Borna Disease Virus Infects Human Neural Progenitor Cells and
Dragan Brnic,a* Vladimir Stevanovic,a* Marielle Cochet,aCécilia Agier,aJennifer Richardson,aClaudia N. Montero-Menei,b,c
Ollivier Milhavet,dMarc Eloit,aand Muriel Coulpiera
INRA, ANSES, ENVA, UMR 1161, Maisons Alfort, Francea; LUNAM Université, Ingénierie de la Vectorisation Particulaire, Angers, Franceb; INSERM, U646, Angers, Francec; and
CNRS UMR-5203, INSERM U661, Universités Montpellier I and II, Institut de Génomique Fonctionnelle, Montpellier, Franced
that infection underlies a wide range of neuropsychiatric disor-
ders. It has been hypothesized that persistent viral infection plays
a role in human mental disorders of unclear etiology ((5, 24, 39).
a behavioral disturbance can be difficult. In these chronic disor-
ders, Koch’s postulate (i.e., proof of a causative relationship by
isolation, propagation outside the original host, and reintroduc-
tion into a new host resulting in disease) may never be demon-
strated. Nevertheless, it is of great interest to investigate the com-
is affected by a mental illness, such as schizophrenia, underscores
the importance of this research.
Borna disease virus (BDV) is a highly neurotropic virus which
persists in the central nervous system (CNS) of infected individu-
als for their entire life span. It is a nonsegmented, negative-sense,
single-stranded RNA virus belonging to the Bornaviridae family
within the order Mononegavirales (3, 10). BDV was originally de-
Germany (36) but later was identified in a wide range of verte-
brates, including sheep, cattle, dogs, cats, shrews, ostriches, and
nonhuman primates (2, 15, 17, 22, 26). Infected hosts develop a
wide spectrum of neurological disorders, ranging from immune-
mediated disease to behavioral alteration without inflammation,
including deficits in learning and social behavior, which are rem-
as schizophrenia, mood disorders, and autism (18, 32). Epidemi-
in humans and that it is related to certain psychiatric diseases (6,
25). In support of this hypothesis, BDV infection was demon-
strated in the brain of a schizophrenic patient (28). However, the
role of BDV infection in human pathology still is under debate
(23). Nevertheless, owing to its implication in neurobehavioral
disorders in animals and its suspected role in mental diseases in
pidemiological analyses of human neuropsychiatric illness, as
well as studies conducted in animal models, have suggested
by which viral infection alters behavior.
BDV primarily infects neurons of the limbic system, notably
the cortex and the hippocampus (14). Other cellular types, how-
ever, such as astrocytes (4) and neural progenitor cells (37, 38),
have been shown to be infected and might be involved in BDV-
induced neuropathogenesis. Indeed, astrocyte dysfunction can
ing neurobehavioral abnormalities have been reported in mice
expressing BDV phosphoprotein (BDV-P) selectively in glial cells
(20). The alteration of progenitor cells and neurogenesis also
would critically affect brain function. In humans, it has been hy-
pothesized that the impairment of adult neurogenesis plays a role
demonstration of a significant reduction in the proliferation of
neural stem cells (NSC) found in schizophrenic patients has pro-
vided support for this new theory (35). In newborn rats, BDV
infection is responsible for intensive neurodegeneration that is
of infection (1, 5, 33). This suggested that the function of imma-
the possibility of tackling this question. Such cultures allow the
investigation of whether BDV, like some other neurotropic vi-
Received 18 July 2011 Accepted 13 December 2011
Published ahead of print 21 December 2011
Address correspondence to M. Coulpier, firstname.lastname@example.org.
*Present address: D. Brnic, Croatian Veterinary Institute, Laboratory for CSF,
Molecular Virology and Genetics, Zagreb, Croatia; V. Stevanovic, Veterinary Faculty
University of Zagreb, Department of Microbiology and Infectious Diseases,
D.B. and V.S. contributed equally to this work.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
jvi.asm.org 0022-538X/12/$12.00Journal of Virologyp. 2512–2522
ruses, including DNA viruses like cytomegalovirus (29) and ret-
roviruses like HIV (27), can infect and damage human NSPCs
To gain an understanding of the involvement of neural stem/
progenitors in BDV-induced neuropathogenesis, we used a cell
stem/progenitor cells, which can differentiate into neurons and
astrocytes. We report that BDV persistently infects these cells
without altering their survival or undifferentiated phenotype. In
contrast, upon the induction of differentiation, BDV is capable of
newly formed neurons. Thus, our study highlights a new mecha-
ity in infected individuals. These results may help us understand
the behavioral disorders associated with BDV infection.
