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Previous Article | Next Article 
Journal of Virology, December 1998, p. 9714-9721, Vol. 72, No. 12
Laboratory of Infectious Diseases, National
Institute of Allergy and Infectious Diseases, National Institutes of
Health, Bethesda, Maryland 208921;
Department of Veterinary Diagnostic and Production Animal
Medicine, College of Veterinary Medicine, Iowa State University, Ames,
Iowa 500112;
Bioqual, Inc., Rockville,
Maryland 208503;
Pathology and
Clinical Laboratories, Rancho Los Amigos Medical Center, Downey,
California 902424; and
Viral
Discovery Group, Abbott Laboratories, North Chicago, Illinois
600645
Received 26 May 1998/Accepted 24 August 1998
Prior to the recent discovery of the swine hepatitis E virus (swine
HEV) in pigs from the midwestern United States, HEV was not considered
endemic to this country. Since swine HEV is antigenically and
genetically related to human strains of HEV, it was important to
characterize this new virus further. The infectivity titer of a pool of
swine HEV in pigs was determined in order to prepare a standardized
reagent and to evaluate the dose response in pigs. Although the
sequence of swine HEV varied extensively from those of most human
strains of HEV, it was very closely related to the two strains of human
HEV (US-1 and US-2) isolated in the United States. The U.S. strains
which were recently recovered from two patients with clinical hepatitis
E in the United States shared Hepatitis E virus (HEV), the
causative agent of hepatitis E, is currently unclassified
(29), although it was once considered to be a member of the
family Caliciviridae (19, 30). In most subtropical and tropical developing countries of Asia and Africa, HEV
is the primary cause of enterically transmitted non-A, non-B hepatitis,
and, hence, hepatitis E is considered an important public health
problem (1, 30, 33). The disease generally affects young
adults and reportedly has a mortality rate of up to 20% in infected
pregnant women (1, 2, 15, 30). Hepatitis E is rarely
diagnosed in industrialized countries, although HEV antibodies
(anti-HEV) have been found in a significant proportion of healthy
individuals (17, 22, 27, 32, 37, 46).
HEV is transmitted primarily by the fecal-oral route, and water-borne
epidemics are characteristic of hepatitis E (30, 33). Strains within large geographical regions tend to be closely related to
each other (6, 30, 43) and distinct from those in distant geographical areas. Recently, we reported the discovery of HEV in pigs
in the United States (23). Swine HEV is antigenically and
genetically related to, but distinct from, most human strains of HEV
(23). However, two cases of acute clinical hepatitis E
recently reported in the United States (34) were caused by virus strains very closely related to the swine HEV. One of the cases
(US-1) involved a patient who had no epidemiological evidence of
exposure to HEV strains from endemic countries (20, 34). The
patient in the second case (US-2) had traveled to Mexico prior to the
diagnosis of the disease (10, 34).
In the present study, we extended genetic comparisons to confirm that
swine HEV recovered from pigs in the midwestern United States and the
U.S. strains of human HEV which caused hepatitis E were closely
related. More importantly, we showed, under experimental conditions,
that swine HEV can cross species barriers and infect nonhuman primates
and that the US-2 strain of human HEV can infect specific-pathogen-free
(SPF) pigs.
Sources of HEV.
Crossbred SPF pigs (Sus scrofa
domesticus) were used throughout this study (24). SPF
pigs were experimentally infected with acute-phase serum from pigs
naturally infected with swine HEV (23, 24). Fecal materials
collected from one of the experimentally infected SPF pigs were used as
the source of swine HEV for most of the subsequent experiments.
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Genetic and Experimental Evidence for Cross-Species
Infection by Swine Hepatitis E Virus
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
97% amino acid identity with swine HEV
in open reading frames 1 and 2. Phylogenetic analyses of different
regions of the genome revealed that swine HEV and the U.S. strains
grouped together and formed a distinct branch. These results suggested
that swine HEV may infect humans. When we inoculated rhesus monkeys and
a chimpanzee, experimental surrogates of humans, with swine HEV, the
primates became infected. Furthermore, in a reciprocal experiment, specific-pathogen-free pigs were experimentally infected with the US-2
strain of human HEV that is genetically similar to swine HEV. These
results provided experimental evidence for cross-species infection by
the swine virus. Thus, humans appear to be at risk of infection with
swine HEV or closely related viruses.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
Generation of a standard pool of swine HEV. Two SPF pigs, 4 weeks of age, were housed in the same room in a BL-2 facility. Blood and fecal samples were collected once prior to inoculation. The two pigs were each inoculated intravenously (i.v.) with 1 ml of eluate from a rectal swab collected from pig A1 in a previous study (24). Blood samples and fecal samples were collected weekly and daily, respectively. Bile was collected on days 13, 14, 17, and 19 postinoculation (p.i.) by an ultrasound-guided technique. Liver biopsies were obtained on days 13, 17, and 19 p.i. Serum, bile, and fecal samples were tested for HEV RNA by reverse transcription-polymerase chain reaction (RT-PCR) (see below). The titer of swine HEV genomes in fecal samples was determined by a sensitive RT-PCR as described previously (24). One genome equivalent (GE) is defined as the number of viral genomes present in the highest 10-fold dilution positive by RT-PCR. A standard infectious pool of swine HEV was prepared in phosphate-buffered saline (PBS) as a 10% suspension from feces of a pig collected at 14 days p.i.
