Journal
of General Virology (1998), 79, 525–536. Printed in Great Britain
...................................................................................................................................................................................................................................................................................
A neuroattenuated ICP34.5-deficient herpes simplex virus type
1 replicates in ependymal cells of the murine central nervous
system
Santosh Kesari,1, 2 Todd M. Lasner,1, 3 Keki R. Balsara,2 Bruce P. Randazzo,1, 4 Virginia M.-Y. Lee,2
John Q. Trojanowski2 and Nigel W. Fraser1
The Wistar Institute1, Department of Pathology and Laboratory Medicine2, Division of Neurosurgery3 and Department of Dermatology4,
University of Pennsylvania School of Medicine, 36th and Spruce Streets, Philadelphia, PA 19104, USA
Herpes simplex virus type 1 (HSV-1) variant 1716 is
deleted in the gene encoding ICP34.5 and is
neuroattenuated after intracranial inoculation of
mice. Although the mechanism of attenuation is
unclear, this property has been exploited to eliminate experimental brain tumours. Previously, it was
shown that infectious 1716 was recoverable for up
to 3 days after intracranial inoculation suggesting
that there may be limited replication in the central
nervous system (CNS). Here it is demonstrated that
1716 replicates in specific cell types (predominantly
CNS ependymal cells) of BALB/c mice, using im-
Introduction
Herpes simplex virus type 1 (HSV-1) is a large neurotropic
DNA virus which infects a wide range of cell types in different
animals. Following primary infection, HSV-1 establishes a lytic
or latent infection in neurons of the central nervous system
(CNS) and peripheral nervous system (PNS). During lytic HSV
infection, the macromolecular machinery of the infected cell is
redirected toward the transcription, replication and assembly
of virus progeny (reviewed by Roizman & Sears, 1996). The
entire process of virus entry, replication, assembly and egress
takes between 18 and 20 h, resulting in cell death (although
neurons may survive acute infection ; Simmons & Tscharke,
1992) and release of progeny virus. During this interval, viral
genes are expressed through a tightly regulated transcriptional
cascade and are designated the α (immediate early, e.g. ICP4) ;
β (early, e.g. TK) ; and γ (late, e.g. VP5, gC, ICP34.5) genes
(Honess & Roizman, 1974, 1975). During latent infection, only
a small of set of latency associated transcripts (LATs) are
expressed (for review see Fraser et al., 1992). In humans, the
natural host, HSV-1 causes a wide range of diseases including
Author for correspondence : Nigel W. Fraser.
Fax ­1 215 898 3849. e-mail Fraser!wista.wistar.upenn.edu
munohistochemical, immunofluorescence, in situ
hybridization and virus titration studies. While
1716-infected mice exhibited no overt signs of
encephalitis, histological analysis showed a persistent loss of the ependymal lining. Thus, although
ICP34.5-deficient viruses are neuroattenuated,
they do retain the ability to replicate in and destroy
the ependyma of the murine CNS. A detailed understanding of the mechanism(s) of neuroattenuation
and limited replication could lead to the rational
design of safe HSV vectors for cancer and gene
therapy in the CNS.
fever, cold sores, keratitis, blindness and encephalitis (reviewed
by Whitley & Gnann, 1993). Most (60–90 %) of the population
is seropositive for HSV-1 by early adulthood, and HSV-1induced disease is usually limited in immunocompetent hosts.
However, when the immune system is compromised, HSV-1
can disseminate to cause severe and life-threatening infections
(Whitley, 1996). For instance, in AIDS patients HSV causes
chronic active systemic infections and brainstem encephalitis
(Hamilton et al., 1995 ; Laskin et al., 1987).
HSV-1 encephalitis is the most serious consequence of CNS
infection, and develops from primary or reactivated infection
(Nahmias et al., 1982 ; Whitley et al., 1982 a). The clinical
manifestations of encephalitis reflect the site of infection in the
brain and may include memory loss, speech deficits, hallucinations and seizures (Whitley et al., 1982 b). Although the nature
of HSV-1 tropism for specific regions of the brain is poorly
understood, studies of the biology of HSV-1 isolates and
mutants thereof in animal models have provided insights into
how viral and cellular factors may affect CNS pathogenesis in
humans (Chrisp et al., 1989 ; Valyi-Nagy et al., 1992, 1994 a, b).
More significantly, these studies have also led to the use of
HSV-derived vectors for cancer and gene therapy of CNS
disorders (Kesari et al., 1995, 1996 ; Lawrence et al., 1995 ;
Martuza et al., 1991).