MATERIALS AND METHODS
HNPC cultures. HNPCs used in this study were prepared from the CNS
of this human fetal CNS tissue were approved and monitored by the
Comité Consultatif de Protection des Personnes dans la Recherche Bio-
medicale of Henri Mondor Hospital, France. Briefly, the cortex was dis-
sected and cut into 1-mm3tissue pieces. After mechanical dissociation,
single-cell suspensions were cultured in Dulbecco’s modified Eagle
medium-F12 (DMEM-F12; 1/1; Invitrogen Life Technologies) supple-
mented with B27 (Invitrogen Life Technologies) and containing epider-
mal growth factor (EGF) and basic fibroblast growth factor (bFGF) at 20
ng/ml (R&D Systems), heparin (5 ?g/ml, Sigma), 100 U penicillin, and
1,000 U streptomycin (Invitrogen Life Technologies). This cell suspen-
(termed neurospheres). Cells were further expanded and maintained in
Dulbecco’s modified Eagle medium-F12 (DMEM-F12 Adv.; Invitrogen
Life Technologies) supplemented with L-glutamine (2 mM; Gibco), apo-
one (6.3 ng/ml; Sigma). Medium, referred to as N2A medium, was
ng/ml; Abcys) were added to maintain undifferentiated cells. For infec-
tion, cells were cultured as monolayers by seeding them in matrigel-
coated dishes (1/1,000; BD Biosciences) in N2A medium. They were sub-
cultured using TrypLE (Invitrogen Life Technologies) when 80%
confluence was reached. Cells were maintained at 37°C in a humidified
atmosphere containing 5% CO2.
Virus infection. Cell-free BDV (strain He80) was prepared by the
focus-forming units [FFU]/ml) on Vero cells using an immunofocus as-
say as described in Danner et al. (7). For both kinetic studies and the
infected at a multiplicity of infection (MOI) of 0.05 FFU/cell. Cells were
then was removed and, after one washing step, replaced by fresh N2A
medium supplemented with growth factors. Persistently infected cells
were maintained with one or two subcultures per week, and BDV infec-
tion then was verified after 2 to 3 weeks of culture for each experiment by
immunofluorescence using an anti-BDV nucleoprotein serum (BDV-
p40; 1/800; a generous gift from D. Gonzalez-Dunia). By 3 weeks postin-
fection, more than 95% of HNPCs were infected, and differentiation was
initiated as described below. The infection of primary cultures of human
astrocytes was performed according to a similar protocol.
Neuronal and glial differentiation. To obtain mixed cultures of hu-
man neurons and astrocytes, BDV-infected and matching noninfected
HNPCs were seeded in 24-well and 6-cm plates at a density of 1.5 ? 105
cells/well or 1.4 ? 106cells/plate, respectively. Differentiation was initi-
ated 1 or 2 days after plating by replacing N2A medium with N2ANBC
without vitamin A; Invitrogen Life Technologies) and withdrawing
growth factors. Differentiation conditions were maintained for up to 21
days, during which time medium was changed 3 times a week. Twenty-
four-well plates were used for immunocytofluorescence, and 6-cm plates
primary cultures of human astrocytes, HNPCs were induced to differen-
tiate in DMEM-F12 medium supplemented with 2 mM L-glutamine and
10% fetal bovine serum for 3 weeks. Medium was changed every 2 to 3
days. Their status as glial fibrillary acidic protein (GFAP)-positive cells
was verified before infection using an antibody directed against GFAP.
Immunocytofluorescence analysis. Standard immunocytofluores-
cence was performed. Undifferentiated and differentiated HNPCs grown
copy Sciences) in phosphate-buffered saline (PBS) for 20 min at room
temperature (RT), rinsed 3 times with PBS, and blocked with 0.1% PBS–
Triton X-100 plus 3% bovine serum albumin (BSA; Sigma) for 45 min at
RT. Primary antibodies were incubated in 0.1% PBS–Triton X-100 plus
at RT with secondary antibodies in 0.1% PBS–Triton X-100 plus 0.3%
BSA. After rinsing with PBS, nuclei were stained with 4=,6-diamidino-2-
antibodies used were directed against nestin (a neural stem cell marker;
1/1,000; AbD Serotec, France), ?-tubulin isotype III (a neuronal precur-
sor marker; 1/1,000; Sigma-Aldrich, France), microtubule-associated
protein 2 (MAP2; a dendritic marker of postmitotic neurons; 1/1,500;
Sigma, France), GFAP (1/1,000; Dako, France), cleaved caspase 3 (an
apoptotic marker; 1/200; Cell Signaling Technology), and BDV nucleo-
protein (BDV-p40; as described above). Secondary antibodies were anti-
rabbit Alexa Fluor 488 and anti-mouse Alexa Fluor 546 (1/1,000; Molec-
ular Probes-Invitrogen Life Technologies, France). For the assessment of
cell death, terminal deoxynucleotidyltransferase-mediated dUTP-biotin
ufacturer’s instructions (Promega, France). Images were acquired using
an ApoTome microscope (Zeiss) equipped with a 20? objective and
AxioVision software. For each experiment, images were acquired from 3
to 4 different wells for each condition and 4 to 9 fields per well. Neurons,
astrocytes, total cells, and apoptotic cells were counted in whole fields
based on ?-tubulin III, GFAP, DAPI, and TUNEL staining. For TUNEL
staining, the percentage of apoptotic events referred to the number of
apoptotic events versus the number of nonapoptotic cells per field. On
average, 600 to 800 cells were counted per well.