Infectivity titration of swine HEV in SPF pigs.
Ten-fold
dilutions of the standard pool of swine HEV were prepared in PBS
buffer. Ten SPF pigs, 4 weeks of age, were housed in separate rooms in
a BL-2 facility. Groups of two pigs each were inoculated i.v. with 1 ml
of a 10
1, 104, 105,
106, or 107 dilution of the standard pool.
Blood and fecal samples were collected twice before and weekly after
inoculation. Serum and fecal samples were tested for viral RNA by a
nested RT-PCR (24). The PCR titer of viral genomes in
positive fecal samples was determined. Anti-HEV (immunoglobulin G
[IgG] and IgM) was assayed by an enzyme-linked immunosorbent assay
(ELISA) standardized for anti-HEV in swine (23, 24). For the
two pigs inoculated with a 101 dilution, levels of liver
enzymes in serum were tested weekly by standard methods, and liver
biopsies were processed for routine histological examination. The liver
biopsy slides were examined under code by a veterinary pathologist with
experience in evaluating swine tissues (P.G.H.). The animals were
monitored for up to 16 weeks p.i.
Experimental inoculation of nonhuman primates with swine HEV. Two rhesus monkeys (Macaca mulatta), housed separately, were each inoculated by the i.v. route with 1 ml of the standard pool of swine HEV containing 104.5 50% pig infectious doses (PID50). Weekly serum samples were tested for viral RNA by nested RT-PCR, for anti-HEV by an ELISA, and for levels of liver enzymes in serum by standard methods. Weekly fecal samples were tested for viral RNA by a nested RT-PCR, and the titer of viral genomes in selected fecal samples was determined by a nested RT-PCR (reference 24; see below). Weekly liver biopsies were processed for routine histological examination. The liver biopsy slides were examined under code by a pathologist (S.G.) with experience in evaluating primate tissues. The animals were monitored for up to 16 weeks.
The standard pool of swine HEV prepared from swine feces appeared to be toxic to nonhuman primates (data not shown). Therefore, a first primate passage 10% fecal suspension containing about 106 GEs/ml of swine HEV was prepared from one of the infected rhesus monkeys (H400) in order to evaluate the susceptibility of chimpanzees (Pan troglodytes) to swine HEV infection. A chimpanzee (chimp 5835) was inoculated by the i.v. route with 1 ml of this suspension. Weekly fecal and serum samples were tested for viral RNA by a nested RT-PCR. Weekly serum samples were tested by an ELISA for anti-HEV and by standard methods for levels of liver enzymes in serum. The baseline liver enzymes of chimp 5835 were 30 U of alanine aminotransferase (ALT) and 248 U of isocitrate dehydrogenase (ICD) (an average of four preinoculation samples).Experimental inoculation of pigs with the US-2 strain of human
HEV.
Four SPF pigs, 4 weeks old, were housed as pairs in two
rooms in a BL-3 facility. One pig in each pair was inoculated i.v. with
0.2 ml of the 10% simian stool suspension containing HEV strain US-2.
The uninoculated pig in each pair served as a contact control. Blood
samples from all four pigs were taken prior to inoculation and weekly
after inoculation. Fecal samples were collected directly from the
rectum twice prior to inoculation and weekly after inoculation. Samples
were tested for anti-HEV, viral RNA, and levels of liver enzymes in
serum as described above.
Serological analyses. ELISAs for anti-HEV in swine and in nonhuman primates were standardized as previously described (23, 39). A high-performance liquid chromatography-purified, 55-kDa truncated form of the putative capsid protein, expressed from a recombinant baculovirus containing open reading frame 2 (ORF2) of a human strain of HEV (Sar-55), was used as the antigen for the ELISAs (38, 39). Sera taken from experimentally inoculated pigs or from primates were tested in duplicate for anti-HEV IgG and IgM. Levels of the liver enzymes alkaline phosphatase, sorbitol dehydrogenase, ALT, and gamma glutamyltransferase and bilirubin in serum were determined weekly for swine sera, and ICD, ALT, and gamma glutamyltransferase for primate sera were determined weekly by standard methods.
RNA extraction and RT-PCR. Total RNA was extracted with TriZol reagent (GIBCO-BRL, Gaithersburg, Md.) from 100 µl of fecal suspension or serum. Total RNA was reverse transcribed with swine HEV-specific primers or HEV degenerate primers and SuperScript II reverse transcriptase (GIBCO-BRL) at 42°C for 1 h; cDNA was amplified by PCR with AmpliTaq Gold polymerase (Perkin-Elmer, Norwalk, Conn.). For titration of swine HEV viral genomes in serum, bile, and fecal samples, the PCRs consisted of 39 cycles of denaturation at 94°C for 1 min, annealing at 52°C for 1 min, and extension at 72°C for 1.5 min, followed by a nested PCR of 39 cycles with 10 µl of the first-round PCR product. The primers used for PCR titration of swine HEV were swine HEV specific (Table 1).
