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Journal of Virology, October 1999, p. 8713-8719, Vol. 73, No. 10
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Replication of Aleutian Mink Disease Parvovirus In
Vivo Is Influenced by Residues in the VP2 Protein
James M.
Fox,*
Mary
A.
McCrackin Stevenson, and
Marshall E.
Bloom
Laboratory of Persistent Viral Diseases,
Rocky Mountain Laboratories, National Institute of Allergy and
Infectious Diseases, Hamilton, Montana 59840
Received 10 May 1999/Accepted 25 June 1999
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ABSTRACT |
Aleutian mink disease parvovirus (ADV) is the etiological agent of
Aleutian disease of mink. Several ADV isolates have been identified
which vary in the severity of the disease they elicit. The isolate
ADV-Utah replicates to high levels in mink, causing severe
Aleutian disease that results in death within 6 to 8 weeks, but does
not replicate in Crandell feline kidney (CrFK) cells. In contrast,
ADV-G replicates in CrFK cells but does not replicate in mink. The
ability of the virus to replicate in vivo is determined by virally
encoded determinants contained within a defined region of the
VP2 gene (M. E. Bloom, J. M. Fox, B. D. Berry, K. L. Oie, and J. B. Wolfinbarger. Virology
251:288-296, 1998). Within this region, ADV-G and ADV-Utah differ at
only five amino acid residues. To determine which of these five
amino acid residues comprise the in vivo replication determinant,
site-directed mutagenesis was performed to individually
convert the amino acid residues of ADV-G to those of ADV-Utah. A virus
in which the ADV-G VP2 residue at 534, histidine (H), was converted to
an aspartic acid (D) of ADV-Utah replicated in CrFK cells as
efficiently as ADV-G. H534D also replicated in mink, causing transient
viremia at 30 days postinfection and a strong antibody response.
Animals infected with this virus developed diffuse hepatocellular
microvesicular steatosis, an abnormal accumulation of intracellular
fat, but did not develop classical Aleutian disease. Thus, the
substitution of an aspartic acid at residue 534 for a histidine allowed
replication of ADV-G in mink, but the ability to replicate was not
sufficient to cause classical Aleutian disease.
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INTRODUCTION |
Aleutian mink disease
parvovirus (ADV) causes both chronic and acute
diseases in mink. The chronic disease, termed Aleutian disease
(AD), is associated with a persistent infection of adult mink and is
characterized by hypergammaglobulinemia, plasmacytosis, increased
CD8+ lymphocytes and an immune complex disorder
(10). Affected animals maintain viremia and high levels of
antiviral antibodies throughout the course of disease. Macrophages have
been identified as sites of restricted virus replication, and infection
of these cells is thought to lead to the immune disturbances (2,
33, 34). The acute disease is a fulminant, fatal interstitial
pneumonitis resulting from permissive ADV infection of type II alveolar
cells in newborn mink. In addition, milder forms of both diseases have been reported and inapparent infections have been recognized (3, 5, 6, 10, 24).
Although host factors contribute to the outcome of ADV infections, the
major determinants of disease variability and severity are virally
encoded (8, 9, 14, 37). Highly virulent isolates of ADV such
as ADV-Utah and ADV-TR cause severe disease in both newborn and adult
mink of either the Aleutian or non-Aleutian genotypes, but have not
been successfully propagated in cell culture (1, 4, 25, 37).
In contrast, ADV-G does not replicate to detectable levels in adult
mink of either genotype, but does replicate permissively in cultures of
Crandell feline kidney (CrFK) cells (1, 4, 14, 37). Thus,
the ability of ADV to replicate either in vitro or in vivo is regulated
by sequences within the viral genome.
The development of full-length infectious molecular clones of ADV-G has
greatly facilitated attempts to identify virally encoded host range and
pathogenicity determinants (7-10). Subgenomic clones have
been used to determine the ADV-Utah sequence and to construct chimeric
viruses between ADV-G and ADV-Utah in an attempt to identify regions of
the viral genome responsible for encoding host range and/or replication
determinants (8, 9). Experiments with these chimeras map
sequences governing in vitro and in vivo viral replication to the VP2
capsid gene (8, 9).