Proliferation test. Proliferation was quantified using the Wst1 kit
(Roche) according to the manufacturer’s instructions. Briefly, HNPCs
were infected as described previously. BDV-infected cells at the 3rd (PI3)
and 10th (PI10) passage following infection and their matching nonin-
fected cells were plated in 6 wells of a 48-well plate precoated with matri-
days 0, 2, 4, and 7 after plating. After 1 h of incubation, 100 ?l of super-
natant was transferred to a 96-well plate to allow the measurement of
absorbance using an enzyme-linked immunosorbent assay (ELISA)
Western blot analysis. Differentiated and nondifferentiated HNPCs
were rapidly lysed in protein lysis buffer containing 20 mM Tris-HCl, pH
8, 137 mM NaCl, 10% glycerol, 2 mM EDTA, 1% NP-40, and a protease
inhibitor cocktail (Sigma). Insoluble material was removed by centrifu-
determined by using the MicroBCAssay protein quantification kit
(Uptima-Interchim) in accordance with the manufacturer’s instructions.
Twenty micrograms of protein per lane was separated by SDS-
polyacrylamide gel electrophoresis and transferred onto a nitrocellulose
membrane (ECL Amersham Hybond membrane; GE Healthcare). Blots
were blocked in Tris-buffered saline containing 0.1% Tween 20 (TBST)
primary antibody overnight at 4°C. After washing with TBST, the blots
Borna Disease Virus Infection of Neural Progenitors
March 2012 Volume 86 Number 5 jvi.asm.org 2513
were incubated for 1 h with either horseradish peroxidase-conjugated
anti-rabbit or anti-mouse antibody (1/25,000; GE Healthcare). After 3
washes with TBST, peroxidase activity was revealed by incubation with
analyzed using a fusion fx5 molecular imager (Vilber Lourmat, France)
measuring the density of the bands and normalizing for actin expression.
For reprobing, blots were stripped for 90 min at RT in 0.1 M acetic acid
and 0.15 M NaCl. Primary antibodies used were anti-? tubulin isotype
III (1/1,000; Sigma), anti-GFAP (1/2,000; Dako), anti-?-actin (1/500;
Sigma), and rabbit polyclonal antibodies raised against the BDV nucleo-
protein (BDV-p40) and the BDV phosphoprotein (BDV-p24) (1/20,000
and 1/10,000, respectively; generous gifts from D. Gonzalez-Dunia).
Reverse transcription and quantitative real-time PCR. Total RNA
was extracted from differentiated and nondifferentiated HNPCs by using
the RNeasy kit for RNA purification (Qiagen) according to the manufac-
turer’s instructions. Isolated RNA was quantified with a NanoDrop spec-
trophotometer, and 500 ng of RNA was reverse transcribed with Super-
random hexamers or a primer specific for viral genomic RNA (5=-TGTT
GCGCTAACAACAAACCAATCAC-3=). Two ?l of cDNA was used to
perform real-time PCR with a LightCycler 1.5 (Roche Applied Science)
using QuantiTect SYBR green PCR master mix (Qiagen) in a 20-?l reac-
tion mixture. For PCR, samples were held for 15 min at 94°C and then
min. Except for those for BDV-p40, primers have been described previ-
ously by Hong et al. (17a) and were the following: F-nestin, 5=-CAGCGT
GATCTGTGAACGCCTTGTCC-3=; F-? tubulin III, 5=-CAACAGCACG
GCCATCCAGG-3=; R-? tubulin III, 5=-CTTGGGGCCCTGGGCCTCC
GA-3=; F-GFAP, 5=-GGCACGTGCGGGAGGCGGCC-3=; R-GFAP, 5=-T
CTCATCACATCCTTGTGC-3=; F-MBP, 5=-AAGGACTCACACCACCC
GGC-3=; and R-MBP, 5=-TTTCAGCGTCTAGCCATGGG-3=. For BDV-
TTGT-3=; and R-BDVp40, 5=-GCAGCGTGCAGTCCTGGGATTA-3=.
the means (SEM). Statistical analyses were performed by employing the
Student’s t test. P ? 0.1 was considered not significant.
HNPCs are multipotent cells capable of differentiating into
the HNPCs utilized in this study. Neurospheres in suspension
additional passages as adherent cells on matrigel-coated dishes.
Cells maintained an undifferentiated or early differentiated state.