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Amplification of nearly the full-length genome of swine HEV. The sequence of ORF2 and ORF3 of swine HEV was previously determined (23). In order to extend this sequence to ORF1 and the terminal noncoding regions (NCRs), we utilized the genome-walking strategy. The complete ORF1 and the 5' and 3' NCRs of swine HEV were amplified by RT-PCR by using one swine HEV-specific primer and one HEV degenerate or consensus primer (Table 1). The PCR conditions used to amplify different regions of the genome varied.
Sequence and phylogenetic analysis. The PCR products were purified by electrophoresis on a 1% agarose gel, followed by extraction with a Geneclean kit (Bio101, La Jolla, Calif.). Both strands were sequenced with an automated DNA Sequencer. The sequences were compiled and analyzed by the GeneWorks program (IntelliGenetics, Inc., Mountain View, Calif.). Sequence alignments were performed with the Clustal W program in the Genetics Computer Group (GCG) package. Phylogenetic analyses were conducted with the aid of the PAUP program in the same package (GCG version 9.1; David L. Swofford, Smithsonian Institute, Washington, D.C.). A bootstrap analysis with 1,000 replications was performed by branch-and-bound searching and midpoint rooting options to produce a 75% majority-rule consensus tree. The resulting phylograms were viewed with TREEVIEW program (26) to create the graphical outputs.
Nucleotide sequence accession number. The genomic sequence of swine HEV reported in this paper has been deposited with the GenBank database under accession no. AF082843.
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RESULTS |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Infectivity titration of swine HEV in SPF pigs. Fecal materials collected from an experimentally infected pig (24) were inoculated i.v. into two additional SPF pigs for amplification. The two inoculated pigs seroconverted to anti-HEV at 2 weeks p.i. Swine HEV RNA was first detected in feces at 3 or 4 days p.i. The standard infectious pool of swine HEV prepared from feces of one pig at 14 days p.i. had a PCR titer of 106 GEs/ml. Swine HEV RNA was also detected in bile in three of the four samples collected (days 13, 14, and 17, but not day 19 p.i.). Since the bile is produced by liver cells, the virus had to have replicated in the liver prior to accumulating in the bile of the gallbladder. One of the pigs had microscopic changes consistent with hepatitis on day 13 p.i., but liver lesions were not observed in the other two biopsy samples (days 17 and 19 p.i.).
The pool of swine HEV was characterized by titration in SPF pigs. Both pigs inoculated with dilutions of 101 and
104, respectively, of the virus pool seroconverted to
anti-HEV and shed virus in feces (Table
2). None of the animals inoculated with
dilutions of 105, 106, or 107
were infected. Therefore, the infectivity titer of the swine HEV
standard pool was 104.5 PID50 per ml, which was
approximately 30-fold lower than the GE titer measured by RT-PCR. In
the two pigs each inoculated with the highest dose (104.5
PID50), seroconversion to anti-HEV occurred 3 weeks p.i.,
and fecal excretion of virus was first detected 1 week p.i. and lasted for 2 to 3 weeks. Mild-to-moderate diffuse hepatocellular swelling and
vacuolation and mild increased numbers of mixed leukocytes within
hepatic sinusoids were observed from 1 to 4 weeks p.i., but hepatic
necrosis was not detected. Levels of liver enzymes in serum were not
significantly elevated in either pig, and clinical signs of hepatitis
were not observed. The pigs inoculated with the lowest infectious dose
of swine HEV (100.5 PID50) took longer to
seroconvert and to excrete virus in feces. In addition, the titer of
viral genomes in the feces of pigs inoculated with 100.5
PID50 was lower than that of pigs inoculated with
104.5 PID50.
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Sequence analysis of ORF1 and terminal NCRs of swine HEV. The ORF1 of swine HEV has been very difficult to amplify, and its sequence has not been previously reported. The ORF1 of swine HEV was sequenced, and the putative functional domains and the hypervariable region (HVR) in the ORF1 were compared with the corresponding regions of other HEV strains (Table 3). The ORF1 of swine HEV and of the US-2 strain contains 5,127 nucleotides (nt), which is 3 nt less than those in the US-1 strain, but 45 and 51 nt more than those in the Asian and the Mexican strains, respectively (data not shown). Swine HEV varied extensively, both at the nucleotide and amino acid levels, from non-U.S. strains of HEV, although it was very similar to the two U.S. strains (see below). The sequence identity in the putative methyltransferase and RNA-dependent RNA polymerase (RDRP) regions between swine HEV and non-U.S. HEV strains varied from 74 to 76% at the nucleotide level and from 84 to 89% at the amino acid level. The GDD tripeptide motif found in all viral RDRP regions is conserved among different strains (data not shown). In the putative helicase region, a slightly higher sequence identity between swine HEV and non-U.S. HEV strains was observed, i.e., 74 to 77% at the nucleotide level and 91 to 92% at the amino acid level. Asian strains of HEV, including Burma (36), Myanmar (3), Pakistan (38), China (45), and Madras (GenBank accession no. X99441) are closely related to each other. The ORF1 of the Mexican strain of HEV (13), like ORF2 and ORF3 (23), also showed much greater sequence divergence from other HEV strains, ranging from 73 to 80% sequence identity at the nucleotide level and from 85 to 94% at the amino acid level. However, the sequence identity between swine HEV and the Mexican strain was as divergent as those between swine HEV or the Mexican strain and non-U.S. strains of HEV.
