Virology Unit, Department of Infectious
Diseases and Immunology, Veterinary Faculty, Utrecht University,
3584 CL Utrecht, The Netherlands
A porcine torovirus (PoTV) was identified and characterized; it is
a novel member of the genus Torovirus (family
Coronaviridae, order Nidovirales), closely
related to but clearly distinct from the already recognized equine
torovirus (ETV) and bovine torovirus (BoTV) representatives.
Immunoelectron microscopy of feces from piglets revealed elongated,
120- by 55-nm particles which were recognized by a torovirus-specific
antiserum. Amplification by reverse transcriptase (RT) PCR with primers
designed to detect conserved regions (on the basis of the
genomes of BoTV strain Breda and ETV strain Berne) resulted in the
identification of the 489-bp nucleocapsid gene, encoding a
18.7-kDa protein. The sequence identity in this region between PoTV
and both ETV and BoTV was only about 68%, whereas the latter two
show 81% identity. Neutralizing antibodies directed against ETV
were found in sera of adult and young pigs. In all 10 herds
sampled, seropositive animals were present, and 81% of
randomly selected adult sows possessed antibodies. A longitudinal study
with RT PCR showed that piglets shed virus in the feces for 1 or
more days, starting 4 to 14 days after weaning.
 |
INTRODUCTION |
Toroviruses are spherical,
oval, elongated, or kidney-shaped enveloped viruses with a
single-stranded RNA genome of positive polarity; they belong to the
second genus in the family Coronaviridae, in the recently
established order Nidovirales (4; for
reviews, see references 8 and
24). Toroviruses resemble coronaviruses in genome
organization and replication strategy but differ in virion
architecture, especially with regard to the nucleocapsid, which is a
tubular structure responsible for the singular particle morphology
(31).
The torovirus genome is nonsegmented, polyadenylated, and 25 to 30 kb
in size. Its 5' two thirds is occupied by two large overlapping open
reading frames, ORF1a and ORF1b. These encode a polyprotein from which
the viral polymerase is derived. Downstream of ORF1b are four smaller
open reading frames which are expressed through a 3'-coterminal nested
set of mRNAs. They code for the following structural proteins (from 5'
to 3'): (i) the 180,000-molecular-weight (180K) precursor of spike
protein S, (ii) the 26K triple spanning integral membrane protein M,
(iii) a 65K class I membrane protein (HE) exhibiting acetylesterase
activity, and (iv) the 19K nucleocapsid protein N (5, 7, 15, 23,
25, 26).
Two torovirus species are recognized, bovine torovirus (BoTV,
originally named Breda virus), evidenced in the feces of diarrheic calves (33), and equine torovirus (ETV, formerly Berne
virus), isolated in cell culture from rectal swabs from a horse
(31). There is serological evidence for the existence of
toroviruses in other mammals (3, 32), including swine.
During a serological survey in Switzerland, ETV-neutralizing antibodies
were detected in the sera of 91 of 112 pigs tested (81%)
(32). Furthermore, several authors have reported
toroviruslike particles in the feces of swine
(10, 20, 22, 35). It is of note, however, that in negatively
stained preparations for electron microscopy (EM), torovirions are
often pleiomorphic and may appear as spherical, oval, elongated, or
kidney-shaped particles (31, 33) carrying either a single or
a double fringe of surface projections (5, 31, 33). Without
additional immunological confirmation, torovirions are difficult to
distinguish from coronaviruses, other viral particles, and even
nonviral fringed particles (1, 9, 33).
In this paper, we present formal evidence for the existence of a
porcine torovirus (PoTV). By using a reverse transcriptase (RT) PCR
targeted to the 3'-nontranslated region (NTR) of the genome, we
detected torovirus RNA in the feces of piglets. Moreover, torovirions
were identified in these samples by immunoelectron microscopy. Virus
shedding, as monitored by RT PCR, started shortly after weaning and
lasted for 1 or more days. Comparative sequence analysis of the
N-protein gene indicated that PoTV is a novel torovirus closely related
to but clearly distinct from BoTV and ETV.
| |
MATERIALS AND METHODS |
Cells, viruses, and antisera.
Equine dermis (EDERM)
cells (American Type Culture Collection) were maintained in Dulbecco's
modified Eagle's medium supplemented with 10% fetal calf serum, 100 IU of penicillin per ml, and 100 µg of streptomycin per ml. ETV
strain Berne (P138/72) was propagated in EDERM cells as described
previously (29). Tissue culture supernatant containing
porcine epidemic diarrhea virus (PEDV) and porcine anti-PEDV serum
(P-
PEDV) were obtained from Ghent University, Ghent, Belgium.
