Am. J. Trop. Med. Hyg., 88(2), 2013, pp. 329–338
Copyright © 2013 by The American Society of Tropical Medicine and Hygiene
High Prevalence of Hepatitis E in Humans and Pigs and Evidence of
Genotype-3 Virus in Swine, Madagascar
Sarah Temmam, Lydia Besnard, Soa Fy Andriamandimby, Coralie Foray, Harentsoaniaina Rasamoelina-Andriamanivo,
Jean-Michel He ´raud, Eric Cardinale, Koussay Dellagi, Nicole Pavio, Herve ´ Pascalis,* and Vincent Porphyre
Centre de Recherche et de Veille sur les Maladies E´mergentes dans l’Oce ´an Indien (CRVOI), Plateforme de Recherche CYROI,
Sainte Clotilde, La Re ´union, France; CNRS, UMR5557 Ecologie Microbienne, Lyon, France; International Center for Agronomical Research for
Development (CIRAD), UMR112 SELMET, Saint Pierre, La Re ´union, France; International Center for Agronomical Research for Development
(CIRAD), UMR15 CMAEE, Saint Denis, La Reunion, France; Institut Pasteur de Madagascar, Virology Unit, Antananarivo, Madagascar;
FOFIFA, Department of Veterinary and Husbandry Research, Antananarivo, Madagascar; Institut de Recherche pour le
De ´veloppement (IRD), Saint Denis, La Re ´union, France; ANSES, Maisons-Alfort, France
contaminated food (mainly pork products), or by contacts with infected animals. Very little data are currently available
regarding the disease in the Southwestern Indian Ocean Islands. We report the first sero- and viro-survey for HEV in
human and swine in Madagascar. A seroprevalence rate of 14.1% (60 of 427) was measured in slaughterhouse workers.
Seroprevalence to HEV in pigs was estimated to 71.2% (178 of 250), strongly suggesting the existence of a zoonotic cycle.
Three out of 250 pig livers (1.2%) tested HEV RNA-positive by quantitative polymerase chain reaction. Phylogenetic
analyses based on 1-kb sequences of the ORF 2-3 identified these viruses as HEV genotype 3. Sequences clustered in a
distinct Malagasy sub-clade, possibly representative of a new sub-genotype, for which the date of emergence was
estimated around 1989. Further studies are needed to confirm other transmission routes of HEV to humans, especially
through non-zoonotic cycles.
Hepatitis E virus (HEV) causes an orofecal disease transmitted through poor hygiene environments,
Hepatitis E virus (HEV), the single member of the family
Hepeviridae, genus Hepevirus, is a small non-enveloped virus
with ~7,200 nucleotide positive-sense, single-stranded RNA
genome1; there are four major recognized and two putative
genotypes of HEV according to previous sequence analyses.
Genotype 1 and 2, restricted to humans, are associated with
epidemics in developing countries with poor hygiene condi-
tions (mainly genotype 1 in Asia and Africa and genotype 2
in Central America and Central Africa).2Genotypes 3 and 4
infect swine and humans, the latter being infected by the
consumption of contaminated food, resulting in sporadic
cases of hepatitis E in both developing and industrialized
countries.3–6Genotype 3 infects the broadest spectrum of
hosts (human, swine, wild boar, deer, rabbit, mongoose, etc.)
and has the greatest genetic diversity, with 10 identified sub-
genotypes (3a to 3j).7Recently, new animal hepeviruses have
been described in rats,8poultry,9–11and bats,12however they
are phylogenetically divergent and likely define new genera
within the Hepeviridae family.
The HEV infection in swine occurs with elevated sero-
prevalences, sometimes higher than 95%, though it remains
more than 30% of pigs and 65% of swine herds.14In humans,
hepatitis E has been reported worldwide.15–20It is considered
a significant cause of acute clinical hepatitis among adults in
Asia, Middle East and Africa.2,21,22High lethality rates have
been reported in some developing countries, especially among
pregnant women.23In addition to the consumption of contam-
inated food, humans can be infected by HEV by direct expo-
there likely exist other important modes of viral transmission.
People workinginswine farms andslaughterhousesare known
to beathigher riskof HEV infection.25,26
In the Southwestern Indian Ocean Islands, little infor-
mation on HEV infection is available, essentially reported
from the French overseas departments of La Re ´union and
Mayotte: a sporadic case of HEV, probably imported, was
reported in Mayotte island, in the Comoros Archipelago.27In
Reunion Island, two human cases were reported in 2012,
three human cases in 2008,28and an imported hepatitis E case
in 2003.29No study has been carried out so far to determine the
epidemiology of HEV infection in human population, nor the
HEV status of the pig herds and risk factors associated with
human contamination in these areas. Hence, the objectives of
this study were 1) to assess HEV infection among humans in
Madagascar by exploring the HEVseroprevalencein ahighrisk
population, i.e., slaughterhouse workers; 2) to explore a possi-
ble pig-to-human zoonotic transmission cycle in Madagascar by
assessing the HEV seroprevalence in swine herds; and 3) to
identify the genotype of HEV circulating in pigs.
MATERIAL AND METHODS
Sampling description. Human sera. Human sera came
from a serobank stored at Institut Pasteur in Madagascar that
was collected between September 27, 2008 and May 27, 2009.
Sera were collected during a national cross-sectional serologic
survey among voluntary slaughterhouse workers within the
administrative center of the district. We selected 427 sera
from workers that lived in the same 18 districts and 11 regions
from where the sampled pigs originated. The demographic
characteristics of the workers included in the study are
presented in Table 1.