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Additional evidence for a new and divergent genotype of HEV. At least three genotypes of HEV have been previously identified, including the first genotype, represented by the Mexican strain; the second genotype, represented by Asian and African HEV strains; and the third genotype, represented by the swine HEV (23). A phylogenetic analysis was performed to determine the relationship of the U.S. and swine HEV strains to other strains. Since complete genomic sequences were not available for all HEV strains, phylogenetic analyses were based on partial regions of the genome (Fig. 2). All phylogenetic trees constructed from different regions of the genome were very similar. The swine HEV and the U.S. strains grouped very closely together and comprised a distinct genotype.
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Taxonomic evidence that the U.S. HEV strains infecting humans are very similar to swine HEV. Further sequence analysis encompassing all three ORFs confirmed that the swine HEV and two U.S. strains were very closely related to each other and differed extensively from other strains of HEV (Table 3). In ORF1, the putative RDRP and the helicase of swine HEV and of the two U.S. strains had 92 to 93% sequence identity at the nucleotide level and 99 to 100% sequence identity at the amino acid level. Similarly, in the putative methyltransferase region, they shared 90 to 91% nucleotide sequence identity and 97% amino acid sequence identity. In the putative capsid gene (ORF2), swine HEV and the two U.S. strains shared 92% sequence identity at the nucleotide level and 98 to 99% sequence identity at the amino acid level. The small ORF3 of swine HEV had 95 to 98% nucleotide sequence and 93 to 97% amino acid sequence identity with the U.S. strains (data not shown). Interestingly, both swine HEV and the two U.S. strains had an in-frame deletion of 3 nt in the ORF3 region compared to other strains (data not shown).
In the putative HVR (37), extensive sequence variations were observed among the three major genotypes of HEV (U.S. strains, Mexican strain, and Asian strains), although the HVR was relatively conserved within each major genotype (Table 3). The lengths of the HVR varied among different HEV strains as follows: 105 amino acid residues for all Asian strains, 119 to 120 amino acids for the U.S. strains and swine HEV, and 103 amino acids for the Mexican strain (data not shown). The amino acid identities within a genotype were 85 to 96% among the Asian strains and 83 to 88% among swine HEV and the two U.S. strains. However, among the three major genotypes, the amino acid sequence identity was less than 50%. The significantly larger size of the HVRs of the swine and U.S. strains compared with those of all other strains, coupled with the high sequence identity among the swine and two U.S. strains, provides compelling evidence that the swine HEV and the U.S. strains are closely related. The very similar swine HEV and US-1 strains were isolated from the same small geographical area (swine HEV from pigs in Illinois [23] and the US-1 strain from a human in the neighboring state of Minnesota [20, 34]), suggesting that they may be variants of the same virus.Infection of nonhuman primates with swine HEV. Because the swine HEV and U.S. strains of human HEV were so similar, it was of interest to determine if swine HEV could infect primates. Two rhesus monkeys were each inoculated i.v. with 104.5 PID50 of swine HEV (Fig. 3). Both primates seroconverted to anti-HEV 4 weeks p.i. and were still seropositive at the end of the 16-week experiment. Fecal excretion of swine HEV, indicative of replication, was detected 1 to 2 weeks p.i. and lasted for 3 to 5 weeks. The peak PCR titer of swine HEV in the feces was 106 GEs/g of feces (data not shown). Viremia lasting for 4 to 5 weeks was detected as early as 1 week p.i. There was a slight elevation of ALT and ICD in serum at 4 weeks p.i. in monkey H400 and at 1 week p.i. in monkey M305. However, the level of liver enzyme elevations in serum was low compared with that in primates experimentally infected with human strains of HEV (38-41). In both primates, focal necroinflammatory changes (1+), consistent with mild acute viral hepatitis were observed in liver biopsy specimens near the time of liver enzyme elevations in serum.
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Infection of pigs with the US-2 strain of human HEV. Both pigs inoculated with strain US-2 seroconverted to anti-HEV at 2 weeks p.i. (Table 4). Fecal excretion of the virus was detected by RT-PCR in both inoculated pigs 1 week p.i. and lasted for about 2 weeks. One uninoculated contact control pig also became infected. This uninoculated pig seroconverted to anti-HEV about 2 weeks after the inoculated pig had seroconverted, and HEV RNA was detected in its feces about 2 weeks after the virus first appeared in the feces of the inoculated pig. The PCR product amplified from the feces of an infected pig was sequenced and confirmed to be specific for the US-2 isolate and distinct from swine HEV (data not shown). Clinically, both inoculated and contact control pigs appeared normal, and we did not observe significant elevation of any of the liver enzymes tested.
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DISCUSSION |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Symptomatic human infection with HEV in the United States and other developed countries is rare. Most of the few cases reported in these countries were in travellers returning from endemic regions (8, 11, 35), although in some cases in Western European countries this risk factor was absent (16, 17, 46). The recent identification of a case of acute hepatitis E in the United States in a patient with no history of travel to endemic regions (20, 34) reiterates that absence of travel to an endemic region does not necessarily exclude the diagnosis of acute hepatitis E infection. The need to explain the source of infection acquired in industrialized countries and to understand the ecology and epidemiology of HEV has led to a hypothesis that an animal reservoir may exist (33). In fact, antibodies reactive with HEV antigens have been detected in a number of animal species, including pigs, sheep, and rats (5, 7, 18, 21, 42). Unfortunately, either virus was not recovered from these species or virus recovered was not sequenced. Our discovery of the swine HEV as a ubiquitous agent in the swine population of the midwestern United States lends credence to the possibility of an animal reservoir.
