Plum Island Animal Disease Center,
Agricultural Research Service, U.S. Department of Agriculture,
Greenport, New York 11944,1 and
Department of Epidemiology and Public Health, Yale
University School of Medicine, New Haven, Connecticut
065202
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INTRODUCTION |
African swine fever (ASF) is a
highly lethal disease of domestic pigs for which animal slaughter and
area quarantine are the only methods of disease control. African swine
fever virus (ASFV), the causative agent of ASF, is a large
double-stranded DNA virus which is the only member of an unnamed family
of viruses. ASFV is the only known DNA arbovirus (4, 6, 12).
The natural arthropod host for ASFV is Ornithodoros porcinus
porcinus (Walton) ticks (40). Some confusion exists in
earlier reports since ticks that should be classified as O. porcinus porcinus are often referred to as either O. moubata
porcinus or simply O. moubata (59).
ASFV can infect hosts through either a sylvatic cycle or a domestic
cycle. In the sylvatic cycle, ASFV infects warthogs (Phacochoerus aethiopicus) and bushpigs (Potamochoerus spp.) as well
as ticks of the genus Ornithodoros (7-10, 36,
55). In sub-Saharan Africa, warthogs occupy burrows which are
frequently infested with large numbers of O. porcinus
porcinus ticks (38, 45, 57, 58), and a correlation,
though not absolute, has been established between ASFV infection of
warthogs and the presence of O. porcinus porcinus ticks in
burrows (57). In ASFV-enzootic areas, adult warthogs are
typically nonviremic, although most are seropositive (28, 41, 46,
53, 58), and virus can usually be isolated only from lymph nodes
(28, 41). Young warthogs, which are confined to the burrow
for the first months of life, are most likely to be infected through
feeding of infected O. porcinus porcinus ticks. Infection in
young warthogs is subclinical, with viremic titers ranging from 2 to 3 log10 50% hemadsorption dose (HAD50)/ml
(56, 57), a level sufficient to infect a low percentage of
naive ticks (42, 58, 30). The sylvatic ASFV cycle is further
maintained by transovarial (43) and venereal (44)
transmission in ticks. In burrows containing ASFV-infected ticks,
infection rates are typically low (<2%), with the highest rate
occurring in adult females (40, 45, 57, 65). The mechanism
of ASFV transmission from the sylvatic cycle in Africa to the domestic
cycle is most likely through feeding of infected ticks on pigs
(41, 58), since direct contact between infected warthogs and
domestic pigs has failed to result in transmission (36, 10, 28,
58), except in a single case (8). The virus may be
transmitted between domestic pigs by either direct or indirect contact
(33).
Various characteristics of ASFV infection have been studied in a number
of Ornithodoros spp. ticks. The first association of ASFV
with a tick was made by Sanchez-Botija (50), who reported isolation of ASFV from O. erraticus, a tick native to the
Iberian peninsula and later considered important to maintenance of ASFV in an enzootic cycle in that region (51). In the first
experimental infection, striking differences were found in the
percentage of O. moubata porcinus ticks infected by two
different ASFV isolates, a low infectious dose for ticks (ranging from
of 0.9 to 4 log10 HAD50) was demonstrated, and
transmission out to 469 days postinfection (p.i.) was successful with
single ticks (42). Experimental ASFV infection and
transmission to pigs has been demonstrated for O. savignyi,
a tick found in Africa (34), O. coriaceus
(23, 25) and O. turicata (25), ticks
indigenous to the United States, and O. puertoricensis
(25, 14), a tick indigenous to the Caribbean. A 40%
mortality rate was found in infected O. coriaceus
(25) and O. puertoricensis ticks (15).
O. marocanus, which was formerly referred to as O. erraticus, transmitted ASFV out to 588 days p.i., although 73%
mortality was reported for infected ticks (16, 17). A number
of reports have not found significant virus-induced mortality in
O. moubata porcinus ticks (22, 40-44). In
contrast, mortality rates were 35% higher in infected O. moubata
porcinus females in the only study to examine mortality during the
gonotrophic cycle (26).
Specific aspects of ASFV infection in the natural host remain poorly
understood. Greig (22) experimentally infected O. moubata porcinus ticks with pathogenic ASFV isolates and used
virus titration and immunofluorescence of dissected tissues to
determine that the midgut was the initial site of viral replication and
the site of longest persistence. Several other tissues were also found to have detectable levels of virus, although the midgut was the only
tissue which was consistently positive. The presence of ASFV has been
demonstrated in hemocytes of infected O. coriaceus ticks by
electron microscopy and immunofluorescence studies, but the presence or
nature of virus replication was not addressed (13).
