Association of Human Herpesvirus-6B with
Mesial Temporal Lobe Epilepsy
Julie Fotheringham1, Donatella Donati1,2, Nahid Akhyani1, Anna Fogdell-Hahn1,3, Alexander Vortmeyer4,
John D. Heiss4, Elizabeth Williams1, Steven Weinstein5, Derek A. Bruce5, William D. Gaillard5,6, Susumu Sato6,
William H. Theodore6, Steven Jacobson1*
1 Viral Immunology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, United States of America, 2 Struttura
Complessa di Microbiologia e Virologia, Azienda Ospedaliera Universitaria Senese, Siena, Italy, 3 Department of Clinical Neuroscience, Division of Neurology, Karolinska
Institutet, Stockholm, Sweden, 4 Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, United
States of America, 5 Children’s National Medical Center, Washington, District of Columbia, United States of America, 6 Clinical Epilepsy Section, National Institute of
Neurological Disorders and Stroke, National Institutes of Health, Maryland, United States of America
Funding: Funding for this study was
provided by the National Institutes
of Health. The funders had no role in
study design, data collection and
analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors
have declared that no competing
Academic Editor: Manuel Graeber,
Imperial College London, United
Citation: Fotheringham J, Donati D,
Akhyani N, Fogdell-Hahn A,
Vortmeyer A, et al. (2007)
Association of human herpesvirus-
6B with mesial temporal lobe
epilepsy. PLoS Med 4(5): e180.
Received: October 25, 2006
Accepted: March 29, 2007
Published: May 29, 2007
This is an open-access article
distributed under the terms of the
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Abbreviations: CNS, central nervous
system; GFAP, glial fibrillary acidic
protein; HHV-6, human herpesvirus-
6; MTLE, mesial temporal lobe
epilepsy; MTS, mesial temporal
sclerosis; PBMC, peripheral blood
mononuclear cells; RT-PCR, reverse
* To whom correspondence should
be addressed. E-mail: jacobsons@
A B S T R A C T
Human herpesvirus-6 (HHV-6) is a b-herpesvirus with 90% seroprevalence that infects and
establishes latency in the central nervous system. Two HHV-6 variants are known: HHV-6A and
HHV-6B. Active infection or reactivation of HHV-6 in the brain is associated with neurological
disorders, including epilepsy, encephalitis, and multiple sclerosis. In a preliminary study, we
found HHV-6B DNA in resected brain tissue from patients with mesial temporal lobe epilepsy
(MTLE) and have localized viral antigen to glial fibrillary acidic protein (GFAP)–positive glia in
the same brain sections. We sought, first, to determine the extent of HHV-6 infection in brain
material resected from MTLE and non-MTLE patients; and second, to establish in vitro primary
astrocyte cultures from freshly resected brain material and determine expression of glutamate
Methods and Findings
HHV-6B infection in astrocytes and brain specimens was investigated in resected brain
material from MTLE and non-MTLE patients using PCR and immunofluorescence. HHV-6B viral
DNA was detected by TaqMan PCR in brain resections from 11 of 16 (69%) additional patients
with MTLE and from zero of seven (0%) additional patients without MTLE. All brain regions that
tested positive by HHV-6B variant-specific TaqMan PCR were positive for viral DNA by nested
PCR. Primary astrocytes were isolated and cultured from seven epilepsy brain resections and
astrocyte purity was defined by GFAP reactivity. HHV-6 gp116/54/64 antigen was detected in
primary cultured GFAP-positive astrocytes from resected tissue that was HHV-6 DNA positive—
the first demonstration of an ex vivo HHV-6–infected astrocyte culture isolated from HHV-6–
positive brain material. Previous work has shown that MTLE is related to glutamate transporter
dysfunction. We infected astrocyte cultures in vitro with HHV-6 and found a marked decrease in
glutamate transporter EAAT-2 expression.
Overall, we have now detected HHV-6B in 15 of 24 patients with mesial temporal sclerosis/
MTLE, in contrast to zero of 14 with other syndromes. Our results suggest a potential etiology
and pathogenic mechanism for MTLE.
The Editors’ Summary of this article follows the references.
PLoS Medicine | www.plosmedicine.orgMay 2007 | Volume 4 | Issue 5 | e1800848
P PL Lo oS S MEDICINE
Human herpesvirus-6 (HHV-6) is a b-herpesvirus first
isolated in 1986 from immunosuppressed patients with
lymphoproliferative disorders and HIV infection . HHV-6
infects most humans between 6 and 12 mo of age , and
more than 90% of the general population is seropositive .
After primary infection, HHV-6 can establish lifelong latency,
with the viral genome persisting in peripheral blood
mononuclear cells (PBMCs), salivary glands , and the
central nervous system (CNS) . HHV-6 DNA has been
detected in the cerebrospinal fluid of children during
primary infection and subsequent to infection, indicating
CNS viral persistence . HHV-6 reactivation may contribute
to disease in immunosuppressed patients following bone
marrow or solid-organ transplantation, and in those with
chronic fatigue syndrome [7,8].
Two variants of the virus have been identified, A and B,
with nucleotide sequence homology between 88% and 96%.
HHV-6A has been implicated in multiple sclerosis, and is
associated with viral persistence and reactivation in the CNS
[9,10]. HHV-6B is primarily associated with symptomatic
infections during infancy and is the causative agent of
exanthem subitum. Although HHV-6B is detected more
frequently in PBMCs of healthy adults in the US population
and may constitute the majority of latent HHV-6 infection,
HHV-6A may be more neurotropic [11–13].
