epi-

Info 2000 v.3.2.2. (CDC, Atlanta, USA).

3. Results

Of the 156 tested animals by RT-PCR, 44 (28.2%)

pigs (all of them between 4 and 14 weeks of age) were

Table 1

Detection of swine HEV RNA and antibody in serum samples of PMWS-

without hepatitis

Age

(weeks)

PMWS-affected pigs

With hepatitis Without hepatitis

Swine HEV

RNA

Anti-HEV

antibodies

Swine HEV

RNA

Anti-HEV

antibodies

4 – – 0/2 (0.0) 0/2 (0.0)

6 2/6 (33.3)a 4/6 (66.7) – –

8 7/25 (28.0) 10/25 (40.0) 2/18 (11.1) 6/19 (31.6)

10 2/10 (20.0) 6/10 (60.0) 1/15 (6.7) 4/16 (25.0)

12 9/14 (64.3) 11/15 (73.3) 1/8 (12.5) 5/8 (62.5)

14 1/4 (25.0) 3/4 (75.0) 0/3 (0.0) 3/3 (100)

16 0/2 (0.0) 2/2 (100) 0/2 (0.0) 2/2 (100)

18–20 0/1 (0.0) 1/2 (50.0) – –

Total 21/62 (33.9) 37/64 (57.8) 4/48 (8.3) 20/50 (40.0

a Number of positive cases/number of total cases tested (percentage o

positive for swine HEV and 30 of them (68.2%) had

lesions of hepatitis. These lesions corresponded to

stages I or II, except for one animal that was in stage

III. Regarding their PMWS status, 25/110 (22.7%)

corresponded to PMWS pigs and 19/46 (41.3%) to

non-PMWS animals. In both groups of pigs (PMWS

and non-PMWS) swine HEV RT-PCR positive results

were significantly associated with the presence of

hepatitis (Odds ratio (OR) = 5.61, CI95% = 1.78–

17.68, p < 0.01 for PMWS animals and OR = 5.17,

CI95% = 1.28–20.82, p < 0.01 for non-PMWS pigs).

To determine whether HEV infection and PMWS

statuses might interact for the development of

hepatitis, a logistic regression was done. Results

indicated that both factors were independently

associated to hepatitis obtaining very similar OR

than above. The age distribution of pigs, presence of

hepatitis and swine HEV RNA detection are summar-

ized in Table 1.

IgG anti-HEV antibodies were detected in 43.8%

(70/160) of the sera tested in the study. Seroprevalence

was 51.9% (40/77) in pigs with hepatitis lesions and

36.1% (30/83) ( p = 0.031) in pigs without hepatitis

(Table 1). Average OD values of sera from pigs with

hepatitis (0.425 � 0.369) were higher than OD values

in pigs without hepatitis (0.252 � 0.173) ( p = 0.002).

In addition, significant differences in OD values were

also observed according to the severity score of

hepatitis: the average ODs for pigs in stages I and II

were 0.413 � 0.100 and 0.502 � 0.259, respectively,

affected and non-affected pigs from 4 to 20 weeks of age with and

Non-PMWS

With hepatitis Without hepatitis

Swine HEV

RNA

Anti-HEV

antibodies

Swine HEV

RNA

Anti-HEV

antibodies

1/1 (100.0) 0/1 (0.0) 2/7 (28.6) 2/7 (28.6)

1/1 (100) 0/1 (0.0) 2/10 (20) 2/10/(20.0)

3/3 (100) 0/3 (0.0) 3/5 (60) 1/5 (20.0)

0/1 (0.0) 0/1 (0.0) 1/3 (33.3) 0/3 (0.0)

4/7 (57.2) 3/7 (42.8) 2/3 (66.6) 2/3 (66.6)

– – – –

– – 0/5 (0.0) 3/5 (60.0)

– – – –

) 9/13 (69.2) 3/13 (23.1) 10/33 (30.3) 10/33 (30.3)

f positive cases).

