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Journal of Clinical Microbiology, September 2001, p. 3040-3046, Vol. 39, No. 9
Center for Molecular Medicine and Infection
Diseases, Department of Biomedical Sciences and Pathobiology, College
of Veterinary Medicine, Virginia Polytechnic Institute and State
University, Blacksburg, Virginia,1 and
Department of Veterinary Diagnostic and Animal Production
Medicine, College of Veterinary Medicine, Iowa State University,
Ames, Iowa2
Received 4 April 2001/Returned for modification 12 June
2001/Accepted 23 June 2001
Hepatitis E virus (HEV) is the major cause of enterically
transmitted non-A, non-B hepatitis in many developing countries and is
also endemic in many industrialized countries. Due to the lack of an
effective cell culture system and a practical animal model, the
mechanisms of HEV pathogenesis and replication are poorly understood.
Our recent identification of swine HEV from pigs affords us an
opportunity to systematically study HEV replication and pathogenesis in
a swine model. In an early study, we experimentally infected
specific-pathogen-free pigs with two strains of HEV: swine HEV and the
US-2 strain of human HEV. Eighteen pigs (group 1) were inoculated
intravenously with swine HEV, 19 pigs (group 2) were inoculated with
the US-2 strain of human HEV, and 17 pigs (group 3) were used as
uninoculated controls. The clinical and pathological findings have been
previously reported. In this expanded study, we aim to identify the
potential extrahepatic sites of HEV replication using the swine model.
Two pigs from each group were necropsied at 3, 7, 14, 20, 27, and 55 days postinoculation (DPI). Thirteen different types of tissues and
organs were collected from each necropsied animal. Reverse
transcriptase PCR (RT-PCR) was used to detect the presence of
positive-strand HEV RNA in each tissue collected during necropsy at
different DPI. A negative-strand-specific RT-PCR was standardized and
used to detect the replicative, negative strand of HEV RNA from tissues
that tested positive for the positive-strand RNA. As expected,
positive-strand HEV RNA was detected in almost every type of tissue at
some time point during the viremic period between 3 and 27 DPI.
Positive-strand HEV RNA was still detectable in some tissues in the
absence of serum HEV RNA from both swine HEV- and human HEV-inoculated
pigs. However, replicative, negative-strand HEV RNA was detected
primarily in the small intestines, lymph nodes, colons, and livers. Our
results indicate that HEV replicates in tissues other than the liver.
The data from this study may have important implications for HEV
pathogenesis, xenotransplantation, and the development of an in vitro
cell culture system for HEV.
Hepatitis E virus (HEV), the
causative agent of hepatitis E, has been recognized as a major cause of
enterically transmitted non-A, non-B hepatitis in many developing
countries (1, 30, 37, 39). Transmission of the virus
occurs primarily by the fecal-oral route through contaminated drinking
water in areas with poor sanitation. The disease affects mainly young
adults, with a reported mortality rate of up to 25% for pregnant women (10, 14, 37, 39). In the United States, sporadic cases of
acute hepatitis E without known risk factors have been documented and
anti-HEV antibodies have been detected in a significant proportion of
healthy individuals (21, 28, 32, 37, 39, 45). HEV is a
positive, single-stranded RNA virus without an envelope. The genome,
which is about 7.5 kb in size, contains three open reading frames
(ORFs) and a short 5' and 3' nontranslated region (13, 18, 31,
38). ORF 1 is the largest of the three and encodes for
nonstructural proteins such as methyltransferase, helicase, and
RNA-dependent RNA polymerase. ORF 2 encodes the putative capsid
protein, and ORF 3, which overlaps with ORF 1 and ORF 2, encodes a
cytoskeleton-associated phosphoprotein (13, 37-39, 55).
HEV was originally classified in the Caliciviridae family.
However, recent studies have demonstrated the unique genomic organization of HEV, and therefore HEV has been declassified and designated "hepatitis E-like viruses" in an unassigned family (3, 18, 35).