Recent work has identified two chimeric ADV viruses, G/U-8 and G/U-10,
that are capable of replicating both in vitro and in vivo
(9). Both of these viruses contain a short segment of the
ADV-Utah VP2 gene (corresponding to amino acid residues 360 to
589) substituted into the ADV-G genome. Both induce viremia, anti-ADV
antibodies, and typical but mild pathological changes. This
segment of VP2 is the minimal ADV-Utah VP2 region necessary to impart
in vivo replication competence to ADV-G. The G/U-8 virus replicated
better in vivo, inducing higher antibody titers and persistent viremia,
whereas the G/U-10 virus produced only transient viremia
(9). The G/U-8 virus contains an additional VP2 mutation, I352V, and a small segment of the ADV-Utah NS1 protein not present in
G/U-10. The G/U-8 and G/U-10 viruses are the first molecularly cloned
ADVs that can replicate both in vitro and in vivo.
In this study, we prepared site-directed mutants of ADV-G to determine
how substitutions at defined locations in the VP2 protein affected in
vivo replication. We tested each virus for the ability to replicate in
vitro, and those that replicated in cell culture were injected into
mink. The ability of each mutant virus to induce viremia, an antibody
response, and pathology was compared to those of ADV-Utah and G/U-10.
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MATERIALS AND METHODS |
Viruses, cells, and plasmids.
Replication-competent mutant
viruses were propagated and assayed in CrFK cells as previously
described (8). The in vivo propagation and assay of ADV-Utah
in adult Aleutian genotype (sapphire) mink have also been described
previously (37).
Cloning and mutagenesis.
Full-length molecular clones were
transformed and amplified in Escherichia coli JC8111
(recBCsbcrecF) by standard techniques (7, 15,
31). Other plasmids used were maintained in either E. coli JM109 or Epicurian Coli XL1-Blue (Stratagene, La Jolla, Calif.). Maintenance of the unstable right-hand hairpin was determined by restriction enzyme analysis as previously described (8, 9).
All site-directed mutagenesis was performed with the QuickChange
Site-Directed Mutagenesis kit (Stratagene). The oligonucleotide primers
used and their corresponding mutations are shown in Fig. 1. The mutated primers were designed so
that each construct also contained noncoding changes that either
introduced or eliminated a restriction enzyme recognition site.
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FIG. 1.
Site-directed mutagenesis of ADV-G. The top line in each
box shows the ADV-G VP2 amino acid sequence with the nucleotide
sequence below it. In the bottom of the box are the corresponding
sequences of the oligonucleotides used for mutagenesis. The mutagenic
nucleotides are shown in boldface type. Restriction enzyme recognition
sequences altered during mutagenesis (for screening) are designated. In
construction of the H395Q mutant, the HpaII site was
created, and in all other mutants, the displayed restriction enzyme
recognition site was destroyed during mutagenesis.
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To facilitate mutagenesis, the
EcoRI-
HindIII
fragment of ADV-G was cloned into pBluescript II SK(+) (Stratagene).
This clone
was denoted p33F-11. Mutagenesis was performed on p33F-11,
and
mutations were confirmed by restriction enzyme digestion and DNA
sequence analysis. The mutated
EcoRV-
HindIII
fragments (contained
within the
EcoRI-
HindIII
fragment) were excised and subcloned
back into the clone pIC4-2. Clone
pIC4-2 is a full-length ADV-G
clone (
7) in which the
EcoRV-
HindIII region has been replaced
with
the 32-bp
EcoRV-
HindIII fragment from pBR322.
The final clones
were transformed into
E. coli JC8111,
amplified, and purified
with Wizard Maxiprep kits (Promega, Madison,
Wis.). The integrity
of the DNA was assessed by agarose gel
electrophoresis, and the
concentration was determined
spectrophotometrically. The DNA was
aliquoted and stored in ethanol at
70°C.