M. Martın et al. / Veterinary Microbiology 122 (2007) 16–2420

Fig. 1. A phylogenetic bootstrapped tree (1000 iterations) based on the nucleotide sequences of a 212 bp region within the ORF2 gene of HEV

was constructed with the aid of the ClustalX program. Only bootstrap values above 50% are shown. Sequences from the present study are shown

in boldface. Other 38 sequences of HEV (genotypes 1–4 and avian HEV) were included for comparison and phylogenetic analysis (GenBank

accession numbers in parentheses). Sequences of porcine origin are shown in italics.

M.

Ma

rtınet

al./V

eterina

ryM

icrob

iolo

gy

12

2(2

00

7)

16

–2

42

1

Table 2

Percentage of nucleotide sequence identity of the swine HEV isolates from Spanish pigs in the current study with other HEV isolates of swine and human origin

Meng SwJ791 Osh205 P143 NLSW28 CV7 CV11 CV18a CV19 E11 VH1a VH2a UAB1 UAB2 UAB3 UAB4 UAB5 UAB6 UAB7 UAB8 UAB9 UAB10 UAB11 UAB12

Meng –

SwJ791 82 –

Osh 205 83 83 –

P143 83 87 85 –

NLSW28 87 86 84 86 –

CV7 84 84 86 87 86 –

CV11 85 80 83 84 83 92 –

CV18a 84 83 86 85 87 99 91 –

CV19 86 82 83 80 90 87 87 88 –

E11 87 84 85 84 90 89 89 89 100 –

VH1a 86 84 85 85 90 89 88 90 93 93 –

VH2a 85 84 85 85 90 91 88 92 93 94 93 –

UAB1 86 86 87 84 89 89 86 89 97 97 92 94 –

UAB2 87 86 86 85 90 90 88 90 100 99 93 95 100 –

UAB3 86 85 86 84 89 89 86 89 97 97 92 93 99 100 –

UAB4 86 83 84 87 86 87 87 86 87 87 88 87 90 90 90 –

UAB5 85 85 86 85 89 91 87 90 93 93 92 95 96 96 96 91 –

UAB6 85 85 85 86 88 91 87 90 91 92 89 94 94 94 94 90 97 –

UAB7 85 84 85 86 88 91 87 89 91 92 90 94 94 94 94 90 97 99 –

UAB8 86 85 86 84 87 88 86 87 96 95 91 92 98 99 99 90 95 94 94 –

UAB9 86 86 87 84 89 89 86 89 97 97 92 94 100 100 99 90 96 94 94 98 –

UAB10 86 86 87 84 88 89 86 88 97 96 91 93 100 100 99 90 96 94 94 98 100 –

UAB11 85 85 85 86 88 91 87 90 91 92 89 94 94 94 94 90 97 100 99 94 94 94 –

UAB12 86 86 86 85 88 89 87 88 97 96 92 93 99 100 99 91 96 94 94 99 99 99 94 –

The corresponding GenBank accession numbers of the strains are shown in Fig. 1.a Spanish human isolates.

M. Martın et al. / Veterinary Microbiology 122 (2007) 16–2422

while animals in stages 0, 3 and 4 had a negative

average (OD < 0.300) ( p = 0.009). Swine HEV

seroprevalence was 50% (57/114) in animals with

PMWS and 28.3% (13/46) in pigs without PMWS

( p = 0.009). Within seropositive animals, no differ-

ences were noticed regarding OD values between

animals with and without PMWS. Moreover, 12

seronegative pigs were found to be HEV viremic.

Phylogenetic analysis based on the ORF2 gene

region revealed that all Spanish swine HEV isolates

from this study were in the same cluster together with

other Spanish human and swine HEV isolates and

belonged to genotype 3 (Fig. 1). Sequence analyses

revealed that all the UAB swine HEV isolates shared

95.5 � 3.5% (89.0–100%) nucleotide sequence iden-

tity to each other and 92.4 � 4.5% (84.6–100%)

nucleotide sequence identity to other Spanish HEV

isolates of either human or porcine origin. The UAB

swine HEV isolates shared 89.1 � 5.7% (77.4–100%)

nucleotide sequence identity with other genotype 3

HEV isolates (Table 2).

