5288 ROBERTSON ET AL. J. VIROL.
ing neurovirulence (i.e., the ability to induce clinical neurologic antibody was a biotinylated goat anti-rat immunoglobulin (Vector Laboratories,
disease). This is a particularly relevant issue in view of the Inc.). This was followed by an incubation with horseradish peroxidase-conjugated
streptavidin (BioGenex) for 15 min at room temperature. Sections were washed
conﬂicting reports on the importance of viral burden in neu- in PBS (three times for 10 min each), developed with AEC as described above,
ropathogenicity of both human immunodeﬁciency virus (1, 2, and counterstained with Mayer’s hematoxylin.
17, 21) and simian immunodeﬁciency virus (41, 43, 46, 48). In Immunohistochemical staining of glial ﬁbrillary acidic protein (GFAP) was
this study, we examined in more detail properties of the CNS performed on parafﬁn-embedded sections (4 m). Sections were deparafﬁnized,
infection by two of the neurovirulent viruses, Fr98 and Fr98/ rehydrated, and washed in PBS prior to incubation with rabbit anti-bovine GFAP
SE, and the nonneurovirulent virus Fr54 in an effort to identify antiserum (Dakopatts) for 1 h at 37°C. Sections were incubated with a biotinyl-
ated goat anti-rabbit immunoglobulin (Vector) for 30 min at 37°C, followed by
features of these infections that correlate with neurovirulence. an incubation with horseradish peroxidase-conjugated streptavidin (BioGenex)
Surprisingly, no differences were found between the neuroviru- for 15 min at room temperature. Sections were developed with AEC as described
lent and nonneurovirulent viruses with respect to the appear- above and counterstained with Mayer’s hematoxylin.
ance of vacuolar degeneration, activation of astrocytes and Cell counts. Fresh, frozen brain tissue was sectioned (5 m), and every eighth
microglial cells, regional distribution of virus, or the cell types section was stained for viral envelope protein by using a polyclonal goat anti-gp70
antiserum as described above. The number of gp70-positive cells in the cerebella
infected in the brain. However, application of more quantita- was determined in a total of ﬁve sections. From these ﬁve sections, the mean
tive measurements of viral protein expression in the brain standard deviation was determined for four mice per virus strain: Fr98/SE (52 to
revealed viral burden to be a consistent correlate of neuroviru- 75 days postinoculation) and Fr54 (52 to 75 days postinoculation). The means
lence in this model. The results suggest that the neurovirulent standard errors of these two groups were compared by using the unpaired
viruses differed from the nonneurovirulent virus in the extent two-tailed Student t test.
of their spread among microglial cells after initial virus entry Double-label indirect immunoﬂuorescence. Mice were anesthetized by inha-
lation of methoxyﬂuorane and perfused with PBS followed by 0.5% paraformal-
into the brain. dehyde-lysine-periodate ﬁxative (PLP) (30). Vibratome sections (50 m) of
PLP-ﬁxed brain were pretreated with 0.1% Triton X-100 for 5 min and then
blocked with 5% bovine serum albumin for 1 h at room temperature. Viral
MATERIALS AND METHODS envelope protein and cell-speciﬁc markers were labeled sequentially. Sections
were incubated with primary antibodies overnight at 4°C and then with secondary
Viruses, animals, and clinical disease. The chimeric viruses were generated as antibodies at room temperature for 1 to 2 h. The sections were washed (three
previously described (19). Brieﬂy, Fr98 and Fr54 contain the 3 pol and env times for 10 min each) in PBS after each antibody incubation step. For localiza-
sequences (SphI-to-ClaI fragment) of FMCF98 (6) and FMCF54 (33), respec- tion of viral protein in astrocytes, sections were incubated with rabbit anti-bovine
tively, within the background of FB29, a nonneurovirulent strain of Friend GFAP antiserum that was detected with a tetramethylrhodamine-5-(and 6) iso-
MuLV (45). Fr98/SE contains the SphI-to-EcoRI fragment of FMCF98 env
thiocyanate (TRITC)-conjugated sheep anti-rabbit immunoglobulin (Cappel).