While associations with adult neurologic disease [14–17]
are thought to involve reactivation of latent HHV-6, primary
HHV-6 infection is associated with febrile seizures in both
infants and young children. The incidence of febrile seizures
with primary HHV-6 infection ranges from 8%–20% in the
US population [18–20].
Epilepsy is one of the most common and severe neuro-
logical disorders, with a prevalence of 0.6%–1%, and a large
social and economic cost . Despite recent diagnostic
advances, the etiology of epilepsy in a high percentage of
patients remains unknown, although a wide range of
infections is associated with the broad category of seizure
disorders. Mesial temporal lobe epilepsy (MTLE) is one of the
most common and intractable forms of seizure disorder.
MTLE usually begins in childhood and is often associated
with a history of prolonged or complex (and possibly simple)
febrile seizures [22,23]. Mesial temporal sclerosis (MTS), with
extensive astrogliosis and neuronal loss, is the most common
pathological finding in MTLE. Imaging studies have found an
association between febrile seizures and MTS [24,25].
Temporal lobectomy is an effective treatment for MTLE,
achieving significantly better seizure control than antiepi-
leptic drugs . In a preliminary study of brain resections
from patients with MTLE, we detected HHV-6B DNA at high
viral loads in hippocampal sections, with viral antigen
colocalized to glial fibrillary acidic protein (GFAP)–positive
cells, confirming the in vivo localization of HHV-6 to
astrocytes . HHV-6 is present in astrocytes in brain tissue
from patients with multiple sclerosis , limbic encephalitis
, and post–bone marrow transplantation encephalitis,
suggesting that astrocytes may be an in vivo reservoir for
HHV-6. In vitro, HHV-6 infects a wide variety of cell types,
including primary human astrocytes [28–31].
Here, we extend our previous investigations on the
prevalence of HHV-6 infection in the hippocampus and
temporal lobe from a new series of patients with MTLE and
non-MTLE–related intractable epilepsy.
Materials and Methods
We studied 22 new patients (Table 1) referred for
evaluation to the Clinical Epilepsy Section, National Institute
of Neurological Disorders and Stroke, National Institutes of
Health (NINDS/NIH; Bethesda, Maryland), or to the Depart-
ment of Neurology Children’s National Medical Center
(CNMC; Washington, D. C.). Of these, 13 had surgery at
NIH and nine had surgery at CNMC. Protocols for patient
evaluation and surgical sample collection were approved by
the NINDS and CNMC Intramural Clinical Research Com-
mittees. Clinical evaluation in each case included ictal video-
EEG monitoring, 3-D–volumetric fast spin echo; and axial T1
and T2, coronal T2, and flair sequences performed on a 1.5
Tesla scanner (General Electric, http://www.gehealthcare.
com). Mean age at seizure onset was 10.8 6 10.7 y, and mean
age at surgery was 21.4 6 12.4 y. The 15 patients with MTLE
had a significantly older age at surgery (27.4 y versus 14.9 y; p
, 0.03) and a nonsignificant trend toward lower seizure onset
age (7 y versus 12 y) than the seven patients without MTLE.
Five patients with MTLE, but no patients without MTLE, had
a history of febrile seizures (Table 1). All the patients with
MTLE, compared with only one patient without MTLE who
had a ganglioglioma, had clinical, electrographic, and/or
imaging characteristics of MTLE [32,33]. We also studied one
additional patient (patient 16) who presented with typical
temporal lobe epilepsy but then developed a diffuse epileptic
syndrome, resulting in a hemispherectomy.
The patient 16 was an 8-y-old, right-handed boy with a
history of febrile seizures presented with sudden onset of
frequent complex partial and rare secondary generalized
seizures, without clear etiology. Complex partial seizures were
characterized were characterized by anxiety and repetition of
a short verbal phrase, followed by staring and aphemia with
preserved comprehension. Initial EEG showed left-sided
slowing; MRI showed left temporal fullness and increased
T2 signal. Cerebrospinal fluid revealed no red and three
white cells, a protein of 13, and glucose of 77 mg/100 ml.
Despite negative PCR for herpes, acyclovir was started.
Seizures persisted despite eight antiepileptic drugs, steroids,
IVIG, ganciclovir, and a vagal nerve stimulator. EEG showed
left temporal periodic epileptiform discharges and left
frontal and right sharp waves. A left mesial temporal
lobectomy led to transient improvement, but within 2 mo
seizure frequency approached baseline levels with semiology
unchanged. MRI was unchanged, and there were no focal
neurological findings. Invasive monitoring revealed left mid/
posterior superior temporal gyrus seizure onset. After
resection, frequent electrographic and clinical seizures arose
from inferior and middle temporal gyrus and frontal lobe.
After several additional focal resections, a hemispherectomy
was performed. The patient has been seizure free without
anticonvulsants for 1 y. He has a right hemiparesis with little
finger function, mild receptive deficits, and more severe
expressive deficits; language is improving.
All patient surgical samples were reviewed by a clinical
neuropathologist before submission for virological study.
Samples from mesial temporal and neocortical resections and
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HHV-6 and Epilepsy
lateral temporal lobe resections were fixed in formalin and
processed for standard pathological examination. All patients
with MTS/MTLE showed variable degrees of neuronal loss and
gliosis preferentially affecting the CA1 and CA3 areas, but
also markedly affecting CA4, CA2, and the dentate gyrus in
more severe cases. There was no evidence of inflammation or
neuronal inclusions in any patient with MTS/MTLE.
