|
Hepatitis
E. Virus: An Emerging
Disease Affecting Multiple Species
Patrick
G. Halbur DVM, PhD
Department of Veterinary Diagnostic and Production Animal Medicine
College of Veterinary Medicine, Iowa State University
Xiang-Jin Meng MD, PhD
Center for Molecular Medicine and Infectious Diseases
College of Veterinary Medicine, Virginia Polytechnic and State University
Introduction
Human hepatitis E virus (human HEV) is the causative agent
of acute non-A, non-B hepatitis in humans.[1]
HEV is a non-enveloped, single-stranded, positive sense RNA virus.
HEV is thought to be
transmitted via the fecal-oral route.
Swine hepatitis E virus (swine HEV) was first detected in pigs in
Illinois in 1997 and is closely related to two recent human HEV isolates
(US-1 and US-2) identified in the United States shortly after the
discovery of swine HEV.[2] Swine
HEV is now believed to be ubiquitous in the global swine population.[2,3]
Our group experimentally confirmed cross species infection
of HEV. We demonstrated that
swine HEV can infect rhesus monkeys and a chimpanzee, and the US-2 strain
of human HEV can infect pigs.[3,4] These
findings suggested that pigs may be an animal reservoir for HEV and
intensified the concern that HEV is a potential zoonotic or xenozoonotic
agent.[5,6] Serological
surveys have confirmed that people with close contact to pigs (pig
farmers, veterinarians, slaughter house workers) have a higher risk of HEV
infection (7, 8, 18). Pig
strains are often homologous to human strains in the same area further
suggesting the possibility of animal-to-human transmission.[3,6,7,8,16]
Until recently, the impression that swine hepatitis E virus
(HEV) is a zoonotic disease was based on the experimental, molecular
virological, and epidemiological evidence summarized above.
Direct evidence of the transmission of swine HEV from pigs or other
animal reservoirs to humans had not been reported.
In 2003 that changed. Strong
clinical evidence of transmission of HEV infection from pig livers to
human patients was documented in people in Japan who intentionally
consumed raw pig livers. Based on genetic analysis, the HEV isolated from one patient
was 100% identical to that of a swine HEV isolate discovered from a
packaged pig liver available at grocery stores where the patients
lived.[9] Further evidence of
the zoonotic potential of HEV was provided by results of an investigation
of an outbreak of hepatitis in people in Japan who had consumed raw deer
meat. This epidemic involved two Japanese families that consumed
raw meat of a wild-caught Japanese Sika deer.
HEV RNA was detected in the left-over portion of the frozen deer
meat and it was genetically identical to the HEV isolated from the
patients that had consumed the meat.[10]
It has now become very important that we understand as much
as possible about HEV from both the veterinary and human health points of
view. This presentation is
intended to review and update the audience on swine HEV research being
conducted at Iowa State University College of Veterinary Medicine in
collaboration with Dr. Xiang-Jin Meng’s group at the College of
Veterinary Medicine at Virginia Polytechnic Institute and State
University.
Comparative
pathogenesis of infection of pigs with hepatitis E virus recovered from a
pig and a human [4]
Three week-old, specific pathogen free (SPF) pigs were
inoculated with hepatitis E virus (HEV) to evaluate the pathogenicity and
determine the sites of viral replication.
Sixty pigs were randomly divided into three groups as follows, pigs
in group #1 (n=19) served as uninoculated control, pigs in group #2 (n=20)
were intravenously inoculated with a swine strain of HEV, pigs in group #3
(n=21) were intravenously inoculated with the US-2 strain of human HEV.
Two to four pigs from each group were necropsied at 3, 7, 14, 20,
27, and 55 days post-inoculation (DPI).
Three pigs from each group were monitored weekly by serum chemistry
profiles (AST, GGT, SDH, and bilirubin) for evidence of liver damage.
