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Journal of Virology, December 1998, p. 10310-10315, Vol. 72, No. 12
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
The African Swine Fever Virus Thymidine Kinase Gene Is Required
for Efficient Replication in Swine Macrophages and for Virulence
in Swine
D. M.
Moore,*
L.
Zsak,
J. G.
Neilan,
Z.
Lu, and
D. L.
Rock
Plum Island Animal Disease Center,
Agricultural Research Service, U.S. Department of Agriculture,
Greenport, New York 11944-0848
Received 15 July 1998/Accepted 2 September 1998
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ABSTRACT |
African swine fever virus (ASFV) replicates in the cytoplasm of
infected cells and contains genes encoding a number of enzymes needed
for DNA synthesis, including a thymidine kinase (TK) gene. Recombinant
TK gene deletion viruses were produced by using two highly pathogenic
isolates of ASFV through homologous recombination with an ASFV p72
promoter-
-glucuronidase indicator cassette (p72GUS) flanked by ASFV
sequences targeting the TK region. Attempts to isolate double-crossover
TK gene deletion mutants on swine macrophages failed, suggesting a
growth deficiency of TK
ASFV on macrophages. Two
pathogenic ASFV isolates, ASFV Malawi and ASFV Haiti, partially adapted
to Vero cells, were used successfully to construct TK deletion viruses
on Vero cells. The selected viruses grew well on Vero cells, but both
mutants exhibited a growth defect on swine macrophages at low
multiplicities of infection (MOI), yielding 0.1 to 1.0% of wild-type
levels. At high MOI, the macrophage growth defect was not apparent. The
Malawi TK deletion mutant showed reduced virulence for swine, producing
transient fevers, lower viremia titers, and reduced mortality. In
contrast, 100% mortality was observed for swine inoculated with the
TK+ revertant virus. Swine surviving TK ASFV
infection remained free of clinical signs of African swine fever
following subsequent challenge with the parental pathogenic ASFV. The
data indicate that the TK gene of ASFV is important for growth in swine
macrophages in vitro and is a virus virulence factor in swine.
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TEXT |
African swine fever virus (ASFV) is
a large icosahedral enveloped DNA virus and is classified as the only
member of a new virus family, the Asfaviridae (3,
11). The structure and replication of its 170- to 190-kbp genome
are similar to those of the poxviruses, but the icosahedral morphology
of the virus more closely resembles that of the iridoviruses (for
reviews see references 9, 43, and
44). African swine fever (ASF) is recognized as an
important disease of domestic swine and is characterized by high
fevers, hemorrhage, shock, and 100% mortality for highly pathogenic
isolates. ASF may range from an acute, highly lethal infection to a
subclinical form, depending on contributing viral and host factors. The
virus infects cells of the mononuclear-phagocytic system, including
highly differentiated fixed-tissue macrophages and specific lineages of
reticular cells in the spleen, lymph node, lung, kidney, and liver.
These tissues show extensive damage with highly virulent strains of
ASFV, and the ability of ASFV to replicate and induce cytopathology in
these tissues in vivo appears to be a critical factor in ASFV virulence
(8, 24, 25, 30, 32). The nature of viral factors responsible
for the virulence and pathogenesis of ASFV remains poorly understood.
Like poxviruses and iridoviruses, ASFV replicates in the cytoplasm and
encodes enzymes for transcription and DNA synthesis (17,
46), since enzymes of the host cell nucleus are unavailable. Also, ASFV encodes enzymes involved in synthesis of deoxynucleoside triphosphates: the thymidine kinase (TK) enzyme (1, 28, 38), the ribonucleotide reductase (RR) enzyme (2, 10), and the thymidylate kinase (TMPK) enzyme (47). These enzymes are
also encoded by other large DNA viruses (29). Herpesviruses
encode a single polypeptide with both TK and TMPK activities
(39). These enzymes are involved in the salvage and de novo
pathways of dTTP synthesis. The ASFV TK gene has been shown to be
nonessential for the growth of ASFV in cultured hamster and monkey
cells (27, 40).