For isolation of PBMCs, blood samples were drawn into
acid citrate dextrose solution A tubes (Becton Dickinson,
http://www.bd.com) and were separated using lymphocyte
separation medium (ICN Biomedicals, http://www.mpbio.
com). Cells were stored in liquid nitrogen prior to DNA
extraction. Serum samples were extracted within 4 h of
collection. Fresh brain material was obtained during epilepsy
brain resection. Brain tissue was stored on ice in Hibernate A
medium, and astrocyte isolation was initiated within 30 min
after obtaining tissue.
DNA and RNA Extraction
DNA and RNA were isolated from fresh brain tissue using
commercially available extraction kits according to manu-
facturers’ instructions. The QIAamp blood kit was used for
DNA extraction from PBMCs, the QIAamp Viral RNA kit was
used for DNA extraction from serum (1.2 ml), and the DNeasy
tissue kit (all kits from Qiagen, http://www.qiagen.com) was
used for DNA extraction from fresh brain tissue. DNA and
RNA were also extracted from cultured primary astroytes
obtained from patient brain resections using DNeasy and
RNeasy extraction kits (Qiagen). For RNA extraction from
tissue, finely minced brain samples were resuspended in 350
ll QIAzol lysis reagent (Qiagen) and stored at ?70 8C until
use. RNA extraction was performed as per the manufacturer’s
directions using the RNeasy lipid tissue mini kit (Qiagen).
Nested PCR for HHV-6 Major Capsid Protein
DNA amplification was performed, using nested primers
specific for a highly conserved sequence corresponding to the
major capsid protein gene (MCP) of HHV-6 [9,34]. The external
primers amplified a 520 bp sequence, and the internal
primers amplified a 258 bp sequence. PCR was performed
using the Taq PCR master mix kit (Qiagen) as per
manufacturer’s instructions. DNA was amplified with 0.5 lM
final primer concentration for 35 cycles using the following
conditions: denaturation at 92 8C for 0.3 min, annealing at 55
8C for 0.3 min, and extension at 72 8C for 0.32 min. A total of
5 ll of primary PCR product was amplified using the internal
primers with the same PCR conditions. A total of 10 ll of PCR
product was subjected to electrophoresis on a 1.5% agarose
gel and visualized by ethidium bromide staining.
Real-Time Quantitative PCR
Viral DNA in patient samples was quantified using TaqMan
PCR with primers specific for HHV-6A and HHV-6B as
described previously [15,35]. The A- and B-specific primers
are located within the immediate early region of HHV-6 and
bind specifically to their respective variants. Control experi-
ments demonstrated that A-specific primers amplified only
the HHV-6A laboratory strains U1102 and GS, and that B-
specific primers amplified the HHV-6B laboratory strain Z29
. Standard and sample DNA were amplified in a 96-well
reaction plate using the following conditions: 50 8C for 2 min
for activation of uracil-N-glycosylase, 95 8C for 10 min to
inactivate uracil-N-glycosylase, and 45 cycles of 95 8C for 15 s
(denaturation) and 60 8C for 1 min (annealing and extension).
Table 1. Clinical Information from NIH/CNMC Epilepsy Patient Cohort
L MTS with cysticercosis
Increased T2, mild atrophy
RF post-ict T2
LF Mag Tra
Dysplasia, no tumour
Mild diffuse HF neuronal loss
Irregular neuronal distribution
HF, hippocampal formation; L, left; NE, neocortical epilepsy, R, right
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HHV-6 and Epilepsy
All standards and samples were assayed in triplicate, and
HHV-6 viral load was normalized to actin.
Primary Astrocyte Culture
Fresh tissue was obtained from epilepsy brain resections
performed at NIH or CNMC. Tissue was kept on ice in
Hibernate A medium (containing 2% B27 supplement, 0.5
mM glutamine, fungicide, and 1% penicillin/streptomycin).
Meninges and blood vessels were removed, and tissue was
minced into small pieces and dissociated in Earl balanced salt
solution (containing 0.01% DNase, 20 U/ml papain, and 1:100
penicillin/streptomycin) in a 37 8C shaking water bath for 1 h.
Digested tissue was mechanically dissociated with sterile
pipettes and passed through a 60 lm filter. The single-cell
suspension was centrifuged at 1,500 rpm for 10 min and
separated on a Percoll gradient for 30 min at 15,000 rpm. The
glial cell layer was isolated, washed, and plated overnight in
poly-L-lysine coated flasks or two-well chamber slides in
astrocytic medium (DMEM/F12 containing 10% FBS, 1%
penicillin/streptomycin, and 0.01% gentamicin). Primary
cultures were allowed to adhere overnight, and fresh medium
was added the following day. After 3–4 wk in culture,
astrocytes were stained for GFAP to determine culture purity.
Primary astrocytes were isolated from patients with MTLE
included in Table 2. Astrocytes from epilepsy patients 15
(Table 1) and patient 2a (previously described as patient 2
) were grown on poly-L-lysine–coated chamber slides for
3–4 wk. Primary antibodies were prepared in PBS and
consisted of 1:100 mouse anti-gp116/54/64 (Advanced Bio-
technologies, http://www.abionline.com), 1:100 rabbit anti-
GFAP (DAKO, http://www.dako.com), and 1:50 mouse anti-
microglia marker CD68 (Santa Cruz Biotechnology, http://
www.scbt.com). Slides were incubated with primary anti-
bodies for 1 h at room temperature and then washed three
times in PBS. Secondary antibodies conjugated to the
appropriate fluorophore (Molecular Probes, http://probes.
invitrogen.com) consisted of 1:100 anti-rabbit IgG FITC
(green), 1:1,000 anti-mouse IgG rhodamine (red), and 1:100
anti-mouse IgG AMCA (blue). Slides were incubated with
secondary antibody for 1 h at room temperature and washed
three times in PBS. Where indicated, slides were counter-
stained with DAPI in mounting media (Molecular Probes).