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S. Kesari and others
There are many cellular and viral factors that determine the
outcome of infection. Although many viral proteins are not
required for the production of infectious virus in cultured cells,
these ‘ non-essential ’ genes play important roles in the life
cycle of the virus in animals (Roizman & Sears, 1996). One viral
gene that is dispensable in vitro but essential in vivo is RL1,
encoding infected cell protein 34.5 (ICP34.5 or γ34.5) (Chou et
al., 1990 ; McGeoch et al., 1991), a protein which has homology
to the cellular genes MyD116 and GADD34 (Chou et al.,
1990 ; Chou & Roizman, 1994 ; McGeoch & Barnett, 1991).
ICP34.5 is thought to act by inhibiting the cessation of protein
synthesis associated with programmed cell death in some nonpermissive, dividing cells (i.e. in SK-N-SH human neuroblastoma cells) by regulating the interferon-inducible PKR
pathway (Chou et al., 1995 ; Chou & Roizman, 1992, 1994). A
more recent tissue culture study (Mohr & Gluzman, 1996)
suggests that other HSV genes are involved in the PKR
pathway and the action(s) of ICP34.5 are likely to be complex.
Another study has shown that the inhibitory action of ICP34.5
can be separated from the virulence phenotype (Markovitz et
al., 1997). However, the studies of ICP34.5 function to date
have focused on in vitro tissue culture characterization, and the
specific mechanism by which the absence of ICP34.5 confers
neuroattenuation in vivo is not clear.
Variants of HSV in which RL1 is deleted or mutated have
been reported to be incapable of replicating in the CNS of mice
and causing encephalitis (Chou et al., 1990 ; MacLean et al.,
1991 ; Taha et al., 1990). From these and other studies, it was
speculated that the replication of HSV-1 ICP34.5 mutants may
be cell cycle dependent, thereby rendering these mutants
incapable of replication in post-mitotic and}or quiescent cells
in the brain (Brown et al., 1994 a ; Chou et al., 1990 ; Kesari et
al., 1995). Previously, we showed that HSV variant 1716,
which has a 759 nucleotide deletion in the RL1, was attenuated
following intracranial (i.c.) inoculation, but infectious virus was
recoverable for up to 3 days after i.c. inoculation (Kesari et al.,
1995, 1996). The present studies demonstrate that 1716
replicates in specific cell types (predominantly ventricular
ependymal cells), using immunohistochemistry (IHC) for the
detection of de novo viral proteins, immunofluorescence for
identification of infected cells, in situ hybridization (ISH) for the
detection of viral transcripts and titration of infectious virus
from brain. These results suggest that although ICP34.5deficient viruses are neuroattenuated, they do replicate in
ependymal cells of the CNS.
Methods
+ Virus stocks. To produce virus stocks, subconfluent monolayers of
baby hamster kidney 21 clone 13 (BHK) cells were infected with HSV
variants 1716 (stock titre 1¬10) p.f.u.}ml), 1771, 1771R or parental 17+
(stock titre 2¬10) p.f.u.}ml). Variant 1716 has a 759 bp deletion which
deletes parts of the genes encoding ICP34.5, LAT and orfP (MacLean et
al., 1991). Variant 1771 has a single stop codon 9 bp downstream of the
ATG for the ICP34.5 ORF, and 1771R is a revertant of 1771 which
FCG
behaves identically to parental 17+ (McKie et al., 1994). Virus was
concentrated from the culture, titred on BHK cells by plaque assay, stored
at ®70 °C in 0±5 ml aliquots of virus culture medium and thawed rapidly
just prior to use as described (Spivack & Fraser, 1987 ; Valyi-Nagy et al.,
1992). Ultraviolet light-inactivated 17+ virus (17+UV) was prepared from
stock 17+ (titre 2¬10) p.f.u.}ml) as described previously (Notarianni &
Preston, 1982) and inactivation was confirmed by plaque assay. Serumfree medium (SFM) was used for control (mock) inoculation studies as
negative controls.
+ Virus inoculation. BALB}c mice (4–6 weeks old) obtained from
HSD (Indianapolis, Ind., USA) were anaesthetized (87 mg}kg ketamine,
13 mg}kg xylazine). Using a Hamilton syringe with a 30 gauge
removable needle, the appropriate amount of virus (5¬10& p.f.u. in 5 µl)
was injected into the right lateral ventricle of the brain using a small
animal stereotactic apparatus (Kopf Instruments). Virus stocks were
serially diluted in culture medium for low dose inoculation (100 p.f.u. in
5 µl). The ventricular injections were performed over 3 min, the needle
was inserted 2±5 mm into the right hemisphere and the inoculum was
injected along the needle tract for 1 mm and then slowly withdrawn over
1 min as described previously (Kesari et al., 1995). The parenchymal
injections were performed over 3 min, the virus (1¬10& in 2 µl) was
injected into the right frontal lobe (less volume was used to reduce the
chance of leakage). Experiments with 17+ and 1716 were performed in
parallel.