In 1997, a novel virus, closely related to human HEV, was discovered in
swine (24). This virus, designated swine HEV, was extensively characterized (9, 24-29). In the United
States, two strains of human HEV identified from patients with acute
hepatitis E (US-1 and US-2) have shown a striking genetic similarity to swine HEV (6, 40). The two U.S. strains of human HEV share Due to the lack of a practical animal model and an in vitro cell
culture system for HEV, the mechanisms of HEV pathogenesis and
replication are poorly understood. With the discovery of swine HEV, we
now have a homologous animal model system to study HEV infection. It
has been suspected that HEV might replicate in tissues and organs other
than the liver (2). Recent results from studies performed
with rats infected with human HEV have suggested that the virus may
replicate extrahepatically (20). The objectives of this
study are to utilize swine as a model system to systematically study
HEV replication and to identify potential extrahepatic sites of HEV replication.
Viruses.
The swine HEV used in this study was recovered from
a pig in Illinois (24). The swine HEV inoculum has an
infectious titer of 104.5 50% pig infectious
doses per ml of inoculum (26). The US-2 strain of human
HEV (kindly provided by Isa Mushahwar of Abbott Laboratories, North
Chicago, Ill.) used in this study was first recovered from a hepatitis
patient in Tennessee and then transmitted to cynomolgus monkeys
(6, 40). The US-2 strain of human HEV has an infectious
titer of 105 50% monkey infectious doses per 0.5 ml of inoculum (R. H. Purcell et al., unpublished data).
Experimental infection.
The experimental infection of pigs
with swine and human HEV and the clinical and pathological findings
from the study have been reported previously (9). Briefly,
54 cross-bred, specific-pathogen-free pigs, 3 to 4 weeks old, were
randomly assigned into three groups. Group 1 consisted of 18 pigs who
were inoculated intravenously (i.v.) with 104.5
50% pig infectious doses of swine HEV. Group 2 consisted of 19 pigs
who were inoculated i.v. with 104.5 monkey
infectious doses of the US-2 strain of human HEV. Group 3 consisted of
17 pigs who were used as uninoculated controls. Two pigs from each
group were necropsied at 3, 7, 14, 20, 27, and 55 days postinoculation
(DPI), respectively. Thirteen different types of tissues and organs
were collected from each necropsied animal including liver, mesenteric,
tracheobronchial, and hepatic lymph nodes, small intestine, colon,
stomach, pancreas, spleen, salivary gland, tonsil, heart, lung, kidney,
and skeletal muscle. Samples of tissues and organs were frozen
immediately at Tissue homogenates.
Samples of each tissue and organ
collected at necropsy were homogenized in 10% (wt/vol) sterile
phosphate-buffered saline. Mesenteric, tracheobronchial, and hepatic
lymph nodes were pooled and homogenized in 10% phosphate-buffered
saline. The tissue homogenates were clarified by centrifugation at
3,000 rpm (Eppendorf centrifuge 5810; Brinkman Instruments Inc.,
Westbury, N.Y.) for 15 min at 4°C. The supernatants of the tissue
homogenates were harvested and stored at RNA extraction and RT-PCR.
Total RNA was extracted with
TriZol reagent (GIBCO-BRL) from 100 µl of 10% tissue homogenates or
serum samples and was resuspended in 11.5 µl of DNase, RNase, and
proteinase-free water (Eppendorf, Inc.). Reverse
transcription (RT) was performed at 42°C for 60 min with 1 µl of
the R1 reverse primer (5'-CTACAGAGCGCCAGCCTTGATTGC-3'), 1 µl of superscript II reverse transcriptase (GIBCO-BRL), 0.5 µl of
0.1 M dithiothreitol, 4 µl of 5× RT buffer, and 1 µl of 10 mM deoxynucleoside triphosphates. Ten microliters of the resulting cDNA
was amplified in a 100-µl nested PCR with AmpliTaq gold
DNA polymerase (GIBCO-BRL). The PCR parameters included an initial incubation at 95°C for 9 min to activate the AmpliTaq gold
DNA polymerase, followed by 39 cycles of denaturation at 94°C for 1 min, annealing at 54°C for 1 min, and extension at 72°C for 1.5 min, with a final incubation at 72°C for 7 min. The first round of
PCR produced an expected fragment of 404 bp using the forward primer F1
(5'-AGCTCCTGTACCTGATGTTGACTC-3') and the reverse primer R1.