Individual 35-mm-diameter culture dishes of CrFK cells were transfected
with 5 µg of purified plasmid DNA and incubated at
31.8°C for 5 days (
7,
8). Cell pellets were collected in
1 ml of medium,
freeze-thawed three times, sonicated, and passaged
to T-25 culture
flasks of CrFK cells. This process was repeated
three more times with
the final two passages being performed in
T-150 culture flasks. Upon
harvesting of the final passage, the
cells were scraped into 5 ml of
PBBS, freeze-thawed three times,
sonicated, and centrifuged at 4,000 rpm for 20 min in a Beckman
J-6 centrifuge. The supernatants were
collected, aliquoted, and
stored at
20°C. Viruses were titrated by
infecting CrFK cells
with supernatants and using immunofluorescent
microscopy with
a fluorescein-conjugated polyclonal anti-ADV mink serum
as previously
described (
9).
In vivo infectivity and pathogenicity.
Adult sapphire
(Aleutian genotype) mink (Mustela vison) obtained from an
ADV-negative mink farm were housed in modified primate cages within
sheds as previously described (37). All experimental procedures involving animals were performed under the guidelines of the
Rocky Mountain Laboratories Animal Care and Use Committee, and animals
were anesthetized with ketamine-acepromazine for all procedures.
Two weeks prior to injection, the animals were distributed into
physically separated groups of four. The ADV-Utah group was
housed in a
separate shed. Immediately prior to injection, sera
(day 0) were
collected. The mink were injected with 10
4 focus-forming
units (FFU) of in vitro-propagated viruses. The
positive control group
was injected with 10
5 50% mink infectious doses of
ADV-Utah. Control mink were injected
with CrFK cell lysates in 0.5 ml
of PBBS diluent. Animals were
bled at 10, 30, 60, 90, and 120 days
postinjection (dpi). Sera
were obtained by jugular venipuncture into
Vacutainer Plus plastic
centrifuge tubes containing SST gel and Clot
Activator (Becton-Dickinson).
Serological responses to ADV infections were assessed by counterimmune
electrophoresis (
13,
37) and determination of the
percent
serum gamma globulin level by serum protein electrophoresis
with a
horizontal thin-layer agarose gel system (Ciba-Corning).
The change in
serum gamma globulin between 0 and 120 dpi was determined
by laser
densitometry (
8).
The presence of viremia was determined with serum-based PCR (
12,
37). This assay, which amplifies a 692-bp fragment in
the VP2
region of the genome, can detect <1 fg of viral DNA (<10
genomes) in
2.5 µl of serum (
37).
Tissue blocks of liver, spleen, kidney, and mesenteric lymph node
collected at necropsy were formalin fixed, embedded in paraffin,
sectioned, and stained with hematoxylin-eosin according to standard
histological procedures. The slides were evaluated for the presence
of
microscopic lesions (
22,
23,
26).
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RESULTS |
Site-directed mutagenesis of ADV-G.
Previous studies
demonstrated that control of ADV replication in vivo localizes to a
region of the VP2 protein (9). Analysis of a sequence
alignment within the defined replication region revealed five amino
acid residues that were highly conserved among pathogenic isolates and
that differed from ADV-G VP2: amino acid residues 352, 395, 434, 491, and 534 (Fig. 2). These five residues were selected for site-directed mutagenesis by a PCR-based method (Fig.
1). Mutant viruses, which contained single ADV-Utah amino acid residues
in the ADV-G background, were constructed as detailed in Materials and
Methods.
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FIG. 2.