Blood and fecal swabs were collected weekly throughout the
experiment. There was no evidence of clinical disease or significant
elevation of liver serum chemistry profiles in any of the groups.
Hepatitis lesions were very mild and multifocal in group 1 pigs,
mild-to-moderate in group 2 pigs, and moderate-to-severe in group 3 pigs.
Multifocal lymphoplasmacytic hepatitis was observed in 9/17, 15/18,
and 16/19 of the pigs in groups 1-3, respectively.
Focal hepatocellular necrosis was observed in 5/17, 10/18, and
13/19 of the pigs in group 1-3, respectively.
Hepatic inflammation and hepatocellular necrosis peaked in severity
at 20 DPI and was still moderately severe at 55 DPI in the group
inoculated with human HEV (group 3).
Swine and human HEV differ in virulence and both induce subclinical,
but morphologically discernable, hepatitis in experimentally-infected SPF
pigs. All HEV-inoculated pigs
seroconverted to anti-HEV IgG, and remained positive for at least 2 months
post inoculation. The virus was present in feces, liver tissue, and bile of
pigs in both HEV-inoculated groups. The
duration of virus shedding in feces was 3-4 weeks. This work suggests that pig livers or cells from the livers
of HEV-infected pigs may represent a risk for transmission of HEV from
pigs to human xenograft recipients. Since
HEV was present in the feces of infected pigs, exposure to such feces
poses a risk for transmission of HEV, and pigs could potentially be an
animal reservoir for HEV infection.
Experimental infection of pregnant gilts with swine
hepatitis E virus [11]
Human HEV has been reported to cause severe disease
and mortality in pregnant women in underdeveloped countries.[1]
The objective of this work was to assess the pathogenicity of swine
HEV in pregnant gilts and to assess the usefulness of the pregnant gilt
model to study human HEV infection. Twelve gilts were intravenously inoculated with swine HEV and
six served as uninoculated controls.
Samples from gilts and their fetuses, and samples from suckling and
growing pigs were tested for swine HEV RNA, anti-HEV antibodies, and liver
chemistry profiles. Pathological
examination was performed on the gilts and their fetuses or offspring at 4
separate days post inoculation. Hepatitis
E virus-inoculated gilts became viremic, shed HEV RNA in feces, and
developed anti-HEV antibodies. There
was no evidence of clinical disease or elevation of liver serum chemistry
profiles in the gilts or their offspring. Mild multifocal lymphohistiocytic hepatitis was observed in 4
of 12 HEV-inoculated gilts. There
was no significant effect of swine HEV on fetal size, fetal viability, or
offspring birth weight or weight gain.
None of the fetal serum or tissue samples contained detectable HEV
RNA. The offspring of the
gilts acquired anti-HEV passive antibodies but remained seronegative after
the passive antibodies waned by 71 days of age.
Swine HEV infection induced subclinical hepatitis in 4 of 12
pregnant gilts but had no effect on the reproductive performance of the
gilts, no effect on the fetuses, and no effect on the offspring.
Vertical transmission of swine HEV from dams to their fetuses or
offspring did not occur. Fulminant
hepatitis associated with HEV infection in some pregnant women was not
reproduced in pregnant gilts, hence pregnant swine may not be an ideal
model for studying HEV-induced disease in humans.
Use
of a swine bioassay and a RT-PCR assay to assess the risk of transmission
of swine hepatitis E virus in pigs [12]
This work was done to assess the risk of transmission of
swine hepatitis E virus to naïve pigs with tissues or feces collected
from pigs experimentally inoculated with swine HEV.
Seventy-five, 3-week-old pigs were randomly assigned to 24 groups
of 3-4 pigs and inoculated with homogenates of tissues (liver, heart,
pancreas, or skeletal muscle) or a suspension of feces from swine HEV-infected
pigs collected at 3, 7, 14, 20, 27, or 55 days post inoculation (DPI).