Here we describe the pathogenesis and persistence of ASFV infection in
O. porcinus porcinus ticks. Our data indicate that initial
ASFV replication occurs in phagocytic digestive cells of the midgut
epithelium, with secondary replication occurring in undifferentiated
midgut cells at later times p.i. Generalization of virus infection from
the midgut to other tick tissues required 2 to 3 weeks. Secondary sites
of virus replication include hemocytes (type I and II), coxal gland,
salivary gland, connective tissue, and reproductive tissue. Successful
tick-to-pig transmission correlated with relatively high viral titers
in salivary and coxal glands. Persistent infection in the tick involves
continuous viral replication in several tissues and is associated with
minimal cytopathology.
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MATERIALS AND METHODS |
ASFV isolates.
Chiredzi/83/1 (Ch1) was isolated from
Ornithodoros spp. ticks collected near Chiredzi, Zimbabwe
(26), and was obtained from the Plum Island Animal Disease
Center reference collection. Pretoriuskop/96/4/1 (Pr4) and
Crocodile/96/1 (Cr1) were isolated from O. porcinus porcinus
ticks collected from warthog burrows in Kruger National Park, Republic
of South Africa, in September 1996. Nooitverwacht/96/6 (No6) was
isolated from O. porcinus porcinus ticks collected from a
warthog burrow in the Northern Province, Republic of South Africa, in
September 1996. For Pr4 and Cr1, virus isolations were made by
homogenizing individual ticks or small pools of ticks in 0.5 ml of
medium (RPMI 1640; Gibco BRL) supplemented with 20% fetal bovine serum
(HyClone Laboratories, Inc.) and 1× antibiotic-antimycotic (Gibco BRL)
in sterilized Ten Broeck grinders. The homogenates were centrifuged at
10,000 × g for 1 min. The supernatants were diluted
1:10 and 1:100, and 100 µl was placed in multiple wells of a 96-well
plate (Primaria) containing porcine peripheral blood mononuclear cells
prepared as previously described (21, 37). After 24 h,
20 µl of diluted (2% [vol/vol]) porcine erythrocytes was added to
each well, and samples were monitored for hemadsorption and cytopathic
effect for a minimum of 7 days. For isolate No6, repeated virus
isolation attempts from approximately 200 ticks, in pools of 3 to 10 ticks, failed to yield a virus isolate. Subsequently, 200 additional
ticks from the same collection were allowed to feed on a naive pig. The
pig developed ASF, and virus was isolated from the spleen and blood.
Restriction endonuclease analysis of genomic DNA from all isolates
confirmed the presence of a double-stranded DNA genome approximately
180 kbp in length (30).
Ticks.
All ticks used in these experiments were O. porcinus porcinus (59). Ticks used for experiments with
Ch1 isolate were from a colony maintained for an indeterminate period
of time at the Plum Island Animal Disease Center. Ticks used for
experiments with ASFV isolates Cr1, Pr4, and No6 were field-collected
nymphs (stage N2 or N3) from the same collections which yielded the
respective virus isolate. Natural ASFV infection rates for each
collection were less than 2%. All ticks were held en masse in
polycarbonate jars with a sand substrate. Jars were maintained in
sealed desiccator boxes over a saturated NaCl solution, which provided
a relative humidity of approximately 78%, at 26°C. Desiccator boxes
were held in a diurnal chamber with 12-h light/12-h dark photoperiod.
A membrane feeding apparatus for individual tick feedings was patterned
after that of Mango and Galun (32). Individual ticks were
fed by placing each one in a polystyrene chamber made from the top end
of a culture tube. This chamber was sealed at one end with stretched
Parafilm M. The sealed end was placed in a well of a 12-well tissue
culture plate which held 0.5 ml of heparinized pig blood. The blood was
maintained at 39°C by floating the plate in a water bath for the
duration of the feeding. Ticks were allowed to feed to repletion before
being removed from the membrane and placed into a sterile multiwell
tissue culture plate for coxal fluid collection. Ticks were observed
for 2 h; if coxal fluid had not been produced by this time, the
ticks were moved to a holding container. If coxal fluid was produced,
it was collected by flushing the well with 0.5 ml of medium. Following
feeding, blood from beneath the membrane was collected; the well and
membrane were washed with an additional 0.5 ml of medium, and this was added to the blood sample. Samples were held at 
70°C until assayed for virus titer as described above.
Infection of ticks.