ADV VP2 protein. (A) Schematic of the VP2 protein
illustrating the location of the previously defined in vivo replication
determinant present in the G/U-10 and G/U-8 viruses (9). aa,
amino acids. (B) Protein sequence alignment of VP2 amino acid residues
341 to 590 of ADV-G and other pathogenic ADV isolates Utah (Ut), Tucker
(Tr), Pullman (Pu), Utah1 kit (U1k), and Zk8. Identical residues are
shown as dots, while divergent residues are shown in the single letter
code. Arrows indicate the amino acid residues targeted for
site-directed mutagenesis in this study.
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In vitro analysis of site-directed mutant ADV clones.
In order
to assess in vitro replication competence, the mutant DNA clones were
individually transfected into CrFK cells. The lysates harvested from
the initial transfections were blindly passaged three successive times
in CrFK cells to determine if virus could be rescued from the clones
and to amplify the viruses for subsequent analysis. After a total of
four passages, the lysates were titrated on CrFK cells by
fluorescent-antibody microscopy. Three of the site-directed mutants,
I352V, N491E, and H534D, replicated to levels equivalent to those of
ADV-G, achieving titers in the 107-FFU/ml range (Fig.
3A). However, two of the clones, H395Q
and N434H, appeared replication defective and did not yield any virus at the end of four passages (Fig. 3A).
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FIG. 3.
Replication of mutant ADV viruses in CrFK cells. (A)
Titration of mutant viruses following DNA transfection and four
passages in CrFK cells. The fourth-passage cell lysates were used to
infect CrFK cells, and 3 days postinfection, the cells were examined
for virus replication by fluorescent-antibody microscopy. (B) The
parental ADV-G molecular clone along with each of the mutant molecular
clones was transfected into CrFK cells and assayed 3 days later for
virus replication by fluorescent-antibody microscopy. Mocks containing
buffer only were used as negative controls.
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To confirm that the H395Q and N434H mutants were replication defective,
the study was repeated and the titers were determined
at each passage
level. Three days after DNA transfection, the
cells were assayed for
viral replication by fluorescent-antibody
microscopy (Fig.
3B). All
clones replicated to similar levels
as the parental ADV-G clone.
Western blot analysis of the lysates
showed that each of the mutants,
as well as the ADV-G parental
virus, expressed the viral proteins NS1,
VP1, and VP2 to similar
levels (data not shown). Thus, all coding
sequences were intact
and our manipulations had not unexpectedly
interrupted the sequences.
However, upon subsequent passage of the
primary cell lysates,
virus could not be detected with H395Q and N434H
mutants (data
not shown). Thus, while these clones are capable of
replicating
when DNA is transfected into cells, they are not able to
produce
virus that is capable of initiating additional rounds of
infection
in
vitro.
Infection of mink with mutant ADV viruses.
To determine if the
replication-competent mutant viruses were infectious for mink, we
injected adult Aleutian mink with the I352V, N491E, and H534D viruses.
In vivo infectivity was assessed by testing for both viremia and
antiviral antibody titers at 10, 30, 60, 90, and 120 dpi. Mink injected
with either the mock control or N491E virus did not develop viremia or
detectable viral antibody levels throughout the 120-day study (Fig.
4). Thus, the N491E virus acted like
ADV-G (8, 9), indicating that the 491 residue alone did not
confer in vivo replication competence. However, mink injected with
I352V and H534D or G/U-10 and ADV-Utah positive controls showed signs
of infection, as evidenced by antiviral antibodies and/or viremia (Fig.
4). Two of the four animals injected with the I352V virus developed an
antibody response, but did not have a detectable viremia (Fig. 4). The
H534D virus induced high titers of antiviral antibodies in all four
animals within the group, and two of the animals had a transient
viremia at 30 dpi (Fig. 4). The G/U-10 virus gave results similar to
those previously published, in that all animals developed high
antiviral antibody titers and two of the animals were transiently
viremic at 10 dpi (Fig. 4). All animals injected with ADV-Utah became
persistently viremic and demonstrated high antiviral antibody titers
throughout the experimental study (Fig. 4).
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FIG. 4.