Each inoculum was prepared as a 10% suspension (W/V) in PBS buffer
and tested by a semi-quantitative RT-PCR for swine HEV RNA and by the
swine bioassay. The
inoculation route was intravenous for liver, heart, and pancreas and via
stomach tube for skeletal muscle and fecal suspension.
For positive controls, 3 groups of pigs were inoculated with a 104.5
PID50, approximately equivalent to 106
genome equivalent (GE)/ml, of swine HEV via oral drop, stomach tube, or
intravenous route, respectively. The
liver homogenate inocula and feces collected at 3-7 DPI and 14-20 DPI were
positive for swine HEV RNA by RT-PCR.
The pigs inoculated intravenously with liver homogenates collected
at 3-7 DPI and 14-20 DPI developed anti-HEV antibodies and swine HEV RNA
was detected in their sera. Pigs
inoculated intravenously with heart, intravenously with pancreas, or via
stomach tube with skeletal muscle homogenates or fecal suspensions did not
develop detectable viremia or anti-HEV antibodies.
Intravenous inoculation (with feces and liver tissue) was
successful; however oral inoculation (with feces and muscle tissue) was
not successful in transmission of HEV. Feces and liver tissue were collected from the same
pigs, and the liver (with a titer of 102 – 104 GE/ml)
was determined to be infectious when inoculated intravenously, whereas the
feces (with a titer of 103 GE/ml) were not infectious when
inoculated orally. The
standard infectious pool (swine feces) of HEV with a titer of 106
GE/ml was not adequate to induce infection orally.
This suggests that the likelihood of transmitting HEV via
consumption of pork from an HEV infected pig is minimal.
These findings indicate that there is a potential risk of
transmission of swine HEV via liver tissues from infected pigs in the
early stages (3-20 DPI) of infection but it is unlikely that pork meat
poses a risk of HEV transmission unless the meat is contaminated with
feces from HEV-infected pigs. The results also suggest that HEV transmission via the
fecal-oral route may require a higher dose compared to the experimental
intravenous route of transmission. The
swine bioassay confirms that HEV detected in liver tissue is infectious
and the RT-PCR assay is an excellent tool to detect HEV infection in pig
tissues and feces.
Determination of the Presence and Infectivity of
Swine Hepatitis E Virus (HEV) in Swine Manure Storage Facilities and
Nearby Water Sources (Kasorndorkbua et al., submitted for publication)
We
recently conducted a field investigation to determine the presence of
swine HEV in pig feces, manure in different types of manure storage
facilities, drinking water on pig farms, and nearby surface water sources. Twenty eight Iowa farms with pig finishing facilities were
visited. Samples of
fresh feces (a pool of feces from 5 pigs per site) collected from
the floor of the finishing barn, pig manure from pits and/or lagoons (when
present on the site), drinking water from the pig barns, and upstream and
downstream surface water from the nearest source was collected (if
applicable) in the late summer and fall of 2002. All samples were processed by appropriate methods as follows;
10% fecal suspension for fresh feces [4], ultracentrifugation for swine
manure [13], and filtration (MD1 Virosorb® and filter housing, CUNO Inc.,
Meriden, CT) organic flocculation and centrifugal concentration (Centricon®,
Millipore, Billerica, MA) from drinking water and surface water [14,15].
The HEV-containing manure or water samples were then subject to
semi-quantitative RT-PCR assay to determine the HEV titer in GE/ml.
Swine HEV RNA was detected in 18 of 28 farms; in fresh feces
collected from the floor of the barns on 7 of 28 farms; in manure
collected from concrete holding pits on 15 of 22 farms; in manure
collected from lagoons on 3 of 8 farms; in drinking water samples on 0/28
farms; in upstream surface water on 0 of 19 farms; in downstream surface
water on 0/24 farms. We are
in the process of performing swine bioassays to determine the infectivity
of the positive feces, pit manure, and lagoon manure samples.