+ Immunohistochemical procedures. Mice were transcardially
perfused with PBS and fixed with 4 % paraformaldehyde (0±1 M PBS
pH 7±4), and the brain and trigeminal ganglia were dissected for
histological and IHC analysis. The methods for tissue processing and
light microscopic IHC analysis were similar to those described elsewhere
(Trojanowski et al., 1993, 1994). Rabbit polyclonal antiserum to HSV-1
which detects the major glycoproteins present in the virus envelope and
at least one core protein (Dako) was used at a dilution of 1 : 2000 to detect
replicating virus (Adams et al., 1984). Although this antiserum had the
ability to detect both structural and non-structural proteins, it did not
detect the small amount of input protein following 17+UV inoculation.
Antigen-expressing cells were detected by the indirect avidin–biotin
immunoperoxidase (Vectastain ABC Kit, Vector Laboratories) and 3, 3«diaminobenzidine as the chromagen. Sixty mice were inoculated with
1716 into the ventricle and sacrificed at different times (five each at days
1, 2, 3, 4, 7, 10, 15, 21, 27, 30, 44 and at month 7). Six mice were
parenchymally inoculated with 1716 and three mice each were sacrificed
at days 3 and 30. Six 17+ ventricle-inoculated mice were sacrificed at days
1, 2 and 3 ; two mice were parenchymally inoculated with 17+ and
sacrificed at day 2.
+ Immunofluorescence detection of double-labelled cells. For
immunofluorescence studies, anti-HSV (1 : 500), anti-GFAP (1 : 10) and
anti-MAP2 (1 : 100) ascites were used alone and in combination for colocalization studies. The secondary antibodies were conjugated with
either fluorescein isothiocyanate (FITC) or Texas red probes. A Nikon
microphot FXA photomicroscope equipped with epifluorescence and
FITC and Texas red filters was used for photomicrography. Bright-field
as well as fluorescent photomicrographs were taken using a Sony 3CCD
colour video camera and Northern Exposure Image analysis program
(Empix Imaging, Release 2±9d). Sections from uninfected mice and
primary antibody omission on infected sections were used as controls.
+ ISH for HSV-1-specific gene expression. Sections of perfused
and fixed tissue were mounted on slides and ISH was performed to detect
viral gene expression as previously described (Valyi-Nagy et al., 1994 b).
The LAT probe BstEII–BstEII subfragment of BamHI B, the BamHI Y
fragment from plasmid pRB113 containing the ICP4 probe, the
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HSV replication in ependymal cells
EcoRI–BamHI fragment I}I (KOS) from plasmid pBR322 containing the
gC probe, and the BamHI fragment a« (KOS) from plasmid pBR322
containing the VP5 probe were isolated from restriction digests by gel
electrophoresis and purified by GeneClean (Bio 101) (Valyi-Nagy et al.,
1992, 1991). DNA probes were nick-translated and then separated from
unincorporated nucleotides by passage through Sephadex G-50 spin
columns (Pharmacia) (Deatly et al., 1988). The specific activities of the
probes were approximately 1–5¬10) c.p.m. per µg DNA. Serial tissue
sections were hybridized with one of the following $&S-labelled HSV
probes : LAT, ICP4, gC or VP5. In all ISH experiments, slides from each
sample were exposed for 5 days. Sections of 17+-infected brains were
used as positive controls and sections from uninfected mice were used as
negative controls. RNase- and DNase-treated slides from 17+-infected
and uninfected mice were used as further controls to ensure specificity of
the probes for HSV RNA.
+ Titration of virus from brain. To titrate virus in brain, mice were
sacrificed by lethal injection of anaesthesia. The brains were dissected
from mice sacrificed at different times post-inoculation (p.i.) (1, 4, 8, 12,
24, 48 and 72 h ; two to five animals per time-point), quick frozen in liquid
nitrogen and stored at ®70 °C. For the ventricular injections, the whole
brain was saved as one sample and titred as one sample. For the
parenchymal injections, the frontal lobe from the posterior border of the
injection site to the anterior border of the olfactory bulb (called anterior
portion) and the rest of the brain posterior to the injection site (called
posterior portion) were saved as separate samples and titred separately.
The samples from the different time-points were rapidly thawed in a
37 °C water bath, and the tissue was homogenized in culture medium at
10 % w}v using a Pyrex Ten Broeck tissue grinder. The homogenates
were centrifuged at 3 000 g for 10 min at 4 °C, the supernatant was
diluted exponentially in media and the virus titre of each sample was
determined by plaque assay on BHK cells (Spivack & Fraser, 1987).