combined with the EcoRI-to-ClaI fragment of FMCF54 env that was also placed
Sections were then incubated with a polyclonal goat anti-MuLV gp70 antiserum
within the background of FB29 (19). Virus stocks were prepared from superna-
tants of conﬂuently infected Mus dunni ﬁbroblast cells as previously described that was detected with ﬂuorescein isothiocyanate (FITC)-conjugated donkey
(38). Virus titers (in focus-forming units/milliliter) were 1.3 106 for Fr98, 3.6 anti-goat immunoglobulin (ICN). For localization of viral protein in oligoden-
106 for Fr54, and 4.2 106 for Fr98/SE, as determined by a previously described drocytes, PLP-ﬁxed vibratome sections (50 m) were incubated with a mouse
focal immunoassay (44) on M. dunni cells. monoclonal antibody to 2 ,3 -cyclic nucleotide 3 -phosphodiesterase (CNP) (50)
Inbred Rocky Mountain White (IRW) mice were bred and raised at Rocky (kindly provided by Bruce Trapp, Cleveland Clinic, Cleveland, Ohio) that was
Mountain Laboratories animal facilities. Neonatal mice ( 24 h old) were inoc- detected with TRITC-conjugated goat anti-mouse immunoglobulin (Pierce).
ulated intraperitoneally with approximately 3 104 focus-forming units of virus Then sections were incubated with a polyclonal rabbit anti-MuLV gp70 that was
per mouse. Mice were observed daily for clinical disease and compared with detected with an FITC-conjugated goat anti-rabbit immunoglobulin G (IgG)
age-matched uninoculated control mice. The ﬁrst signs of neurologic dysfunction (Cappel). Viral protein localization in microglia was performed either on PLP-
were hyperactivity and bilateral imbalance when climbing up the side of the box ﬁxed vibratome sections (50 m) or on frozen sections (4 to 6 m) ﬁxed with
(19, 39). Clinical disease progressed to hindlimb weakness, ataxia, and reﬂex 3.7% formaldehyde in PBS. Microglia were labeled with a rat monoclonal anti-
abduction of the hindlimbs, at which time the mice were euthanized. All in vivo body to Mac-1 detected with Texas red-conjugated goat anti-rat IgG (Southern
experiments were done in accordance with the standards established by the Biotechnology Associates, Inc.) followed by a polyclonal rabbit anti-MuLV gp70
Rocky Mountain Laboratories Animal Care and Use Committee and the Na- (kindly provided by Roland Friedrich) detected with an FITC-conjugated goat
tional Institutes of Health. anti-rabbit IgG (Cappel). For all dual-labeling schemes, each secondary antibody
Histopathology. Animals were anesthetized by inhalation of methoxyﬂuorane was shown not to bind in the absence of primary antibody or cross-react with
and perfused through the left ventricle of the heart with phosphate-buffered
primary antibodies of different species. Double-labeling immunoﬂuorescence
saline (PBS; 10 mM Na2HPO4, 10 mM NaH2PO4, 130 mM NaCl [pH 7.4])
was performed on brains from at least three mice per virus strain: Fr98 (20 to 34
followed by 4% paraformaldehyde in phosphate buffer (0.1 M Na2HPO4, 0.1 M
NaH2PO4 [pH 7.4]). Brains were removed and ﬁxed in 4% paraformaldehyde- days postinoculation), Fr98/SE (48 to 73 days postinoculation), Fr54 (48 to 73
phosphate buffer for an additional 24 to 48 h, after which time they were days postinoculation), and uninoculated controls (20 to 73 days of age). Immun-
dehydrated through graded ethanols and embedded in parafﬁn (Polysciences, oﬂuorescence-labeled sections were analyzed by using a Nikon Microphot SA
Inc.). Coronal or sagittal sections (4 m) were stained with hematoxylin and ﬂuorescence microscope.