In the present study, a standardized infectious pool of swine HEV was
prepared and characterized. In our previous studies, we found only
subclinical infections in naturally infected young pigs (23)
or young pigs experimentally infected with an unquantified but
relatively low dose of virus (24). In the present study, pigs infected with 104.5 PID50 also did not
have clinical or biochemical evidence of hepatitis, although HEV was
detected in bile, indicating that swine HEV replicated in the liver. In
previous studies, cynomolgus macaques infected with a human strain of
HEV developed biochemical evidence of significant hepatitis (ALT 100
U/liter) when the infecting dose was around 103.5 50%
monkey infectious doses or higher but had little or no biochemical evidence of hepatitis when inoculated with lower doses (40). The rhesus macaques infected with 104.5 PID50
of swine HEV had mild focal necroinflammatory changes in liver biopsy
specimens and a slight elevation of ALT and ICD levels in serum, which
could indicate mild hepatitis and virus replication in the liver. The
chimpanzee infected with 106 GEs of swine HEV was
clinically normal, although fecal excretion of swine HEV and
seroconversion to anti-HEV IgG were detected. This inoculum has not yet
been titrated for infectivity; however, based on the ratio of GE to
infectivity of the original pool of swine HEV in swine feces
(106 GE/104.5 PID50), the
infectious dose may have been the same as that given to the rhesus
monkeys. Additional studies must be performed to determine if a larger
dose will induce clinical disease. The patterns of appearance of
viremia, anti-HEV, and fecal excretion of virus in primates infected
with swine HEV were similar to those observed in infected pigs.
Infection of primates with swine HEV demonstrated that swine HEV can
cross species barriers, at least under experimental conditions,
suggesting the possibility of human infection with swine HEV. The
extremely high prevalence of swine HEV in pigs (23) and its
ability to cross species barriers may put swine practitioners, swine
producers, and other pig handlers at possible risk of zoonotic
infection by the virus.
Balayan et al. (5) previously reported that Russian domestic swine were experimentally infected with a Central Asian strain of HEV isolated from a naturally infected patient. However, the virus infecting the pigs in this experiment was not sequenced to confirm its identity; thus, it is not clear whether the pigs were infected with the virus in the inoculum or were fortuitously infected by a swine virus circulating among the pigs at the time. We were unable to infect crossbred SPF pigs with strains of human HEV representing two major genotypes (24), Mex-14 (Mexican) and Sar-55 (Pakistan), even though the Sar-55 strain is from the same geographic region as the strain used by Balayan et al. and even though high doses of infectious virus (105 monkey infectious doses) were administered (24). Similarly, Platt et al. (28) failed to infect SPF pigs experimentally with the Mexican strain (Mex-14) of human HEV. Since the swine HEV shares only about 75% nucleotide sequence homology with either of the two human strains of HEV tested, our failure to infect swine with these strains (24) was likely due to a difference in susceptibility of pigs to different HEV strains. In contrast, when we inoculated SPF pigs with the US-2 strain of human HEV, the inoculated pigs became infected and, in one case, virus even spread to the uninoculated pig housed in the same room. Since the virus recovered from the infected pig had a unique sequence identical to that of the virus in the inoculum, these data provided conclusive experimental evidence for cross-species infection by HEV. Like pigs infected with swine HEV, swine infected with the US-2 strain of human HEV (either by inoculation or by contact) remained clinically normal, and levels of liver enzymes in serum were not significantly elevated. The rapid seroconversion of pigs infected with the US-2 strain of human HEV further suggested that the US-2 strain is already competent to replicate in swine and may be of swine origin. These data strongly suggest that a swine virus strain or one very similar to swine HEV infects and causes hepatitis in rare cases in humans in the United States and perhaps in other countries. The results suggested that pigs could well be an animal reservoir for HEV in the United States.
We have amplified and sequenced nearly the full-length genome of the swine HEV. Sequence analyses revealed that, in all three ORFs and the 3' NCR, swine HEV and the two U.S. strains of HEV are very closely related. However, both swine HEV and the U.S. strains diverged extensively from other strains of HEV, especially in the HVR. Phylogenetic analyses have shown that genotypes of HEV generally have unique geographic distributions. Swine HEV and the closely related U.S. strains were all isolated in the United States. In general, the many Asian strains isolated are related both genetically and geographically. The African strains appear to be similar to but distinguishable from Asian strains. The Mexican strain is the only strain that forms a single branch. However, exceptions to this geographic distribution of HEV strains are the two unique strains of HEV, G-20 and G-9, recovered from patients in China (14). These two Chinese strains do not appear to group with the other Asian strains. The observed genetic diversities among different strains of HEV may affect the development of HEV vaccines. Although the experimental HEV vaccines are very promising (31, 41), their efficacy must be evaluated for protection against these novel and divergent strains.