For the second round of PCR, the forward primer F2 (5'-GCTCACGTCATCTGTCGCTGCTGG-3') and the reverse primer R2
(5'-GGGCTGAACCAAAATCCTGACATC-3') were used to produce an
expected product of 266 bp. The PCR parameters for the nested PCR were
essentially the same as those in the first-round PCR. The primers were
designed in conserved regions of the ORF 2 to amplify both swine HEV
and the US-2 strain of human HEV.
Standardization of a negative-strand-specific RT-PCR. (i) Cloning
of an ORF 2 fragment.
To standardize a negative-strand-specific
RT-PCR to detect replicative, negative-strand HEV RNA in infected
tissues, a negative strand of HEV RNA transcript had to be generated
for use as a positive control. Briefly, total RNA was extracted from
100 µl of feces collected from a specific-pathogen-free pig
experimentally infected with swine HEV (25). The Qiagen
1-step RT-PCR kit was used to amplify an ORF 2 fragment of swine HEV
according to the protocol supplied by the manufacturer. Total RNA was
reverse transcribed, and PCR was performed with 10 µl of resulting
cDNA in a 100-µl reaction mixture. The PCR was carried out for 31 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1.5 min, followed by a final
incubation period at 72°C for 7 min. Primers F5526
(5'-GGGGGATCCAGCTCCTGTACCTGATGTTGACTC-3') and
R5955 (5'-GGCCTCGAGCTACAGAGCGCCAGCCTTGATTGC-3')
were used in the RT-PCR. The sense primer F5526 has an introduced
BamHI restriction site, and the antisense primer R5955 has
an introduced XhoI restriction site (introduced restriction
sites underlined) to facilitate the subsequent cloning steps.
The resulting PCR product (418 bp) was excised from an agarose gel and
purified with the glass milk procedure using the GENE CLEAN II Kit (Bio 101, Inc.). The purified PCR product was first ligated into a TA vector
using T4 DNA ligase (Stratagene) at 12°C overnight. The recombinant
plasmid was transformed into DH5 (ii) In vitro transcription of negative-strand HEV RNA.
Recombinant PSK II plasmid containing the ORF 2 fragment of swine HEV
was linearized by restriction enzyme digestion with BamHI.
Synthetic negative-stranded RNA was transcribed in vitro by activation
of the T7 promoter of the PSK II plasmid using Ampliscribe T7
high-yield transcription kit (Epicentre Technologies) according to the
manufacturer's protocol. The transcribed negative-strand RNA was
separated in a 1.5% agarose gel, and the expected RNA band was excised
and purified using the RNaid isolation kit (Bio 101, Inc.) to remove
plasmid DNA. The gel-purified RNA was further treated with DNase for 60 min at 37°C. The RNA was then extracted with TriZol reagent, and only
the top half of the aqueous layer was collected to further eliminate
potential plasmid DNA contamination. A nested PCR with no RT step using
AmpliTaq gold DNA polymerase and the external F1 and R1 set
of primers and the internal F2 and R2 set of primers was performed to
ensure that no plasmid DNA remained in the purified synthetic
negative-strand RNA.
Specificity and sensitivity of the negative-strand-specific
RT-PCR.
To standardize a negative-strand-specific RT-PCR for HEV,
the synthetic negative-strand HEV RNA was serially diluted from 100 ng
to 10 ag. Negative-strand-specific RT-PCR was performed on each
dilution. RT was performed with the forward primer F1, 1 µl of
superscript II reverse transcriptase, 0.5 µl of 0.1 M dithiothreitol,
4 µl of 5× RT buffer, and 1 µl of 10 mM deoxynucleoside triphosphates. A nested PCR with AmpliTaq gold DNA
polymerase was subsequently performed with 5 µl of the cDNA in a
50-µl reaction mixture. The first-round PCR used primers F1 and R1
with an expected product of 404 bp. The second-round PCR used 5 µl of
the first-round PCR product in a 50-µl reaction mixture with internal
primers F2 and R2 with an expected product of 266 bp. For
specificity, the negative-strand-specific RT-PCR was performed with RNA
extracted from serum samples known to contain positive-stranded HEV RNA.