eosin by using standard histology techniques. Brain sections were evaluated for Immunoblotting. Mice were anesthetized by inhalation of methoxyﬂuorane
neuropathology in four mice inoculated with Fr98 (21 days postinoculation), ﬁve and exsanguinated by axillary vessel incision. Brains were taken from infected
mice inoculated with Fr98/SE (57 to 74 days postinoculation), eight mice inoc- mice at 3 and 8 weeks postinoculation with age-matched uninoculated mice.
ulated with Fr54 (55 to 159 days postinoculation), and seven uninoculated con- Extracts from mouse cerebellum were prepared by making 10% (wt/vol) homog-
trols (21 to 106 days of age). enates in 0.5% Nonidet P-40 in TNE (0.01 M Tris base, 0.15 M NaCl, and 0.001
Immunohistochemistry. Immunohistochemical staining was performed on M EDTA [pH 7.4]) and were clariﬁed by centrifugation as described elsewhere
fresh frozen sections (4 to 6 m), collected on glass slides, and ﬁxed in 3.7% (39). Immunoblots were performed as previously described (11). Brieﬂy, proteins
formaldehyde in PBS for 5 min at room temperature. Sections were blocked for were separated by sodium dodecyl sulfate–12% polyacrylamide gel electrophore-
30 min at 37°C with either 5% bovine serum albumin or 3% normal serum of the sis (SDS-PAGE) and transferred to Immobilon P membranes (Millipore). Cap-
same species as the secondary antibody. Sections were washed (three times for 10 sid protein (p30) was detected with rabbit anti-p30gag antiserum (26) followed by
min each) in PBS after each antibody incubation. For detection of viral protein horseradish peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad Laboratories)
and determination of virus distribution, sagittal sections were incubated with a and developed with the ECL (enhanced chemiluminescence) system (Amer-
polyclonal goat anti-MuLV gp70 antiserum (kindly provided by Roland
Friedrich, Institute of Medical Virology, Geissen, Germany) for 1 h at 37°C and
The relative amounts of viral protein were determined by immunoblot anal-
then incubated with horseradish peroxidase-conjugated rabbit anti-goat immu-
noglobulin (ICN Biochemicals Inc.) for 30 min at room temperature. Sections yses of twofold serial dilutions of cerebellar homogenates. Samples from three
were developed with 3-amino-9-ethylcarbazole (AEC) in 0.1 M acetate buffer mice per virus strain per time point were analyzed in pairs on the same immu-
(pH 5.0) and were counterstained with Mayer’s hematoxylin. noblot as follows: (i) Fr98 compared with Fr54, both at 3 weeks postinoculation;
For detection of activated microglia/pericytes, the same immunohistochemical (ii) Fr98/SE compared with Fr54, both at 3 weeks postinoculation; (iii) Fr98/SE
staining procedure was used except that the primary antibody was a rat mono- compared with Fr54, both at 8 weeks postinoculation; and (iv) Fr98 at 3 weeks
clonal antibody directed to Mac-1 (CD11b complement receptor) (47). Mac-1 is compared with Fr98/SE at 8 weeks postinoculation. The comparisons of the
a speciﬁc marker of microglia/macrophages in the brain, and upregulation of this relative viral protein content were made only between paired samples analyzed
protein is associated with microglia/macrophage activation (27). The secondary on the same immunoblot.
VOL. 71, 1997 CORRELATES OF POLYTROPIC MuLV NEUROVIRULENCE 5289
RESULTS an intense astrogliosis throughout the brain, as indicated by the
increased expression of the astrocytic marker GFAP (39).