Mounted slides were visualized using a fluorescence micro-
scope (Carl Zeiss, http://www.zeiss.com) at 203, 323, or 403
HHV-6 Infection of Primary Astrocytes
After 2–3 wk in culture, primary astrocytic cells from
patient 20 were infected with HHV-6A and HHV-6B.
Infection was performed as described previously . Briefly,
cultures were infected with freshly thawed cell-free super-
natant from HHV-6A–infected JJahn or HHV-6B–infected
SupT-1 cells at a ratio of 103DNA viral copies/cell (quantified
by TaqMan PCR). Mock infections were performed using
culture medium from uninfected SupT1 or JJahn. Cultures
were incubated for 3 h at 37 8C in 5% CO2, cultures were
washed three times with PBS, and fresh medium was added.
Cells were harvested for experiments 5 d after infection.
Reverse Transcription-PCR and TaqMan for EAAT-2
RNA extracted from primary astrocytes was reverse tran-
scribed using reagents from Applied Biosystems (http://www.
appliedbiosystems.com) according to manufacturer’s instruc-
tions. cDNA was amplified using primers specific for EAAT-2
. PCR for EAAT-2 was run for 35 cycles using the
following conditions: 95 8C for 45 s, 55 8C for 1 min, and 72 8C
for 1 min. cDNA was also amplified using TaqMan primer/
probe sequences specific for EAAT-2 and HPRT (synthesized
from cDNA sequences by Synthegen, now Integrated DNA
Technologies, http://www.idtdna.com). cDNA was amplified in
a 96-well reaction plate using the following conditions: 50 8C
for 2 min for activation of uracil-N-glycosylase, 95 8C for 10
min to inactivate uracil-N-glycosylase, and 45 cycles of 95 8C
for 15 s (denaturation) and 60 8C for 1 min (annealing and
Quantitative Detection of HHV-6B DNA in Brain Tissue
HHV-6 type–specific analysis amplified HHV-6B but not
HHV-6A from a subset of patients with MTLE (Figure 1). The
Figure 1. High Levels of HHV-6 DNA Are Detected in Brain Resections
from Patients with MTLE
HHV-6B DNA was quantitated in PBMCs, serum, and fresh brain (hipp,
hippocampus; LTL, lateral temporal lobe) from epilepsy brain resections
by TaqMan PCR. Viral load was normalized as DNA copies/106cells (brain
and PBMCs) or DNA copies/ml (serum). Data from brain, serum, and
PBMCs are represented graphically from patients with and without MTLE.
Means are shown by black bars.
Table 2. HHV-6 DNA Detection in Temporal Lobe Resections
from NIH/CNMC epilepsy cohort (n¼38)
MTLE (n ¼ 24)
Non-MTLE (n ¼ 14)
Current cohort n ¼ 23; cohort published in Neurology 2003 , n ¼ 15.
ap , 0.001.
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HHV-6 and Epilepsy
range of HHV-6 in normal PBMCs has been reported to be
?100 copies/106cells , and no statistical difference was
observed in quantitative HHV-6B levels in PBMCs isolated
from patients with MTLE compared with those in patients
without MTLE. In contrast, HHV-6B DNA levels were
significantly higher in resected brain tissue from patients
with MTLE compared with patients with epilepsy without
MTLE (p , 0.001; Figure 1). Mean HHV-6B DNA levels in
MTLE brain tissue (hippocampus and temporal lobe) were 1.5
3 103copies/106cells and 3.9 3 102copies/106cells
respectively (Figure 1), while patients without MTLE had
undetectable levels of HHV-6 DNA in brain resections.
Notably, three hippocampus specimens resected from pa-
tients with MTLE, and four specimens from patients without
MTLE were negative for HHV-6B DNA. The negative viral
loads assessed by TaqMan were also confirmed by negative
nested PCR findings.
Characterization of HHV-6B Active Infection on Cultured
Astrocytes from Patients with MTLE
From brain resections where sufficient quantities of tissue
were available (five patients with MTLE and two patients
without MTLE), primary glial cells were cultured and tested
for purity of astrocyte populations by immunofluorescent-
staining GFAP. Each culture demonstrated variable astrocyte
purity by expression of GFAP (blue-stained cells in Figure 2A
and 2B; green-stained cells in Figure 2C and 2D). Primary
cultures were negative for the neuronal marker Tuj1 (b-
tubulin III; lack of red staining in Figure 2A and 2B) and for
the microglial marker CD68 (lack of red staining in Figure
2D), supporting the characterization of these cells as primary
astrocytes. Culture conditions did not support growth of
neurons or oligodendrocytes.