Samples from pigs. A total of 20,000 pigs were estimated
to be slaughtered at the four abattoirs of Antananarivo, the
capital city of Madagascar, during the 3 months of surveys. To
detect an estimated prevalence of HEV viral contamination
of 2% in the swine population (with a confidence level
at 99%), blood sera and liver samples were collected from
*Address correspondence to Herve ´ Pascalis, CRVOI, Plateforme de
Recherche CYROI, 2 rue Maxime Rivie `re BP 80005, 97491 Sainte
Clotilde cedex, REUNION (France). E-mail: firstname.lastname@example.org
250 animals (M/F = 111/139) from November 2010 to January
2011. Animals were all more than 6 months of age at the
date of slaughtering. Blood samples were collected at the
bleeding post and a small piece of liver tissue was cut from
the left medial lobe close to the gallbladder, as previously
described14; all biological samples were transported in a cool
box at 4°C, and then frozen and stored at −80°C. Because the
absence of animal traceability in Madagascar excludes the
possibility of the identification of an individual pig’s farm at
source, we considered the district and region of production as
areas of origin. The sampled animals originated from 18 dis-
tricts from 11 of 22 administrative regions.
Ethical considerations. The human serobank used in this
study was constituted under a human research protocol
approved by the National Ethical Review Committee from
the Malagasy Ministry of Public Health. (Authorization
no. 228 – SANPFPS July 3, 2008) and amended for the current
study (Authorization no. 039-SANPFPS June 4, 2012). The
does not, at this time, have a committee to review and approve
scientific research protocols involving animals.
Serological analysis. Detection of anti-HEV in human sera.
A commercial kit (EIAgen HEV Ab, Adaltis, Milano, Italy)
was used, which detects immunoglobulin G (IgG) and IgM
antibodies to the four HEV genotypes. The test was carried
out according to the manufacturer’s instructions. Samples
were considered positive when the ratio between the OD450
values of the sample to that of the cutoff was higher than 1.1
(cutoff = mean of the negative control + 0.350).
Detection of antibodies to HEV in pig sera. A commercial
test validated for veterinary analysis was used: HEV ELISA
4.0v (MP Diagnostics, Illkirch, France). This test is a double
sandwich ELISA detecting IgG, IgM, and IgA antibodies to a
proprietary recombinant antigen, which is highly conserved
between different HEV strains (genotypes 1 to 4). Analyses
were performed according to the manufacturer’s instructions
except that 10 mL of sera were used.30Samples were considered
positive when the OD450 value of the sample was superior to
the cutoff value (cutoff = mean of the negative control + 0.300).
Molecular detection of HEV in pig liver. A small piece of
liver tissue (~1 mm3) was dissected on ice from the frozen
samples and homogenized in DMEM medium using the
TissueLyser (Qiagen GmbH, Hilden, France) and a 3 mm
tungsten bead for 2 min at 25 Hz. Total nucleic acids were
extracted from the mixture supernatant using the EZ1 Viral
mini kit v2.0 and the EZ1 BioRobot (Qiagen), according to
the manufacturer’s recommendations. The complementary
DNA (cDNA) was generated by GoScript Reverse Transcrip-
tase using random hexamers (Promega Corporation, Madison,
WI). TaqMan HEV quantitative polymerase chain reaction
i.e., primers and probe targeting a portion of the open reading
frame (ORF) 3 region of HEV genotypes 1 to 4 in animals and
Sequencing. A sequence of 1,059 bp within the ORF 2-3
region was generated using a semi-nested PCR. The first
round PCR was conducted on 5 mL of cDNA in a final volume
of 25 mL containing 2X GoTaq Hot Start Mastermix, and
200 nM of primers JVHEV-F,31and TqRev.32Cycling condi-
tions were as follows:95°C for 2 min, and then 35 cycles of 95°C
for 15 sec/60°C for 30 sec/72°C for 2 min followed by a final
elongation step at 72°C for 7 min. Second round PCR was
performed on 4 mL of primary PCR product in a final volume
of 50 mL containing 200 nM of primers JVHEV-F and
3159N.33Cycling conditions were the same as the first round
PCR except that 40 cycles were performed. Positive amplifi-
cations were purified on a 1% (w/v) agarose gel electrophore-
sis with the QIAquick gel extraction kit (Qiagen), cloned in
a pGEM-T Easy vector (Promega), and finally sequenced
(GATC Biotech, Konstanz, Germany). Sequences were primers
trimmed and assembled for analysis using the Geneious Pro
5.3.4 software package.34
Phylogenetic analyses. All alignments were generated in
Geneious Pro using the MUSCLE alignment method.35The
DNA substitution model that best fitted the data was
performed by the software jModelTest 0.1.136and was consid-
ered for all phylogenetic analyses. We selected different
models of nucleotide substitution using the corrected Akaike
Molecular evolutionary distances. Evolutionary distances
between sequences were calculated using MEGA 5,37taking
into account the best model of sequences evolution allowing
correction of the estimates of evolutionary distance. Because
MEGA does not contain the general time-reversible (GTR)
model proposed, we used the Tajima-Nei correction,38which
is the nearest model to those proposed by jModelTest.
Genotyping and sub-typing. For the determination of HEV
genotype of the Malagasy strains, a total of 40 GenBank
sequences of full-genome HEV were selected according to
their origin (Asia, Europe, Africa, and America), their host
(human, swine, rabbit, mongoose, and wild boar) and their
Socio-demographic characteristics of slaughterhouse workers
included in the study
Characteristics of individuals Number (%)
Age group (years)
Duration of activity (years)
Fianarantsoa Urban (10)
TEMMAM AND OTHERS
genotype and sub-genotype. Avian HEV was defined as
outgroup. Phylogenetic trees were constructed using the gen-
erated sequence of ~1-kb by maximum likelihood (ML)
within PhyML.39Nodal support was evaluated by 1,000 boot-
strap replicates. Bayesian phylogenetic inference (BI) was
carried out using MrBayes,40with two independent runs of
four incrementally heated, Metropolis-coupled Markov chain
Monte Carlo (MCMC) starting from a random tree. The
MCMC were run for 1,000,000 iterations and associated
model parameters being sampled every 200 generations. The
initial 1,000 trees in each run were discarded as burning sam-
ples and the harmonic mean of the likelihood was calculated
by combining the two independent runs.