Three groups of ticks were fed on
separate pigs infected by intramuscular injection with 2 log10 HAD50 of the ASFV isolate Ch1. Viremic
titers on the day of tick feedings were 7.7 ± 0.2, 8.0 ± 0.3, and 7.9 ± 0.2 log10 HAD50/ml for the
three feedings on Ch1-infected pigs, 8.3 ± 0.3 log10
HAD50/ml for the feeding on the Pr4-infected pig, 8.3 ± 0.3 log10 HAD50/ml for the feeding on the
Cr1-infected pig, and 7.8 ± 0.2 log10
HAD50/ml for the feeding on the No6-infected pig. Tick
feedings were conducted on pigs anesthetized with xylazine and ketamine
HCl (1 mg/lb of body weight, intravenously) 5 to 6 days p.i. During the
feeding, ticks were contained in chambers, made from sections of
polyvinyl chloride pipe with wire mesh glued to either side, which were held in contact with the medial surface of the hindlimb. Only fully fed
ticks were used for subsequent experiments.
Virus titrations.
Individual whole ticks were ground in 0.5 ml of medium in sterilized Ten Broeck grinders. The samples were stored
at 70°C. Immediately prior to titration, samples were thawed at
37°C, sonicated for 1 min, and centrifuged for 1 min at 10,000 × g. Supernatants were serially diluted, and replicate
samples were added to porcine peripheral blood mononuclear cells as
described above. Titers were calculated by the method of Reed and
Muench (49).
To determine the virus titers in isolated organs, ticks were dissected
under a binocular microscope and tissue samples were taken for
determination of virus titer as follows. Individual ticks were weighed
on an analytical balance so that titers could be reported on a
per-milligram-of-tick-body-weight basis. Ticks were placed in a petri
dish containing black dissecting wax ventral side down in a depression
melted into the wax surface and held in place by forcing the legs into
the softened wax. Immobilized ticks were covered with 100 µl of
Grace's insect cell culture medium. After an incision was made through
the cuticle and around the lateral edge, the dorsal cuticle was
removed. After removal of the cuticle, as much of the 100 µl of
medium as could be collected was withdrawn and saved for assay of virus
titer; this sample contained hemolymph, leaked gut and rectal sac
contents, and any coxal fluid produced by the tick as it was being
attached. The tick was then immersed in fresh medium, and any remaining
leaked gut contents and rectal sac contents were washed away to
facilitate dissection. The following organs were removed and saved for
assay of titer: the midgut and all of the attached ceca, paired
salivary glands, paired coxal glands (without the associated coxal
accessory gland), the gonads (testis, ovary, or the immature organ from preadult ticks, excluding the accessory gland or Gene's organ), and
the synganglion. Following removal, each tissue was washed three times
in sterile Grace's insect cell culture medium and placed in an
Eppendorf tube with 100 µl of medium. Following dissection, the
tissues were ground with sterile plastic pestles (Pellet pestle; Kontes). The cuticle and any remaining tissues (including but not
limited to the rectal sac, Malpighian tubules, tracheal system, muscles, and connective tissues) were saved and processed as described above. Viral titers were determined as described above.
To determine the viremic titer of infected pigs, heparinized blood
drawn from the anterior vena cava was held at 70°C until used,
thawed at 37°C, sonicated for 1 min on ice, serially diluted, and
added to cells as described above.
Histological and ultrastructural procedures.
At times p.i.,
ticks were immobilized in paraffin wax, surrounded by a pool of
ice-cold fixative, and sliced in half along the sagittal plane to allow
infiltration of the fixatives and embedding resins (19). To
ensure complete infiltration of the embedding medium a small portion of
the front and back cuticle was removed. Ticks were fixed with a
solution containing 2.5% glutaraldehyde in 0.1 M sodium cacodylate
buffer (pH 7.4) for 2 h at 4°C and further processed with 2%
osmium tetroxide for 2 h at 4°C followed by 2% aqueous uranyl
acetate overnight at 4°C. Ticks were dehydrated in ethanol and/or
ethanol with propylene oxide and embedded in either Spurr's resin or
Embed 812 (Electron Microscopy Sciences, Port Washington, Pa.). For
ultrastructural studies, 70- to 90-nm sections were collected on single
slot grids coated with Formvar, stabilized with carbon (Electron
Microscopy Sciences), and photographed with a Philips 410 electron
microscope operated at 80 kV. Nymphal ticks were sampled most
extensively in the front half, which contained two-thirds of the midgut
and its associated diverticula, salivary glands, coxal glands,
synganglion, and portions of the Malpighian tubules. At least four
sections from each of two ticks were sampled for each time point. For
adult ticks, reproductive organs were isolated by dissection then
processed and examined as described above.