Histopathological features of Fr98- and Fr54-infected As noted earlier, Fr54 infects the CNS but fails to induce
brains. Neonatal IRW mice inoculated with Fr98 exhibit clin- clinical neurologic disease in neonatally inoculated IRW mice
ical signs of neurologic disease 16 to 30 days postinoculation (19). To determine the relationship of glial activation and
(19, 39). As previously reported, the histopathology associated vacuolar degeneration to neurologic disease, we analyzed the
with Fr98 infection was characterized by mild vacuolar lesions pathological changes associated with Fr54 infection compared
most prevalent in the lateral cerebellar nuclei (dentate nu- with that induced by the neurovirulent viruses, Fr98 and Fr98/
cleus) and thalamus (39), with no evidence of inﬂammatory SE. Interestingly, immunohistochemical staining of Fr54-in-
inﬁltrates. The predominant pathologic feature, however, was fected brain for GFAP revealed an intense astrogliosis in a
FIG. 1. Reactive astrogliosis and microglial activation induced by Fr98 and Fr54. (A to C) Immunohistochemical staining for astrocytes on parafﬁn sections, using
anti-GFAP antiserum. Panels show the lateral cerebellar nuclei (dentate nucleus) in brains taken from uninoculated (A), Fr54-inoculated (B), and Fr98-inoculated (C)
mice. Notice the dark staining astrocytes in panels B and C not seen in panel A. Reactive astrogliosis was a consistent ﬁnding in all infected mice analyzed: Fr98 (ﬁve
mice, 35 to 45 days postinoculation), Fr54 (eight mice, 55 to 159 days postinoculation), and Fr98/SE (ﬁve mice, 57 to 74 days postinoculation [not shown]). (D to F)
Immunohistochemical staining for microglia/pericytes on cryostat sections of brain, using anti-Mac-1 monoclonal antibody. Panels show Mac-1-positive cells (arrows)
in white matter tracts of the cerebella of brains taken from uninoculated (D), Fr54-inoculated (E), and Fr98-inoculated (F) mice. Microglial activation was a consistent
ﬁnding in brains of all infected mice examined: Fr98 (six mice, 25 to 34 days postinoculation), Fr54 (three mice, 26 to 73 days postinoculation), and Fr98/SE (three
mice, 48 to 73 days postinoculation [not shown]). The Fr98-inoculated mice analyzed exhibited severe clinical signs of neurologic disease. Fr54-inoculated mice were
clinically indistinguishable from age-matched uninoculated controls. Sections were viewed by differential interference microscopy on a Nikon Microphot SA microscope.
Bar, 80 m.
5290 ROBERTSON ET AL. J. VIROL.
FIG. 2. Localization and morphology of infected cells in brains of Fr98-, Fr54-, and Fr98/SE-infected mice. Shown is immunohistochemical staining of frozen
sections of brain for viral envelope glycoprotein with an polyclonal goat anti-gp70 antiserum. These panels show the cerebella of mice inoculated with Fr98 (33 days
postinoculation) (A), Fr54 (B), and Fr98/SE (C), (both 48 days postinoculation) and uninoculated (48 days of age) (D). Note the immunohistochemical staining of cells
with bipolar or highly ramiﬁed morphology (arrows and panel A, inset) as well as staining associated with blood vessels (panels B and C, arrowheads). This ﬁgure is
representative of consistent ﬁndings in four mice per virus strain: Fr98 (18 to 33 days postinoculation), Fr54 (26 to 75 days postinoculation), and Fr98/SE (52 to 75 days
postinoculation). Sections were viewed by differential interference microscopy.
distribution similar to that previously reported for Fr98-in- clonal anti-MuLVgp70 antiserum which recognizes the enve-
fected brain (Fig. 1A to C). In addition to astrogliosis, we lope proteins of all three viruses. Brains of Fr98- and Fr98/SE-
looked for activation of microglia, which has been found in inoculated mice were analyzed at the time clinical disease was
other retrovirus-induced neurologic diseases and may play an apparent (3 to 4 weeks and 6 to 8 weeks, respectively). Fr54-
important role in neuropathogenesis (13). Activated microglia, inoculated mice were analyzed at 6 to 8 weeks postinoculation.
which stained positive with anti-Mac-1 monoclonal antibody, The most predominant gp70 staining for both neurovirulent
were found in both Fr98- and Fr54-infected brain and were
localized primarily in the white matter tracts of the cerebellum
(Fig. 1E and F, arrows), internal capsule, and corpus callosum.