To determine whether primary astrocytes isolated from
MTLE brain tissue contained HHV-6, cells were analyzed for
the expression of the nonvariant specific HHV-6 surface
glycoprotein gp116/54/64 by immunofluorescence. As shown
in Figure 2, detection of HHV-6 gp116 in primary astrocytes
from patients with MTLE demonstrated HHV-6 viral protein
expression that colocalized with GFAP-positive astrocytic
cells (green staining in Figure 2A and 2B; red staining in
Figure 2C). The astrocytes in Figure 2 were cultured from the
hippocampus/temporal lobe of MTLE brain resections, each
of which was positive for HHV-6 by PCR and typed as HHV-
6B by TaqMan (unpublished data). Examples of primary
astrocytes from additional patients with MTLE expressing
HHV-6 antigen and containing HHV-6 viral DNA is shown in
Figure S1. By contrast, primary astrocyte cell lines cultured
from HHV-6–negative patients without MTLE remained
HHV-6 PCR negative and did not express HHV-6 gp116 by
immunofluorescence (unpublished data).
Longitudinal Detection and Characterization of HHV-6B
Between February 2004 and January 2005, patient 16 at
CNMC had four brain resections for uncontrolled seizures,
including a hemispherectomy in January 2005. Although
patient 16 was not classified with classic MTLE, early
symptoms indicated MTLE that progressed into a more
severe syndrome eventually requiring hemispherectomy. In
tissue from each brain resection, HHV-6 was detected by PCR
(unpublished data) and was quantified and subtyped as HHV-
Figure 2. Primary Astrocytes Isolated and Cultured from HHV-6B–Positive MTLE Brain Resections Express Viral Antigen
Primary astrocytes were isolated from fresh brain material obtained during epilepsy brain resection. Cells were cultured for 3–4 wk and costained for the
nonvariant specific HHV-6 gp116 surface glycoprotein and GFAP as a marker for astrocytes (A–C), the neuronal marker Tuj1 (A–B), or the microglial
marker CD68 (D).
(A) Epilepsy patient 2a: GFAP ¼ blue; HHV-6 ¼ green; Tuj1 ¼ red, 203.
(B) Epilepsy patient 2a: GFAP ¼ blue; HHV-6 ¼ green; Tuj1 ¼ red, 323.
(C) Epilepsy patient 15; GFAP ¼ green, HHV-6 ¼ red; DAPI ¼ blue, 403.
(D) Epilepsy patient 15; GFAP ¼ green; CD68 ¼ red; DAPI ¼ blue, 403.
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HHV-6 and Epilepsy
6B by TaqMan PCR (Figure 3). The highest HHV-6 viral load
was detected in the hippocampus following the first brain
resection (2.533103/106cells; Figure 3). Sufficient quantity of
brain material was obtained following hemispherectomy in
January 2005 for isolation and culture of primary cells.
Astrocyte cultures isolated from frontal/parietal and tempo-
ral lobes were HHV-6 gp116/54/64 positive and colocalized
with GFAP (Figure 2A), indicating the presence of viral
antigen in astrocytes in vitro. In addition, these cells were
positive for HHV-6 DNA by PCR (unpublished data), and
viral DNA was quantified and subtyped as HHV-6B (Figure 3).
Notably, TaqMan HHV-6B viral DNA levels in these primary
astrocyte cultures were higher than those quantified from
DNA isolated from the corresponding regions of fresh brain,
indicating the possibility of further viral reactivation during
primary culture (Figure 3). DNA extracted from freshly
resected temporal and frontal/parietal lobes following hemi-
spherectomy was also positive for HHV-6 by PCR (unpub-
lished data), and was quantified and typed as HHV-6B with
viral loads of 1.77 copies/106cells and 93 copies/106cells,
respectively. In contrast, DNA extracted from freshly resected
frontal and occipital lobes was negative for HHV-6 by both
PCR and TaqMan (unpublished data), indicating that viral
infection was localized and not spread throughout the brain.
In patients with MTLE, HHV-6 infection was localized
primarily to the temporal lobe, with highest viral load
detected in the hippocampus. Similarly, the first hippocampal
resection of patient 16 demonstrated the highest viral load
detected. Detection of HHV-6B DNA in the frontal/parietal
lobe following hemispherectomy supports the finding that
the syndrome progressed from early MTLE to a chronic
encephalitis, and may have lead to spread of HHV-6 infection
in the brain of this patient. Finally, we determined expression
of HHV-6 RNA using a set of HHV-6–specific primers specific
for HHV-6 U67 and U100 late genes. HHV-6 RNA was
demonstrated in primary astrocytes isolated from the frontal/
parietal lobe (Figure 3B), consistent with the detection of
high levels of HHV-6B DNA (Figure 3A) and HHV-6 antigen
by IFA (Figure 2A).
Association of Active HHV-6B Infection and Impaired
EAAT-2 mRNA Transcription in Cultured Astrocytes
We measured transcription of the astrocytic glutamate
transporter EAAT-2 mRNA in four brain regions and
cultured primary astrocytes isolated from patient 16 by
reverse transcription PCR (RT-PCR). All four brain regions
expressed mRNA for EAAT-2, while primary astrocytes
isolated from the frontal/parietal lobe expressed low levels
of EAAT-2 and mRNA was undetectable in primary astrocytes
isolated from the temporal lobe (Figure 4A). We compared
mRNA expression of EAAT-2 in primary astrocytes isolated
from patient 16 with primary astrocytes isolated from a
patient who was HHV-6B DNA negative. Primary astrocytes
from patient 20 (HHV-6B DNA negative) were also infected
in vitro with HHV-6A and HHV-6B to compare EAAT-2
expression in astrocytes infected in vitro with astrocytes
cultured from HHV-6–positive brain tissue. While EAAT-2
mRNA was expressed in mock-infected primary astrocytes
from patient 20, EAAT-2 expression was dramatically
reduced in primary astrocytes infected with either HHV-6A
(U1102) or HHV-6B (Z29) (Figure 4A). These observations
were confirmed by TaqMan, showing low levels of EAAT-2
expression in both primary astrocytes isolated from patient
16 and in the HHV-6A– and HHV-6B–infected primary
astrocytes from patient 20 compared with the expression
levels of EAAT-2 in mock-infected primary astrocytes (Figure
4B). Viral load in HHV-6A– and HHV-6B–infected primary
astrocytes from patient 20 were 1.193108copies/106cells and
1.68 3 106copies/106cells, respectively.