Results
IHC localization of infected cells after ventricular and
parenchymal inoculation
In order to assess whether there were any cells in the mouse
CNS that were permissive for 1716, we performed IHC
analyses on brains of BALB}c mice infected via i.c. inoculation
into the ventricle at different times (5¬10& p.f.u.}5 µl ; days 1,
2, 3, 4, 7, 10, 15, 21, 27 and 30). Except for the first 5–7 days
when piloerection was observed, these mice did not show any
obvious signs of disease or encephalitis. However, on gross
dissection and histology some of the mice infected with 1716
had enlarged ventricles, and this was not observed in
uninfected mice. Some SFM- and 17+UV-inoculated mice were
seen by histology to have slightly dilated ventricles at day 1
(Fig. 1 A).
Using a polyclonal HSV antibody which detects late viral
proteins, sections from animals were screened for virus-infected
cells. At day 1 after 1716 inoculation, immunopositive cells
were detected only in the ependymal cells lining the ventricles
(Fig. 1 B). At day 2 almost all the ependymal cells were
immunopositive for HSV with some positive cells in the
subependymal region (Fig. 1 C). Histologically, these cells had
the characteristic features of herpes-infected cells, including
rounding-up and the presence of inclusion bodies. HSVimmunopositive cells were not observed in uninfected, SFM-
inoculated and 17+UV-inoculated mice at days 1, 2, 7 and
30 p.i.) (Fig. 1 A), suggesting that most of the immunopositivity is due to detection of de novo synthesis of viral
proteins in infected cells and not due to detection of input
virion proteins on the surface or on extracellular virus. Since
most of the positive cells lined the lateral ventricles with some
around the third and fourth ventricles, this may reflect the
spread of virus by the flow of cerebrospinal fluid from the
lateral to the third and fourth ventricles (Fig. 1 E). By day 4,
ependymal cells throughout the ventricular system were
positive for virus antigens. Immunopositive cells were also
detected in the subependymal region (Fig. 1 D, E, G), choroid
plexus (Fig. 1 H) and the parenchyma (Fig. 1 I). By day 7, only
a few immunopositive cells were detected in the ependyma
and adjacent brain parenchyma. From day 10 (Fig. 1 F) through
to day 30, no HSV-immunopositive cells were detected in
1716-inoculated mice. In contrast, in mice ventricularly
inoculated with parental 17+ (10' p.f.u.}5 µl, and also at lower
doses) both ependymal and widespread parenchymal cells
were positive as early as day 1 (Fig. 1 J, K, L).
In contrast to ventricle inoculation, parenchymal inoculation with 1716 (5¬10& p.f.u.) showed immunopositive cells
at the site of inoculation but not in the ependyma (day 2 ; Fig.
2 A, B). Parenchymal inoculation with 17+ (10' p.f.u.) resulted
in infection of parenchyma at the site of inoculation (Fig. 2 D)
and also widespread infection of ependyma and other
parenchymal sites (Fig. 2 C). Inoculation of mice with 17+
(doses ranging from 10# to 10' p.f.u.) produces encephalitis,
and 100 % of the mice die by 3–10 days p.i., the time
depending on the dose (unpublished observations). The lethal
dose (50 %) is less than 10 p.f.u. for 17+ (McKie et al., 1994).
Inoculation with 17+ induced a perivascular inflammatory
response and in some cases enlarged ventricles were present
(data not shown).
Similar findings to those for 1716 were observed in mice
infected with 1771, which has a point mutation in the ORF of
ICP34.5 (McKie et al., 1994). Thus, the properties of 1716 and
1771 are probably due to the specific absence of ICP34.5 and
not to changes in other unidentified, overlapping genes in the
region of ICP34.5. The behaviour of the revertant of 1771
(called 1771R) was indistinguishable from 17+. The ependymal
infection and loss, as well as ventricular enlargement, were
dose-dependent. For example, a low dose ventricular inoculation of 1716 (10# p.f.u.) did not result in the loss of
ependyma and enlargement of ventricles, and analyses at early
time-points showed fewer infected cells compared to the
results of a high dose of 1716 (10& p.f.u.) (Fig. 1).
Determination of the cellular phenotype of 1716infected cells by morphology and immunofluorescence
co-localization
HSV has been shown to infect both neuronal and nonneuronal cells during productive and latent infections (Tenser
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S. Kesari and others
Fig. 1. For legend see facing page.
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HSV replication in ependymal cells
Fig. 2. Detection of HSV-infected cells
after parenchymal inoculation of 1716
and 17+. Mice were stereotactically
inoculated into the parenchyma (frontal
lobe) with either 5¬105 p.f.u. 1716
(three mice each sacrificed at days 2 and
30) or 1¬106 p.f.u. 17+ (two mice
sacrificed at day 2). HSV-positive cells in
the parenchyma are localized at the site
of inoculation of 1716 at day 2 (A). Note
that both neurons and non-neuronal cells
are infected in 1716-infected mice (B).