Immunofluorescence demonstrated that more than 95% of cells
expressed the neuroepithelial stem cell marker nestin (Fig. 1A,
left). Upon growth factor withdrawal, cells grown in matrigel-
coated plates differentiated into neurons and astrocytes. The two
phenotypes were distinguished on the basis of morphology and
myelin basic protein (mbp) gene was not detectable by quantita-
tive real-time PCR (data not shown).
susceptibility of HNPCs to BDV infection, we examined whether
the cells to replication and propagation. Cells were plated on
matrigel-coated 96-well plates and infected at a low MOI of 0.05.
in HNPCs then was evaluated by immunofluorescence on days 2,
albeit a small number, stained positive for BDV-p40, revealing
their permissivity to BDV infection. The observation of cultures
replicates in HNPCs and disseminates in a very efficient manner.
The relative content of BDV-p40 RNA (genomic and transcripts)
was examined next (Fig. 1E). Viral RNA increased dramatically
from day 2 (1 arbitrary unit) until day 12 (396 ? 108 arbitrary
of the culture.
BDV infection persists in HNPCs without altering their un-
differentiated phenotype or capacity for survival. HNPCs were
infected at low MOI (0.05) as described above and further sub-
cultured, with an average of one passage a week. After infec-
2, 3, 4, 6, and 10 for infection, protein level, and morphology,
and at passages 3 and 8 for proliferation. After 2 to 3 subcul-
tures, which corresponded to approximately 12 days of infec-
tion, more than 96% of the cells were infected as determined by
the detection of the viral nucleoprotein p40 by immunofluo-
rescence (Fig. 2A, BDV-PI2). The percentage of infected cells
then remained stable during the duration of the experiment up
to 10 passages (Fig. 2A, BDV-PI10), which corresponded to
about 70 days of infection. The content of viral proteins, as
determined by Western blotting using antibodies directed
against phosphoprotein p24 and nucleoprotein p40, was stable
in fully infected cultures from passages 2 to 10 (Fig. 2B). These
results demonstrated that BDV establishes a persistent infec-
tion in HNPCs. At every passage at which cells were examined
using phase-contrast microscopy, BDV infection had no cyto-
pathic effect. Also, BDV infection did not alter the morphology
of HNPCs. Early after infection, at passage 2 (PI2), both BDV-
infected and noninfected cells stained with an anti-nestin an-
tibody presented the typical morphology of neural progenitor
cells (Fig. 2C). At later passages, it was not unusual to observe
observed in both infected and noninfected cultures and there-
fore was not due to BDV infection but rather to natural differ-
entiation occurring in HNPCs grown on matrigel-coated
plates. The level of expression of nestin, as determined by
Western blotting, did not differ in BDV-infected and nonin-
fected cells. This was observed from passage 3 following infec-
tion (Fig. 2D) to passage 10 (not shown). However, a general
decrease in nestin expression was observed in later passages
the immature state of HNPCs was affected by serial passaging
on matrigel-coated plates, but no alteration of the undifferen-
tiated phenotype, based on morphology or expression of nestin
level, was observed in relation to BDV infection. Moreover,
mitochondrial dehydrogenase activity was similar in nonin-
fected and BDV-infected cultures (Fig. 2E), showing that BDV
infection did not alter the capacity of HNPCs to proliferate.
Therefore, although BDV establishes a full viral cycle in
HNPCs, it does not alter its undifferentiated phenotype.
infection. We then sought to determine whether BDV could im-
Brnic et al.
jvi.asm.orgJournal of Virology
pair the process of neural differentiation by examining the initial
steps when, upon growth factor withdrawal, proliferation is ar-
rested and differentiation is initiated. At this time, nestin and
cd133, two neural stem cells markers, are downregulated. Fully
infected controls were seeded at the same density on matrigel-
coated 6-cm dishes and induced to differentiate for 4 days by
growth factor withdrawal. Nestin and cd133 mRNA were quanti-
fied by quantitative real-time PCR. As expected, the levels of nes-
tin and cd133 mRNA decreased in our noninfected cultures (Fig.
3A and B). A similar decrease was observed in BDV-infected cul-
tures (Fig. 3A and B), revealing that BDV infection does not alter
HNPCs in their capacity to lose their immature stage and com-
mence a differentiation program.
BDV impairs neural differentiation by inducing cell death.
We then wondered whether BDV could impair differentiation
at later steps. Fully infected HNPCs (after PI2) and their
14 days by growth factor withdrawal (Fig. 4A). In noninfected
cultures, differentiated cells exhibited a typical pattern of
mixed cultures of astrocytes, visible as gray and protoplasmic
cells, and neurons, visible as small-sized refringent cells, as
observed by phase-contrast microscopy. The two cell types
were uniformly distributed in culture dishes. In contrast, in
infected cultures an atypical pattern was observed, with nu-
merous dying cells being present in groups and being sur-
rounded by healthy cells of both neuronal and astrocytic mor-
phologies. Cell death was further observed by TUNEL and
DAPI stainings. DAPI staining revealed nuclei of two sizes in
noninfected cultures, corresponding to astrocytes (14.80 ?