Tissue distribution of positive-stranded HEV RNA.
All pigs
from both HEV-inoculated groups seroconverted to anti-HEV
(9). Positive-strand HEV RNA was detected from 3 to 27 DPI
in various tissues including livers, lymph nodes, colons, small
intestines, stomachs, spleens, kidneys, tonsils, salivary glands, and
lungs from pigs inoculated with swine HEV (Table
1). Viral RNA was not detected from
tissues collected at 55 DPI. In the absence of viremia, swine HEV RNA
was still detectable in tissues from 20 to 27 DPI (Table 1). Swine HEV
viremia disappeared after 14 DPI, and positive-strand viral RNA was not
detectable from 20 to 55 DPI in the sera of swine HEV-inoculated pigs.
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.9.3040-3046.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Evidence of Extrahepatic Sites of Replication of
the Hepatitis E Virus in a Swine Model
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
97% amino acid identity with swine HEV in ORFs 1 and 2 but are genetically distinct from other known strains of HEV worldwide (26). It has been shown that the US-2 strain of human HEV
infects pigs and that swine HEV infects nonhuman primates (9,
26). Similar findings were reported in Taiwan, where a novel
strain of swine HEV was isolated from Taiwanese pigs. This Taiwanese strain of swine HEV shared 97.3% nucleotide sequence similarity with a
strain of human HEV isolated from a retired Taiwanese farmer, but it is
genetically distinct from the U.S. strain of swine HEV and other HEV
strains worldwide (12). Numerous genetically distinct strains of human HEV have also been identified in many other
industrialized and developing countries (41, 42, 49, 50).
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
80°C until used for analysis.
80°C until use.
-competent Escherichia coli cells (GIBCO-BRL). Plasmids containing the insert were
identified and confirmed by restriction enzyme digestions. The insert
was subsequently subcloned into PSK II plasmid (Stratagene) by
directional cloning using BamHI and XhoI. The
recombinant PSK II plasmid containing the ORF 2 fragment was isolated
and confirmed by DNA sequencing.
RESULTS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Detection of positive-strand HEV RNA in pigs inoculated
with swine HEV and the US-2 strain of human HEV
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Standardization of the negative-strand-specific RT-PCR.
The
negative-strand-specific RT-PCR for HEV was standardized with the
synthetic negative-strand HEV RNA transcript. By using the synthetic
negative-strand RNA as a positive control, we showed that the test can
detect negative-strand HEV RNA to the dilution level of 10 pg in the
first round of the PCR and 1 fg in the second round of the nested PCR
(Fig. 2). The dilution level of 1 fg
correlates to approximately 4,500 viral genome copies. The
negative-strand RT-PCR is specific as it fails to detect
positive-strand viral RNA from positive serum samples that contain only
positive-strand HEV RNA.
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Evidence for extrahepatic replication of HEV.
Detection of
positive-strand HEV RNA in various tissues of inoculated pigs does not
mean that HEV replicates in these sites. Therefore, a
negative-strand-specific RT-PCR was performed to retest all the tissues
that had tested positive for positive-strand HEV RNA in the traditional
RT-PCR. For swine HEV-inoculated pigs, replicative, negative-strand
viral RNA was detected in the livers, lymph nodes, colons, small
intestines, and spleens between 7 and 27 DPI (Table
2). For pigs inoculated with the US-2
strain of human HEV, negative-strand HEV RNA was detected in the
livers, lymph nodes, colons, small intestines, stomachs, spleens,
kidneys, tonsils, and salivary glands between 3 and 27 DPI (Fig.
3) (Table 2).
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DISCUSSION |
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Introduction
Materials and Methods
Results
Discussion
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The first animal strain of HEV, swine HEV, was genetically identified and characterized from a pig in the United States (24). Subsequently, several other strains of swine HEV were identified from pigs in Taiwan (12, 53). In Spain, strains of HEV from patients with acute hepatitis E were found to have a 92 to 94% nucleotide sequence identity with a strain of HEV recovered from slaughterhouse sewage which was primarily of swine origin (34). A variant strain of HEV has also been recovered from tissues and fecal samples of rodents trapped in the wild in Nepal. Phylogenetic analysis revealed that the HEV strain recovered from these rodents is most closely related to human strains of HEV from hepatitis patients in Nepal (48).