Injection of mink with ADV mutant viruses. Mink were
injected with mock, I352V, N491E, H534D, G/U-10, and ADV-Utah viruses
and monitored for 120 dpi. At each time point, sera were collected and
analyzed for the presence of viral antibody and viremia. Open circles
represent antibody titers, and solid circles indicate viremia. Each
circle represents one animal. One animal in each of the G/U-10 and
ADV-Utah groups died of irrelevant causes early in the study.
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To compare the viral antibody titers of the different virus groups, the
geometric mean titers (GMTs) were calculated and plotted
(Fig.
5). Clear trends were observed, but the
small group size
and individual variability between animals did not
allow for statistical
differences between all groups. The I352V virus,
although variable,
gave a final GMT of 1:6 at 120 dpi. The H534D and
G/U-10 viruses
gave relatively high GMTs, with both final titers being
1:257
at 120 dpi. ADV-Utah resulted in a characteristically high GMT
with a final value of 1:1,024 at 120 dpi. Thus, the H534D virus
is
capable of replicating to equivalent levels as G/U-10 in vivo.
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FIG. 5.
Geometric mean viral antibody titers. The GMT is plotted
for each animal group at the time points of serum collection during the
study. The standard mean error is displayed with error bars. The
symbols and the corresponding viruses used to plot the graph are shown
in the upper left-hand corner. The final GMT is printed on the
right-hand side of the graph.
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A hallmark of progressive AD is pronounced hypergammaglobulinemia. To
determine if any of the viruses induced elevated gamma
globulin levels,
sera from each animal at 0 and 120 dpi were compared.
Only ADV-Utah
induced a hypergammaglobulinemia (Table
1). Thus,
in spite of clear evidence of
in vivo infectivity, the H534D and
G/U-10 viruses did not induce
the hypergammaglobulinemia characteristic
of progressive AD.
Throughout the course of the study noticeable signs of infection were
observed in the animals infected with ADV-Utah, G/U-10,
and H534D.
Weight loss, changes in pelt condition, and lethargy
were evident in
animals within these groups. The animals injected
with the other mutant
viruses did not show these clinical signs
of infection. These results
suggested that the animals infected
with these three viruses were being
impacted by the viral
infections.
To more precisely determine if the animals had lesions typical of AD,
the liver, spleen, kidney, and mesenteric lymph node,
harvested at 120 dpi, were examined. The livers of animals infected
with ADV-Utah showed
typical AD pathology, including widespread
lymphocyte and plasma cell
infiltration (Fig.
6). In addition,
plasma cell infiltration and mesangial thickening of the glomeruli
within the kidney, erythropoiesis within the spleen, and infiltration
of plasma cells within the paracortex of the mesenteric lymph
nodes
were all evident (data not shown).
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FIG. 6.
Liver pathology. Liver sections that have been
hematoxylin and eosin stained are shown for a representative animal
from each virus group. All pictures were taken at an original
magnification of ×20, except for the H534D and Utah panels, which were
taken at an original magnification of ×40. Notice the intracellular
fat accumulation in the H534D virus frames.
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Consistent with the previous report, the G/U-10 virus induced lesions
typical of mild AD (
9). These included mild lymphocytic
infiltration of the liver portal tracts (Fig.
6) and paracortex
of the
mesenteric lymph nodes (data not shown). Animals injected
with the mock
control, I352V, and N491E viruses did not show any
significant
pathological findings in the livers (Fig.
6) or other
tissues.
The tissues of H534D virus-infected animals did not show typical signs
of AD, but the livers of these animals displayed microvesicular
steatosis, a hepatocellular condition not previously reported
with ADV
and not seen in the livers of other animals in this study
(Fig.
6). It
was noted during necropsy of these animals that the
livers were mildly
swollen and yellow, with brown mottling over
the entire external
surface of the tissue. Thus, while the H534D
mutant virus did not cause
typical symptoms of AD, it induced
a pathological change that had not
been previously reported for
ADV.