The inactivation of the TK gene in poxviruses and herpesviruses showed
the gene to be nonessential for growth in cultured cells (12, 21,
26, 36), but TK viruses exhibited a reduction in
virulence and pathogenicity in experimental animal hosts. A
104 to 105 reduction in the 50% lethal dose
(LD50) was noted for mice injected with TK
vaccinia or ectromelia virus (4, 23), and TK
herpes simplex virus and marmoset herpesvirus showed similar losses of
virulence in mice (13, 22). Cell culture conditions were
found to be important, as serum-starved cultures showed reduced growth
of TK, but not of TK+, herpesviruses
(13, 21). Also, TK herpes simplex virus showed
a reduced ability to infect highly differentiated neuronal tissues
(13, 42). In similar fashion, highly differentiated murine
macrophages, an important type of target cell in the pathogenesis of
ectromelia virus infection in mice, failed to support the growth of
TK ectromelia virus (23).
In this study, we have constructed TK gene deletion mutants of
pathogenic ASFV isolates in order to examine the role of this gene in
viral pathogenesis and virulence. Experiments using primary swine
macrophages failed to produce stable TK deletion mutants, suggesting
that the ASFV TK gene might be important for viral replication in this
cell type (31). Two pathogenic ASFV isolates, partially
adapted to Vero cells, were used successfully to construct TK deletion
viruses on Vero cells. The loss of TK from these viruses impaired their
growth on swine macrophages in vitro and reduced their virulence in
vivo. Thus, TK could be considered an important gene for growth in
target macrophage cells and possibly in other tissues involved in the
pathogenesis of lethal ASFV infections.
Construction of recombinant ASFV TK gene deletion mutants and
TK-positive revertants.
The pathogenic tissue culture-adapted ASFV
Malawi LiL-20/1V (Vero Malawi) and ASFV Haiti H811 (H811) were obtained
from the Plum Island Animal Disease Center ASFV reference collection
and grown on Vero cells, and viral DNA was obtained (45).
ASFV recombinant viruses were generated by homologous recombination
between parental ASFV genomes and engineered recombination vectors as
previously described (34, 48), except that Vero cells were
used for transfection/infection and isolation of -glucuronidase
(GUS)-positive virus foci. The exchange vector (p5.3) was produced by
the sequential cloning into plasmid pBluescript II KS (Stratagene) of
PCR-derived DNA of left (892 bp) and right (956 bp) TK flanking
sequences, deleting the central region of the TK gene (315 bp; codons
55 to 158) but leaving the N-terminal and C-terminal TK coding regions
intact and inserting a 2.4-kb reporter cassette (p72GUS) consisting of the ASFV p72 gene promoter linked to the GUS gene (34) (Fig. 1). GUS-positive foci were picked from
agarose overlays containing 100 µg of
5-bromo-4-chloro-3-indolyl--D-glucuronic acid (X-Gluc) per ml (48) and were plaque purified four to six times until PCR and Southern blot analysis (41) showed no evidence of
contamination with the parental virus.
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FIG. 1.
Diagram of the TK region of the Malawi LiL-20/1 genome,
showing the placement and orientation of the TK gene and adjacent genes
p10 (DNA binding protein 5-AR) (33), K205R, K145R, and K421R
(Ba71V open reading frames) (46); structure of the TK gene
deletion transfer vector, p5.3, showing insertion sites into the
pBluescript multiple cloning site; and structure of plasmid pLR-TK,
containing a 2,164-bp PCR fragment of the intact TK gene and flanking
ASFV DNA.
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Virus stocks were amplified on Vero cells, and viral DNA was prepared.