Serological studies also support the theory of an animal reservoir(s) for HEV. Anti-HEV antibodies have been identified in pigs from industrialized countries such as the United States (25), Canada (27), Korea (27), Taiwan (12, 52, 53), and Australia (4). Anti-HEV antibodies have also been detected in pigs from regions where HEV is endemic, such as Nepal, China, and Thailand (5, 27). In addition to pigs, 77% of rats in Maryland and 90% of rats in Hawaii are positive for anti-HEV antibodies (17). The prevalence of antibodies in rats occurs in both urban and rural areas and increases in proportion to the estimated ages of the animals (8, 17). Anti-HEV antibodies were also detected in rhesus monkeys caught in the wild (47). In Vietnam, where HEV is endemic, anti-HEV antibodies have been detected in 44% of chickens, 36% of pigs, and 27% of dogs (46). Approximately 42 to 67% of sheep and goats have tested positive for anti-HEV antibodies in Turkmenistan, where HEV is also endemic (7). In the United States, anti-HEV antibodies have been detected in about 1 to 2% of blood donors, with up to 20% detected in areas such as Baltimore, Md. (21, 22, 32, 45). Similar results have been reported in other industrialized countries such as The Netherlands (54), Italy (56), Greece (36), England (23, 30), Spain (15), Germany (19), Sweden (16), Finland (30), and Taiwan (11, 33, 52). Although the sensitivity and specificity of these various serological tests are unknown, these results suggest that the prevalence of HEV infection may be underestimated, especially in industrialized countries, and that hepatitis E may be a zoonosis.
The lack of a practical animal model has hindered HEV research. The identification of swine HEV in pigs affords us an opportunity to study HEV pathogenesis and replication in a swine model. The U.S. strain of swine HEV has shown the ability to infect nonhuman primates (26). In an earlier report, we showed that pigs could be experimentally infected with both swine HEV and the US-2 strain of human HEV (9). The clinical and pathological findings of HEV infection in pigs were reported previously (9). In the present study, we attempted to use the samples of tissues and organs collected from this previous study to identify the potential extrahepatic sites of HEV replication.
It has been hypothesized that liver damage induced by HEV infection may be due to the immune response to the invading virus and may not be a direct cause of viral replication in hepatocytes (37). Since HEV is presumably transmitted by the fecal-oral route, it is unclear how the virus reaches the liver, and an extrahepatic site(s) of replication would be a possible explanation (2, 37). Primary hepatocytes are the only known sites of HEV replication (43, 44). In a preliminary study with naturally infected pigs, we found that positive-strand HEV RNA was detectable in a number of tissues, even after viremia was cleared (X. J. Meng et al., unpublished data). Furthermore, in experimentally infected pigs, we also found that the relative genomic titer of HEV in the feces was at least 10-fold higher than that in bile collected on the same and prior days (Meng et al., unpublished), suggesting that HEV may replicate in the gastrointestinal tract.
In this study, we first tested by RT-PCR for the presence of positive-strand HEV RNA from various tissues and organs of both swine HEV- and human HEV-infected pigs. We showed that positive-strand HEV RNA was detectable in numerous tissues. The positive-strand viral RNA detected in tissues from 3 to 14 DPI in swine HEV-inoculated pigs and from 3 to 20 DPI in human HEV-inoculated pigs may not be attributable to the replicating virus, since viral RNA was also detected in the sera. However, after viremia disappeared, positive-strand viral RNA was still detectable in various tissues between 20 to 27 DPI in swine HEV-infected pigs and at 27 DPI in human HEV-infected pigs. Detection of positive-strand HEV RNA from various tissues and organs in the absence of detectable serum viral RNA indicated that the virus detected from these tissues represents replicating virus and is not due to contamination of the tissue samples by virus circulating in the blood.