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DISCUSSION |
In this study, we identified a single amino acid residue, aspartic
acid 534 (D534), that when present in ADV-G, results in a virus capable
of inducing a persistent antibody response and transient viremia. The
replication of this virus resembled that seen with the previously
reported G/U-10 virus, an ADV-G-like virus containing four amino acid
changes from ADV-Utah in the VP2 protein (9). The similar
antibody response and transient viremia induced by both the H534D and
G/U-10 viruses suggested that D534 is the major determinant within
G/U-10 that allows it to replicate in vivo. If the additional three
amino acid changes present in the G/U-10 VP2 protein play a role in in
vivo replication, it is minimal. Furthermore, the data suggest that
D534 is a major determinant for ADV-Utah replication in vivo.
The I352V virus induced a persistently low antibody response in two of
the four animals injected. While this response was low relative to the
H534D, G/U-10, or ADV-Utah viruses, it appeared relevant when compared
to either the N491E or ADV-G (8, 9) viruses, neither of
which induced an antibody response. The I352V mutation was the only
difference within the VP2 protein between the previously reported G/U-8
and G/U-10 viruses (9). It is not known if the I352V VP2
mutation or three additional mutations in the NS1 protein are
responsible for the enhanced in vivo replication and pathogenicity of
G/U-8. However, our data suggested that the I352V mutation may be a
reason the G/U-8 virus replicates better than the G/U-10 virus
(9). Experiments are under way to determine if the I352V
mutation is the reason for enhanced in vivo replication and to
determine what role, if any, the mutations in the NS1 protein have on
virus replication and pathogenicity.
In vivo replication of the H534D virus was transient as demonstrated by
the lack of persistent viremia. Furthermore, H534D induced a lower
overall antibody response than ADV-Utah. These differences from
ADV-Utah suggest there are other determinants within ADV-Utah, not
present in H534D, contributing to persistent virus replication. We are
interested in mapping these differences and defining their role in ADV
replication and pathogenicity. The differences may lie within the
nonstructural proteins NS1 and/or NS2 or within other regions of the
VP2 protein. There is precedent for determinants of parvovirus host
range residing in both the nonstructural and structural proteins. The
NS2 protein of minute virus of mice is responsible for determining its
virus host range (16, 35). In addition, the NS1 protein and
capsid proteins together determine the host range of porcine parvovirus (47). Finally, amino acid residues within the VP2 protein of both canine parvovirus and feline panleukopenia virus are responsible for determining the ability of the viruses to differentially replicate in various host cells (17, 30, 40, 41, 45, 46). There are
few reports on the analysis of the nonstructural proteins of ADV with
regard to host range or replication determinants. One report by Bloom
et al. (8) demonstrated that replacement of the majority of
the ADV-G NS1 and NS2 protein coding sequences with ADV-Utah sequences
resulted in viruses that were ADV-G like (i.e., replicated in vitro but
not in vivo). However, none of these viruses had H534D, a required VP2
residue needed for in vivo replication.
It is interesting to speculate about the possible mechanistic
differences between ADV-Utah and H534D virus replication and disease
induction. A hallmark of ADV infection is the pronounced immune
disturbances that occur as a result of persistent virus infection
(22, 23, 26). It is believed that many of the immune
disturbances are a direct consequence of persistent ADV replication
occurring within macrophages (2, 27, 33, 34). It is believed
ADV gains entry into macrophages via Fc-Fc receptor-mediated interactions, a process termed antibody-dependent enhancement (21,
28, 38). Thus, antibodies raised against the virus assist the
virus in entering the target cell where replication occurs. One
difference between ADV-Utah and H534D may be a different immune
response induced by the two viruses. Since ADV-Utah contains several
additional amino acid differences in the VP2 protein, there may be
significant antigenic or structural variation on the virion surface
that results in a differing immune response relative to H534D. If the
H534D virus induces a different response, the types of antibodies
needed to infect macrophages and persistently replicate may not be
generated. Recent work on the structure of ADV suggests many of these
various amino acid residues are in regions of VP2 that are
immunodominant (11, 32). Thus, it seems possible that
changes in these regions could alter the antigenicity in a subtle fashion.