Transmissible gastroenteritis virus (TGEV), strain Purdue, was
propagated in PD5 (Philips Duphar) porcine kidney cells. Preparation of
rabbit antiserum against BoTV (Ra-BoTV) has been described elsewhere
(14). An ascitic fluid sample (A40) from a cat that had
succumbed to feline infectious peritonitis was used for the
immunodetection of TGEV (19).
Collection of porcine serum and feces field samples.
Serum
samples were obtained from four piglets at a commercial breeding and
fattening farm in Belgium; the samples were obtained from the piglets
at 2 to 11 weeks of age at 2-week intervals (13). In
addition, serum samples were collected from 10 to 12 randomly selected
adult sows at each of 10 commercial breeding farms in The Netherlands.
At one of these, sequential serum samples were also collected from nine
piglets in three litters (three samples per litter). The first samples
were taken 1 to 3 days after birth (day 0), followed by bleedings at
days 14, 21, 35, and 49. The piglets were weaned at day 21. Serum
samples were also obtained from the sows after farrowing (day 0).
Fecal samples were collected from each piglet at days 14, 21, 23, 25, 27, 29, 31, 35, and 49.
Serum neutralization assay.
Serial twofold dilutions of
heat-inactivated porcine sera were incubated with 100 50% infective
doses of ETV for 1 h at 37°C. The mixtures were used to
inoculate EDERM cell monolayers grown in 96-well microtiter plates. The
cell cultures were examined for cytopathic effects (CPE) at 2 days
after infection, and the neutralization titers were calculated by use
of the Spearman-Kärber formula.
RT PCR and sequence analysis.
RNA was extracted from fecal
samples and concentrated by a modification of the guanidinium
isothiocyanate-silica protocol of Boom et al. (2), and an RT
PCR was performed as described previously (11). Details of
the primers used for RT PCR and for sequence analysis of the PCR
products are given in Table 1. Torovirus
RNA was detected by use of an RT PCR assay targeted to the NTR of the
viral genome with oligonucleotides 293 and 294 as primers. For RT PCR
amplification of the PoTV N-protein gene, oligonucleotides 294 and 253 and oligonucleotides 620 and 542 served as primers. The resulting
products were cloned into vector pGEM-T (Promega Corp., Madison, Wis.)
and sequenced with a T7 sequencing kit (Pharmacia Biotech) according to
the manufacturer's instructions. The nucleotide sequence was
determined for both orientations with two or more independent clones.
Nucleic acid and amino acid sequence data were analyzed with PC-DOS
HIBIO DNASIS and PROSIS software from Pharmacia Biochemicals
(Milwaukee, Wis.).
EM and immunoelectron microscopy.
Immediately after
collection, fecal samples were suspended in phosphate-buffered saline
(approximately 1:2), and the suspension was centrifuged for 1 min at
12,000 × g. From the supernatant, 10-µl droplets
were deposited on Parafilm, and a 300-mesh collodion- and carbon-coated
copper grid was placed on top. Incubation was done for 5 min at room
temperature. The preparations were negatively stained with 2%
phosphotungstic acid (pH 6.9) for 1 min. For immunoelectron microscopy,
the fecal suspension was spun through a 20% (wt/wt) sucrose solution
onto a cushion of 50% (wt/wt) sucrose for 2 h at 150,000 × g and 4°C. The interphase was collected and used to
prepare grids as described above. Preparations from TGEV and PEDV cell
culture supernatants were used as controls. Blocking and immunostaining
were performed as described previously (28); Ra-BoTV and
P-PEDV sera were used at a 1:100 dilution in phosphate-buffered saline containing 0.5% bovine serum albumin (fraction V; Sigma), 1% acetylated bovine serum albumin (Aurion) or ascites A40
(diluted 1:200), and a protein A-colloidal gold (5 nm) conjugate
(Aurion). The grids were examined with a Philips CM10 electron
microscope at an accelerating voltage of 80 kV.
| |
RESULTS |
Serological evidence for torovirus infection in swine.
A
serological survey was performed to study the occurrence of torovirus
infections in swine. Sera collected from adult sows at 10 breeding
farms in The Netherlands were tested for cross-reacting antibodies in
an ETV neutralization assay. On each of the farms, seropositive animals
were identified (Table 2). Of the 118 pigs tested, 96 (81.4%) had ETV-neutralizing antibody titers of 
10; 13 pigs (11.9%) had titers of 160.