HEV is a positive-strand RNA virus, and therefore HEV replication produces an intermediate, negative-strand RNA. To further confirm that the HEV RNAs detected in extrahepatic tissues are due to HEV replication, we retested by the negative-strand-specific RT-PCR assay all tissues that were positive for the positive-strand viral RNA. The negative-strand RT-PCR was standardized to detect only the replicative, negative-strand HEV RNA in infected tissues. We showed that the negative-strand-specific RT-PCR is sensitive and specific for the replicative, negative-strand HEV RNA. Our results indicate that extrahepatic replication of HEV does occur. We were able to detect negative-strand HEV RNA in a variety of extrahepatic tissues. It appears that pigs inoculated with the US-2 strain of HEV had positive results in more tissues than did pigs inoculated with swine HEV. No replicating virus was detected in the kidney, stomach, pancreas, lung, heart, muscle, salivary gland, or tonsil tissues of pigs inoculated with swine HEV. Similarly, negative-strand HEV RNA was also absent in pancreas, lung, heart, and muscle tissues from human HEV-inoculated pigs. The detection of replicating virus primarily in small intestine and colon tissues supports our earlier preliminary observations that the feces contain more virus than bile does.
For human HEV-infected pigs, HEV replication was found in tonsil, small intestine, and colon tissues as early as 3 DPI. For swine HEV-infected pigs, replicative viral RNA was first detected in small intestines, colons, lymph nodes, and livers. Both swine HEV and human HEV replicate for a longer time in liver, lymph node, small intestine, and colon tissues than in other tissues. Other extrahepatic tissues such as kidney, tonsil, and salivary gland had detectable replicative HEV RNA for only 1 or 2 weeks. It appears that lymph nodes and the intestinal tract are the main extrahepatic sites of replication. The i.v. route of inoculation used in this study prevents us from identifying the initial site of HEV infection, since the natural route of HEV infection is presumably fecal-oral. Our earlier studies showed that a much higher infectious titer of HEV is required to initiate an infection by the oral route of inoculation (C. Kasorndorkbua et al., unpublished data). The i.v. route of inoculation has been almost exclusively used in animal experiments with HEV and the hepatitis A virus.
The significance of identifying extrahepatic sites of HEV replication is unclear at this time. Since we demonstrated that tissues other than the liver support HEV replication, this could help identify an in vitro cell culture system to propagate HEV. Extrahepatic replication of HEV also raises additional concerns involving xenotransplantation with pig organs. Xenotransplantation is a potential solution for the shortage of human organs for transplantation. Pigs have been identified as favorable organ donors because they have anatomic and metabolic characteristics similar to those of humans (51). The possibility of transmission of swine HEV to immunosuppressed xenograft recipients has been a concern, especially in situations that would involve liver transplants. The findings from this study indicate that xenotransplantation with other organs may also pose potential risk for HEV xenozoonosis. In addition, damages to these extrahepatic tissues and organs resulting from HEV infection may render them useless for xenotransplantation.
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ACKNOWLEDGMENTS |
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We thank Prem Paul for technical advice and use of laboratory equipment, Amy Weaver and Crystal Gilbert for assistance with the RT-PCR testing, Robert Purcell and Suzanne U. Emerson of the National Institutes of Health, Bethesda, Md., for providing HEV inocula and supporting the study, and Roger Avery of Virginia Polytechnic Institute and State University College of Veterinary Medicine for his support.
This study was supported by grants (to X.J.M.) from the National Institutes of Health (AI01653 and AI46505) and in part by grants (to P.G.H.) from the National Pork Producers Council and the Healthy Livestock Initiatives.
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FOOTNOTES |
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* Corresponding author. Mailing address: Center for Molecular Medicine and Infectious Diseases, Virginia Polytechnic Institute and State University, 1410 Prices Fork Rd., Blacksburg, VA 24061-0342. Phone: (540) 231-6912. Fax: (540) 231-3476. E-mail: xjmeng{at}vt.edu.
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Journal of Clinical Microbiology, September 2001, p. 3040-3046, Vol. 39, No. 9
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.9.3040-3046.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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