The H534D virus did not cause pathological lesions typical of AD.
However, infected animals showed weight loss and lethargy and developed
rough coats, indicating virus infection was having a negative impact on
the animals. The livers of mink injected with this virus consistently
displayed microvesicular steatosis (Fig. 6). This lesion, an
accumulation of small fatty deposits within hepatocytes, has not been
previously noted for ADV-infected animals and is distinct from
ADV-Utah-induced lesions (Fig. 6). Microvesicular steatosis has been
reported to be associated with influenza virus B infections of mice,
murine adenovirus infections of SCID mice, and arctic squirrel
hepatitis virus infections of arctic ground squirrels (19, 20,
42-44). These reports have compared the liver lesions resulting
from the virus infections with Reye's syndrome in humans. While
microvesicular steatosis has not been reported to be associated with
any parvovirus infections, associations between parvovirus infections
and liver dysfunction have been reported (18, 29, 36, 39,
48). The human parvovirus B19 infection is associated with
fulminant liver failure and associated aplastic anemia. In addition,
the cellular receptor for B19 can be detected in liver tissue. It is
interesting that the single H534D mutation in ADV-G allowed it to
replicate in vivo in a similar manner to the G/U-10 virus, but the
disease that each induces is dramatically different. The additional
three amino acid mutations present in the G/U-10 virus, while not
controlling in vivo replication, may be involved in classical AD
induction. Another explanation is the possibility that the H534D
mutation altered the host range of the virus to be hepatotropic.
Two of the mutants we constructed, H395Q and N434H, were unable to
replicate in CrFK cells. Currently it is not understood why these
viruses are replication defective in vitro. The transfected full-length
clones of both viruses are capable of expressing the viral proteins
NS1, VP1, and VP2 at similar levels to ADV-G in CrFK cells. However, it
is not possible to infect subsequent cells from lysates of these
primary transfected cells. While these mutations abolish CrFK
infectivity when present alone, they do not abolish infectivity when
present together with the other divergent amino acid residues in either
the G/U-8 or G/U-10 viruses (9). Both the G/U-8 and G/U-10
viruses replicate, albeit poorly, in CrFK cells. Perhaps the structural
alterations being formed by the presence of single mutations H395Q and
N434H can be partly compensated for by the presence of other mutations.
It is possible that these residues contribute to ADV in vitro
replication competence and when present with the H534D mutation would
result in a virus that behaves more like ADV-Utah. Currently studies
are under way to determine where the infection cycle of these mutants
is disrupted.
In summary, we have identified a single VP2 amino acid residue, D534,
that plays a role in determining in vivo replication competence. The
D534 residue confers on ADV-G the ability to replicate in mink and
induce a persistent antibody response. This virus clone will provide a
starting point to determine which other features of ADV-Utah contribute
to its viral persistence and pathogenicity. In addition, the H534D
virus causes a disease that results in lesions, microvesicular
steatosis of the liver, not previously seen with ADV. A more detailed
study of this virus' replication and pathogenic potential will further
our understanding of ADV replication and its relationship to viral pathogenicity.
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ACKNOWLEDGMENTS |
We thank William Hadlow for assistance in evaluating the
pathological results in this study. We thank Cynthia Favara for the preparation, sectioning, and staining of tissue blocks. We thank James
Wolfinbarger and Julie Buss for technical assistance and Dirk Whitsitt,
Don Dale, and Ralph Larson for assistance with the care and handling of
the animals.
Mink for this study were provided by the Mink Farmers Research Foundation.
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FOOTNOTES |
*
Corresponding author. Mailing address: 903 S. 4th St.,
Hamilton, MT 59840. Phone: (406) 363-9284. Fax: (406) 363-9286. E-mail: jfox{at}niaid.nih.gov.
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Journal of Virology, October 1999, p. 8713-8719, Vol. 73, No. 10
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