4. Discussion

In pigs, infectious hepatitis can be caused by

viruses, bacteria, and parasites (Sims, 1996). Among

those agents, PCV2, the etiological agent of PMWS,

and HEV are among the most recently recognised

causes of infectious hepatitis (Meng et al., 1997;

Hamel et al., 1998). Hepatitis is considered as one of

the hallmark pathological lesions in PMWS (Rosell

et al., 1999), although approximately one third of the

PMWS-affected pigs with stages I and II hepatic

lesions are negative for PCV2 detection in the liver

(Rosell et al., 2000). The results from the present study

indicate that HEV is associated with hepatitis in pigs

and this association is not affected by the PMWS

status of the animals. Therefore both agents (HEV and

PCV2) probably may act independently in the

development of the hepatitis lesions observed.

The fact that seroprevalence against HEV was

significantly higher in animals with hepatitis reinforce

the idea that HEV is involved in the development of

hepatitis in pigs. Also, higher ELISA OD values were

detected in pigs with stages I or II of hepatitis compared

either with animals having higher scores or having no

hepatitis lesions. Moreover, the hepatic lesions

described for swine HEV infection under both natural

and experimental conditions (Meng et al., 1997; Halbur

et al., 2001) are similar to the lesions of hepatitis

described in this study for the HEV positive pigs. It has

been hypothesized that liver lesions observed in human

HEV infection may be attributable to an immune

response against the virus rather than to a direct effect of

virus replication (Jameel, 1999; Nagasaki et al., 2005).

In our case, the animals with compatible stages I or II

lesions were mostly seropositive while pigs with other

lesional stages were seronegative. However, in the

present study the average of positive OD for animals

with hepatitis was 0.425, a value too low to support this

hypothesis in pigs. All animals with lesional stages III

and IV were PMWS-affected pigs. The fact that the

presence of PCV2 in stages III and IV hepatic lesions is

systematic and usually massive (Rosell et al., 2000),

indicates that those cases with higher lesion scores are

probably associated only with PCV2 infection.

In the non-PMWS group, samples other than serum

were available including liver, bile, lymph nodes and

faeces. In some of those animals HEV could be

detected in some of the samples but not the others,

being bile and lymph nodes the most frequently

positive ones. In both PMWS and non-PMWS pigs,

some seronegative animals with hepatitis were HEV

positive by RT-PCR. These results are in accordance

with de Deus et al. (2006) and emphasize the need for

defining a sampling and analysis strategy based not

only in antibodies or HEV RNA detection in serum.

All HEV viremic pigs from this study were

approximately 4–14 weeks of age, which is consistent

with earlier reports in other countries (Meng et al.,

1997; Huang et al., 2002; Meng, 2003; Takahashi

et al., 2003). Seroprevalence to anti-HEVantibodies is

age-dependent and varies depending on the ages of

pigs (Meng et al., 1997).

Thus far all swine HEV isolates identified from pigs

worldwide belong to either genotypes 3 or 4, and all

the Spanish swine HEV isolates from this study

belonged to genotype 3, which further confirms earlier

reports in our country (Pina et al., 2000; Clemente-

Casares et al., 2003).

Taking together, the data from this study suggest

that HEV can be a primary agent of subclinical

hepatitis (stages I or II) in pigs and also that PCV2 can

be a hepatitis inducing agent PMWS-affected pigs.

Furthermore, HEV and PCV2 do not seem to be

M. Martın et al. / Veterinary Microbiology 122 (2007) 16–24 23

synergistic in the development of hepatitis lesions in

young pigs.

References

Banks, M., Heath, G.S., Grierson, S.S., King, D.P., Gresham, A.,

Girones, R., Widen, F., Harrison, T.J., 2004. Evidence for the

presence of hepatitis E virus in pigs in the United Kingdom. Vet.

Rec. 154, 223–227.

Choi, C., Chae, C., 2003. Localization of swine hepatitis E virus in

liver and extrahepatic tissues from naturally infected pigs by in

situ hybridization. J. Hepatol. 38, 827–832.

Choi, I.-S., Kwon, H.-J., Shin, N.-R., Yoo, H.S., 2003. Identification

of swine hepatitis E virus (HEV) and prevalence of anti-HEV

antibodies in swine and human populations in Korea. J. Clin.

Microbiol. 41, 3602–3608.