To implement a maximum of sequences from the 10 HEV-3
sub-genotypes, phylogenies were restricted to a fragment of
~300 bp inside the previous set of the ORF 2-3 region, as
previously described by Lu and collaborators.7A total of 233
sequences were used. The BI was conducted as previously
described for genotyping analyses except that MCMC were
run for 10,000,000 iterations, associated model parameters
was sampled every 1,000 generations and the initial 100,000
trees in each run were discarded as burning samples.
Date estimations. The same set of GenBank sequences as
the one for sub-typing analyses was used, except that we
retained only sequences for which the year of sampling was
available (N = 65). The Bayesian MCMC analyses were per-
formed using BEAST v. 1.6.1 (http://beast.bio.ed.ac.uk) under
a strict molecular clock setting. GTR + I + G were used, as
proposed by jModelTest. An exponential-growth coalescent
model was chosen as a prior on the tree. We ran a chain length
of 100,000,000 by sampling trees every 1,000 generations.
Convergence, Bayes factors, and burning were assessed
using Tracer v1.4.1b (http://tree.bio.ed.ac.uk/software/tracer).
The maximum clade credibility phylogeny for analyzing
the MCMC data set was annotated by TreeAnotator in the
BEAST package. The tree was visualized using FigTree v1.2.2.
Statistical and spatial analyses. For serological data from
pigs, statistical analyses were performed in R software version
188.8.131.52The c2test was run to test the significance of the
differences observed in seroprevalence repartition by gender
and to compare the human and pig spatial seroprevalence
distributions by district and region. Confidence intervals
(CIs) based on these variables were also determined. For
human serological data, same analyses were used to compare
prevalence by age, sex, and duration of activity in slaughter-
houses. The Mantel-Haenszel test was used to carry out
adjustment for age when testing the association between
duration of activity and serological status. The c2test and
confidence intervals for human variables were performed on
STATA/IC11.1 (StataCorp, College Station, TX). Human
and pig data were geographically displayed with ArcGIS
10.0 (ESRI, Redlands, CA).
Seroprevalence to HEV in slaughterhouse workers. Of 427
human sera, 60 were positive for HEV, leading to a sero-
prevalence of 14.1%, (CI 95% [10.9–17.7]). There was no sig-
40–50 years, and 50–60 years, respectively, P value = 0.464)
P value = 0.223). The seropositivity rates in slaughterhouse
workers ranged from 5.3% (district of Bealanana, region of
Sofia) to 31.4% (district of Fianarantsoa, region of Haute
Hepatitis E virus (HEV) seroprevalence among swine and slaughterhouse workers according to geographical repartition - HEV seroprevalence
and confidence intervals (CI 95%) are expressed in percentage
HEV seroprevalence according to district repartition (%) HEV seroprevalence according to region repartition (%)
% CI 95%% CI 95%%CI 95%% CI 95%
Fianarantsoa Urban (10)
Port Berger (16)
Itasy 21.1 13.4–30.677.1 59.9–89.6
Vatovavy-Fitovinany 11.11.4–34.7 70.034.8–93.3
Hepatitis E virus (HEV) seroprevalence among swine and slaughter-
house workers according to the duration of professional activity
Age of workers
Duration of activity
P value (a vs. b)
< 5 years (a)
³ 5 years (b)
HEV DETECTED IN MALAGASY HUMAN AND PIGS
Matsiatra) (Table 2A, Figure 1). The duration of professional
activity of workers in slaughterhouses before blood sampling
was 11 years on average (CI 95% [1–56]). Seroprevalences
are presented in Table 2B according to the age of workers
and the duration of their professional activity. There was no
significant difference between age groups for a given dura-
tion of activity (P value between 0.09 and 0.50). However,
a significant difference was observed between people that
worked for < 5 years, compared with those who worked for
longer periods (P value = 0.03), suggesting a correlation
between the time of exposure and the likelihood of infection
irrespective of the workers age.
Seroprevalence to HEV in swine populations. Of 250 sera
collected from pigs at the slaughterhouse, 178 tested positive
for HEV. Seroprevalence was estimated at 71.2% (CI 95%
[65.2–76.7]). There was no significant difference between
male and female (72.1% and 70.5%, respectively, P value =
0.786). The highest seroprevalence in pigs was observed in the
district of Mianarivo, region of Itasy (88.9% [51.8–99.7]), and
the lowest in the district of Befandriana-Nord, region of Sofia
(33.3% [7.5–70.1]) (Table 2A, Figure 1).
Spatial distribution of HEV seropositivity among swine and
slaughterhouse workers. All 18 districts covered by the study
were found positive for infection in swine, whereas only 12 of
alence is displayed in red, and swine HEV seroprevalence with pie-charts. Location of virus-positive livers is mentioned in labels.
Geographical distribution of hepatitis E virus (HEV) seroprevalence in human and swine. Slaughterhouse workers HEV seroprev-
TEMMAM AND OTHERS
them had evidence of infection in humans. There were signif-
icant differences between the spatial distributions of human
and swine HEV seroprevalences when tested at the district
and the region levels (P value < 0.0001, Table 2A).