The data presented in this study suggested that swine may serve as an animal reservoir for HEV. Young pigs infected naturally (23) or experimentally (24) with swine HEV appear not to have clinical symptoms, and primates showed negligible or minimal evidence of hepatitis. Therefore, subclinical infection of humans with swine HEV could explain the relatively high prevalence of anti-HEV in apparently healthy individuals in the United States and other industrialized countries (16, 17, 22, 37, 46). However, anti-HEV was also found in healthy individuals in the United States who seem to have had no contact with swine or other farm animals (22, 37). Thus, it is likely that other animal species may also serve as reservoirs for HEV. These putative animal reservoirs of HEV could be a source of contamination in regions where HEV is endemic and may also be responsible for the anti-HEV detected in healthy individuals in nonendemic regions.
Recently, xenotransplantation has become the focus of intensive research for a solution to the shortage of organ donors for transplantations (4). Swine are relatively easy to breed and maintain; therefore, xenotransplantation with pig organs has received considerable attention (4, 44). However, xenozoonoses, the transmission of pathogens from pigs to human recipients, is of major concern in xenotransplantation (25, 44). Many swine viruses are known to infect humans (9). Our results proved that swine HEV also has the ability to cross species barriers and therefore might infect humans. Although swine HEV appeared to be nonpathogenic for pigs and primates in experimental infections, it might become pathogenic in immunosuppressed xenotransplantation recipients. Therefore, it is important to develop sensitive and easy-to-perform assays to screen for swine HEV in donor pigs used for xenotransplantation. In addition, adequate diagnostic reagents are needed not only for screening of xenotransplantation donors but for epidemiologic studies as well. With the recent diagnosis in the United States of acute hepatitis E that was not associated with travel to endemic regions and the recovery from such a case of HEV that is closely related to swine HEV, HEV should now be considered as a possible etiologic agent in persons with acute hepatitis in the United States.
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ACKNOWLEDGMENTS |
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We thank R. Royer for assistance in the swine transmission studies; E. Riedeseo for expert help in ultrasound-guided bile collection; and D. Wong, L. Rasmussen, Y. Huang, R. Engle, and H. Nguyen for assistance in the laboratory studies.
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FOOTNOTES |
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* Corresponding author. Mailing address: Laboratory of Infectious Diseases, NIAID, National Institutes of Health, Bldg. 7, Rm. 206, 7 Center Dr. MSC 0740, Bethesda, MD 20892-0740. Phone: (301) 496-6227. Fax: (301) 402-0524. E-mail: xjmeng{at}atlas.niaid.nih.gov.
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REFERENCES |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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| 1. |
Arankalle, V. A.,
M. S. Chadha,
S. A. Tsarev,
S. U. Emerson,
A. R. Risbud,
K. Banerjee, and R. H. Purcell.
1994.
Seroepidemiology of water-born hepatitis in India and evidence for a third enterically-transmitted hepatitis agent.
Proc. Natl. Acad. Sci. USA
91:3428-3432 |
| 2. | Arankalle, V. A., S. A. Tsarev, M. S. Chadha, D. W. Alling, S. U. Emerson, K. Banerjee, and R. H. Purcell. 1995. Age-specific prevalence of antibodies to hepatitis A and E viruses in Pune, India, 1982 and 1992. J. Infect. Dis. 171:447-450[Medline]. |
| 3. | Aye, T. T., T. Uchida, X. Ma, F. Iida, T. Shikata, M. Ichikawa, T. Rikihisa, and K. M. Win. 1993. Sequence and gene structure of the hepatitis E virus isolated from Myanmar. Virus Genes 7:95-109[Medline]. |
| 4. | Bach, F. H. 1998. Xenotransplantation: problems and prospects. Annu. Rev. Med. 49:301-310[Medline]. |
| 5. | Balayan, M. S., R. K. Usmanov, D. I. Zamyatina, and F. R. Karas. 1990. Brief report: experimental hepatitis E infection in domestic pigs. J. Med. Virol. 32:58-59[Medline]. |
| 6. | Chatterjee, R., S. A. Tsarev, J. Pillot, P. Coursaget, S. U. Emerson, and R. H. Purcell. 1997. African strains of hepatitis E virus that are distinct from Asian strains. J. Med. Virol. 53:139-144[Medline]. |