This study demonstrates persistent HHV-6B infection in
most patients with MTS-MTLE, but no detectable infection in
patients with other pathology and constitutes the first report
of primary isolation and maintenance of virus-infected
astrocytes from the human brain. In addition, we provide
Figure 3. Longitudinal Characterization of HHV-6 Infection in One
Patient with Multiple Brain Resections Followed by Hemispherectomy
(A) HHV-6B viral load was quantitated from fresh tissue obtained from
three consecutive brain resections (13 February 2004, 18 March 2004,
and 15 June 2004) followed by hemispherectomy (19 January 2005)
using variant-specific TaqMan PCR. Primary astrocytes were cultured
from tissue obtained at hemispherectomy from frontal/parietal and
temporal lobes. HHV-6B viral load was quantitated by Taqman in these
astrocyte cultures (1 March 2005) approximately 6 wk after surgery. All
viral loads are represented as DNA copies/106cells.
(B) Expression of viral RNA was determined by RT-PCR using primers for
three HHV-6 genes (from top to bottom: U86, immediate early [primers
specific for variants A and B], U67, late; U100, late). Lane 1, patient 16
frontal/parietal lobe astrocytes; lane 2, patient 16 temporal lobe
astrocytes; lane 3, negative control temporal lobe; lane 4, patient 16
frontal/parietal lobe; lane 5, patient 16 temporal lobe; lane 6, patient 16
occipital lobe; lane 7, patient 16 frontal lobe; lane 8, uninfected JJahn
(negative control); lane 9, U1102-infected JJahn (positive control).
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HHV-6 and Epilepsy
direct evidence for an etiological link of a ubiquitous human
herpesvirus in a subset of patients with intractable MTLE
with MTS, a syndrome of unknown origin. Active HHV-6
infection was confirmed by the detection of viral DNA,
mRNA viral transcripts, and viral protein expression in
human primary astrocytes isolated and cultured from
resected brain tissue from patients with MTLE. HHV-6B in
MTS-MTLE is unlikely to be a consequence of nonspecific
inflammation or seizures, since none of the patients without
MTLE patients, all of whom had intractable epilepsy, were
positive for virus. Moreover, there was no evidence for
inflammatory changes in any of the HHV-6B–positive
Studies associating HHV-6 with neurologic disorders,
including epilepsy, are based on detection of viral DNA,
RNA, and antigen, suggesting pathogenic reactivation of
latent HHV-6. Establishment of HHV-6 latency in the CNS
can follow primary childhood infection [37,38]. Although
HHV-6 reactivation in the brain appears to occur predom-
inately in immunosuppressed adults and is associated with
neurological complications following bone marrow and stem
cell transplantation, viral reactivation may also play a role in
disorders such as multiple sclerosis and epilepsy. Several
studies have demonstrated the presence of low levels HHV-6
DNA (typically nested PCR) in normal human brains that can
range between a frequency of 0%–75% [15,37–40]. These
discrepancies can be attributed to different patient popula-
tions, methods of detection, and sensitivities and specificities
of reagents. Studies demonstrating HHV-6 DNA in the
normal human brain have typically used nested PCR,
indicating that levels of viral DNA are low, supporting the
observations by Blumberg et al. that HHV-6 may be present
as a latent commensal pathogen that is able to infect the
human brain without causing any apparent neurological
disease . The result in this study demonstrating high levels
of viral DNA detected in MTLE specimens by non-nested
TaqMan PCR, together with the presence of viral antigen and
RNA, suggests association with disease. Detection of HHV-6
DNA by PCR in 25%–50% of patients with temporal lobe
epilepsy indicates an association of viral reactivation with a
neurological disorder that may be independent of immuno-
suppression [15,42–44]. The results in this study demonstra-
ted higher HHV-6 levels in hippocampal tissue than in the
surrounding temporal neocortex. High viral loads in the
hippocampus from patients with MTLE could reflect viral
reactivation in a specific brain region associated with latency
resulting from early childhood infection.
Alternatively, persistent noninflammatory encephalitis may
also lead to the development of epilepsy. Several lines of
evidence, including clinical, imaging, and neuropsychological
data, suggest that MTS/MTLE is a progressive disorder [45,46].