1.27 ?m) and neurons (8.25 ? 0.98 ?m). In BDV-infected
numerous and colocalized with dying cells observed with
phase-contrast light and TUNEL staining. The occurrence of
cell death in infected cultures was confirmed by the determi-
nation of the total number of cells based on DAPI staining (Fig.
4C), as well as by the determination of the percentage of apop-
totic events based on TUNEL staining (Fig. 4D). At 14 days of
differentiation, a 26.7% decrease in total cell number was ob-
served in BDV-infected cultures (165 ? 7.8 cells per field in
noninfected cultures and 121 ? 7.1 in BDV-infected cultures).
We verified that a similar number of cells had been plated in
noninfected and BDV-infected cells by counting them at day 0
(not shown). Therefore, the decrease in cell number could not
FIG 1 HNPCs differentiate into neurons and astrocytes (A and B) and are fully permissive to BDV infection (C, D, and E). (A) Immunofluorescence
labeling of human neural progenitor cells (HNPCs) and their derivatives. On the left is a photomicrograph of the undifferentiated cells immunostained
with nestin antibody (orange). On the right is a photomicrograph of cells differentiated into neurons (N) and astrocytes (As) immunostained with
antibodies against ?-tubulin III (?-tub III; orange) and GFAP (green). Nuclei were stained with DAPI (blue). (B) Western blot showing an increase in
neuronal and astroglial markers upon differentiation. N ? As, mixed cultures of neurons and astrocytes. (C) The susceptibility of HNPCs to BDV
infection was analyzed with an antibody directed toward the viral nucleoprotein p40 at day 2, 4, 8, and 12 postinfection. Nuclei were stained with DAPI.
(D) Based on BDV immunostaining, the percentage of infected HNPCs was determined from day 2 to day 12 following infection. Data represent means ?
SEM from one experiment performed in triplicate. Similar results were obtained in two independent experiments. NI, noninfected. (E) The expression
of the gene coding for viral nucleoprotein p40 was analyzed by quantitative real-time reverse transcription-PCR on 500 ng of total RNA. Data are
representative of two independent experiments performed in duplicate. Statistical analysis was performed by employing the Student’s t test. ***, P ?
Borna Disease Virus Infection of Neural Progenitors
March 2012 Volume 86 Number 5jvi.asm.org 2515
be attributed to an experimental artifact but rather demon-
strates that BDV diminishes cell survival during the process of
neural differentiation. This was confirmed by the dramatic in-
crease in the percentage of apoptotic events in BDV-infected
cells (89.4% ? 20%) compared to that of the noninfected one
(5.1% ? 0.9%). By examining cells at an earlier time point, the
kinetics of cell death could be established. At day 4 of differen-
tiation (Fig. 4B, C, and D), the number of cells in noninfected
and BDV-infected cultures was similar (Fig. 4C). At this time
point, a fraction of the cell population was apoptotic (Fig. 4B
and D). This was independent of BDV infection and was due to
natural death occurring in the initial step of neural differenti-
cultures, showing that BDV infection contributed to cell death
at this early time point. Thus, while apoptosis was transitory in
uninfected cells, apoptotic events in BDV-infected cultures
markedly increased between days 4 and 14, indicating that
BDV-induced death occurred mainly after the initial steps of
BDV infection strongly impairs neuronal differentiation.
We next examined whether BDV impaired neurogenesis or
gliogenesis. To evaluate the effect of BDV on neurogenesis,
HNPCs that had undergone differentiation for 4 and 14 days
were fixed and immunostained with an antibody directed
against the neuronal marker ?-tubulin III. Four days following
the onset of differentiation, the examination of noninfected
and BDV-infected cultures revealed that numerous ?-tubulin
III-positive cells had been generated at this early time point
positive cells were heterogeneous, presenting either no neu-
rites, short neurites, or more elongated ones, showing that cells
were at different states of neuronal differentiation. Alteration
in neurite length or branching was not observed in infected
cultures, indicating that BDV does not interfere with neurito-
genesis. The enumeration of ?-tubulin III-positive cells
showed an average of 84 ? 2.0 neurons per field in noninfected
cultures (Fig. 5B), whereas 66 ? 7.3 neurons were present in
BDV-infected cultures (Fig. 5B). Thus, although numerous
neurons had been generated in both noninfected and BDV-
infected cultures, a 22% loss occurred in infected cultures. At
that time, no difference was observed in the level of either
?-tubulin III mRNA or protein in the two cultures (Fig. 5C and
D). Fourteen days after the induction of differentiation, the
number of ?-tubulin III-positive cells had increased by 20% in
noninfected cultures (106 ? 7.4 neurons per field) (Fig. 5B),
showing that neurons had continued to be generated. In the
FIG 2 BDV infection persists in HNPCs without altering their undifferentiated phenotype, survival, and proliferation. HNPCs were infected and analyzed at
by Western blotting; (C) morphology, by immunofluorescence using an anti-nestin antibody; (D) nestin level, by Western blotting; and (E) proliferation. Data
represent the means ? SEM from 3 independent experiments performed in quintuplicate. Statistical analysis was performed by employing the Student’s t test.
ns, not significant.