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RESULTS |
Infection of O. porcinus porcinus ticks with ASFV.
Nymphal O. porcinus porcinus ticks (stage N2 or N3) were fed
on domestic pigs infected with ASFV isolate Ch1. Titration of individual ticks immediately after feeding indicated they were infected
with a mean dose of 5.7 log10 HAD50 of ASFV/mg
of tick body weight. At times p.i. ranging from 6 h to 290 days,
whole ticks or dissected tick tissues were titrated for infectious
virus (n = 3) and examined ultrastructurally for
evidence of ASFV replication (n = 2). Virus titration
data are shown in Fig. 1, and
ultrastructural findings are summarized in Table
1. All ticks examined over the course of
this study (n = 78) were found to be ASFV infected.
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FIG. 1.
ASFV titers in Ch1-infected ticks. Whole ticks and
dissected tick midguts (A) plus additional tissues from the same ticks
(B) were titered at times p.i. Values are expressed as mean titers ± standard errors of the means. Unsuccessful () and successful (+)
tick-to-pig transmission attempts are indicated.
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ASFV infection of O. porcinus porcinus was not associated
with a significant increase in tick mortality. A group of ASFV infected ticks (n = 60) held separately for the duration of the
study (290 days) exhibited a cumulative mortality rate of 6.6%,
compared to 2.9% for an age- and feeding status-matched control group
(n = 35). Additionally, no mortality was observed for
ticks used in the pathogenesis experiments.
Initial ASFV replication occurs in phagocytic digestive cells of
the midgut.
Titration of individual whole ticks at 1, 2, 3, 6, 9, and 15 days p.i. demonstrated that virus levels decreased slightly, to
4.8 log10 HAD50/mg at 6 days p.i., before
increasing to 6.2 log10 HAD50/mg at 15 days
p.i. (Fig. 1A). Within 24 h postfeeding, ultrastructural analysis
demonstrated phagocytic digestive midgut cells containing erythrocytes
within phagolysosomes. Virions were observed adsorbed to erythrocytes,
both within the phagolysosomes and in the midgut
lumen (Fig. 2A, B, and D). Beginning at 4 days p.i., nascent virus factories were observed in the cytoplasm of phagocytic digestive cells (Fig. 2C). Virus factory structures were
characterized by a small number of curvilinear (partially formed) viral
capsids in electron-lucent regions of the cytoplasm (Fig. 2E). After 9 days p.i., virus factories were consistently observed in the phagocytic
digestive cells of the midgut (Table 1). At 9 to 15 days p.i., infected
cells containing large numbers of viral particles were projecting into
the lumen (Fig. 3). Sloughed infected
epithelial cells were also observed in the midgut lumen (data not
shown). At 21 days p.i., infection of less differentiated midgut
epithelial cells was noted (Fig. 4A).
Peak numbers of infected midgut cells were observed at 21 days p.i.
These data indicate that phagocytic midgut epithelial cells are the
initial site of virus replication, replication in less differentiated
cell types occurs at later times, and the midgut contains the majority
of ASFV through 28 days p.i.
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FIG. 2.
ASFV uptake and early stages of replication in
midgut digestive cells. (A) At 24 h postfeeding, intact
erythrocytes have been taken into phagolysosomes (p) from the blood
meal in the lumen (L). Hematin crystals (arrows) are scattered
throughout the cytoplasm. Undifferentiated epithelial cells (asterisks)
occur on either side of the digestive cell. N, nucleus of digestive
cell. Bar, 5 µm. (B) Phagolysosome (p) of a digestive cell 72 h
postfeeding. Ingested erythrocyte has an enveloped ASFV particle
associated with it (arrow). Bar, 0.5 µm. (C) Nascent virus factory in
digestive cell at 96 h postfeeding. Curvilinear forms of
assembling virus particles occur in a more electron-lucent region of
the cytoplasm. p, phagolysosomes; bar, 0.5 µm. (D) High-magnification
view of ASFV particle (arrow) in panel B showing possible deterioration
of virus structure. Bar, 0.2 µm. (E) High-magnification view of
nascent virus factory in panel C. Bar, 0.2 µm.
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FIG. 3.