Imaging studies have shown that febrile seizure history and
epilepsy duration are associated with increasing hippocampal
atrophy, independent of seizure frequency [24,47,48]. A
history of febrile seizures may be difficult to establish
retrospectively, many years after the event. In our overall
cohort of 38 patients, which represents our collective
experience at NIH and the CNMC (Table 2), five of nine
patients with a definite history of febrile seizures (all in the
MTS-MTLE group), compared with nine of 28 reporting no
history of febrile seizures, were positive for HHV-6B (a clear
history could not be obtained for one HHV-6B–positive
patient with MTS-MTLE), suggesting a trend towards HHV-
6B–positive patients with MTLE having febrile seizure
history. The long latency between childhood febrile seizures
and the appearance of persistent unprovoked seizures
suggests these patients may have chronic HHV-6 infection
rather than reactivated virus. The presence of chronic viral
infection in these patients would be supported by the
progression of hippocampal atrophy. Collectively, these data
suggest an ongoing process; the latency between occurrence
of an early risk factor such as febrile seizures and onset of
chronic epilepsy is consistent with either persistent or
reactivated infection [25,32].
We also had the unique opportunity to follow longitudi-
nally a patient who experienced recurrent seizures after three
focal resections and a hemispherectomy for a period of 11
mo. Consistent detection of high levels of HHV-6B DNA in
each of the three resections and in frontal and temporal/
parietal lobes following hemispherectomy suggests this
patient had a persistent and widespread infection with
HHV-6B. Following hemispherectomy, this patient has been
seizure-free without anticonvulsant treatment for more than
Figure 4. Decreased Expression of the Glutamate Transporter EAAT-2 in
(A) EAAT-2 mRNA was detected by RT-PCR in fresh brain from patient 16,
in astrocytes cultured from patient 15, and in astrocytes cultured from
patient 20, and infected with HHV-6 in vitro. Samples from the two
different patients were run in two separate PCR reactions (on different
days) using the same set of EAAT-2 primers but different actin primers.
The products of these PCR reactions were run on the same gel. Lane 1,
patient 16 frontal/parietal lobe; lane 2, patient 16 temporal lobe; lane 3,
patient 16 occipital lobe; lane 4, patient 16 frontal lobe; lane 5, patient 16
frontal/parietal lobe astrocytes; lane 6, patient 16 temporal lobe
astrocytes; lane 7, uninfected astrocytes cultured from patient 20; lane
8, HHV-6A (strain U1102)–infected astrocytes cultured from patient 20;
lane 9, HHV-6B (strain Z29)–infected astrocytes cultured from patient 20;
lane 10, negative control (JJhan T cells); lane 11, positive control (U251
(B) EAAT-2 mRNA was detected by quantitative TaqMan and normalized
to expression of HPRT from astrocytes cultured from frontal/parietal (F/P)
and temporal (TL) lobes of patient 16. Astrocytes from patient 20 were
mock-infected or were infected with HHV-6A (strain U1102) or HHV-6B
PLoS Medicine | www.plosmedicine.org May 2007 | Volume 4 | Issue 5 | e1800854
HHV-6 and Epilepsy
1 y. It is intriguing to speculate that these data demonstrate a
widespread and persistent HHV-6 infection that was asso-
ciated with epilepsy, and that once the viral infection was
removed, the patient became seizure-free.
An association between chronic epilepsy and persistent or
reactivated HHV-6 infection of astrocytes suggests the
possibility that viral infection of astrocytes are associated
with changes in cell function that may contribute to disease.
Astrocytes are known to interact closely with neurons and are
critical in modulating synaptic transmission . Astrocytes
can modulate neurotransmission by maintaining low concen-
trations of extracellular glutamate by the glial glutamate
transporters EAAT-1 and EAAT-2 . Elevated extracellular
glutamate, the main excitatory neurotransmitter, may be
involved in epilepsy by triggering excitotoxicity through loss
of glutamine synthetase , an enzyme that metabolizes
glutamate in astrocytes, and/or by malfunctioning astrocytic
glutamate transporters . The CA1 and CA3 neurons lost
in MTS/MTLE are particularly susceptible to glutamatergic-
mediated cell death. Sclerotic hippocampi from temporal
lobe epilepsy demonstrate reduced EAAT-2 immunoreactiv-
ity , and are prone to alternative EAAT-2 mRNA splicing
. A unique finding in this study is the isolation ex vivo of
cultured astrocytes from patients with MTLE who are
infected with HHV-6. These primary HHV-6–infected astro-
cytes demonstrated low levels of EAAT-2 mRNA. In support
of our ex vivo findings, astrocytes infected with HHV-6 in
vitro also demonstrated a remarkable decrease in EAAT-2
mRNA. Detection of high levels of HHV-6 DNA in MTLE
brain tissue, isolation of HHV-6 from primary astrocytes
isolated from MTLE brain tissue, and decreased expression of
EAAT-2 mRNA demonstrates an association between HHV-6
infection and astrocytic dysfunction. Functional changes in
virus infected glia or in glia harboring reactivated virus may
lead to secondary injury of the exquisitely sensitive hippo-
campal neuron, and ultimately to development of MTLE and
epilepsy. The potential relationship between HHV-6 astro-
cytic infection and MTLE deserves further investigation.
Overall, our cumulative experience at the NIH Clinical
Center and the CNMC consists of a large cohort of patients
with epilepsy, from which 60% with clinically defined MTLE
had detectable HHV-6B sequences in surgical brain resec-
tions. The highest HHV-6 viral loads were demonstrated in
the hippocampus. This appeared to be specific for MTLE,
since non-MTLE epilepsy material had no detectable levels of
HHV-6 DNA by TaqMan or nested PCR. The statistically
significant correlation of HHV-6 and MTLE (Table 2) in a
large cohort of patients with epilepsy and dysregulation of
glutamate transporter expression by HHV-6 suggests a novel
pathophysiological mechanism of disease.
