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Journal of General Virology (1990), 71, 1835 1838. Printedin Great Britain
Production of infectious swine vesicular disease virus from cloned c D N A in
mammalian cells
Toru Inoue, ~* Shigeo Yamaguchi, 1 Takakiyo Saeki I and Kiichi Sekiguchi 2
1Exotic Disease Research Division, National Institute of Animal Health, Jyousuihonchyo, Kodaira-shi, Tokyo 187 and
2Epizootic Research Station, Equine Research Institute, Kokubunji-machi, Shimotsuga-gun, Tochigi-ken 329-04, Japan
Full-length cDNA clones of the swine vesicular disease
virus (SVDV) were constructed from subgenomic
cDNA clones in the expression vector pSVL
(pSVLS00). The direct transfection of mammalian
cells with plasmid pSVLS00 results in the production
of infectious virus. The recovered virus was neutralized
completely by anti-SVDV guinea-pig serum, but did
show a difference in plaque morphology from the
parental virus.
Swine vesicular disease (SVD) was first observed in Italy
in 1966 (Nardelli et al., 1968). It is an infectious disease
of pigs characterized by the appearance of vesicles on the
tongue, in the mouth and on the feet and hocks. These
lesions are indistinguishable from those caused by footand-mouth disease virus (FMDV).
The causative agent, SVD virus (SVDV) belongs to the
enterovirus genus, of the Picornaviridae family and has a
close serological relationship to coxsackievirus B5
(Graves, 1973). Like other picornaviruses, the genome of
SVDV is a single-stranded R N A with a positive polarity.
The RNA genome is 7400 nucleotides long, excluding
the poly(A) tract and encodes a single polyprotein. The
predicted amino acid sequence shows close homology to
those of coxsackievirus B and poliovirus (Inoue et al.,
1989).
Full-length infectious cDNA constructs of the R N A
genomes of members of the family Picornaviridae were
described first by Racaniello & Baltimore (1981). This
feature, coupled with addition of a DNA-dependent
RNA polymerase promoter (T7 or SP6), has significantly
contributed to the molecular characterization of poliovirus and has subsequently been widely applied to many
other virus genomes, mainly of human and plant
pathogens. Recently, the development of engineered
poliovirus chimeric vaccines using infectious poliovirus
cDNA has been reported (Burke et al., 1988, '1989;
Murray et al., 1988; Evans et al., 1989). The construction
of infectious cDNA from the RNA genomes of livestock
pathogens will enable not only the analysis of genetic
functions leading to virulence but also the develoPment
of new types of vaccine.
We report here the construction and the properties of a
full-length cDNA clone of SVDV from subgenomic viral
cDNA clones. The constructed full-length SVDV cDNA
clone generated infectious virus which was antigenically
identical to the parental SVDV.
For the construction of full-length SVDV cDNA,
clones MPS671, pBRS14, MPS453 and pBRS19 were
used (Inoue et al., 1989). The plasmid pUCS825
containing Y-terminal cDNA (nucleotides 1 to 65) was
newly prepared by the method of Geliebter (1987) with
several modifications. These five clones overlap with
each other. On the basis of these overlapping restriction
sites, the construction steps were subdivided into three
blocks: (i) from the SVDV Y-terminal end to the first
SacI site (nucelotide position 747) composed of
pUCS825, MPS671 and pBRS14, (ii) from the second
SacI site (nucleotide position 5932) to the 3' poly(A) tract
composed of MPS453 and pBRS19 and (iii), from the
first SacI site to the second SacI site composed of
pBRS14. The reconstruction step of each portion was
performed in parallel and pSVLS00 was finally obtained
(Fig. 1). Plasmid pSVLS00, based on pSVL (Pharmacia),
harbours the full-length SVDV cDNA copy at the
XbaI/BamHI cloning site which is located within the
simian virus 40 (SV40) VP1 transcriptional unit. The
orientation and composition of all inserts were confirmed by restriction digestion with several enzymes.
DNA transfection of mammalian cells was performed
with the CellPhect transfection kit (Pharmacia). Three
~tg of pSVLS00 was transfected into Cos-7 cells or IBRS2 cells grown to 20 to 50% confluence in 60 mm dishes. In
IBRS-2 cells, distinct c.p.e, was observed 2 days after
transfection of the plasmids and the infectious viruses
were recovered from culture fluids. In Cos-7 cells,
0000-9535 © 1990SGM
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1836
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Fig. 1. Schematic diagram of steps used in the assembly of full-length SVDV cDNA clones. Plasmid designations of identities are
shown in the centre of each plasmid. Numbered positions within each plasmid refer to nucleotide positions within the SVDV sequence
(Inoue et al., 1989). DNA fragments indicated by thick lines in each plasmid were ligated using the common restriction sites as
indicated. A, Asp718; B, BamHI; E, EcoRI; H, I-IindIII; P, PstI; SC, SacI; SL, SalI; SM, Sinai; SP, SphI; X, XbaI.
infectious virus was recovered but no c.p.e, was
observed. The kinetics of virus production after transfection of IBRS-2 cells or Cos-7 cells with pSVLS00 is
presented in Fig. 2. Inoculated SVDV (m.o.i. of 1)
showed a typical one-step growth curve in IBRS-2 cells
and Cos-7 cells. When SVDV genomic R N A (1 pg) was
transfected into IBRS-2 cells or Cos-7 cells, virus
expression began immediately, as with the virus inoculations. In the D N A transfections, the production of
viruses was delayed more than 24 h in both IBRS-2 and
Cos-7 cells relative to R N A transfections or virus
inoculations. However, once viruses were expressed,
they multiplied efficiently in both cells. The level of virus
production in Cos-7 cells seemed to lag 6 to 12 h behind
in IBRS-2 cells.