Replication of ASFV in midgut digestive cells at 15 days
postfeeding. (A) A digestive cell with an extensive virus factory
projects into the midgut lumen (L) and is separated from
undifferentiated epithelial cells (asterisks) via septate junctions
(arrowheads). H, hemocoel; M, muscle; bar, 5 µm. (B)
High-magnification view of the mature virus factory in panel A showing
virus in various stages of assembly. Virions (arrows) are budding into
the lumen (L). Bar, 0.5 µm.
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FIG. 4.
Replication and generalization of ASFV in
undifferentiated midgut cells at 21 days postfeeding. (A) Three
undifferentiated epithelial cells with virus factories (large arrows).
M, muscle; H, hemocoel; L, lumen; bar, 5 µm. (B) Accumulation of ASFV
under the basal lamina (Bl). Arrow, virus free in the hemocoel (H);
bar, 1 µm.
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Generalization of ASFV infection requires 15 to 21 days.
Before 15 to 21 days p.i., only midgut tissues exhibited
ultrastructural evidence of virus infection and replication. At 21 days
p.i., undifferentiated cells of the midgut contained numerous virus
factories. A large number of mature virions were observed both adjacent
to and within the basal lamina (BL) of the midgut, with an occasional
virion on the hemocoel side of the BL, suggesting a possible route of
movement of virus from the primary site of replication, the midgut,
across the BL into the hemocoel (Fig. 4B). At 15 to 21 days p.i.,
ultrastructural analysis demonstrated the first appearance of viral
replication in both type I and type II hemocytes and connective tissue
(Fig. 5). At this time point, and all
subsequent time points, infected hemocytes were observed with virions
budding from the plasma membrane (Fig. 5B). Virus titration data from
individual ticks tissues at 21 days p.i. support the ultrastructural
data described above. At 21 days p.i., high viral titers were detected
only in the midgut (Fig. 1A). However, small amounts of virus (2 log10 HAD50/mg of tick) were consistently found
in the salivary gland at this time, indicating that virus infection of
this tissue had already occurred (Fig. 1B). These data suggest that
critical early events in midgut are necessary for successful
generalization of ASFV infection in O. porcinus porcinus
ticks.
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FIG. 5.
ASFV in hemocytes. (A) Type I hemocyte containing an
extensive virus factory with many crystalline arrays at the periphery.
Bar, 1 µm. (B) Type II hemocyte with virus factory and budding virus
particles (arrows). Bar, 0.5 µm.
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Generalization and persistence of ASFV infection in O. porcinus porcinus ticks.
Whole tick titers, which after 21 days p.i. were calculated by summation of the titers of all dissected
and undissected tissues, reached their peak (6.4 log10
HAD50/mg of tick) at 28 days p.i. and were maintained at
this approximate level throughout the 290-day sampling period (Fig.
1A). Virus titers in the midgut increased to 6.1 log10
HAD50/mg of tick at 28 days p.i., the highest titer detected for this tissue, and then persisted at titers of approximately 5.0 log10 HAD50/mg of tick for the duration of
the study.
Coxal gland titers of ASFV rose to peak levels of approximately 4.5 log10 HAD50/mg of tick by 70 days p.i. and
persisted at this level throughout the 290-day experimental period
(Fig. 1B). Virus replication was observed in both cells of the
filtration membrane and cells of the collecting tubule of the coxal
gland, and numerous virions were observed budding into the lumen of the filtration membrane (Fig. 6). Viral
titers in the reproductive tissue continued to rise through 28 weeks
p.i. In males, virus was observed budding from the accessory gland and
the connective tissue lining the sperm ducts. In females, active virus
replication was observed in nurse cells and in developing oocytes (data
not shown).
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FIG. 6.
ASFV in coxal gland. (A) A small virus factory (Vf),
mature particles (arrows), and a condensed-chromatin-containing nucleus
in the proximal tubule of the coxal gland. Lt, lumen of tubule; bar, 1 µm. (B) Cell of the filtration membrane portion of coxal gland with a
virus factory (Vf) and released virions (arrows). Bar, 0.5 µm.
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ASFV titers in salivary gland increased 10,000-fold between 21 and 112 days p.i., reaching a peak titer of 6.1 log10
HAD50/mg of tick. Similar high ASFV titers were detected at
later sampling points (Fig. 1B). Virus replication was first observed
in the connective tissue of the salivary gland at 21 days p.i. (Fig. 7B). At 42 days p.i., virus factories and mature virions were observed
in the granular cells of the salivary gland, with virions accumulating
in secretory granules (Fig. 7C). At later
times, these observations were more pronounced.
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FIG. 7.