Molecular detection of HEV in pig liver. A total of 250 livers
were collected from the same pigs, which were serologically
tested. Only three livers (1.2%) were found positive for
HEV viral RNA, namely swHEV-MG-104, swHEV-MG-121,
and swHEV-MG-190 (GenBank accessions nos.: JX507128–
130).The three livers were sampledin December 2010(onemale
and two female pigs). Two of these pigs originated from
the Fianarantsoa and Mianarivo districts (located in the
central highlands of Madagascar), and the third one from
the Port-Berger district in the north-west (Figure 1). Inter-
estingly, these regions had the highest seroprevalence rates
toswineHEVand thehighestratestohuman HEVinslaugh-
terhouse workers (district of Fianarantsoa), reflecting a
probably active viral circulation in swine with an elevated
risk of infection in humans. Of note, two animals detected
positive for HEV viral RNA were concomitantly seroposi-
tive to HEV.
Genotyping, sub-typing and phylogenetic analysis of HEV
in Malagasy pigs. Whencomparedwithhomologoussequences
2(ORF 2-3 sequences of 1-kb length) from human and swine
HEV genotypes 1 to 4 deposited in the GenBank database
the Malagasy HEV strains could be identified as genotype 3
(Figure 2). The three sequences branch together, forming a
distinct group, with a high nodal support (posterior probabil-
ity P = 0.998, ML bootstrap = 100). Within this group,
swHEV-MG-104 and swHEV-MG-190 sequences fall into a
P value of 77 (Figure 2). Genetic distances between Malagasy
HEV strains range between 7.4% (±0.97) and 9.8% (±1.09).
Sub-typing of the 3 HEV strains was performed by phylo-
genetic analysis of a small fragment (300 bp) of the ORF 2-
3 region, as previously described.7The Malagasy sequences
also clustered in a distinct group, located at the root of clades
formed by sub-genotypes 3c and 3i (Figure 3), with a strong
nodal support (P = 0.827), suggesting a shared history
between sub-genotypes 3c-, 3i-, and Malagasy-HEV. Table 3
shows the genetic distances between genotype 3 HEV sub-
genotypes, as defined in Figure 3. Genetic distances between
the Malagasy HEV clade and other sub-genotypes were in the
same range as those observed between each other sub-type
(15.6–22.4%), suggesting that the Malagasy HEV strains may
form a distinct sub-genotype. It is most closely related to sub-
type 3a (15.6%), and most distantly related to sub-type 3f
(22.4%) according to the genetic distances.
Estimation of date of emergence of genotype 3 Malagasy
HEV strains. The Bayesian MCMC analysis estimates the
time to the most recent common ancestor (TMRCA) of each
genotype and sub-genotype, with a 95% highest probability
density, and is presented in Figure 4. The resulting phyloge-
netic tree topology is the same as the one obtained by the
were used to fix tree topologies. Sequences are colored according their genotype (pink, green, orange, and blue for genotypes 1, 2, 3, and 4,
respectively). Phylogenetic clusters containing Malagasy sequences are outlined. Only major nodal junctions are presented (at the origin of
each genotype): Posterior probabilities (PP) in italic (P > 0.90) and, where nodes coincided, maximum likelihood bootstrap values (ML) in bold
(ML > 70). Arrow marks established Hepevirus gender. Scale bar indicates the number of nucleotide substitution per site.
Phylogenetic analysis of Malagasy hepatitis E virus (HEV) sequences based on a 1-kb fragment in the ORF 2-3. Bayesian analyses
HEV DETECTED IN MALAGASY HUMAN AND PIGS
Bayesian method for sub-typing (Figure 3). High posterior
probabilities support the nodes defining each genotype and
sub-type of HEV (P = 1.0 except for 3i-HEV for which P =
0.97). The TMRCA of HEV-3, HEV-4, HEV-1, and HEV-2
were estimated at 1915, 1923, 1957, and 1986, respectively.
The estimated dates of emergence of the common ancestor
and 3i was estimated to appear in 1965. The Malagasy HEV
clade apparently diverged from the 3c-3i cluster around 1989.
Interestingly, TMRCA of sub-type 3c is also estimated to
emerge around 1989, suggesting that the common ancestor of
sub-types 3c-, 3i-, and Malagasy-HEV had emerged in 1965,
described by Lu and others.4Sequences are colored according their genotype (pink, green, orange, and blue for genotypes 1, 2, 3, and 4,
respectively). Phylogenetic clusters containing Malagasy sequences are outlined. Only major nodal junctions are presented: Posterior probabilities
(PP) are noted in italic (P > 0.70). * Indicates a cluster with no clear sub-genotyping, including 3b- and 3d-HEV sequences. The HEV-3 sub-
genotypes are mentioned in dashed lines. Inset: enlarged representation of Malagasy-, 3c- and 3i-HEV subtypes. Scale bar indicates the number
of nucleotide substitution per site.