As we suspected infection to take place early in life, we tested sera
obtained sequentially from four piglets at a breeding and fattening
farm in Belgium. Two of the animals had ETV-neutralizing antibodies at
2 weeks of age. Titers then declined and, by 7 to 9 weeks, had dropped
to values below 10; they then rose again, and at 11 weeks of age, three
animals had ETV-neutralizing antibody titers between 10 and 90 (Fig.
1). In a more extensive longitudinal experiment on a breeding farm in The Netherlands, 9 piglets belonging to three litters (three animals per litter) were monitored until 7 weeks of age. Sera were obtained at regular intervals, and fecal samples also were collected. At the first sampling, at an age of 1 to 3 days, all piglets possessed ETV-neutralizing antibodies with titers
ranging from 90 to 900 (Fig. 2). In each
case, the titers in the piglets exceeded those in the sows. Again,
titers declined rapidly in the following weeks; in a number of piglets, a moderate rise was noted at 2 to 4 weeks after weaning (Fig. 2).
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FIG. 2.
Serum neutralization titers against ETV in sera from
three different sows (sows A, B, and C; one serum sample each at day 0)
and nine piglets (three piglets per sow). On day 0, the piglets were 1 to 3 days old; on day 21, they were weaned. Note that the scales on the
ordinate are different for the three graphs.
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Torovirus detection in pig feces.
The serological findings
indicated that torovirus infections are common in swine, with primary
infection taking place shortly after weaning. To corroborate this
observation and to obtain genetic evidence for the existence of a
genuine PoTV, the fecal samples from the nine piglets were assayed with
an RT PCR targeted to the NTR of the viral genome. The oligonucleotide
primers had been designed according to conserved sequences in the
genomes of BoTV (strain Breda) and ETV (strain Berne). For eight of the
nine piglets, RT PCR yielded a product of the anticipated size of 135 bp. Sequence analysis confirmed that the product was torovirus
specific, displaying 88.2 and 87.1% sequence identities with the NTRs
of ETV and BoTV, respectively. Virus shedding, as monitored by RT PCR,
started 4 to 14 days after weaning. Virus shedding was shown to last
for 1 to 9 days in four of the eight PCR-positive piglets. Since no samples were collected between 14 and 28 days after weaning, the exact
date of termination of shedding could not be determined for the other
four animals (Table 3). During this
period, the animals appeared healthy and passed normal stools.
Direct evidence for torovirus shedding in the feces was obtained by EM.
In fecal samples that had tested positive by RT PCR, torovirus-like
particles were indeed observed. In fresh material they were mainly
elongated, approximately 120 nm in length and approximately 55 nm in
width, the surface projections excluded. Upon repeated freezing and
thawing, the virions became more pleiomorphic, and round, kidney- and
torus-shaped particles were found. Two sets of surface projections were
discerned. The longer projections were drumstick or petal shaped,
extending for approximately 19 nm. The short spikes measured about 6 nm
(Fig. 3a).
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FIG. 3.
Morphology of PoTV and immunogold labeling (controls
include TGEV and PEDV) with different sera. (a) PoTV particles in a
fresh fecal sample from a piglet 1 week after weaning. (b to h)
Immunogold staining of PoTV with anti-BoTV serum and protein A-5-nm
colloidal gold conjugate (b); PoTV with A40, specific for TGEV (c);
PoTV with anti-PEDV serum (d); PEDV with anti-PEDV serum (e); PEDV with
anti-BoTV serum (f); TGEV with A40, specific for TGEV (g); and TGEV
with anti-BoTV serum (h). The bar applies to all of the micrographs.
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Formal proof that the particles represented torovirions was obtained by
immunoelectron microscopy. Ra-BoTV specifically recognized the
particles. This serum did not bind to virions of TGEV or PEDV, other
members of the Coronaviridae family frequently found in porcine feces. Moreover, immunogold labeling of the particles did not
occur when rabbit preimmune serum or antiserum against TGEV or PEDV was
used (Fig. 3b to h).
Genetic analysis of PoTV.