The structure of the TK deletion mutant (ASFV v5.3)
was revealed by
Southern blotting, yielding the predicted
BglII
5.1-kb band
representing the deletion of TK sequences and the
insertion of the
2.4-kb p72GUS cassette (Fig.
2A and B).
PCR analysis
of v5.3 DNA with appropriate primer pairs revealed only a
1,250-bp
recombinant-specific fragment (data not shown). These results
indicated the isolation of a virus, derived from a double-crossover
recombination, in which the p72GUS cassette replaced the central
part
of the TK gene. In similar fashion, a recombinant virus was
produced by
using the pathogenic, Vero-adapted H811 ASFV isolate
and the TK
deletion recombination vector (p5.3). This virus (ASFV
vH53) yielded
the predicted 4.3-kb
BglII fragment, compared to
the 2.2-kb
uninterrupted TK fragment of the parental virus (Fig.
2C).
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FIG. 2.
Southern blot analysis of parental, recombinant, and
revertant ASFV DNA digested with BglII. ASFV Malawi (A and
B) and ASFV Haiti (C) were probed with a TK probe
(HindIII fragment of pLR-TK) (A and C) or a GUS probe
(SmaI/SacI fragment of the p72GUS cassette)
(34) (B). Lanes 1, swine spleen isolate; lanes 2, Vero-adapted isolates; (lanes 3, TK deletion mutant viruses; lanes 4, revertant virus.
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The difficulty in isolating GUS-positive TK
recombinant
viruses on macrophages and initial growth analysis of ASFV recombinant
v5.3 on macrophages suggested a growth deficiency in macrophages.
Therefore, a positive growth selection method was attempted in
order to
rescue TK
+ revertants. A 2,164-bp fragment spanning the TK
gene was amplified
by using the left flank 5'-forward and the right
flank 3'-reverse
primer (described above) to construct the TK knockout
vector and
cloned into pBluescript, resulting in plasmid pLR-TK. This
plasmid
contained the complete TK gene with 734 bp of ASFV TK-flanking
sequences upstream and 865 bp downstream (Fig.
1). Swine macrophage
cultures (
16,
34) were infected with ASFV v5.3, transfected
with plasmid pLR-TK, blind passaged three times on swine macrophages,
and purified by endpoint dilution, resulting in virus v5.3R. Virus
stocks were made on swine macrophages, and viral DNA was prepared.
Southern blot analysis showed the presence of the native 3.0-kb
TK
fragment and no reactivity with the GUS probe (Fig.
2A and
B, lanes 4),
indicating restoration of the native TK
genotype.
TK is important for in vitro growth of ASFV on porcine
macrophages.
Growth properties of the Malawi (v5.3) and Haiti
(vH53) TK deletion mutants were compared to those of the respective
parental Vero-adapted viruses on swine macrophage and Vero cell
cultures. At a low multiplicity of infection (MOI = 0.01), the
parental isolates grew well on both Vero and macrophage cultures (data not shown). The TK deletion mutants grew well on Vero cells (data not
shown) but grew poorly on macrophages; Malawi v5.3 and Haiti vH53 grew
to 0.1 and 1% of parental levels, respectively (Fig. 3A and
B). However, at high MOIs (10 to 20), the
growth defect of the TK deletion mutants was not apparent (Fig. 3C and
D).
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FIG. 3.
Growth characteristics of ASFV Malawi and Haiti parental
(closed circles) and recombinant (open circles) viruses on primary
swine macrophages. Cells were infected at MOIs of 0.01 (A and B) and 10 to 20 (C and D) with the appropriate viruses (which were absorbed for
2 h at 37°C), rinsed twice with growth medium, and incubated. At
the indicated times, cultures were harvested and lysates were titrated
for total virus yield on Vero cells by the immunoperoxidase method
(49). (A and C) Growth of ASFV Malawi (parent) and TK
deletion mutant v5.3; (B and D) growth of ASFV Haiti H811 (parent) and
TK deletion mutant vH53. Data represent the TCID50
titers ± standard errors of the means (14) assayed for
two or three independent experiments.