The finding of swine HEV in feces and pig manure on several
randomly chosen farms further confirms that swine HEV is widespread in
pigs in Iowa. The presence of
HEV in pit/lagoon samples indicates that the virus is stable in pit manure
and such manure may pose a risk as a direct source of human infection or
potentially as a source of water contamination and subsequent infection of
humans or other species. However,
we did not find evidence of HEV in drinking water or surface water on any
of the farms and it remains to be determined if HEV detected in manure
samples is viable and infectious to pigs, humans, or other species.
Transmission routes of swine hepatitis E virus in pigs (19)
Human hepatitis E virus has long
been considered to be fecal-orally transmitted. It seems logical that swine HEV is similarly transmitted.
We have recently demonstrated that intravenous
inoculation (with feces and liver tissue) was successful; however, oral
inoculation (with feces and muscle tissue) was not successful in
transmission of HEV.[12] Transmission
of HEV in humans by blood transfusions in endemic areas has been
confirmed.[19] Other
possible routes of transmission of swine HEV have not been investigated.
This study was designed to further investigate fecal-oral
transmission, transmission by direct contact, transmission in tonsillar
and nasal and ocular secretions, and transmission by contaminated needles.
Three positive control pigs were infected intravenously with our
infectious pool of swine HEV and served as the source of HEV inocula or
exposure as follows. The
positive control pigs were snared and a swab was used to aggressively rub
the palatine tonsilar surface, nasal mucosa, and conjunctiva.
The same procedure was done with those same swabs on naïve pigs
(n=3) penned separately in another room.
The positive control pigs were injected in the neck with Mycoplasma
hyopneumoniae vaccine using a 1-inch 18 gauge needle, and the same
needle was used to inject pigs (n=3) penned separately in another room.
The third group (n=3, penned separately in another room) was orally
inoculated with approximately 15 grams of freshly-collected pooled feces
obtained from the three swine HEV-shedding positive control pigs.
All the inoculation procedures were performed for three consecutive
days. Three pigs were placed
in the same pen with the positive control pigs to confirm that the virus
shed by those positive controls was infectious and could be transmitted by
direct contact. Infection
status was determined by detection of swine HEV RNA in feces and serum by
RT-PCR and/or demonstration of anti-HEV IgG seroconversion by ELISA.
All positive control pigs shed the virus in feces but only 2 of 3
had detectable viremia. The
direct contact pigs all became infected.
None of the tonsil or nasal or conjunctival swabs from the positive
control pigs contained detectable HEV RNA and the HEV-naïve pigs exposed
to these swabs remained free of HEV.
One of 3 pigs in the fecal-oral exposure group shed the virus in
feces at 12 days after exposure and the shedding persisted for two weeks
and anti-HEV was detected in this pig.
None of the pigs injected with the “contaminated” needles
became viremic or shed the virus in feces or developed anti-HEV
antibodies. The findings
indicate that fecal-oral route is most likely the primary route of swine
HEV transmission and it is less likely that swine HEV is transmitted via
exposure to saliva or nasal or ocular secretions or injection with
blood-contaminated needles.
Future
Work in Progress
We have begun further work to better understand the
survivability of HEV in different food processing and handling procedures
and in different environmental and production management regimens.
We are also investigating whether pigs can replicate HEV from other
species such as rats and chickens and deer.
We are also interested in better understanding the mechanism of HEV-induced
hepatic lesions. Work also
needs to be done to determine if there is a synergistic effect of HEV with
other common swine pathogens such as PRRS virus, circovirus, Salmonella
sp. and others.
Summary
The recent cases of HEV in humans following consumption of
raw pig livers provides strong evidence further supporting the hypothesis
that swine HEV is a zoonotic disease and pigs are a reservoir for HEV
infection of humans. The
primary source for swine HEV transmission is most likely feces shed from
pigs infected with HEV. There
remains little doubt that consumption of pig livers, particularly raw or
undercooked, can serve as a means for transmission of hepatitis E virus
from pigs to humans. Our
results to date indicate no evidence of contamination of drinking water on
swine farms or contamination of surface water near swine farms with HEV.