Focal collections of activated microglia associated with blood TABLE 1. Quantitation of cells expressing gp70 in the cerebella of
vessels in the brainstem and cerebral cortex were also noted. Fr98/SE- and Fr54-infected mice at 8 weeks postinoculation
Mac-1 staining was not observed in uninoculated controls (Fig. No. of infected cellsa Group mean
1D). Fr98/SE induced similar astrocytic and microglial activa- Virus Animal
(mean SD) SEM
tion (data not shown). In addition to the glial activation, mild
vacuolar degeneration in the lateral cerebellar nuclei and/or Fr98/SE 1 192.8 32.5
thalamus was observed in brains of Fr98-inoculated mice (four 2 196.4 26.1 186.0 6.8
3 166.2 34.3
of four), Fr98/SE-inoculated mice (three of ﬁve), and Fr54- 4 188.6 50.4
inoculated mice (ﬁve of eight) (not shown). Thus, we were
unable to detect any qualitative differences in the pathology Fr54 1 102.2 7.2
induced by neurovirulent and nonneurovirulent retroviruses. 2 111.8 19.0 104.6 11.6
Localization of virus and identiﬁcation of virus-infected 3 74.6 11.6
cells in the brain. Since we were interested in ﬁnding corre- 4 130.0 21.3
lates of neurovirulence, it was important to determine if there a
Determined by counting the number of gp70-positive cells in ﬁve 4- m-thick
were any differences in regional distribution of these viruses in sections of the cerebella per animal. Comparison of the two groups by using
the brain. Sagittal sections of brain were stained with a poly- Student’s t test gave a P value of 0.001.
VOL. 71, 1997 CORRELATES OF POLYTROPIC MuLV NEUROVIRULENCE 5291
nuclei and thalamus, where parenchymal cells reminiscent of
microglia were detected (see below).
In the absence of any obvious differences in regional distri-
bution of virus, we examined the speciﬁc cell types in brains
infected by these viruses. The cells staining for viral envelope
protein in brains of Fr98-, Fr54-, and Fr98/SE-infected mice
exhibited similar morphologies (Fig. 2). Infected cells were
either associated with blood vessels (endothelia and/or peri-
cytes) or localized in the CNS parenchyma and characterized
by a bipolar or highly ramiﬁed morphology typical of activated
microglia (Fig. 2, inset). It was apparent that the distribution of
viral envelope protein closely paralleled that of Mac-1-positive
microglia (compare Fig. 2 with Fig. 1E and F). To conﬁrm this
observation and identify any other infected cell type(s), we
performed dual-label immunoﬂuorescence using the poly-
clonal anti-MuLV gp70 antiserum in conjunction with antibod-
ies directed against various cell-speciﬁc markers. Since viral
antigens were concentrated primarily in white matter without
neuronal infection, antibodies to the glial cell markers Mac-1
(activated microglia), CNP (oligodendrocytes), and GFAP (ac-
tivated astrocytes) were used. In mice inoculated with either
neurovirulent or nonneurovirulent virus, viral antigen colocal-
ized primarily with cells expressing the microglial cell marker,
Mac-1 (Fig. 3A to D). In addition to microglial cells, a small
number of the infected cells were identiﬁed as oligodendro-
cytes (Fig. 3E and F). Despite the pronounced astroglial acti-
vation, no viral antigen colocalized with GFAP-positive astro-
cytes in brains infected with Fr98 (Fig. 4), Fr54, or Fr98/SE.