No HSV-positive cells were detected at
day 30 in 1716-infected mice (Fig. 7
and corresponding text). In contrast 17+
spreads extensively from site of
inoculation (D) to distant locations away
from the inoculation site including the
cortex (C), brainstem and ependyma.
Scale bar : (A, C), 118±5 µm ; (B, D),
59±3 µm.
et al., 1991). But different strains or mutants of HSV can have
a different host-cell range (in the same host) which is dependent
on a number of viral, immune and cellular factors (Fraser &
Valyi-Nagy, 1993). Thus, to determine the cellular phenotype
of HSV-infected cells, morphological characterization and
double-immunofluorescence co-localization experiments were
performed using markers for astroglial (GFAP) and neuronal
(MAP2) cells (Fig. 3). Only a few HSV-immunopositive
parenchymal cells (! 5 per section) were positive for GFAP in
both 1716- and 17+-infected mice ; HSV is red, GFAP is green
and cells that co-localize (arrows) are yellow (Fig. 3 A–F). In
1716-infected mice (at day 4 when parenchymal cells were
positive for HSV), most of the HSV-immunopositive cells were
also MAP2-positive and had neuronal morphology ; HSV is
red, MAP2 is green and cells that co-localize (arrows) are
yellow (Fig. 3 G–I). Likewise, in 17+-infected mice (at day 2),
most of the HSV-positive cells also were MAP2-positive with
neuronal morphology (Fig. 3 J–L). Although 1716 predominantly infected ependymal cells (which are glial-derived), the
small number of infected parenchymal cells were mostly
neurons. Thus, similar to 17+, 1716 infects both ependymal
and parenchymal cells, but the degree of infection of
parenchymal cells is significantly less in the case of 1716. This
suggests either that spread of 1716 from ependyma to
parenchyma is inefficient, or that infection of parenchymal cells
is reduced in relation to 17+.
Virus productive cycle transcripts are detectable
during 1716 infection
HSV genes are expressed through a tightly controlled
transcriptional cascade during productive infection and are
termed immediate early, early and late genes in order of their
appearance (Honess & Roizman, 1974). To determine whether
the HSV 1716-immunopositive cells were undergoing a
productive infection, ISH was performed to detect these
different classes of viral transcripts. Serial sections were probed
for RNA of LAT, ICP4 (an immediate early gene), gC (a late
gene) and VP5 (a late gene). Tissue sections from 17+ ventricleinfected mice (1¬10' at day 2) were used as positive controls
(Fig. 4 A, C, E). For 17+, in both ventricle- and parenchymainfected brains, both ependymal and parenchymal cells were
Fig. 1. Detection of HSV-infected cells after ventricle inoculation with 1716 and 17+. BALB/c mice (4–6 weeks old) obtained
from HSD (Indianapolis, Ind., USA) were stereotactically inoculated into the right ventricle with either 5¬105 p.f.u. 1716 ;
1¬106 p.f.u. 17+ ; or 1¬106 p.f.u. 17+UV and sacrificed at different times p.i. as described under Methods. Representative
photomicrographs are shown from some sections of brains after IHC for HSV antigens. No HSV-positive cells were detected in
the ependyma of the right lateral ventricle from a 17+UV (1¬106 p.f.u.)-infected mouse, and the ventricles were slightly
dilated at day 1 (A). Ventricles of 1716 (5¬105 p.f.u.)-infected mice at different days p.i. : days 1 (B) ; 2 (C) ; 4 (D) and 10
(F) showing the lateral ventricle, and day 4 (E) showing the third ventricle. During the acute stage of 1716 infection (! 10
days at 5¬105 p.f.u.), HSV-immunopositive cells were found in the ependyma (day 4, G), choroid plexus (day 4, H) and
subependymal parenchyma (day 4, I) and no antigen-positive cells were detectable after 7–10 days (F). Sections from 17+
(1¬106 p.f.u.)-infected mice were used as positive controls and show infection of ependymal and subependymal cells (J),
brainstem neurons (K) and parenchymal cells (L) as early as day 1. Scale bar : (A–D, J), 296 µm ; (E, F, K), 118±5 µm ; (G–I,
L), 59±3 µm.
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S. Kesari and others
Fig. 3. Double immunofluorescence staining and morphological identification of HSV-immunopositive cells. Sections from brains
infected with either 17+ (day 2, 1¬106 p.f.u.) or 1716 (day 4, 5¬105 p.f.u.) after ventricular inoculation were stained by
immunofluorescence for HSV (1 : 500 ; red) and GFAP (1 : 10 ; green) or MAP2 (1 : 100 ; green). The images obtained from
each section indicate staining and the combined images show double-labelled cells (yellow). Some cells double-labelled
(yellow in B and E) for HSV (red in A and D) and GFAP (green in C and F) in both 17+- and 1716-infected mice (arrows in
A–F). The rest of the HSV+/GFAP− non-ependymal cells in this field (A) were identified as being MAP2+. Most non-ependymal
cells in both 17+- and 1716-infected mice that labelled for HSV also labelled for MAP2 (G–L) and had neuronal morphology.