References
1. Purcell RH: 1996.
Hepatitis E virus. In: Fields BN, Knipe, DM, Howley, PM, eds. Fields
Virology 3rd ed. Vol 2. Philadelphia: Lippincott-Raven
Publishers: 2831-2843.
2.
Meng XJ, Purcell RH, Halbur PG,
et al.: 1997. A novel virus in swine is closely related to the
human hepatitis E virus. Proc Natl Acad Sci USA. 94:9860-9865.
4.
Halbur PG, Kasorndorkbua C, Gilbert C, et al.: 2001. Comparative
pathogenesis of infection of pigs with hepatitis E viruses discovered from
a pig and a human. J. Clin.
Microbiol. 39:918-923.
5. Meng XJ,
Halbur PG, Shapiro MS, et al.: 1998. Genetic and experimental evidence
for cross-species infection by swine hepatitis E virus. J. Virol. 72:9714-9721.
6. Yoo D and Giulivi A:
2000. Xenotransplantation and
the potential risk of xenogenic transmission of porcine viruses. Can.
J. Vet. Res. 64:193-203.
9.
Yazaki Y, Mizuo H, Takahashi M, et al.: 2003. Sporadic acute or
fulminant hepatitis E virus in Hokkaido, Japan, may be food-borne, as
suggested by the presence of hepaitits E virus in pig liver as food. J.
Gen. Viol. 84:2351-2357.
10.
Tei S, Kitajima N, Takahashi K, and Mishiro S: 2003. Zoonotic
transmission of hepatitis E virus from deer to human beings. Lancet
362:371-373.
11.
Kasorndorkbua C, Thacker BJ, Halbur PG, et al.: 2003. Experimental
infection of pregnant gilts with swine hepatitis E virus. Can.
J. Vet. Res. 67:303-306, 2003.
12.
Kasorndorkbua C, Halbur PG, Thomas PJ, et al.: 2002. Use of a swine
bioassay and a RT-PCR assay to assess the risk of transmission of swine
hepatitis E virus in pigs. J. Virol. Methods 101:71-78.
13.
Pina S, Jofre J, Emerson SU, et al.: 1998. Characterization of a
strain of infectious hepatitis E virus isolated from sewage in an area
where hepatitis E virus is not endemic. Appl. Environ. Microbiol. 64:4485-4488.
14.
Environmental Protection Agency: 1990. Detection of enteric
viruses. Standard Methods
:87-102.
15.
Abbaszadegan M, Stewart P and LeChevallier M: 1999. A strategy for
detection of viruses in ground water by PCR. Appl.
Environ. Microbiol. 65:444-449.
16.
Banks M, Bendall R, Grierson S et al.:
2004. Human and porcine hepatitis E virus strains, United Kingdom.
Emerg. Infect. Dis. 10
(5):953-955.
17.
Nishizawa T,
Takahashi M, Mizuo H, et al.: 2003. Characterization of Japanese swine
and human hepatitis E virus isolates with 99% identity over the entire
genome. J. Gen. Virol. 84:1245-51.
18. Siochu A,
Froesner G., Tassis PD et al.: 2004. Forst report of the prevalence of
anti-hepatitis E virus (anti-HEV) IgG in serum of blood donors,
slaughtermen, and swine farmers in Greece.
In Proceeding of the 18th
International Pig Veterinary Society Congress, Hamburg, Germany, Vol.
1. p367.
19.
Kasorndorkbua C, Guenette DK,
Huang FF, Thomas PJ, Meng XJ, Halbur PG: 2004.
Routes of transmission of swine hepatitis E virus in pigs.
J. of Clin. Microbiol., 42(11):5047-52.
|