Comparisons of relative viral burden in the brain. Because
the cerebellum appeared to be the primary target of infection
for these viruses and because the clinical disease is consistent
with cerebellar dysfunction (i.e., imbalance), quantiﬁcation of
viral burden focused on this region of the brain. In a previous
study, Fr98, Fr54, and Fr98/SE were shown to have similar
plasma viremia levels, indicating that these viruses did not
differ in the ability to replicate in the periphery (19). Although
viral capsid (CA) protein (p30) was ﬁrst detected in the brain
at 2 weeks postinoculation for all three viruses (19), immuno-
blot analyses suggested that the amount of viral protein in the
cerebella of Fr98-infected mice was greater than that in cere-
bella of Fr54- and Fr98/SE-infected mice (19). To quantify this
FIG. 3. Identiﬁcation of Fr98- and Fr54-infected cells in brain. Shown is dual
immunoﬂuorescent labeling of cryostat sections of brains taken from Fr98-
inoculated (A and B) and Fr54-inoculated (C and D) mice. Panels A and C show
immunoﬂuorescent labeling of viral envelope protein (anti-gp70), with corre-
sponding labeling of the microglial cell marker, anti-Mac-1, shown in panels B
and D. Viral antigen colocalized with the microglial cell marker in both Fr98-
and Fr54-infected brains. Localization of virus in oligodendrocytes was shown by
dual immunoﬂuorescent labeling of viral envelope protein (E) with the oligo-
dendrocyte marker anti-CNP (F) in vibratome sections of brain taken from
Fr98-infected mice. Analyses of Fr54- and Fr98/SE-infected brains revealed
similar results (data not shown). Bars in panels A and C, 20 m; bar in panel E,
and nonneurovirulent viruses was detected in the white matter
of the cerebellum and consisted of both vascular and paren-
chymal cells. Infected vascular and parenchymal cells were
observed in the corpus callosum and internal capsule but were
less frequent than in the cerebellum. Focal areas of infected FIG. 4. Dual immunoﬂuorescent labeling of infected cells and astrocytes.
cells were also observed within the brainstem, colliculus, cere- Vibratome sections were dual labeled for viral envelope protein (green; arrow)
and GFAP (red; arrowhead). As is represented in this photo of cells in the white
bral cortex, hippocampus, thalamus, and cerebellar nuclei. In- matter tract of the cerebellum, no colocalization of these two antigens was
fection in grey matter areas appeared to be restricted to cells detected in brains of Fr98-infected mice. Analyses of brains from Fr54- and
associated with small blood vessels except in the cerebellar Fr98/SE-inoculated mice revealed similar results. Bar, 20 m.
5292 ROBERTSON ET AL. J. VIROL.
Moreover, the amount of viral protein in brains from clinically
ill Fr98/SE-infected mice at 8 weeks postinoculation had
achieved levels indistinguishable from those seen in clinically
ill Fr98-infected mice at 3 weeks postinoculation (Fig. 5C).
Together, these results suggested that entry of Fr54 and
Fr98/SE into the brain occurred with comparable efﬁciencies
but that the spread of Fr98/SE within the brain was more rapid
than that of Fr54. Furthermore, these data suggested that
induction of clinical neurologic disease may require a critical
threshold of viral protein expression.
The differences observed in the relative levels of viral pro-
tein expression between Fr54 and Fr98/SE at 8 weeks could
have been a consequence of differences in the frequency of
infected cells or in the amount of viral protein expressed on a
per-cell basis. This issue was examined by counting infected
cells in sections of cerebellum stained for viral envelope pro-
tein in Fr54- and Fr98/SE-inoculated mice at 8 weeks postin-
oculation. The results indicated that the number of infected
cells in Fr98/SE-inoculated mice was 1.5- to 2-fold higher than
that in Fr54-inoculated mice (Table 1). This difference is in the
same range as that detected by immunoblot analysis (Fig. 5B),
suggesting that the differences in viral burden were reﬂective of
differences in frequency of virus-infected cells within the brain.
The comparative studies of coisogenic polytropic MuLV
strains differing in neurovirulence revealed that the amount of
virus infection in the CNS appeared to be an important factor
correlating with clinical neurologic disease. In contrast, there
was no correlation between disease and the regional distribu-
tion of virus in the CNS, the types of brain cells infected, or the
neuropathology induced by these viruses.