ASFV in salivary gland. (A) Mock-infected salivary gland
at 30 days postfeeding to show tissue structure. The salivary gland is
composed of agranular (Ag) and granular (Gr) portions. Connective
tissue cells occur at the edge and in the center of the gland. D,
cuticle-lined ducts. (B) At 30 days p.i., replicating ASFV is found
only in the connective tissue cells in the center of the gland.
Secretory granules (arrows) do not contain virus. (C) At 42 days p.i.,
virus is observed budding into secretory granules (arrows). Bars: panel
A, 5 µm; panels B and C, 1 µm.
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In addition to cells of specific organs, connective tissue cells
surrounding most organs and tissues were observed to contain replicating virus, which may have contributed to the viral titers of
dissected tissues. Most notably, the connective tissue surrounding the
synganglion contained replicating virus, although virus was never
observed in the nervous tissue proper. In addition, evidence of virus
replication was not detected in either the Malpighian tubules or
skeletal muscle.
Successful tick-to-pig transmission of ASFV correlates with high
viral titers in salivary and coxal glands.
Virus transmission was
first attempted at 21 days p.i. by feeding 19 ticks on an anesthetized,
naive pig (Table 2). The pig failed to
demonstrate any signs of ASFV infection over a 21-day observation
period. Subsequent attempts at virus transmission via tick feeding at
48, 71, 92, and 187 days p.i. were successful, with characteristic ASF
disease in infected pigs. The time to onset of febrile response ranged
from 2 to 4 days, with death occurring from 6 to 8 days postfeeding
(Table 2). These transmission results correlate well with the dramatic
rise of viral titers in salivary and coxal glands that occurred between
21 days p.i., a time when transmission was unsuccessful, and the time
of successful transmission at 48 days p.i. (Fig. 1B). Additionally,
ASFV virions were first observed in secretory granules of the salivary
gland and in the coxal gland of infected ticks at 42 days p.i. (Fig. 6
and 7C).
To determine the potential route(s) of virus transmission and to
quantitate the level(s) of virus excreted, ticks were individually fed
on artificial membrane feeders. Titratable virus was detected in both
the salivary secretion and the coxal fluid excreted following feeding
(Table 3). At 56 days p.i., salivary
secretions from 4 of 13 (31%) ticks contained titratable virus, with
titers ranging from 0.8 to 2.0 log10 HAD50. All
coxal fluid samples collected (n = 6) at this time
contained virus, with titers ranging from 1.0 to 4.8 log10
HAD50. At 84 days p.i., salivary secretions from 10 of 18 (56%) ticks contained virus, with titers ranging from 1.0 to 4.0 log10 HAD50. All coxal fluid samples collected
(n = 8) contained virus, with titers ranging from 1.8 to 4.3 log10 HAD50.
Infection of O. porcinus porcinus with ASFV field
isolates.
Experiments similar to those described for the ASFV
isolate Ch1 in colonized O. porcinus porcinus ticks were
performed with ASFV isolates and O. porcinus porcinus ticks
that were obtained from the same warthog burrow. Groups of ticks were
fed on anesthetized pigs previously infected with 2 log10
HAD50 of the Pr4, Cr1, and No6 virus isolates. As with Ch1,
tick infection rates with the Pr4, Cr1, and No6 isolates were 100%. At
227 days p.i., the cumulative mortality rates for Pr4-, Cr1-, and
No6-infected ticks were 6.8% (n = 44), 1.9%
(n = 52), and 11.0% (n = 63),
respectively. Individual ticks (n = 3) were dissected
at 3, 7, and 13 weeks p.i. Titration of dissected tissues revealed a
pattern of virus infection and persistence similar to that found with
Ch1-infected ticks (Fig. 8). At 21 days
p.i., the midgut contained 5.4 to 6.5 log10
HAD50/mg of tick body weight, while the synganglion,
salivary gland, coxal gland, and reproductive tissue contained from 1 to 3 log10 HAD50/mg of tick body weight. By 7 weeks p.i., salivary gland titers had increased over 100-fold, coxal
gland titers had increased 10- to 50-fold, reproductive tissue titers
had increased 100-fold, and synganglion titers had increased 50-fold.
On average, midgut titers at 7 weeks p.i. were the highest of any
tissue examined, with values ranging between 5.2 and 6.3 log10 HAD50/mg of tick body weight. At 13 weeks
p.i., salivary gland titers had increased further to 4.9 to 5.2 log10 HAD50/mg of tick body weight. Ticks infected with Pr4, Cr1, and No6 isolates (n = 20/isolate) successfully transmitted virus to naive pigs at 56 days
p.i. A typical ASF disease course was observed for infected animals.