Parts (J–L) are from a field away from the ventricle shown in (D) to illustrate the striking neuronal morphology of most of the
HSV- and MAP2-positive cells. This pattern was reproducible from at least two sections from two animals from each group.
Sections from uninfected mice and primary antibody omission on infected sections were used as controls. Scale bar, 71±1 µm.
positive for ICP4 (Fig. 4 C), LAT (Fig. 4 E), gC and VP5. In 1716
ventricle-infected mice, a subset of the immunopositive
ependymal cells (Fig. 4 B) was also positive for ICP4 (Fig. 4 D),
LAT (Fig. 4 F), gC and VP5 transcripts. Also, in both 1716
FDA
ventricle- and parenchyma-inoculated brains, transcript-positive cells were detected in the parenchyma. The detection of
late RNA and protein expression in these infected ependymal
cells suggests that there is no block in the characteristic
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HSV replication in ependymal cells
Fig. 4. Detection of productive cycle viral
gene expression in the ependyma by ISH.
BALB/c mice were stereotactically
inoculated into the right ventricle with
either 5¬105 p.f.u. 1716 (day 4 ; B, D,
F) or 1¬106 p.f.u. 17+ (day 2 ; A, C, E).
Both sets of mice were sacrificed and the
brains were removed and processed as
described under Methods. After
confirming the presence of antigenpositive cells (A, B), serial sections were
then individually probed for the different
classes of viral transcripts by ISH. As
expected, 17+-infected ependymal and
parenchymal cells express ICP4 (C), LAT
(E), gC (not shown) and VP5 (not
shown) transcripts. Likewise, 1716infected ependymal and parenchymal
cells also express all four transcripts ;
including ICP4 (D) and LAT (F). No in
situ positive signals were detected in
uninfected, SFM-inoculated or 17+UVinoculated mice and were used as
negative controls. Scale bar : (A, C, E),
118±5 µm ; (B, D, F), 59±3 µm.
productive gene and protein expression and that these cells
may be undergoing a full productive infection (see below).
After ventricular inoculation with 1716, infectious
virus is recovered
Although there is no block to viral gene and protein
expression in 1716-infected cells, infectious virus may not be
produced. To determine whether the infection observed by
IHC and ISH resulted in the production of infectious virus,
titrations were performed after i.c. inoculation at two different
sites : ventricular versus parenchymal inoculations (Fig. 5 A).
The results of recovery of infectious 1716 or 17+ after i.c.
inoculation into the ventricle is shown in Fig. 5 (B). The titre of
1716 (Fig. 5 B) drops at 8 h and then increases over time,
levelling off between 24 and 48 h, and virus is not detectable
after 72 h. The rapid drop and increase in the first 24 h is
characteristic of a single-step growth curve. The flattening of
the curve between 24 and 48 h and the decrease thereafter
suggest that at least one round of replication and no more than
2–3 replication cycles are occurring, assuming 18–20 h per
cycle as seen in tissue culture (Roizman & Sears, 1996). In
contrast, the recovery of 17+ from ventricular inoculations
showed a higher slope and no decrease after 24 h (Fig. 5 B).
Furthermore, no infectious virus was recoverable after i.c.
inoculation of 17+UV or from uninfected mice (data not
shown). Thus, infection of ependymal cells and}or parenchymal cells must lead to the production of infectious virus.
To determine whether the infectious virus recovered was
from ependymal and}or parenchymal infection, a modified
protocol was used (see legend to Fig. 5 A). Fig. 5 (C) shows the
recovery of 1716 and 17+ after parenchymal inoculation. Virus
was inoculated into the parenchyma and the brain was
separated into two parts, anterior (containing the inoculation
site) and posterior (rest of the brain posterior to the inoculation
site) (Fig. 5 A), and titred separately. At time 0 (input), 1716
was recovered in the anterior portion, but was not detected
thereafter in either the anterior or posterior portions (Fig. 5 C).