Bayesian phylogenetic analysis of Malagasy hepatitis E virus (HEV) sequences based on a 300 bp fragment in the ORF 2-3, as
Estimates of evolutionary distances among hepatitis E virus (HEV)-3 sub-genotypes*
3a-HEV3c-HEV 3e-HEV 3f-HEV3g-HEV 3i-HEVMalagasy HEV
*Genetic distances are expressed in percentages and are displayed below the diagonal. Standard error estimates, in italic, are shown above the diagonal. Analyses were conducted in MEGA537
using the Tajima-Nei model.42
TEMMAM AND OTHERS
and then diversified in 3i-HEV around 1980 and in 3c- and
Hepatitis E is widespread all over the world, both in human
and swine populations. In industrialized countries, sporadic
human infection mainly occurs by the consumption of contami-
are operative in developing countries (such as Madagascar):
poor hygiene environments, exposure to infected swine, or
swine effluents.2,3As humans may be infected by zoonotic or
environmental routes, multiple HEV genotypes may circulate
among human and swine populations.33,43,44To investigate a
possible swine-to-human route of infection, we report here the
first sero- and viro-survey among slaughterhouse workers and
Antibodies to HEV were detected in Malagasy slaughter-
house workers with a global prevalence of 14.1%. Previous
studies have identified workers in slaughterhouses and pig
handlers as populations at high risk of infection, because of
their frequent contacts with organs, manure, and blood from
animals.45,46In Madagascar, we found that HEV seropreva-
lence among slaughterhouse workers reached 33.3% in the
district of Fianarantsoa, but the average seropositivity (14.1%)
could be considered as modest when compared with other
studies. Indeed, studies in Spain,47Germany,48Switzerland,49
and in the United States24,50have shown that HEV seropreva-
lence in exposed human populations, such as veterinarians and
slaughterhouse workers, can reach more than 35%, whereas
in non-exposed humans (i.e., blood donors) the seroprevalence
is significantly lower. Further studies are needed to measure
the seroprevalence of HEV in the Malagasy general popula-
tion, and to determine if pig breeders are at higher risk of
300 bp within ORF 2-3 of the 3 Malagasy HEV sequences and 65 globally representative GenBank sequences. Arrows indicate date positions. (A)
Representation of the global diversification of HEV genotypes. Solid arrow represents the most recent common ancestor (TMRCA) for each
defined genotype; dashed arrow represents TMRCA for non-distinct HEV genotype. (B) Sequences are colored by genotype (pink, green, orange,
blue, and black for genotypes 1, 2, 3, 4, and 5, respectively). The HEV-3 sub-genotypes are mentioned in dashed lines. Standard deviation bars
represent the 95% confidence interval of the mean estimated TMRCA. Scale bar indicates the number of years.
Date estimations of hepatitis E virus (HEV) genotypes apparition and circulation. BEAST analysis is based on the fragment of
HEV DETECTED IN MALAGASY HUMAN AND PIGS
contamination. Indeed, in developing countries, several
routes that are not linked to a direct contact with infected
swine are commonly described for human contamination with
HEV. In a resource-poor country, contamination of village
water sources by HEV51or human-to-human HEV transmis-
sion are possible routes, but in-depth analysis of the natural
history of the disease and identification of associated risk
factors are still lacking.
Our study shows that HEV circulates in Madagascar within
the pig population, which strongly supports the role of an
animal-to-human cycle in the country. A high seroprevalence
rate of 71.2% on the average was found in pigs. Large differ-
ences were observed between districts (seroprevalences rang-
ing from 33.3% to 88.9%) and regions (from 50.0% to
83.3%); however, because no traceability is operational in
Madagascar to identify the individual pig farm at its source,
no solid conclusion could be drawn with regard to the geo-
graphical distribution of HEV in the country, or the related
on-farm risk factors. Hence, further studies targeting breeding
areas are needed to more precisely measure the rates of infec-
tion and HEV burden in herds, and identify risks factors of
contamination. Interestingly, because pigs fed on kitchen res-
idue were recently reported to be more frequently infected
than those fed on complete feed,52on-farm practices, espe-
cially in animal feeding and hygiene management, need to be
considered to promote biosecurity measures adapted to the
In Madagascar, we found more than 70% of pigs sampled
in slaughterhouses were seropositive for HEV, but that only
1.2% of livers were detected RNA virus positive. This rela-
by the age of swine at which the animals are slaughtered. In
fact, pigs are usually infected during the early stages of the
breeding (3–4 months of age), and the viral load in organs and
In Madagascar, pigs are usually slaughtered at older ages (6 to
12 months), and one may consider that only a few pigs remain
infectious at that age.2Therefore, pig farm breeders might be
more exposed to HEV infection than slaughterhouse workers,
as noted by Geng.55
Phylogenetic analyses revealed that genotype 3 HEV is
circulating among swine populations in Madagascar. Recent
studies21,56have shown that HEV isolate TLS40 (GenBank
accession no. EU495232) is at the root of sub-types 3c and 3i,
as our Malagasy sequences. Unfortunately, we were not able
to cluster this strain with the Malagasy HEV (data not
shown), or to classify the Malagasy strains within an already
known sub-genotype, probably caused by the shortness of the
sequences used for this analysis. Full-genome analysis may
allow us to determine whether the Malagasy HEV strains
may form a new sub-type, and to better understand the origins
of these strains.
Nakano and others57recently estimated the TMRCA of
HEV genotypes and sub-genotypes. Our observations slightly
differ from theirs, probably caused by different sequences
used in the data set. We estimated the divergence of strictly
human HEV (genotypes 1 and 2) and zoonotic HEV (geno-
types 3, 4, and 5) to be in 1794. Then in 1837, genotype 4-HEV
diverge from genotypes 3–5 clusters. Interestingly, genotype 4
is mainly found in Asian countries, and not in Old (Europe
and Africa) or New World countries. We estimated the
emergence of genotype 3-HEV around 1915 and that of the
Malagasy HEV around 1989. Moreover, a clear diversification
of genotype 3 seems to have occurred after the 70s (TMRCA
of sub-types 3b, 3f, 3a, 3e, 3i, 3c estimated to appear in 1970,
1974, 1978, 1980, 1980, and 1989, respectively). The phyloge-
netic cluster formed by 3c-, 3i-, and Malagasy-HEV sub-types
After 1965 (date of emergence of TMRCA of this cluster),
a diversification occurred and resulted in a distinct European
3i-HEV sub-type, and two other groups formed by sequences
both from Europe and Africa (3c-HEV and Malagasy-HEV).
Because international commercial exchanges and importation
of live animals have been regularly reported from Europe
(France, Germany, Belgium) since the 60s, and because few
animals were recently imported in Madagascar since the late
90s, our findings suggests that the HEV strains circulating in
Madagascar may have a common history with European and
Our study is the first reported so far on HEV infection in
humans and swine in Madagascar, and the circulation of the
virus in swine. The HEV strains circulating in Madagascar
among pigs are of genotype 3 and may have a common history
with European and African HEV strains. Further studies are
in preparation to explore the role of environmental contami-
nation, the role of other transmission routes, and the role of
the wild fauna in the viral cycle.