Brnic et al.
jvi.asm.orgJournal of Virology
meantime, extensive neurite outgrowth had occurred, as
shown by ?-tubulin III immunostaining (Fig. 5A, b), indicat-
ing that neurons had acquired a more mature morphology. At
that time, a lower density of ?-tubulin III-positive cells was
clearly observed in BDV-infected cultures (Fig. 5A, d). Quan-
tification confirmed that a dramatic decrease in the number of
neurons had occurred (Fig. 5B). The average per field was 54 ?
5.4, showing that 50% of the neurons had been lost compared
to levels in noninfected cultures. The decrease in the quantity
III protein (Fig. 5D), as shown by quantitative real-time PCR
and Western blotting, respectively, confirmed the loss of
onset of differentiation. The careful examination of double
DAPI and ?-tubulin III staining in BDV-infected cultures re-
vealed apoptotic nuclei surrounded by ?-tubulin III staining
(Fig. 6A), demonstrating that BDV-induced neuronal loss was
attributable to neuronal apoptosis. To increase precision con-
was initiated, we performed immunostaining for MAP2, a den-
dritic marker of postmitotic neurons (Fig. 6B). Four days fol-
lowing the induction of differentiation, the overall MAP2
staining was weak in both noninfected and BDV-infected cul-
tures, except for a few cells in which neuritic extensions were
highly stained (Fig. 6B, a and c). This recapitulated the obser-
vation made with ?-tubulin III staining and further confirmed
that, at that time, the majority of neuronal cells were poorly
differentiated. At 14 days of differentiation, all neuronal cells
had acquired neuritic extensions which formed a network im-
munostained with MAP2. Neuronal cells had matured and ac-
quired a postmitotic phenotype. However, almost all of them
exhibited a uni- or bipolar morphology (Fig. 6B, b, arrow-
heads, and C, top), showing that they had not reached a fully
matured stage, which is characterized by a complex multipolar
staining was observed in BDV-infected cultures but not in the
noninfected one, demonstrating that dying cells were post-
mitotic neurons. Our results, therefore, showed that BDV-
D) occurred when neurons were poorly differentiated. As neu-
ronal death became massive (Fig. 4A and D), by day 14 of
differentiation more matured postmitotic neurons were af-
fected. To further elucidate the mechanism of neuronal death,
noninfected and BDV-infected cells differentiated for 14 days
were analyzed for cleaved caspase 3, another marker of apop-
tosis. In infected cultures, all small-sized atypical nuclei, as
revealed by DAPI, expressed cleaved caspase 3 (Fig. 6C), dem-
onstrating that caspase 3 activation was substantially higher in
infected than in noninfected cultures, in which only a few cells
were positive for cleaved caspase 3. This confirmed that cells
Furthermore, cleaved caspase 3 was also observed along degen-
erating neurites (Fig. 6C), which provided additional evidence
that, at 14 days of differentiation, dying neurons had grown
BDV infection does not alter glial differentiation. To exam-
ine whether BDV could also influence glial differentiation, we
analyzed HNPCs induced to differentiate using an antibody
directed against GFAP, an astroglial marker. Immunostaining
revealed that numerous astrocytes were formed 14 days after
the onset of differentiation in both noninfected and BDV-
infected cultures. No morphological differences were observed
in the two cultures (Fig. 7A). This suggested that astroglial
differentiation occurred normally in infected cultures. This
was confirmed by the enumeration of cells, which showed that
a similar number of astrocytes was generated in infected
(58.8 ? 9.82 per field) and noninfected cultures (53.9 ? 5.61
per field) (Fig. 7B). In addition, the quantification of GFAP at
the mRNA level by quantitative real-time PCR (Fig. 7C) or at
the protein level by Western blotting (Fig. 7D) showed a strong
upregulation of transcripts and proteins by 21 days of differen-
tiation but no statistically significant difference between in-
fected and noninfected cells. Clearly, BDV infection did not
alter astroglial differentiation. Whether this could be explained
by a lack of permissivity to the BDV infection of cells differen-
tiated into astrocytes was evaluated. The evidence that viral
replication, as assessed by the quantification of viral genomic
RNA, is slightly increased during the differentiation process
found that all progenies of infected HNPCs, whether they were
committed to glial or neuronal lineages, were themselves
after the induction of differentiation (Fig. 7E). Similar obser-
vations were made at 4 and 21 days of differentiation (not
FIG 3 BDV-infected HNPCs lose their immature phenotype upon the initia-
tion of differentiation. Fully infected HNPCs and their matched noninfected
controls were induced to differentiate for 4 days by growth factor withdrawal.