FIG. 5. Western blot analyses of p30 capsid protein in Fr98-, Fr54-, and
Fr98/SE-infected brains. Total cerebellar protein homogenates clariﬁed with The regional distribution of polytropic MuLV-infected cells
low-speed centrifugation (10%, wt/vol) from mice neonatally inoculated with was notably restricted in comparison to the widespread CNS
Fr98, Fr54, or Fr98/SE and age-matched uninoculated controls were separated distribution of previously studied ecotropic MuLV such as
by SDS-PAGE and probed with anti-p30 antiserum. Twofold serial dilutions of FrCasE (25). Cells infected by the polytropic MuLV were lo-
the homogenates were analyzed to compare the relative amounts of viral protein
in the brain. (A) Comparison of Fr98-, Fr54-, and Fr98/SE-infected brains at 3 calized primarily to the white matter of the cerebellum, corpus
weeks postinoculation, showing that Fr98-infected mice contained two- to four- callosum, and subcortex. In contrast, neuroinvasive ecotropic
fold-greater amounts of viral capsid protein than either Fr54- or Fr98/SE-in- MuLV infect cells throughout the grey and white matter of the
fected mice. (B) At 8 weeks, the amount of viral capsid protein in samples from brain, brainstem, and spinal cord. The restriction of the poly-
Fr98/SE-infected mice exceeded that in age-matched Fr54-infected mice and (C)
reached levels equivalent to those of Fr98-infected mice at 3 weeks postinocu- tropic viruses is likely a function of the envelope gene, since
lation. These results were a consistent ﬁnding in the analysis of brain homoge- the ecotropic virus FrCasE mentioned above shares long ter-
nates from three mice per virus strain per time point. The clinical status of the minal repeat, gag, and pol sequences with the polytropic viruses
mice at each time point is presented at the right ( , clinical neurologic disease; reported here. Since retrovirus infection requires cell division
, clinically normal).
(18), a possible explanation for the restricted distribution of
the polytropic viruses is that they are less efﬁcient at reaching
the CNS at a time when target cells in the brain are still actively
apparent difference, we compared the relative levels of viral dividing. It is also possible that the differences in virus distri-
protein in a dilution series of 10% cerebellar homogenates bution reﬂect differential expression of the polytropic and eco-
from Fr98-, Fr54-, and Fr98/SE-infected mice. Homogenates tropic receptors in the brain.
were analyzed in pairs by immunoblot analysis after separation The predominant target cells in the CNS for both neuro-
by SDS-PAGE, using a polyclonal anti-capsid (CA) protein virulent and nonneurovirulent viruses were vascular endothe-
(p30) antiserum (Fig. 5) (26). At 3 weeks postinoculation, the lial cells/pericytes and activated microglia. Astrocytes were
level of CA protein in the cerebella of Fr98-inoculated mice consistently negative for viral protein. Since the astrocyte
was two- to fourfold higher than that in either Fr98/SE- or marker, GFAP, used in this analysis may identify only the
Fr54-inoculated mice (Fig. 5A). At this time, Fr98-inoculated population of astrocytes that is activated, it is possible that
mice were exhibiting clinical disease whereas Fr98/SE-inocu- there were productively infected GFAP-negative astrocytes.
lated mice were asymptomatic. At 3 weeks, the contents of CA However, this possibility is unlikely for two reasons. First, in
protein observed in Fr54- and Fr98/SE-inoculated mice were double-label immunoﬂuorescence studies, the vast majority of
indistinguishable (Fig. 5A). cells expressing viral protein were Mac-1-positive microglia.
At 8 weeks postinoculation, a time when Fr98/SE-inoculated Second, the morphology of the infected cells in the CNS pa-
mice were developing ataxia (19), immunoblot analysis re- renchyma was most consistent with that of activated microglia
vealed that expression of viral protein in the brains of Fr98/ (Fig. 2).