The results of transfection of IBRS-2 or Cos-7 cells
with the plasmids are summarized in Table 1. At least 1
pg of plasmid was necessary to produce infectious virus
in IBRS-2 cells. N o virus was recovered from culture
fluids of Cos-7 cells transfected with the vector pSVL.
When pSVLS00 was transfected into Cos-7 cells, 0.1 ng
of plasmid was sufficient to produce infectious virus.
This input was 1/10000 of that required for IBRS-2 cells.
Our results clearly show that the constructed plasmid
was infectious in Cos-7 cells or IBRS-2 cells. For
pSVLS00, 1 ~tg of D N A generated infectious SVDV in
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I
9
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Virus neutralization test
T a b l e 2.
AVirus/IBRS-2
/
DNA/IBRS-2
7
Source
/@
,/,Virus~Cos-7
Virus titre (TCIDs0/0"05 ml)
Template
DNA
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?RNA/Cos-7
pSVLS00
pSVLS00
H/3 '76:~
5
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Negative serum*
Antiserumt
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6.5
3.5
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< 0.5
< 0.5
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1837
* Normal guinea-pig serum (1 : 100 dilution).
t Anti-SVDV guinea-pig serum (1 : 100 dilution).
Parental strain of SVDV.
j//~
d
t
1
2
Time post-inoculation (days)
Fig. 2. The time course of virus production in mammalian cells. Cells
grown in 60 mm plastic dishes were transfected with 1 Hg of SVDV
genome R N A or 3 p-g of the plasmid construct pSVLS00. As a control,
cells were infected with the parental H/3 '76 strain of SVDV at an
m.o.i, of l. R N A transfections were performed by the calcium
phosphate method.
IBRS-2 cells. These results are similar to those obtained
by Racaniello & Baltimore (1981) and Omata et al. (1984)
with poliovirus. When plasmid pSVLS00 was transfected into Cos-7 cells, the transfection efficiency was
greatly increased (0.1 to 0.01 ng of plasmid D N A could
produce infectious SVDV), a result consistent with those
of Semler et al. (1984) and Kean et al. (1986) using
poliovirus. Plasmid pSVLS00, which is based on pSVL,
harbours full-length SVDV cDNA within the SV40 VP1
translational unit and carries the SV40 origin of
replication. Presumably Cos cells replicate pSVLS00.
Although the mechanism of expressing infectious virus
from cDNA is not understood, a delay in expression
following transfection suggests some unknown steps are
required for expressing infectious virus from cDNA
(Racaniello & Baltimore, 1981).
Virus recovered from DNA transfections was characterized for its biological, physical and structural properties. The virus growth in IBRS-2 cells, the buoyant
density in CsC1 and the genomic R N A size of recovered
T a b l e 1.
Fig. 3. Comparison of plaque size. (a) SVDV strain H/3 '76. Cells were
fixed and stained 2 days after inoculation with virus. (b) The recovered
virus. Cells were fixed and stained 3 days after inoculation.
virus were identical to those of the parental virus. To
examine the identity of the virus produced in transfected
ceils, a virus neutralization test was performed and all of
the recovered virus was neutralized with anti-SVDV
serum (Table 2). The genomic R N A of recovered virus
was extracted and analysed with respect to its in vitro
translated products and by sequencing the 5'-terminal
region. The in vitro translation of recovered viral R N A
was performed using a rabbit reticulocyte lysate translation system (NEN) and analysed on 10~ SDSpolyacrylamide gels. Autoradiography of in vitro translation products did not demonstrate any detectable
Transfection of cultured mammalian cells with various plasmid DNAs
Transfected dose (ng)
Template D N A
pSVLS00
pSVLS00
pSVL
Cells
1000
100
10
1
0.1
0.01
Cos-7
IBRS-2
Cos-7
+*
+
-
+
4-
+
- ;~
+
-
+
+t
Minimum transfected
dose to yield
infectious virus (ng)
0.01-0-1
100-1000
* Infectious virus can always be recovered. Cells were maintained in 12-weU plastic dishes. Infectious virus was
assayed at 6 days post-transfection.
t Virus can be recovered sometimes.
Infectious virus cannot be recovered.
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differences between the recovered and parental virus
(data not shown). The RNA sequence of the 5'-terminal
90 sequence (nucleotides 1 to 90) of recovered virus was
also identical to that of parental virus (data not shown).
The only difference observed was in plaque size. The
recovered virus formed smaller plaques than the parental
virus. Fig. 3 depicts typical plaques of the parental
SVDV strain and the recovered virus. This phenotype
was stable for multiple replications of the virus in IBRS-2
cells, so the phenotype is genetically encoded. Whether
the small plaque phenotype originated by using a cDNA
clone from a mutant with an unusually small plaque size
and/or from mutations arising during multiple manipulations of the cDNA during the cloning and construction of
full-length cDNA is unknown. Further studies are
necessary to clarify the mechanisms or genes regulating
plaque size.
The reported infectious cDNA of SVDV may contribute to a virus vector for developing chimeric vaccines
for livestock. The cDNA may also act as a powerful tool
for analysing the pathogenesis of vesicular disease
caused by SVDV and FMDV.
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(Received 14 March 1990 ; Accepted 9 May 1990)
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