Clemente-Casares, P., Pina, S., Buti, M., Jardi, R., Martın, M.,

Bofill-Mas, S., Girones, R., 2003. Hepatitis E virus epidemiol-

ogy in industrialized countries. Emerg. Infect. Dis. 9, 448–454.

de Deus, N., Seminati, C., Pina, S., Mateu, E., Martin, M., Segales,

J., 2006. Detection of hepatitis E virus in liver, mesenteric lymph

node, serum, bile and faeces of naturally infected pigs affected

by different pathological conditions. Vet. Microbiol. 21 [Epub

ahead of print].

Drobeniuc, J., Favorov, M.O., Shapiro, C.N., Bell, B.P., Mast, E.E.,

Dadu, A., Culver, D., Iarovoi, P., Robertson, B.H., Margolis,

H.S., 2001. Hepatitis E virus antibody prevalence among per-

sons who work with swine. J. Infect. Dis. 184, 1594–1597.

Emerson, S.U., Purcell, R.H., 2003. Hepatitis E virus. Rev. Med.

Virol. 13, 145–154.

Emerson, S.U., Anderson, D., Arankalle, A., Meng, X.-J., Purdy, M.,

Schlauder, G.G., Tsarev, S.A., 2004. Hepevirus. In: Fauquet,

C.M., Mayo, M.A., Maniloff, J., Desselberger, U., Ball, L.A.

(Eds.), Virus Taxonomy, VIIIth Report of the ICTV. Elsevier/

Academic Press, London, pp. 851–855.

Garkavenko, O.A., Obriadina, A., Meng, J., Anderson, D.A.,

Benard, H.J., Schroeder, B.A., Khudyakov, Y.E., Fields, H.A.,

Croxson, M.C., 2001. Detection and characterisation of swine

hepatitis E virus in New Zealand. J. Med. Virol. 65, 525–529.

Halbur, P.G., Kasorndorkbua, C., Gilbert, C., Guenette, D., Potters,

M.B., Purcell, R.H., Emerson, S.U., Toth, T.E., Meng, X.-J.,

2001. Comparative pathogenesis of infection of pigs with hepa-

titis E viruses recovered from a pig and a human. J. Clin.

Microbiol. 39, 918–923.

Hamel, A.L., Lin, L.L., Nayar, G.P., 1998. Nucleotide sequence of

porcine circovirus associated with postweaning multisystemic

wasting syndrome in pigs. J. Virol. 72, 5262–5267.

Huang, F.F., Haqshenas, G., Guenette, D.K., Halbur, P.G., Schom-

mer, S.K., Pierson, F.W., Toth, T.E., Meng, X.J., 2002. Detection

by reverse transcription-PCR and genetic characterization of

field isolates of swine hepatitis E virus from pigs in different

geographic regions of the United States. J. Clin. Microbiol. 40,

1326–1332.

Jameel, S., 1999. Molecular biology and pathogenesis of hepatitis E

virus. Expert. Rev. Mol. Med. 1999, 1–16.

Meng, X.-J., 2003. Swine hepatitis E virus: cross-species infection

and risk in xenotransplantation. Curr. Top. Microbiol. Immunol.

278, 185–216.

Meng, X.-J., Purcell, R.H., Halbur, P.G., Lehman, J.R., Webb, D.M.,

Tsareva, T.S., Haynes, J.S., Thacker, B.J., Emerson, S.U., 1997.

A novel virus in swine is closely related to the human hepatitis E

virus. Proc. Natl. Acad. Sci. USA 94, 9860–9865.

Meng, X.J., Halbur, P.G., Shapiro, M.S., Govindarajan, S., Bruna,

J.D., Mushahwar, I.K., Purcell, R.H., Emerson, S.U., 1998.

Genetic and experimental evidence for cross-species infection

by swine hepatitis E virus. J. Virol. 72, 9714–9721.

Meng, X.J., Wiseman, B., Elvinger, F., Guenette, D.K., Toth, T.E.,

Engle, R.E., Emerson, S.U., Purcell, R.H., 2002. Prevalence of

antibodies to hepatitis E virus in veterinarians working with

swine and in normal blood donors in the United States and other

countries. J. Clin. Microbiol. 40, 117–122.