Received October 4, 2012. Accepted for publication October 31, 2012.
Published online December 3, 2012.
Acknowledgments: We deeply thank the technical staffs of the
FOFIFA-DRZV, and especially Samuel Rakotonindrina, for their
invaluable work in pig slaughterhouses. We are grateful to Jean
The ´ophile Rafisandrantantsoa, Institute Pasteur of Madagascar, from
the Virology Unit, for his involvement in the serological analysis.
Equally, we thank warmly Elodie Barnaud, ANSES Maison-Alfort,
for her technical assistance. We also thank D. A. Wilkinson for proof-
reading the text.
Financial support: The main financial support was provided by the
Regional Council of La Re ´union, the European Regional develop-
ment Funds(ERDF) andFrench
QualiREG research network in Indian Ocean (www.qualireg.org).
We are grateful to Institut Pasteur in Madagascar for funding the
human study component. We warmly thank the French Agency for
Food, Environmental and Occupational Health & Safety (ANSES)
for funding the serological analysis work in swine.
Authors’ addresses: Sarah Temmam, Centre de Recherche et de
Veille sur les Maladies E´mergentes dans l’Oce ´an Indien (CRVOI),
Plateforme de Recherche CYROI, Sainte Clotilde, La Re ´union,
France, and CNRS, UMR5557 Ecologie Microbienne, Lyon, France,
E-mail: email@example.com. Lydia Besnard, Coralie Foray, and Eric
Cardinale, Centre de Recherche et de Veille sur les Maladies
E´mergentes dans l’Oce ´an Indien (CRVOI), Plateforme de Recherche
CYROI, Sainte Clotilde, La Re ´union, France, and International
Center for Agronomical Research for Development (CIRAD), UMR15
CMAEE, Saint Denis, La Re ´union, France, E-mails: lydiabesnard@
gmail.com, firstname.lastname@example.org, and email@example.com. Soa
Fy Andriamandimby and Jean-Michel He ´raud, Institut Pasteur de
Madagascar, Virology Unit, Antaananarivo, Madagascar, E-mails:
firstname.lastname@example.org and email@example.com.
Rasamoelina-Andriamanivo, FOFIFA, Department of Veterinary
and Husbandry Research, Ministry of Agriculture, Antananarivo,
Madagascar, E-mail: firstname.lastname@example.org. Koussay Dellagi and
Herve ´ Pascalis, Centre de Recherche et de Veille sur les Maladies
E´mergentes dans l’Oce ´an Indien (CRVOI), Plateforme de Recherche
pour le De ´veloppement (IRD), Saint Denis, La Re ´union, France,
E-mails: email@example.com and firstname.lastname@example.org. Nicole
Pavio, ANSES, Maisons-Alfort, France, E-mail: npavio@vet-
alfort.fr. Vincent Porphyre, International Center for Agronomical
TEMMAM AND OTHERS
Research for Development (CIRAD), UMR112 SELMET, Saint
Pierre, La Re ´union, France, E-mail: email@example.com.
1. Meng XJ, Anderson DA, Arankalle VA, Emerson SU, Harrison
TJ, Jameel S, Okamoto H, 2011. Family Hepeviridae. King
AM, Carstens EB, Lefkowitz EJ, ed. Virus Taxonomy: 9th
Report of the International Committee on Taxonomy of
Viruses. San Diego, CA: Elsevier, 1021–1028.
2. Purcell RH, Emerson SU, 2010. Hidden danger: the raw facts
about hepatitis E virus. J Infect Dis 202: 819–821.
3. Pavio N, Meng X-J, Renou C, 2010. Zoonotic hepatitis E: animal
reservoirs and emerging risks. Vet Res 41: 46.
4. Zhang W, Shen Q, Hua X, Cui L, 2011. Novel hepatitis E virus
genotype in Norway rats, Germany. Emerg Infect Dis 17:
5. Cossaboom C, Co ´rdoba L, Dryman B, Meng X-J, 2011. Hepa-
titis E virus in rabbits, Virginia, USA. Emerg Infect Dis
6. Colson P, Borentain P, Queyriaux B, Kaba M, Moal V, Gallian P,
Heyries L, Raoult D, Gerolami R, 2010. Pig liver sausage as a
source of hepatitis E virus transmission to humans. J Infect Dis
7. Lu L, Li C, Hagedorn CH, 2006. Phylogenetic analysis of global
hepatitis E virus sequences: genetic diversity, subtypes and
zoonosis. Rev Med Virol 16: 5–36.
8. Johne R, Heckel G, Plenge-Bo ¨nig A, Kindler E, Maresch C,
Reetz J, Schielke A, Ulrich R, 2010. Novel hepatitis E virus
genotype in Norway rats, Germany. Emerg Infect Dis 16:
9. Haqshenas G, Shivaprasad HL, Woolcock PR, Read DH, Meng
X, 2001. Genetic identification and characterization of a
novel virus related to human hepatitis E virus from chickens
with hepatitis-splenomegaly syndrome in the United States.
J Gen Virol 82: 2449–2462.
10. Marek A, Bilic I, Prokofieva I, Hess M, 2010. Phylogenetic
analysis of avian hepatitis E virus samples from European
and Australian chicken flocks supports the existence of a differ-
ent genus within the Hepeviridae comprising at least three dif-
ferent genotypes. Vet Microbiol 145: 54–61.
11. Zhao Q, Zhou EM, Dong SW, Qiu HK, Zhang L, Hu SB, Zhao
FF, Jiang SJ, Sun YN, 2010. Analysis of avian hepatitis E virus
from chickens, China. Emerg Infect Dis 16: 1469–1472.