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FIG. 8.
Tissue titrations of ASFV-infected O. porcinus
porcinus ticks. O. porcinus porcinus ticks (stages N2
to N4) collected from natural habitat were infected with ASFV isolates
(Pr4, Cr1, and No6). At times p.i., ticks were dissected and tissue
titers were determined. Values are expressed as mean titers ± standard errors of the means. Saliv., salivary; gl., gland.
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DISCUSSION |
In this report, we have described characteristics of ASFV
infection, replication, dissemination, and transmission in O. porcinus porcinus ticks. Most work on the pathogenesis and
dissemination of arboviruses within their vectors has been done with
mosquitoes (64). However, ticks differ fundamentally from
other hematophagous insects in their life history, feeding behavior,
and digestive physiology and therefore pose different questions
regarding virus-vector interactions. Few studies have addressed
characteristics of arboviruses pathogenesis in ticks (1, 2, 5, 11,
22, 35).
After infection, the initial site of ASFV replication is in phagocytic
digestive cells of the midgut epithelium. Similarly, Dugbe virus
targets differentiated digestive gut cells of Amblyomma variegatum ticks soon after feeding, although hemocytes are
considered to be the predominant cell supporting virus replication
later in infection (2). In Qalyub virus infection of
O. erraticus ticks, replication was detected only in midgut
cells, and dissemination to other tissues was rarely observed
(35). Powassan virus was detected exclusively in the midgut
of Dermacentor andersoni ticks until 17 days p.i.
(5). In contrast, midgut cells were infected in 20% or less
of Thogoto virus infected Rhipicephalus appendiculatus ticks, and the synganglion appeared to be the main organ involved with
virus replication and persistence (1). Midgut titers of these viruses were at least 1,000-fold lower than those observed for
ASFV in this study.
Mechanisms of ASFV entry into midgut epithelial cells have not been
previously described. Observations made here of ASF virions adsorbed to
intact erythrocytes within phagolysosomes of midgut epithelial cells
suggest that initial virus entry may be via erythrocyte phagocytosis.
Notably, almost all ASFV field isolates are hemadsorbing (31), and a significant proportion of the viremia in
infected pigs is associated with the erythrocyte fraction (3, 20, 27, 39, 60). However, a second mechanism of virus entry is also
likely to exist since undifferentiated epithelial cells become infected
after hemolysis of the blood meal is complete. Receptor-mediated
endocytosis of free virus in the midgut lumen at later times after
infection may be an additional route of virus entry. A possible source
of virus for infection of undifferentiated midgut cells is virions
released from the initially infected midgut cells, which subsequently
slough into the midgut lumen, releasing a large number of infectious
particles. Isolation of nonhemadsorbing ASFV from ticks collected in
warthog burrows (57) suggests the possibility of an entry
mechanism that does not involve erythrocyte phagocytosis. Also,
O. porcinus porcinus ticks have been experimentally infected
with ASFV by feeding on cell-free virus preparations on an artificial
membrane (52).
Interestingly, and in contrast to the report of Greig (22),
a 15- to 21-day delay in generalization of ASFV infection in the tick
was observed, suggesting a midgut barrier to generalization of
infection. Such a barrier has been described for some arboviruses of
mosquitoes (64). As with other arboviruses, the gut barrier to ASFV generalization may involve translocation of virus across the BL
of the midgut into the hemocoel. The extensive replication and
accumulation of ASF virions under the BL, similar to that seen in
several arbovirus infections of mosquitoes (29, 62, 63), and
the observation of virions within and immediately adjacent to the BL on
the hemocoel side suggest that ASFV dissemination involves movement of
virus across the BL. The delay observed may be due to the inefficiency
of movement across the BL and/or to a requirement for high viral titers
in proximity to the BL, a condition likely requiring extensive virus
replication and thus time to achieve. The observation of ASFV
replication in connective tissue and hemocytes early in generalized
infection indicates their significance as secondary sites of virus
amplification.