| 7. | Clayson, E. T., B. L. Innis, K. S. A. Myint, S. Narupiti, D. W. Vaughn, S. Giri, P. Ranabhat, and M. P. Shrestha. 1995. Detection of hepatitis E virus infections among domestic swine in the Kathmandu Valley of Nepal. Am. J. Trop. Med. Hyg. 53:228-232. |
| 8. | Dawson, G. J., I. K. Mushahwar, K. H. Chau, and G. L. Gitnick. 1992. Detection of long-lasting antibody to hepatitis E in a US traveller to Pakistan. Lancet 340:426-427[Medline]. |
| 9. | de Groen, P. C. 1997. Hepatitis E in the United States: a case of "hog fever"? Mayo Clin. Proc. 72:1197-1198[Medline]. |
| 10. | Erker, J. C., S. M. Desai, G. G. Schlauder, G. J. Dawson, and I. K. Mushahwar. Hepatitis E variant in the United States: molecular characterization and transmission in cynomologus macaques. Submitted for publication. |
| 11. | Herrera, J. L., S. Hill, J. Shaw, M. Fleenor, T. Bader, and M. S. Wolfe. 1993. Hepatitis E among US travelers, 1989-1992. Mortal. Morbid. Weekly Rep. 42:1-4. |
| 12. | Hsieh, S. Y., P. Y. Yang, Y. P. Ho, C. M. Chu, and Y. F. Liaw. 1998. Identification of a novel strain of hepatitis E virus responsible for sporadic acute hepatitis in Taiwan. J. Med. Virol. 55:300-304[Medline]. |
| 13. | Huang, C.-C., D. Nguyen, J. Fernandez, K. Y. Yun, K. E. Fry, D. W. Bradley, A. W. Tam, and G. R. Reyes. 1992. Molecular cloning and sequencing of the Mexico isolate of hepatitis E virus (HEV). Virology 191:550-558[Medline]. |
| 14. | Huang, R., N. Nakazono, K. Ishii, O. Kawamata, R. Kawaguchi, and Y. Tsukada. 1995. Existing variations on the gene structure of hepatitis E virus strains from some regions of China. J. Med. Virol. 47:303-308[Medline]. |
| 15. | Hussaini, S. H., S. J. Skidmore, P. Richardson, L. M. Sherratt, B. T. Cooper, and J. G. O'Grady. 1997. Severe hepatitis E infection during pregnancy. J. Viral. Hepat. 4:51-54[Medline]. |
| 16. | Jardi, R., M. Buti, F. Rodriguez-Frias, and R. Esteban. 1993. Hepatitis E infection in acute sporadic hepatitis in Spain. Lancet 341:1355-1356[Medline]. |
| 17. | Johansson, P. J., I. K. Mushahwar, G. Norkrans, O. Weiland, and E. Nordenfelt. 1995. Hepatitis E virus infections in patients with acute hepatitis non-A-D in Sweden. Scand. J. Infect. Dis. 27:543-546[Medline]. |
| 18. | Karetnyi, Y. V., D. I. Dzhumalieva, R. K. Usmanov, I. P. Titova, Y. I. Litvak, and M. S. Balayan. 1993. Possible involvement of rodents in the spread of hepatitis E. Zh. Mikrobiol. Epidemiol. Immunobiol. 4:52-56. |
| 19. |
Koonin, E. V.,
A. E. Gorbalenya,
M. A. Purdy,
M. N. Rozanov,
G. R. Reyes, and D. W. Bradley.
1992.
Computer-assisted assignment of functional domains in the nonstructural polyprotein of hepatitis E virus: delineation of an additional group of positive-strand RNA plant and animal viruses.
Proc. Natl. Acad. Sci. USA
89:8259-8263 |
| 20. | Kwo, P. Y., G. G. Schlauder, H. A. Carpenter, P. J. Murphy, J. E. Rosenblatt, G. J. Dawson, E. E. Mast, K. Krawczynski, and V. Balan. 1997. Acute hepatitis E by a new isolate acquired in the United States. Mayo Clin. Proc. 72:1133-1136[Medline]. |
| 21. | Maneerat, Y., E. T. Clayson, K. S. A. Myint, G. D. Young, and B. L. Innis. 1996. Experimental infection of the laboratory rat with the hepatitis E virus. J. Med. Virol. 48:121-128[Medline]. |
| 22. | Mast, E. E., I. K. Kuramoto, M. O. Favorov, V. R. Schoening, B. T. Burkholder, C. N. Shapiro, and P. V. Holland. 1997. Prevalence of and risk factors for antibody to hepatitis E virus seroreactivity among blood donors in Northern California. J. Infect. Dis. 176:34-40[Medline]. |
| 23. |
Meng, X.-J.,
R. H. Purcell,
P. G. Halbur,
J. R. Lehman,
D. M. Webb,
T. S. Tsareva,
J. S. Haynes,
B. J. Thacker, and S. U. Emerson.
1997.
A novel virus in swine is closely related to the human hepatitis E virus.
Proc. Natl. Acad. Sci. USA
94:9860-9865 |
| 24. | Meng, X.-J., P. G. Halbur, J. S. Haynes, T. S. Tsareva, J. D. Bruna, R. L. Royer, R. H. Purcell, and S. U. Emerson. 1998. Experimental infection of pigs with the newly identified swine hepatitis E virus (swine HEV), but not with human strains of HEV. Arch. Virol. 143:1405-1415[Medline]. |
| 25. |
Murphy, F. A.
1996.
The public health risk of animal organ and tissue transplantation into humans.
Science
273:746-747 |
| 26. |
Page, R. D. M.
1996.
TREEVIEW: an application to display phylogenetic trees on personal computers.
Comput. Appl. Biosci.