This suggests that either there is no production of infectious
virus in the parenchyma or there is a low level which is below
the detection limit of the assay. Although immunopositive
cells were detected in the parenchyma in both the ventricular
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S. Kesari and others
Fig. 5. Quantification of infectious virus in BALB/c mouse brain after i.c.
inoculation into ventricle or parenchyma. (A) To establish whether
infectious 1716 was produced in the CNS, BALB/c mice were injected
with 1716 into one of two locations shown in the sagital diagram of the
mouse brain (V, ventricular or P, parenchyma). The parenchymal injections
were performed over 3 min, virus (1¬105 p.f.u. in 2 µl) was injected into
the frontal lobe. The vertical line between P and V in the brain diagram
represents the separation of the anterior and posterior parts of the brain
for parenchymal inoculation titrations. For the ventricular injections, the
whole brain was saved as one sample and titred as one sample. For the
parenchymal injections, the frontal lobe from the posterior border of the
injection site to the anterior border of the olfactory bulb (called anterior)
which contains the inoculation site, and the rest of the brain posterior to
the injection site (called posterior) were saved as separate samples and
titred separately. (B) Virus titre after ventricular inoculation of 1716 (U)
and 17+ (*). No virus was recovered from mice infected with 17+UV or
SFM-inoculated mice (not shown). (C) Titre following parenchymal
inoculation of 1716 in the anterior (U) and posterior (_) portions of the
brain, and of 17+ in the anterior (D) and posterior (*) portions. 17+infected mice were morbid and died 3–4 days p.i. Each point is the mean
of two to five mice with standard error of the mean (SEM) bars.
and parenchymal injections (Figs 1 and 2), it is not clear
whether this is a productive or an abortive infection. No
ependymal infection was detected in 1716 parenchymalinoculated mice (Fig. 2). Contamination with 17+ was ruled out
by performing PCR for the 1716 deletion on DNA isolated
from infected brains at different days p.i. (data not shown).
Infection with 17+ was used as a positive control (Fig. 5 C). The
titre of 17+ increased with time as expected in both the anterior
and posterior portions due to replication and spread of 17+
from the initial inoculation site.
Long-term sequelae of ependymal infection and
detection of latent virus
The long-term consequences of ependymal infection are
ependymal loss, ventricular enlargement and inflammation.
FDC
Although detailed behavioural studies have not been performed, the mice seem normal except for some hyperactivity.
The enlarged ventricles persisted from early days (days 2 and
10 ; Fig. 6 B, C) to 7 months (Fig. 6 D) after 1716 inoculation.
This was not observed in SFM- and 17+UV-inoculated mice
(day 30 ; Fig. 6 A). Also, in all 1716-infected mice sacrificed
after 10 days, there were no detectable HSV-immunopositive
cells, but in most of these cases the ependyma was almost
completely denuded (Fig. 6 D, E). The choroid plexus was
preserved in all mice (Fig. 6 C, F). Perivascular inflammation
was present in both 1716- and 17+-infected mice. In mice
where the virus was inoculated into the parenchyma without
leakage into ventricles, the ependyma was not denuded and
there was no enlargement of the ventricles, suggesting that the
ventricular enlargement is due to ependymal infection and not
to another effect of HSV infection of non-ependymal cells.
To determine whether 1716 established a latent infection
after ventricle inoculation, ISH was performed for the detection
of LATs in the absence of other productive viral transcripts
(ICP4, gC and VP5) and viral proteins. In ventricle-inoculated
mice, at day 30, surprisingly few LAT-expressing cells were
detected in the brainstem (Fig. 7 A, B). This is in contrast to a
previous study which showed widespread latent infection of
the CNS after parenchymal inoculation (Kesari et al., 1996).
This may be due to reduced infection of the parenchyma after
ventricle inoculation and}or to secondary effect of ependymal
loss}ventricle enlargement on the expression of LATs.
Discussion
The data presented here suggest that although ICP34.5deficient viruses are neuroattenuated, they do retain the ability
to replicate in ependymal cells. In the CNS there is a variety of
cell types having diverse functions, including neurons, oligodendrocytes and astrocytes as well as ependymal, subependymal, endothelial and microglial cells. Differences in the
molecular phenotype of these cells in different areas of the
brain may determine the contrasting patterns of infection,
spread and pathogenicity of different viruses and of different
strains or variants of viruses. Although the reason for the
greater vulnerability of the ependyma than the parenchyma to
1716 infection is unclear, it may be related to the proliferative
ability or maturational state of different CNS cell types. It is
clear that there are dividing progenitor cells in the subependymal region of the adult CNS of mammals, including
mice and humans, which give rise to neurons and glia
(Reynolds & Weiss, 1992). There are also dividing cells in the
hippocampus (Gage et al., 1995). How the proliferative capacity
of different populations of adult CNS cells affects the ability of
different strains of HSV to replicate is unknown, but this may
be important in understanding the vulnerability of specific
brain areas to certain diseases, such as the limbic system in HSV
encephalitis. In the present studies, almost 100 % of the
ependymal cells were infected by 1716, suggesting that
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HSV replication in ependymal cells
Fig. 6. Ependymal loss and ventricular dilatation persists for at least 7 months. Representative photomicrographs from
haemotoxylin and eosin-stained sections showing ventricular region. (A) In mock (SFM)-inoculated animals (at day 30) the
ventricle is slightly dilated. The ventricular enlargement after 1716 (5¬105 p.f.u.) infection is observed as early as day 2 (B),
progresses over time to day 10 (C) and persists for up to 7 months (D, E, F). Ependymal loss (D, E, F) also persists for 7
months. The choroid plexus is intact in all mice at all times (C, F). Scale bar : (A–D), 296 µm ; (E), 59±3 µm ; (F), 118±5 µm.