After dissemination and infection of a number of different tissues, a
viral titer of over 6 log10 HAD50/mg of body
weight was maintained through out the 290-day sampling period. The
total tick titers reported here correlate well with ASFV titers found in some naturally infected ticks (30, 45, 56, 57, 65). The
titers of dissected salivary glands and reproductive tissue, which were
the highest of any tissues studied after 91 days p.i., were found to
rise to 5 to 6 log10 HAD50/mg and persisted at
this level through the sampling period. Maintenance of high ASFV titers in the salivary gland throughout infection is in contrast to
observations made for ixodid ticks infected with other arboviruses, in
which salivary gland viral titers increase after commencement of
feeding (1, 2, 5, 11). This difference is likely
necessitated by the different biology and life cycles of these two
families of ticks. Unlike ixodid ticks, which feed on a vertebrate host for a number of days, thus allowing virus replication to be regulated in direct response to a transmission opportunity, O. porcinus porcinus ticks feed rapidly (in 1 h or less), requiring that
virus already be present in tissues in quantities sufficient for
transmission.
Successful transmission of ASFV to pigs by infected O. porcinus
porcinus ticks coincided with high viral titers in salivary and
coxal glands and the presence of virus in their secretions. Both
salivary secretions and coxal fluid have previously been suggested as
sources of virus for tick-pig transmission of ASFV (42). The
observation here that coxal secretions were more often positive for
virus than salivary secretions and that they contained relatively high
titers of virus suggests that this may be a more important source of
virus for transmission than previously recognized. Infection via coxal
fluid may occur through contamination of abrasions and/or tick-feeding
lesions at the skin surface. Ultrastructural visualization of ASF
virions in secretory granules of the salivary gland indicates that this
is a likely route of transmission during tick feeding. Although high
titers of ASFV were present in tick midgut at all times p.i., the
presence of a proventricular valve at the esophagal terminus in argasid
species precludes regurgitation as an operative transmission mechanism
for ASFV (54).
Overall, our data indicate that ASFV is highly adapted to O. porcinus porcinus ticks. High levels of viral replication are present in diverse tissues and cell types, with little evidence of
cytopathology and no significant increase in tick mortality. These data
are consistent with observations made for ASFV-infected O. porcinus porcinus ticks under field conditions (30, 45, 56,
57, 65). The relatively constant high titers of virus maintained
in the tick over long periods of time suggest that there is a mechanism
for regulating viral replication at levels that are compatible with
host survival. In the swine host, differentiated tissue macrophages and
reticular cells are the major viral targets in vivo and, it has been
suggested that the stage of monocyte differentiation may influence cell
susceptibility to ASFV infection (24, 31, 61). Observations
here of extensive replication in differentiated midgut epithelial cells
and hemocytes suggest that viral replication might be regulated in part
by the availability of cells at the appropriate state of
differentiation. Alternatively, an immune-like host response to viral
infection might be involved. Although no antiviral host responses have
yet been described in arthropods (18, 48), it is reasonable
to assume that they exist. ASFV infection of nonadapted European and
North American Ornithodoros spp. results in significant tick
mortality (14-17, 23, 25). Pathogenesis experiments with
these ticks have not been done; thus, a direct comparison with our data
on ASFV infection of O. porcinus porcinus cannot be made.
However, it is possible that the inability of a nonadapted tick host to
control virus replication and/or virus-induced pathology is responsible
for the observed mortality.
In agreement with a previous study (22), the work presented
here demonstrates that the midgut is the initial site of virus replication. However, a number of results described here for ASFV infection of O. porcinus porcinus ticks differ significantly
from those reported by Greig (22). In these earlier
experiments, which used an infectious dose (~5.0 log10
HAD50) and virus isolation procedures similar to those used
in our studies, tick infection rates varied from 54 to 97%, depending
on the ASFV isolate and the collection of O. porcinus
porcinus ticks used. Viral titers of infected tick tissues
declined dramatically over the sampling period of 140 to 350 days, and
salivary gland and reproductive tissues titers were never consistently
higher than 2 log10 HAD50. It is unlikely that
these differences are due to experimental design and/or random
variation; rather, they likely reflect the degree of virus-host
adaptation. The four ASFV isolates examined in the present study were
all of tick origin, and infection studies with three of them (Pr4, Cr1,
and No6) were conducted with ticks from the same collection from which
the virus was isolated. The significance of virus-host adaptation to
infection outcome is also suggested by a previous study where markedly
different infection rates (5% versus 95%) were observed when ticks
from the same collection were infected with two different ASFV isolates
(42).
In summary, our results demonstrate that O. porcinus
porcinus ticks fed on viremic pigs develop a long-term, persistent
infection with continuous and high levels of virus replication,
experience minimal mortality, and efficiently transmit virus to pigs.
Given these characteristics, it is likely that ASFV infection in its natural O. porcinus porcinus host represents a well-adapted
and possibly coevolved biological system.
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Abstract
Introduction