12:357-358 |
| 27. | Paul, D. A., M. F. Knigge, A. Ritter, R. Gutierrez, T. Pilot-Matias, K. H. Chau, and G. J. Dawson. 1994. Determination of hepatitis E virus seroprevalence by using recombinant fusion protein and synthetic peptides. J. Infect. Dis. 169:801-806[Medline]. |
| 28. | Platt, K. B., K.-J. Yoon, and J. J. Zimmerman. 1998. Susceptibility of swine to hepatitis E virus and its significance to human health, p. 125-126. In 1998 Swine Research Report. Iowa State University, Ames, IA. |
| 29. | Pringle, C. 1998. Minutes of the 27th International Committee on Taxonomy of Viruses Meeting. Arch. Virol. 143:1449-1459[Medline]. |
| 30. | Purcell, R. H. 1996. Hepatitis E virus, p. 2831-2843. In B. N. Fields, D. M. Knipe, P. M. Howley, et al. (ed.), Fields virology, 3rd ed., vol. 2. Lippincott-Raven Publishers, Philadelphia, Pa. |
| 31. | Purdy, M. A., K. A. McCaustland, K. Krawczynski, J. Spelbring, G. R. Reyes, and D. W. Bradley. 1993. Preliminary evidence that a trpE-HEV fusion protein protects cynomolgus macaques against challenge with wild-type hepatitis E virus (HEV). J. Med. Virol. 41:90-94[Medline]. |
| 32. | Quiroga, J. A., T. Cotonat, I. Castillo, and V. Carreno. 1996. Hepatitis E virus seroprevalence in acute viral hepatitis in a developed country confirmed by a supplemental assay. J. Med. Virol. 50:16-19[Medline]. |
| 33. | Reyes, G. R. 1997. Overview of the epidemiology and biology of the hepatitis E virus, p. 239-258. In R. A. Willsen (ed.), Viral hepatitis. Marcel Dekker, Inc., New York, N.Y. |
| 34. | Schlauder, G. G., G. J. Dawson, J. C. Erker, P. Y. Kwo, M. F. Knigge, D. L. Smalley, J. E. Rosenblatt, S. M. Desai, and I. K. Mushahwar. 1998. The sequence and phylogenetic analysis of a novel hepatitis E virus isolated from a patient with acute hepatitis reported in the United States. J. Gen. Virol. 79:447-456[Abstract]. |
| 35. | Skidmore, S. J., P. O. Yarbough, K. A. Gabor, A. W. Tam, and G. R. Reyes. 1991. Imported hepatitis E in UK. Lancet 337:1541[Medline]. |
| 36. | Tam, A. W., M. M. Smith, M. E. Guerra, C. C. Huang, D. W. Bradley, K. E. Fry, and G. R. Reyes. 1991. Hepatitis E virus (HEV): molecular cloning and sequencing of the full-length viral genome. Virology 185:120-131[Medline]. |
| 37. | Thomas, D. L., P. O. Yarbough, D. Vlahov, S. A. Tsarev, K. E. Nelson, A. J. Saah, and R. H. Purcell. 1997. Seroreactivity to hepatitis E virus in areas where the disease is not endemic. J. Clin. Microbiol. 35:1244-1247[Abstract]. |
| 38. |
Tsarev, S. A.,
S. U. Emerson,
G. R. Reyes,
T. S. Tsareva,
L. J. Legters,
I. A. Malik,
M. Iqbal, and R. H. Purcell.
1992.
Characterization of a prototype strain of hepatitis E virus.
Proc. Natl. Acad. Sci. USA
89:559-563 |
| 39. | Tsarev, S. A., T. S. Tsareva, S. U. Emerson, A. Z. Kapikian, J. Ticehurst, W. London, and R. H. Purcell. 1993. ELISA for antibody to hepatitis E virus (HEV) based on complete open-reading frame-2 protein expressed in insect cells: identification of HEV infection in primates. J. Infect. Dis. 168:369-378[Medline]. |
| 40. | Tsarev, S. A., T. S. Tsareva, S. U. Emerson, P. O. Yarbough, L. J. Legters, T. Moskal, and R. H. Purcell. 1994. Infectivity titration of a prototype strain of hepatitis E virus in cynomolgus monkeys. J. Med. Virol. 43:135-142[Medline]. |
| 41. | Tsarev, S. A., T. S. Tsareva, S. U. Emerson, S. Govindarajan, M. Shapiro, J. L. Gerin, and R. H. Purcell. 1997. Recombinant vaccine against hepatitis E: dose response and protection against heterologous challenge. Vaccine 15:1834-1838[Medline]. |
| 42. | Usmanov, R. K., M. S. Balayan, O. V. Dvoinikova, D. B. Alymbaeva, N. A. Zamiatina, LuA. Kazachkov, and V. I. Belov. 1994. An experimental infection in lambs by the hepatitis E virus. Vopr. Virusol. 39:165-168[Medline]. |
| 43. | van Cuyck-Gandre, H., H. Y. Zhang, S. A. Tsarev, N. J. Clements, S. J. Cohen, J. D. Caudill, Y. Buisson, P. Coursaget, R. L. Warren, and C. F. Longer. 1997. Characterization of hepatitis E virus (HEV) from Algeria and Chad by partial genome sequence. J. Med. Virol. 53:340-347[Medline]. |
| 44. | Weiss, R. A. 1998. Transgenic pigs and virus adaptation. Nature 391:327-328[Medline]. |
| 45. | Yin, S., R. H. Purcell, and S. U. Emerson. 1994. A new Chinese isolate of hepatitis E virus: comparison with strains recovered from different geographical regions. Virus Genes 9:23-32[Medline]. |
| 46. | Zaaijer, H. L., E. P. Mauser-Bunschoten, J. H. ten Veen, H. P. Kapprell, M. Kok, H. M. van den Berg, and P. N. Lelie. 1995. Hepatitis E virus antibodies among patients with hemophilia, blood donors, and hepatitis patients. J. Med. Virol. 46:244-246[Medline]. |
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