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To confirm the macrophage growth deficiency of ASFV TK deletion
mutants, high-titer Vero cell stocks of the mutant and parental
viruses
were serially passaged four times on macrophage cultures
by using large
inoculum volumes. Cultures were observed for cytopathic
effect (CPE),
and cell lysates were monitored for GUS expression
(GUS-positive
mutants) and titrated for ASFV. As expected, the
parental ASFVs caused
complete CPE through each passage and maintained
high titers on
macrophage cell passage (Fig.
4).
Positive CPE
and GUS expression were observed for ASFV v5.3 on the
first passage,
and trace levels were observed on the second passage;
ASFV vH53
showed CPE on the first and second passages, and low levels
of
GUS were present through the third passage. Infectivity titers
of
the TK deletion mutants dropped successively at each passage
to low
levels, with somewhat higher titers of Haiti vH53 remaining
(Fig.
4B).
The viruses were also compared in a plaque assay on
swine macrophages.
Dilutions of virus were plated and overlayered
as described above for
isolation of recombinants, but without
X-Gluc in the overlays. At 6 days, the cultures were fixed with
formalin, the agar was removed, and
the cells were stained with
crystal violet. Figure
5 shows that for each virus, the plaque
size was substantially reduced for the respective TK
mutants.
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FIG. 4.
Virus titers obtained on successive passages of parental
(closed circles) and TK deletion mutant (open circles) viruses on
primary swine macrophages. (A) ASFV Malawi viruses; (B) ASFV Haiti H811
viruses. Cells in T-25 flasks were inoculated with 1 ml (passage
[pass] 1) or 2 ml (passages 2, 3, and 4) of virus (which was absorbed
for 2 h), rinsed twice with growth medium, and incubated for 2 to
4 days. Cultures were observed for CPE, and cells and medium were
harvested at 2 days (passage 1) or 4 days (passages 2, 3, and 4); then
culture lysates were examined for GUS expression, and
TCID50 titers were determined by the immunoperoxidase
method. Titers ± standard errors of the means are the results of
three independent experiments.
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FIG. 5.
Plaques formed on primary swine macrophages by parental
Malawi (A) and Haiti (B) ASFV and by TK deletion mutants Malawi v5.3
(C) and Haiti vH53 (D).
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Amounts of viral DNA synthesized by Vero Malawi and the ASFV
TK
mutant v5.3 grown on Vero cell and macrophage cultures
were examined
by dot blot hybridization (
41). Cells were
infected at MOIs
of 1 to 2, DNA was isolated from cells at 24 h
postinfection (p.i.),
and dilution sets of DNA were blotted onto Zeta
Probe membranes
(Bio-Rad) and probed with a
32P-labelled
2,164-bp TK region DNA fragment (
SalI/
NotI-cut
DNA
from pLR-TK). On macrophages, the parental ASFV Malawi consistently
produced large amounts of viral DNA. For v5.3 no viral DNA was
detected
in two experiments, and in a third, a small amount of
v5.3 DNA was
present over background levels measured at 2 h p.i.
In comparison,
both the Vero-adapted ASFV Malawi and the TK
mutant v5.3
synthesized large amounts of viral DNA on Vero cells
(data not
shown).
Cellular levels of TK are very low in quiescent, nondividing cells but
increase dramatically during cell division (
37),
and TK
activity in primary macrophage cultures is very low, even
during
prolonged incubation in complete growth medium (
31).