The expression of nestin (A) and CD133 (B) mRNA was analyzed by quanti-
tative real-time PCR at day 0 and day 4 of differentiation. Data represent the
means ? SEM from three independent experiments performed in duplicate.
Statistical analysis was performed by employing the Student’s t test. ns, not
significant. **, P ? 0.01; ***, P ? 0.001.
Borna Disease Virus Infection of Neural Progenitors
March 2012 Volume 86 Number 5jvi.asm.org 2517
shown). Finally, the full permissivity of astrocytes to BDV in-
Cells were infected at an MOI of 0.05, and the capacity of the
virus to infect, replicate, and disseminate was evaluated by im-
munofluorescence on days 4 and 9 following infection using an
On day 4, few cells were immunostained for BDV-p40 (Fig. 7G,
a), revealing that the virus could enter the cells. Their numbers
had considerably increased by day 9 (Fig. 7G, b), demonstrat-
ing that the virus could replicate and disseminate in human
The mechanisms by which BDV alters the behavior of infected
FIG 4 BDV interferes with cell survival during neural differentiation. Fully infected HNPCs and their matched noninfected controls were induced to
differentiate for 14 (A) or 4 (B) days by growth factor withdrawal. Differentiated cells were observed by phase-contrast microscopy, TUNEL, and DAPI
staining. Similar observations (bright-field/DAPI) were made in 7 independent experiments at 14 days of differentiation and 3 independent experiments
at 4 days of differentiation. Note that TUNEL staining matches with fragmented nuclei stained with DAPI. (C) Total number of cells based on DAPI
staining. Four to 9 fields and a minimum of 700 cells per well were counted. Data represent mean values ? SEM from one experiment performed in
ns, not significant. d4, day 4; d14, day 14. (D) Percentage of apoptotic events based on TUNEL staining. Three to 4 fields and a minimum of 400 cells per
well were counted. Data represent mean values ? SEM from one experiment performed in triplicate. Statistical analysis was performed by employing the
Student’s t test. *, P ? 0.1; **, P ? 0.05.
Brnic et al.
jvi.asm.org Journal of Virology
synaptic plasticity (34, 40, 41), but it is highly probable that be-
havior impairment also results from BDV-induced neuronal loss.
infected newborn rats (19), but the underlying mechanisms are
uration at the time of infection, we suspected that BDV was capa-
ble of interfering with neurogenesis. In this study, we took
advantage of the availability of human brain-derived progenitor
cells to investigate whether BDV can impair neurogenesis.
In infected newborn rats, BDV-induced cell death occurs at
a time when neurons continue to be generated from precursor
are highly vulnerable to BDV infection (32, 42). Consistently
with this hypothesis, progenitor cells had been shown to be
infected in different brain areas (37). In the present study, us-
ing HNPCs, we investigated whether neural progenitors were
indeed particularly vulnerable to BDV infection. We showed
that HNPCs are highly permissive to BDV. The virus enters the
cells, replicates, and propagates very efficiently. The rapidity of
of neurons from newborn rats (16) and is much greater than
propagation in Vero cells (our unpublished observation),
which are commonly used for virus titration or purification.
However, although HNPCs are highly permissive to the virus,
they did not appear to be particularly vulnerable. As has been
observed in other cell types, including primary cultures of neu-
rons and astrocytes from rats (16, 44) and other cell lines, BDV
infection proceeds without an overt pathogenic effect in
HNPCs, impairing neither their survival nor their undifferen-
vulnerability of immature neural cells to BDV infection.
Although BDV-infected HNPCs were not damaged at an
immature stage, a substantial fraction of cells died in infected
cultures upon the induction of differentiation by growth factor
withdrawal. BDV infection may have inhibited the differentia-
tion of neural progenitors, inducing an instability which cul-
minated in cell death, as has been observed for another neu-
differentiation and induces apoptosis in human neural precur-
sor cells (29). Our results, however, demonstrate that BDV
infection did not block the differentiation of HNPCs, as a sim-
ilar decrease in the expression of the neural stem cell markers
cd133 and nestin was observed in noninfected and BDV-
infected cultures. The absence of alteration in astroglial differ-
entiation confirmed that progenitors were not blocked in an
undifferentiated stage and further indicated that only cells en-
gaged along the neuronal pathway were affected by the pres-
ence of the virus. Thus, neuronal cells were generated and died
sometime after their birth. BDV-induced death was first ob-
served as early as 4 days after the induction of differentiation.
they were poorly differentiated, as shown by the paucity of
neuritic extension immunostained with ?-tubulin III and
noninfected and BDV-infected cultures. Data represent mean values ? SEM from one experiment performed in triplicate. Similar results were obtained from 3
independent experiments. ?-Tubulin III mRNA and protein were analyzed by quantitative real-time PCR (C) and Western blotting (D). Data represent mean
were performed by employing the Student’s t test. ns, not significant; *, P ? 0.1; ***, P ? 0.005.