To study the relationship of
putative PoTV with BoTV and ETV, its N-protein gene was amplified by RT
PCR. A schematic outline of the strategies for the RT PCR assay and
sequence analysis is shown in Fig. 4. The
N-protein gene of PoTV was found to be 489 bp in length and to encode
an 18.7-kDa protein. The sequence variation in the N-protein gene
between PoTVs sampled from two piglets was 0.2%. Comparison to the
N-protein genes of BoTV and ETV (23) revealed sequence
identities of 68.7 and 68.3%, respectively. In contrast, the N-protein
genes of BoTV and ETV show 81.2% sequence identity. At the amino acid
sequence level, the PoTV N protein showed 68.3 and 66.9% identities
with the N proteins of BoTV and ETV, respectively. The BoTV and ETV N
proteins are 82% identical (Table 4). An
alignment is shown in Fig. 5.
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FIG. 4.
Genomic organization of toroviruses and a schematic
outline of the strategies used for amplification and sequencing of the
3' genomic region. The upper panel shows a schematic representation of
the torovirus genome, with the genes represented by boxes. The genes
for the polymerase (Pol 1a, Pol 1b), the spike protein (S), the
membrane protein (M), the hemagglutinin-esterase (HE), and the
nucleocapsid protein (N) are indicated. The lower panel shows a
schematic outline of the RT PCR assay targeted to the NTR and the
N-protein gene. The positions and orientations of the oligonucleotides
on the PoTV genome are shown, as are the lengths of the products of the
PCRs. Primer 593 was used only in the sequence analysis.
|
|
| |
DISCUSSION |
In this report, we describe the identification of a torovirus of
swine and initial morphological and genetic data. The virus was
detected in the feces of piglets by immunoelectron microscopy and RT
PCR. In fresh fecal samples, PoTV particles appeared elongated, measuring 120 nm in length and 55 nm in width. Two types of surface projections were observed, the longer of which was petal shaped and 18 to 20 nm in length and most likely represented oligomers of the S
protein (12). The shorter spikes were 6 nm long and presumably represented HE. Surface projections of this size are also
seen in BoTV; they are absent in ETV (5), where the HE gene
is truncated at its 5' end (27). Preliminary observations from RT PCR amplification and sequence analysis indicate that, like
BoTV, PoTV contains an intact HE gene (17).
Comparative sequence analysis of the torovirus N-protein
genes showed that BoTV and ETV were closely related, with 81% sequence identity in this region. In contrast, PoTV showed only 68%
sequence identity with the other two viruses for this region. The NTRs of PoTV, BoTV, and ETV were highly conserved, with sequence identities of about 88%. We conclude that PoTV is antigenically and genetically related to but clearly distinct from the bovine and equine
representatives of the torovirus genus and should therefore be
considered a new member.
In a heterotypic in vitro neutralization assay, >80% of the adult
sows in The Netherlands were positive for torovirus antibody; similar
observations had been reported for Switzerland (32). Torovirus infections are obviously as common and widespread in pigs as
in cattle and horses (16, 31, 32, 34). Piglets are infected
shortly after weaning, when protection by maternal antibodies and/or
lactogenic immunity has waned. In this study, virus shedding in the
feces, as monitored by RT PCR, lasted between 1 and 9 days, suggesting
that PoTV predominantly causes acute enteric infections.
The high percentage of seropositive animals and the early occurrence of
infection (shortly after weaning, when immunological protection has
declined) are indicative of PoTV endemicity. The virus would persist in
a herd because of the continuous presence of susceptible piglets and
reinfection of partly immune animals. Also, chronically infected
carriers may exist, as has been demonstrated for other members of
the Nidovirales, such as coronaviruses (6, 11) and arteriviruses (for a review, see reference
21). A more sensitive nested RT PCR targeted to the
conserved NTR may allow the identification of long-term shedders among
the adult pig population. The physical stability of toroviruses in the
environment is as yet unknown; ETV is surprisingly stable at a low pH
but is less thermostable than TGEV (18, 30).
Piglets passed normal stools during PoTV shedding and did not show any
sign of disease. As our longitudinal study included only a few animals
and monitored only one herd, we cannot exclude the possibility that the
virus causes disease at a low incidence, in other circumstances, or in
combination with other agents. Future research will focus on the
epidemiology and pathogenic potential of PoTV.
We thank M. B. Pensaert and K. van Reeth, Laboratory of
Virology, Faculty of Veterinary Medicine, University of Ghent, Ghent, Belgium, for providing the sequential sera from piglets, PEDV, and
anti-PEDV serum. We also thank W. F. Voorhout for helping with the
electron microscopy, A. A. P. M. Herrewegh for
assistance with the RT PCR, and the Hargeerds Farm (Markelo, The
Netherlands) for permission to take and help in taking samples on the
premises.
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Abstract
Introduction