With
pools of nucleoside triphosphates low in macrophages, the
loss of viral
TK activity might be expected to have a significant
effect on viral DNA
synthesis and subsequent production of viral
progeny. The TK, RR, and
TMPK enzymes have all been implicated
in attenuation of poxviruses
(
4,
7,
19) and herpesviruses
(
5,
20,
22). For
ASFV, the levels of expression of these
enzymes in different isolates
of ASFV are not known, and each
may contribute to viral replication in
quiescent cells such as
macrophages. Here, in virus growth experiments,
the growth defect
of the TK mutants was overcome at high MOIs. The
levels of RR
found in ASFV-infected Vero cells were shown to be
proportional
to the MOIs, and inhibition of DNA replication did not
alter the
levels of RR present (
10). Thus, early
transcription of RR at
high MOIs could supplement the TK deficiency by
providing an alternate
source of
deoxythymidylate.
In the growth curve experiments, the TK deletion mutants grew poorly at
low MOIs compared to the parental viruses (Fig.
3A
and B). However,
since there was some increase in virus titers
over the first several
days, we cannot formally exclude the possibility
that, after a single
round of replication, subsequent poor growth
was due to failure of the
virus to be released and to infect adjacent
cells. However, the nature
of growth defects of TK deletion mutants
of other DNA viruses is linked
to nucleotide metabolism and subsequent
viral DNA synthesis.
Furthermore, the passage experiments performed
here showed that despite
harvest of virus from cell lysates and
reapplication to new cells,
virus titers diminished (Fig.
4).
The results of the growth experiments
and the diminished levels
of DNA synthesized at low MOIs with v5.3
suggest that TK is important
in macrophages for replication of viral
DNA in the cytoplasm of
these
cells.
TK affects ASFV virulence for swine.
To assess the importance
of the TK gene in viral virulence, two separate groups of four
Yorkshire pigs were inoculated intramuscularly with 104
50% tissue culture infective doses (TCID50) of the
revertant ASFV v5.3R or the TK mutant ASFV v5.3 and were
observed for clinical signs of ASF: fever, anorexia, lethargy,
shivering, cyanosis, and recumbency. A dose of 104
TCID50 of pathogenic ASFV strains represents a challenge of
1,000 to 10,000 LD100s (34, 48). Results of the
experiment are shown in Table 1. The
animals inoculated with the revertant virus showed signs typical of
acute ASF, with onset of fever at day 4 and all animals dying between
days 9 and 13. The swine inoculated with the mutant virus v5.3 showed
only transient fever responses, and one animal, which had the most
persistent fever, died at day 15. Except for transient fevers, the
remaining animals were clinically normal throughout the experiment.
Maximum viremia titers in the revertant virus group reached
106 to 107 TCID50, whereas the
titers for the mutant v5.3 group ranged from <103.5 to
106 TCID50, with viremia onset delayed 4 to 5 days. DNA from virus isolated at the peak of viremia from the
recombinant virus group was examined by PCR and found to be that of the
recombinant virus, with no wild-type virus DNA detected (data not
shown). By using a virus isolation-PCR-blot detection method
(6), virus was detected in only one animal of the ASFV v5.3
group at day 30 p.i. and none of the animals was positive at day
56 p.i. (data not shown). At day 56 p.i., the three surviving
v5.3-infected swine were challenged intramuscularly with
104 TCID50 of the parental Vero-adapted Malawi
virus. A fever response was noted in two of the three animals on days 4 to 6, and one animal remained febrile following this time. That animal
developed an infected abscess at the inoculation site which advanced to a severe cellulitis (the animal was sacrificed on day 21). Maximum viremia titers ranged from undetectable levels to levels lower than
104 TCID50/ml, and all animals remained free of
clinical symptoms for the experiment's duration.
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TABLE 1.
Swine survival, viremia, and fever response following
infection with ASFV Malawi revertant and TK deletion
mutant v5.3a
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The results of the animal experiment indicate that loss of the TK gene
reduced the virulence of ASFV for domestic swine and
that swine that
recovered from infection with the TK
ASFV v5.3 were
protected from developing ASF on virulent virus
challenge. Recently,
another ASFV gene, 23-NL-S, was shown to
be a virulence-associated gene
for ASF (
48). However, ASFV 23-NL-S
was nonessential for
growth in vitro in Vero cells and swine macrophages,
but deletion of
the gene significantly reduced virulence for swine,
suggesting that the
gene may serve as a host range gene in swine.