SE-infected mice had exceeded the viral burden in age- As has been shown for the neuropathogenic ecotropic
matched Fr54-infected mice by two- to fourfold (Fig. 5B). MuLV (3, 25), human immunodeﬁciency virus (24, 49), and
VOL. 71, 1997 CORRELATES OF POLYTROPIC MuLV NEUROVIRULENCE 5293
other animal lentiviruses (43), polytropic MuLV appear pri- ciency of spread within the microglial compartment. The coiso-
marily to infect cells of the microglia/macrophage lineage in genic nature of these viruses identiﬁes the envelope gene as an
the CNS parenchyma. The importance of microglial cell infec- important determinant of this phenotype. It is noteworthy that
tion in retrovirus-induced neuropathogenesis has been dem- the viral burden in the cerebella of Fr98-infected mice reached
onstrated by engraftment experiments where microglial cells levels two- to fourfold higher than those in cerebella of both
infected with a neurovirulent ecotropic MuLV induced focal Fr54- and Fr98/SE-infected mice at 2 (19) and 3 (Fig. 5) weeks
spongiform degeneration in the brains of mice (27), while the postinoculation, suggesting that Fr98 may be more efﬁcient at
implantation of infected cells of neuroglial origin (oligoden- infecting endothelial cells in addition to microglial cells.
drocytes and astrocytes) failed to induce neuropathology (28). Although the neuropathologic changes examined in this
Microglial cells may mediate neuropathogenesis, in part, by the study did not correlate with neurovirulence, the results of these
production of nonviral neurotoxins, the effects of which have studies were interesting in several respects. First, the neuro-
been demonstrated in in vitro culture systems (15, 16). In light virulent viruses Fr98 and Fr98/SE induced only mild spongi-
of these ﬁndings, it is particularly interesting that both the form degeneration in the brain, despite the dramatic clinical
nonneurovirulent polytropic virus Fr54 and the neurovirulent neurologic deﬁcits exhibited by infected mice. The ﬁnding of
Fr98 and Fr98/SE exhibited microglial tropism. These results similar lesions in Fr54-inoculated mice, which appeared clini-
demonstrate that microglial tropism per se was not sufﬁcient cally normal, implies that these vacuolar lesions might not be
for induction of disease. Thus, envelope gene variations may clinically relevant in this model. The lack of obvious disease-
inﬂuence neurovirulence in this model by altering other qual- speciﬁc structural CNS damage suggests that the nature of the
itative and/or quantitative aspects of CNS infection. insult leading to clinical disease may involve disruption of
The results of this study indicated that there was a direct physiological processes in the brain which may not be detect-
correlation between neurovirulence and the level of virus in- able at the light microscopic level. Second, reactive astrogliosis
fection in the brain. Viral burden was measured by both the and microglial activation were induced by all three viruses
amount of viral capsid protein and the number of infected regardless of differences in neurovirulence and viral burden.
cells. It is noteworthy that the levels of viral protein in the Thus, the glial response appeared to correlate with virus in-
cerebella of Fr98/SE- and Fr54-infected mice were indistin- fection in the CNS rather than clinical disease. However, it is
guishable at 2 (19) and 3 (Fig. 5) weeks postinoculation yet possible that the glial response induced by virulent and aviru-
differed signiﬁcantly at 8 weeks postinoculation. In the com- lent viruses is qualitatively or quantitatively different. For ex-
parison of the two neurovirulent viruses, Fr98 and Fr98/SE, ample the patterns or quantity of cytokines expressed by reac-
there was a direct correlation between kinetics of viral protein tive astrocytes and activated microglia may vary among
accumulation in the brain and incubation period of clinical different virus strains. To begin to address this issue, we are
disease. Furthermore, the viral burden in the cerebella of Fr98- currently comparing the cytokine responses in the brains of
and Fr98/SE-infected mice reached comparable levels, albeit mice infected with neurovirulent and nonneurovirulent viruses.
at different times, coincident with the onset of clinical disease,
suggesting that a critical threshold of viral burden may be ACKNOWLEDGMENTS
required to induce neurologic disease. Similar to previous
We thank Richard Bessen for helpful discussions regarding this
studies of the neurovirulent ecotropic virus tsMoBA (4), these
study, and we thank Bob Evans and Gary Hettrick for assistance with
results suggest that the capacity of Fr98 and Fr98/SE to infect graphics.
more target cells in the brain than Fr54 may alone explain their This work was supported in part by a National Institutes of Health
neurovirulence. It should be noted that the differences in viral Predoctoral Intramural Research Training Award fellowship (S.J.R.).
burden revealed here, though consistent and signiﬁcant, were
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