Fig. 7. Detection of latent 1716 in the
CNS after ventricle inoculation by ISH.
ISH was performed on sections from
1716 (5¬105 p.f.u.)-infected brains at
day 30 and later. 17+ (1¬106 p.f.u.)infected sections (day 2) were used as
positive controls for the ISH.
Representative photomicrographs of
sections from 1716-infected mice at day
30 showing the presence of a few LATexpressing cells in the brainstem (A, B).
Serial sections showed the absence of
HSV proteins and productive viral
transcripts at this latent time (" 30
days). Scale bar : (A), 118±5 µm ; (B),
59±3 µm.
factor(s) specific in ependymal cells (i.e. expression of complementing and}or inhibiting factors such as GADD34}
MyD116 homologues) complement the defect in 1716. Thus,
these data show that the ependyma is the major site for 1716
replication in the CNS in immunocompetent mice. Also, it
would be of interest to study the biology of this virus in an
immuno-incompetent mouse to determine if and how the
immune system plays a role in restricting replication in the
CNS.
Although the ependyma plays an important homoeostatic
role in the foetal CNS, the function of the ependyma in the
adult CNS is not clear (Del Bigio, 1995). Over the course of
these studies there was no lethality associated with CNS
infection by 1716, confirming that the ependyma in the adult
mouse is not essential for survival. But the enlarged ventricles
(hydrocephalus) suggest that there is pathology associated
with this loss. Hydrocephalus has been shown to be produced
in foetal and neonatal animals by a variety of viruses (Johnson,
1967, 1975) and has been shown to be produced in adult mice
by influenza virus (Johnson & Johnson, 1972) and HSV
(Hayashi et al., 1986). Hydrocephalus often develops as a
sequelae to ependymal damage in humans (Del Bigio, 1993,
1995 ; Sarnat, 1995). Hydrocephalus is characterized by
accumulation of cerebrospinal fluid within the ventricle
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FDD
S. Kesari and others
associated with an elevated i.c. pressure. However, the crania
of mice (unlike those of humans) are capable of expanding with
elevated pressure, as seen in animals with i.c. tumours (Kesari
et al., 1995). Thus, several processes may be involved in the
ventricular enlargement seen here, such as hydrocephalus, cell
death, demyelination, inflammation or a combination of these
processes.
The replication of herpesviruses is an extremely inefficient
process ; less than 10 % of the viral DNA is assembled into
virus particles and a large excess of viral proteins is produced
(Johnson, 1982). The complex interaction of many viral and
cellular proteins is involved in determining the final fate of the
infected cell and the output of virus. The products of some
genes are dispensable in certain situations (i.e. in vitro), or are
needed only for efficient production of virus. The RL1
(ICP34.5) gene was identified genetically as a neurovirulence
gene in mice (Chou et al., 1990 ; MacLean et al., 1991).
Subsequent in vitro studies have been performed in cultured
cells to study the function of this gene product (ICP34.5)
(Brown et al., 1994 a, b ; Chou et al., 1994 ; Chou & Roizman,
1992, 1994). However, it is not clear if these in vitro studies will
lead to the elucidation of the role of ICP34.5 in neurovirulence,
as neurovirulence is an in vivo phenotype. In contrast, the in
vivo studies described here using ICP34.5-deficient viruses
show that limited replication of these mutant viruses can be
detected in ependymal cells after direct ventricular injection
but not after parenchymal injection. In light of the in vivo data
documenting a large complement of HSV gene and protein
expression in the CNS of 1716-infected mice, it is speculated
that ICP34.5 is needed either for efficient virion production
and}or for the cell-to-cell propagation or spread of HSV in
vivo, or that factor(s) specific to ependymal cells complement
the defect in 1716.
The further elucidation of the CNS cell-type-specific
replication and gene expression of these and other attenuated
herpesviruses in the CNS is important in understanding the
natural biology and pathogenesis of HSV in humans. With
increasing interest in using viral vectors for therapy of CNS
diseases, study of the biology of HSV and mutants in the CNS
will be important in evaluating its pathogenicity and will
suggest ways to circumvent potential problems that may arise
in the clinical setting. More significantly, such information is
critical for the design of HSV vectors for use in cancer and gene
therapy in the CNS.
This work was supported in part by grants from the Albert R. Taxin
Brain Tumor Center, NINDS (NS29390), NIMH (MH10915) and NCI
(CA-36245).
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