Work with tissue
culture-adapted isolates of ASFV established
that TK was nonessential
for growth in Vero cells (
27,
40).
However, the present
report shows the TK gene to be important
for growth on macrophages in
vitro and to be a virulence factor
in vivo. Thus, TK should be
considered an essential gene for the
target lymphoreticular tissues
involved in the pathogenesis of
lethal ASFV infections. However, it is
not known at this time
if loss of the TK gene affects the ability of
the virus to infect
lymphoreticular tissues and organs involved in the
pathogenesis
of lethal ASF (
8,
24,
25,
30,
32) or if a
reduction
in virus load in swine may allow time for the development of
host
defenses.
The TK locus has been used to demonstrate the feasibility of
constructing ASFV recombinant viruses (
15,
27,
40), but
these experiments used highly adapted, cell-cultured viruses no
longer
virulent for swine, so the role of TK in the virulence
and pathogenesis
of ASFV in swine could not be assessed. Several
low-passage-number
isolates of ASFV retaining their infectivity
for swine have been
engineered with chromogenic marker genes inserted
into the TK locus,
and their use in tropism, pathogenicity, and
latency studies has been
suggested (
18). We have observed CPE,
the expression of GUS
driven by an ASFV late gene promoter, and
positive hemadsorption (late
expression of the CD2 gene) with
TK deletion mutants in swine
macrophages, but the virus grows
poorly and shows reduced virulence in
swine. The present work
shows that the TK gene itself plays a
significant role in virus
virulence and that this site would not be
suitable for the insertion
of markers to monitor pathogenesis or as an
insertion site for
other viral genes to assess their role in virus
virulence.
We have demonstrated that the selective macrophage growth advantage of
TK
+ ASFV may be used to engineer other ASFV gene deletions
through
TK
+ rescue of the ASFV mutant v5.3 (
35).
Plasmids in which the
intact TK gene with its upstream promoter
sequence replaced a
specific gene of interest were engineered.
Preliminary experiments
indicate that stable GUS
+
TK
+ recombinants which could be directly isolated through
selective
growth on macrophage cultures were produced. These results
demonstrate
the utility of using the TK gene as a selective factor in
genome
manipulations of pathogenic isolates of ASFV and also confirm
that the growth defect of ASFV v5.3 in macrophages was directly
due to
the loss of the TK gene
function.
Animals previously infected with the TK deletion mutant were resistant
to challenge with virulent ASFV Malawi, although the
level of
attenuation of the TK deletion mutant would not render
it suitable for
use as a vaccine. Better understanding of the
role of TK, RR, and TMPK
enzymes in supplying pools of deoxynucleoside
triphosphates for viral
DNA replication may define how these viral
enzymes support the
replication of the virus in vivo. Modifications
of the viral genes
controlling DNA metabolism in infected cells
may be a valuable
component in the design of multiple-gene-deletion,
attenuated live
virus vaccines for
ASFV.
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ACKNOWLEDGMENTS |
We thank G. Kutish for assistance with sequence and statistical
data analysis, T. Burrage for assistance in titration experiments, E. Kramer and R. Mireles for swine macrophage cell cultures, and the PIADC
animal care staff for assistance with animal experiments.
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FOOTNOTES |
*
Corresponding author. Mailing address: Plum Island
Animal Disease Center, Agricultural Research Service, U.S. Department
of Agriculture, P.O. Box 848, Greenport, NY 11944-0848. Phone: (516) 323-2500, ext. 306. Fax: (516) 323-2507. E-mail:
dmoore{at}asrr.arsusda.gov.
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Journal of Virology, December 1998, p. 10310-10315, Vol. 72, No. 12
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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