12. Drexler JF, Seelen A, Corman VM, Fumie Tateno A, Cottontail
V, Melim Zerbinati R, Gloza-Rausch F, Klose SM, Adu-
Sarkodie Y, Oppong SK, Kalko EK, Osterman A, Rasche A,
Adam A, Mu ¨ller MA, Ulrich RG, Leroy EM, Lukashev AN,
Drosten C, 2012. Bats worldwide carry hepatitis E-related
viruses that form a putative novel genus within the family
Hepeviridae. J Virol 86: 9134–9147.
13. Satou K, Nishiura H, 2007. Transmission dynamics of hepatitis E
among swine: potential impact upon human infection. BMC
Vet Res 3: 9.
14. Rose N, Lunazzi A, Dorenlor V, Merbah T, Eono F, Eloit M,
Madec F, Pavio N, 2011. High prevalence of Hepatitis E virus
in French domestic pigs. Comp Immunol Microbiol Infect Dis
15. Montalvo Villalba M, Lay L, Chandra V, Bello Corredor M,
Frometa S, Moreno A, Jameel SM, 2008. Hepatitis E virus
genotype 1, Cuba. Emerg Infect Dis 14: 1320–1322.
16. Despierres L-A, Kaphan E, Attarian S, Cohen-Bacrie S, Pelletier
J, Pouget J, Motte A, Charrel R, Gerolami R, Colson P, 2010.
Neurologic disorders and hepatitis E, France. Emerg Infect Dis
17. Escriba ` J, Nakoune E, Recio C, Massamba P-M, Matsika-Claquin
M, Goumba C, Rose AM, Nicand E, Garcı ´a E, Leklegban C,
Koffi B, 2008. Hepatitis E, Central African Republic. Emerg
Infect Dis 14: 681–683.
18. Lewis H, Boisson S, Ijaz S, Hewitt K, Ngui S, Boxall E, Teo CG,
Morgan D, 2008. Hepatitis E in England and Wales. Emerg
Infect Dis 14: 165–167.
19. Li R-C, Ge S-X, Li Y-P, Zheng Y-J, Nong Y, Guo Q-S, Zhang J,
Ng M, Xia N, 2006. Seroprevalence of hepatitis E virus infec-
tion, rural southern People’s Republic of China. Emerg Infect
Dis 12: 1682–1688.
20. Rutjes S, Lodder W, Lodder-Verschoor F, van den Berg H,
Vennema H, Duizer E, Koopmans M, de Roda Husman AM,
2009. Sources of hepatitis E virus genotype 3 in the Netherlands.
Emerg Infect Dis 15: 381–387.
21. Legrand-Abravanel F, Mansuy JM, Dubois M, Kamar N, Peron
JM, Rostaing L, Izopet J, 2009. Hepatitis E virus genotype 3
diversity, France. Emerg Infect Dis 15: 110–114.
22. Purcell RH, Emerson SU, 2008. Hepatitis E: an emerging aware-
ness of an old disease. J Hepatol 48: 494–503.
23. Boccia D, Guthmann J, Klovstad H, Hamid N, Tatay M,
Ciglenecki I, Nizou J, Nicand E, Guerin P, 2006. High mortal-
ity associated with an outbreak of hepatitis E among displaced
persons in Darfur, Sudan. Clin Infect Dis 42: 1679–1684.
24. Meng XJ, Wiseman B, Elvinger F, Guenette DK, Toth TE, Engle
RE, Emerson SU, Purcell RH, 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.
25. Pe ´rez-Gracia M, Mateos M, Galiana C, Ferna ´ndez-Barredo S,
Garcı ´a A, Go ´mez M, Moreira V, 2007. Autochthonous hepati-
tis E infection in a slaughterhouse worker. Am J Trop Med
Hyg 77: 893–896.
26. Di Martino B, Di Profio F, Martella V, Di Felice E, Di Francesco
C, Ceci C, Marsilio F, 2010. Detection of hepatitis E virus
in slaughtered pigs in Italy. Arch Virol 155: 103–106.
27. Epelboin L, Nicand E, Roussin C, Lernout T, Pettinelli M-E,
Tesse S, Ali R, Aubry P, 2011. A sporadic case of genotype 3f
hepatitis E in Mayotte. Med Mal Infect 41: 392–394.
28. Nicand E´, Bigaillon C, Tesse ´ S, 2009. Hepatitis E in France:
surveillance data for human cases, 2006–2008. Bull E´pide ´miol
Hebd 31–32: 337–342.
29. Milon A, 2004. He ´patite E a ` la Re ´union. Bull Soc Pathol Exot
30. Barnaud E, Rogee S, Garry P, Rose N, Pavio N, 2012. Thermal
inactivation of infectious hepatitis e virus in experimentally
contaminated food. Appl Environ Microbiol 78: 5153–5159.
31. Jothikumar N, Cromeans TL, Robertson BH, Meng XJ, Hill VR,
2006. A broadly reactive one-step real-time RT-PCR assay for
rapid and sensitive detection of hepatitis E virus. J Virol
Methods 131: 65–71.
32. Gyarmati P, Mohammed N, Norder H, Blomberg J, Bela ´k S,
Wide ´n F, 2007. Universal detection of hepatitis E virus by two
real-time PCR assays: TaqMan and Primer-Probe Energy
Transfer. J Virol Methods 146: 226–235.
33. Cooper K, Huang FF, Batista L, Rayo CD, Bezanilla JC, Toth TE,
Meng XJ, 2005. Identification of genotype 3 hepatitis E virus
(HEV) in serum and fecal samples from pigs in Thailand and
Mexico, where genotype 1 and 2 HEV strains are prevalent in
the respective human populations. J Clin Microbiol 43: 1684–1688.
34. Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M,
Sturrock S, Buxton S, Cooper A, Markowitz S, Duran C,
Thierer T, Ashton B, Meintjes P, Drummond A, 2012.
Geneious basic: an integrated and extendable desktop software
platform for the organization and analysis of sequence data.
Bioinformatics 28: 1647–1649.
35. Edgar RC, 2004. MUSCLE: a multiple sequence alignment
method with reduced time and space complexity. BMC Bio-
informatics 5: 113.
36. Posada D, 2008. jModelTest: phylogenetic model averaging. Mol
Biol Evol 25: 1253–1256.
37. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S,
2011. MEGA5: molecular evolutionary genetics analysis using
maximum likelihood, evolutionary distance, and maximum
parsimony methods. Mol Biol Evol 28: 2731–2739.
38. Tajima F, Nei M, 1984. Estimation of evolutionary distance
between nucleotide sequences. Mol Biol Evol 1: 269–285.
39. Guindon S, Gascuel O, 2003. A simple, fast, and accurate algo-
rithm to estimate large phylogenies by maximum likelihood.
Syst Biol 52: 696–704.
40. Ronquist F, Huelsenbeck JP, 2003. MrBayes 3: Bayesian phylo-
genetic inference under mixed models. Bioinformatics 19:
41. R Development Core Team, 2008. R: a language and environ-
ment for statistical computing. Vienna. Available at: http://
www.r-project.org. Accessed January 22, 2012.
HEV DETECTED IN MALAGASY HUMAN AND PIGS
42. Tajima F, Nei M, 1984. Estimation of evolutionary distance Download full-text
between nucleotide sequences. Mol Biol Evol 1: 269–285.
43. Vivek R, Kang G, 2011. Hepatitis E virus infections in swine and
swine handlers in Vellore, southern India. Am J Trop Med Hyg
44. Zhang W, Yang S, Ren L, Shen Q, Cui L, Fan K, Huang F, Kang
Y, Shan T, Wei J, Xiu H, Lou Y, Liu J, Yang Z, Zhu J, Hua X,
2009. Hepatitis E virus infection in central China reveals no
evidence of cross-species transmission between human and
swine in this area. PLoS ONE 4: e8156.
45. Perez-Gracia MT, Mateos ML, Galiana C, Fernandez-Barredo S,
Garcia A, Gomez MT, Moreira V, 2007. Autochthonous hepa-
titis E infection in a slaughterhouse worker. Am J Trop Med
Hyg 77: 893–896.
46. Adjei AA, Aviyase JT, Tettey Y, Adu-Gyamfi C, Mingle JA,
Ayeh-Kumi PF, Adiku TK, Gyasi RK, 2009. Hepatitis E virus
infection among pig handlers in Accra, Ghana. East Afr Med J
47. Galiana C, Fernandez-Barredo S, Garcia A, Gomez MT, Perez-
Gracia MT, 2008. Occupational exposure to hepatitis E virus
(HEV) in swine workers. Am J Trop Med Hyg 78: 1012–1015.
48. Krumbholz A, Mohn U, Lange J, Motz M, Wenzel JJ, Jilg W,
Walther M, Straube E, Wutzler P, Zell R, 2012. Prevalence of
hepatitis E virus-specific antibodies in humans with occupational
exposure to pigs. Med Microbiol Immunol (Berl) 201: 239–244.
49. Kaufmann A, Kenfak-Foguena A, Andre C, Canellini G,
Burgisser P, Moradpour D, Darling KE, Cavassini M, 2011.
Hepatitis E virus seroprevalence among blood donors in south-
west Switzerland. PLoS ONE 6: e21150.
50. Withers MR, Correa MT, Morrow M, Stebbins ME, Seriwatana J,
Webster WD, Boak MB, Vaughn DW, 2002. Antibody levels
to hepatitis E virus in North Carolina swine workers, non-
swine workers, swine, and murids. Am J Trop Med Hyg 66:
51. Toole MJ, Claridge F, Anderson DA, Zhuang H, Morgan C, Otto
B, Stewart T, 2006. Hepatitis E virus infection as a marker for
contaminated community drinking water sources in Tibetan
villages. Am J Trop Med Hyg 74: 250–254.
52. Xiao P, Li R, She R, Yin J, Li W, Mao J, Sun Q, 2012. Prevalence
of hepatitis E virus in swine fed on kitchen residue. PLoS ONE
53. Di Bartolo I, Martelli F, Inglese N, Pourshaban M, Caprioli A,
Ostanello F, Ruggeri FM, 2008. Widespread diffusion of geno-
type 3 hepatitis E virus among farming swine in Northern Italy.
Vet Microbiol 132: 47–55.
54. Nakai I, Kato K, Miyazaki A, Yoshii M, Li TC, Takeda N,
Tsunemitsu H, Ikeda H, 2006. Different fecal shedding pat-
terns of two common strains of hepatitis E virusat three Japanese
swine farms. Am J Trop Med Hyg 75: 1171–1177.
55. Geng J, Wang L, Wang X, Fu H, Bu Q, Liu P, Zhu Y, Wang M,
Sui Y, Zhuang H, 2011. Potential risk of zoonotic transmission
from young swine to human: seroepidemiological and genetic
characterization of hepatitis E virus in human and various
animals in Beijing, China. J Viral Hepat 18: 583–590.
56. Adlhoch C, Wolf A, Meisel H, Kaiser M, Ellerbrok H, Pauli G,
2009. High HEV presence in four different wild boar
populations in East and West Germany. Vet Microbiol 139:
57. Nakano T, Takahashi K, Pybus OG, Hashimoto N, Kato H,
Okano H,KobayashiM,FujitaN,Shiraki K,Takei Y,Ayada M,
epidemic history and population dynamics of Japan-indigenous
genotype 3 hepatitis E virus inferred by molecular evolution.
TEMMAM AND OTHERS