Nagasaki, F., Ueno, Y., Kanno, N., Okamoto, H., Shimosegawa, T.,

2005. A case of acute hepatitis with positive autoantibodies who

actually had hepatitis E virus infection. Hepatol. Res. 32, 134–137.

Pina, S., Buti, M., Cotrina, M., Piella, J., Girones, R., 2000. HEV

identified in serum from humans with acute hepatitis and in

sewage of animal origin in Spain. J. Hepatol. 33, 826–833.

Purcell, R.H., Emerson, S.U., 2001. Hepatitis E virus. In: Knipe,

D.M., Howley, P.M. (Eds.), Fields Virology. Lippincott Williams

& Wilkins, Philadelphia, pp. 3051–3061.

Rosell, C., Segales, J., Plana-Duran, J., Balasch, M., Rodriguez-

Arrioja, G.M., Kennedy, S., Allan, G.M., McNeilly, F., Latimer,

K.S., Domingo, M., 1999. Pathological, immunohistochemical,

and in-situ hybridization studies of natural cases of postweaning

multisystemic wasting syndrome (PMWS) in pigs. J. Comp.

Pathol. 120, 59–78.

Rosell, C., Segales, J., Domingo, M., 2000. Hepatitis and staging of

hepatic damage in pigs naturally infected with porcine circovirus

type 2. Vet. Pathol. 37, 687–692.

Saitou, N., Nei, M., 1987. The neighbor-joining method: a new

method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4,

406–425.

Segales, J., Domingo, M., 2002. Postweaning multisystemic wasting

syndrome (PMWS) in pigs. Vet. Q. 24, 109–124.

Segales, J., Rosell, C., Domingo, M., 2004. Pathological findings

associated with naturally acquired porcine circovirus type 2

associated disease. Vet. Microbiol. 98, 137–149.

Sims, L.D., 1996. The liver. In: Sims, L.D., Glastonbury, J.R.W.

(Eds.), Pathology of the Pig. A Diagnostic Guide. The Pig

Research and Development Corporation and Agricultural Vic-

toria, Victoria, pp. 109–126.

Takahashi, M., Nishizawa, T., Miyajima, H., Gotanda, Y., Iita, T.,

Tsuda, F., Okamoto, H., 2003. Swine hepatitis E virus strains in

Japan form four phylogenetic clusters comparable with those of

Japanese isolates of human hepatitis E virus. J. Gen. Virol. 84,

851–862.

Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Hig-

gins, D.G., 1997. The ClustalX windows interface: flexible

strategies for multiple sequence alignment aided by quality

analysis tools. Nucleic Acids Res. 25, 4876–4882.

van der Poel, W.H., Verschoor, F., van der Heide, R., Herrera, M.I.,

Vivo, A., Kooreman, M., de Roda Husman, A.M., 2001. Hepa-

M. Martın et al. / Veterinary Microbiology 122 (2007) 16–2424

titis E virus sequences in swine related to sequences in humans,

the Netherlands. Emerg. Infect. Dis. 7, 970–976.

Williams, T.P., Kasorndorkbua, C., Halbur, P.G., Haqshenas, G.,

Guenette, D.K., Toth, T.E., Meng, X.J., 2001. Evidence of

extrahepatic sites of replication of the hepatitis E virus in a

swine model. J. Clin. Microbiol. 39, 3040–3046.

Withers, M.R., Correa, M.T., Morrow, M., Stebbins, M.E., Seriwa-

tana, J., Webster, W.D., Boak, M.B., Vaughn, D.W., 2002.

Antibody levels to hepatitis E virus in North Carolina swine

workers, non-swine workers, swine, and murids. Am. J. Trop.

Med. Hyg. 66, 384–388.

Yoo, D., Willson, P., Pei, Y., Hayes, M.A., Deckert, A., Dewey, C.E.,

Friendship, R.M., Yoon, Y., Gottschalk, M., Yason, C., Giulivi,

A., 2001. Prevalence of hepatitis E virus antibodies in Canadian

swine herds and identification of a novel variant of swine

hepatitis E virus. Clin. Diagn. Lab. Immunol. 8, 1213–1219.