Borna Disease Virus Infection of Neural Progenitors
March 2012 Volume 86 Number 5jvi.asm.org 2519
MAP2 antibodies. Neuronal death then was dramatically in-
creased in the next days, as neurons matured and acquired a
postmitotic phenotype, as shown by extensive neurite out-
growth and MAP2 immunostaining in neuritic extensions.
These data argue that BDV-induced neuronal death occurs in a
progressive manner beginning at an early time of neuronal
differentiation and being highly intensified through neuronal
a fully mature stage, as shown by morphological immaturity.
Human neurons, indeed, require longer culture times to spon-
in our cultures proceeds during neuronal maturation, before
the acquisition of a fully differentiated stage. Our results led us
to conclude that the presence of the virus during neuronal
differentiation induces neuronal death by interfering with sig-
naling pathways which are important for the proper matura-
tion of neurons. Importantly, the effect of BDV on neurons is
restricted to developmental stages, since it has been repeatedly
shown in previous works that fully differentiated neurons in
cultures were not affected by BDV infection (16, 31). The BDV
impairment of neurogenesis was already suggested in a study
by Friedl et al. (12), who used an elegant ex vivo model of
organotypic hippocampal slice cultures from newborn rat
pups. In that study, infection was performed early, before neu-
ronal maturation. The authors found that BDV-induced neu-
ronal death was an event that started late in neuronal matura-
tion, after axons and synapses were formed. Taking these
results together with ours, we demonstrate that the BDV im-
pairment of neurogenesis is a robust mechanism in neurons of
both rat and human origins. The differences in the timing at
which neuronal apoptosis is initiated might be due either to
species or environmental particularities. It is possible that, in a
complex environment such as the one provided by organotypic
slices, survival signals compete with apoptotic signals, allowing
vitro system. In our cultures, the exact mechanisms involved in
the BDV impairment of neurogenesis remain to be elucidated.
Further studies will be needed to determine whether neuronal
apoptosis results from an altered neuron-glial cross-talk or
whether an intrinsic program of neuronal differentiation is
impaired. As both neuronal and glial cells derived from in-
fected HNPCs are themselves highly infected, one or the other
mechanism might occur.
The selective loss of neurons in experimentally infected new-
to be noncytolytic in differentiated neurons in cultures (16, 31).
Our findings support the hypothesis that BDV-induced neurode-
generation is due to BDV interference with neurogenesis. In sup-
port of this theory, similar caspase 3-dependent apoptotic mech-
anisms have been demonstrated in vitro and in vivo (43). We
cannot, however, exclude that other mechanisms also contribute
to BDV-induced neuropathogenesis in vivo. The observation of
activated microglia in areas of intense neurodegeneration (42),
preceding neuronal apoptosis (31), suggested that microgliosis
could trigger the demise of infected neurons. Although this hy-
pothesis was not supported by an in vitro study in which the BDV
infection of a coculture of differentiated neurons, astrocytes, and
microglia led to microglial activation without neurotoxicity (30),
it is plausible that microglia exacerbates dysfunction initiated by
direct virus infection in the in vivo setting. Further studies will be
needed to determine the precise mechanisms involved in vivo.
Nevertheless, HNPCs in culture offer a unique in vitro model to
unravel at least some of the mechanisms leading to BDV-induced
Several neurotropic viruses are capable of modulating the fate
of NPCs both in vivo and in vitro (for a review, see reference 8).
FIG 6 Neurons die by a caspase-3 dependent pathway. Fully infected HNPCs
and 14 (A, B, and C) days by growth factor withdrawal. (A) Cells were immu-
nostained for ?-tubulin III (orange) and DAPI (blue; nuclear staining). Ar-
rows show small-sized atypical nuclei surrounded by ?-tubulin III immuno-
staining, indicating that dying cells have a neuronal phenotype. (B) Cells were
immunostained for MAP2 (red), TUNEL (green), and DAPI (blue). Arrow-
mitotic neurons. (C) Cells were immunostained for ?-tubulin III (orange),
cleaved caspase 3 (green), and DAPI (blue). Note that cleaved caspase 3 stain-
neurites are positive for cleaved caspase 3 (arrows).
Brnic et al.
jvi.asm.org Journal of Virology
Damage might occur either through common pathways, as was
suggested by De Miranda et al. (11), or through specific interac-
tions, such as the inhibition of proliferation, the inhibition of
neuronal or glial differentiation, progenitor death or persistence,
or, as for BDV, the impairment of the survival of newly formed
rogenesis contributes to behavioral disorders (13, 21), we specu-
late that a basis of BDV-induced neurobehavioral disorder is the
impairment of neurogenesis.
This work was supported financially by the French National Institute for
Agricultural Research (INRA). D.B. and V.S. were supported by grants
from the French Embassy in Croatia.
We are most grateful to D. Gonzalez-Dunia for his generous gift of
antiviral phosphoprotein and nucleoprotein antibodies and his critically
reading the manuscript.
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