Figure S1. Primary Astrocytes Isolated and Cultured from HHV-6B–
Positive MTLE Brain Resections Express Viral DNA and Antigen
Primary astrocytes were isolated from fresh brain material obtained
during epilepsy brain resection.
(A) Cells were cultured for 3–4 wk and stained for GFAP (green),
DAPI (blue; nuclei), and the nonvariant specific HHV-6 antigen
gp116/54/64 (red). Representative immunofluorescence images show
primary astrocyte cultures from four epilepsy brain resections
(patients 14, 15, 5, and 6). All images were acquired with a 203
(B) Cells were scraped from fixed slides (patients 5 and 6), DNA was
extracted, and DNA for HHV-6 U57 (major capsid protein) was
detected by nested PCR. Negative and positive controls used were
uninfected SupT1 T cells and HHV-6B (strain Z29)–infected SupT1 T
Found at doi:10.1371/journal.pmed.0040180.sg001 (36 KB JPG).
The GenBank (http://www.ncbi.nlm.nih.gov/) accession numbers for
the viruses and transporter discussed in this paper are HHV-6A
(NC_001664), HHV-6B (NC_000898), and EAAT-2 (NM_004171).
The authors gratefully acknowledge Dr. Robert Bonwetsch for
assistance in processing samples and immunofluorescence. Julie
Fotheringham was supported by a postdoctoral fellowship from the
Multiple Sclerosis Society of Canada.
Author contributions. DD, AFH, JDH, WDG, WHT, and SJ designed
the study. JF, DD, NA, AFH, AV, WHT, and SJ analyzed the data. JDH,
SW, DAB, WDG, SS, and WHT enrolled patients. JF, DD, NA, AFH,
AV, JDH, EW, WDG, WHT, and SJ contributed to writing the paper.
DD, JF, NA, and AFH performed the experiments (sample processing,
DNA extraction, astrocyte isolation, IFA staining). AV performed
procurement and histologic evaluation of resected tissues. JDH is the
Principal Investigator for the clinical protocol, Research Study of
Specimens Obtained During Epilepsy Surgery, 02-N-0014, and was
involved in the design of the experiments, the acquisition of surgical
tissue specimens, and the care of the patients who donated the
research specimens. DAB was the neurourgeon who operated on the
children who underwent seizure surgery. SS participated in the
selection of surgical candidates and of surgical plans, performed
intraoperative electrocorticography and extraopertive invasive (sub-
dural) recording in order to determine the location of seizure foci,
and interpreted these results and discussed and determined actual
resection plans with a neurosurgeon. WHT was responsible for
clinical evaluation and neuroimaging. SJ participated in study design
and data analysis.
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HHV-6 and Epilepsy
Editors’ Summary Download full-text
Background. Epilepsy is a common brain disorder caused by a sudden,
excessive electrical discharge in a cluster of neurons—the cells that
transmit electrical messages between the body and the brain. Its
symptoms depend on which part of the brain is affected by this electrical
firestorm and how far the disturbance spreads. When only part of the
brain is affected (a partial seizure or fit), patients may see or smell strange
things, recall forgotten memories, or have part of their body jerk
uncontrollably. When the electrical disturbance spreads across the whole
brain (a generalized seizure), there may be loss of consciousness and/or
the whole body may become rigid or jerk. Epilepsy is usually controlled
with anti-epileptic drugs or, in very severe focal cases, surgery to the area
of the brain where the seizure starts. Although head injuries or brain
tumors can trigger epilepsy, the cause of most cases of epilepsy is
Why Was This Study Done? Knowing what causes epilepsy might lead
to better treatments for it. One possibility is that infections trigger
epilepsy. The researchers in this study asked whether infections with
human herpesvirus 6B (HHV-6B) are associated with a common type of
epilepsy called mesial temporal lobe epilepsy (MTLE). Patients with MTLE
often have extensive scarring in the hippocampus, a brain region
responsible for memory that lies deep within a bigger region called the
temporal lobe. Hippocampal scarring and MTLE are associated with a
history of fever-induced fits, and HHV-6B infection can cause such fits in
young children. Most people become infected with HHV-6B (or the
closely related HHV-6A) early in life. The virus then remains latent for
years within the brain and elsewhere. Given these facts and a previous
investigation that showed that brain tissue from several patients with
MTLE contained HHV-6B, the researchers reasoned that it was worth
investigating HHV-6B as a cause of MTLE.
What Did the Researchers Do and Find? The researchers first looked
for HHV-6B DNA in brain tissue surgically removed from patients with
MTLE or another type of epilepsy. Tissue from 11 of 16 patients with
MTLE (but from 0 of 7 control patients) contained HHV-6B DNA. When
the researchers grew astrocytes (a type of brain cell) from some of these
samples, only those from HHV-6B DNA-positive samples from patients
with MTLE expressed an HHV-6-specific protein. Next, the researchers
investigated in detail a patient with MTLE who had four sequential
operations to control his epilepsy. This patient’s hippocampus, which
was removed in his first operation, contained a higher level of HHV-6B
DNA than the tissues removed in later operations. After the fourth
operation (which removed half of his brain and cured his epilepsy),
astrocytes grown from the temporal lobe and the frontal/parietal lobe (a
brain region next to the temporal lobe) but not the frontal and occipital
lobes contained HHV-6B DNA and expressed a viral protein. The
researchers also measured the production by these various astrocytes
of a substance that moves glutamate (an amino acid that also acts as a
neurotransmitter) across cell membranes—MTLE has been associated
with a glutamate transporter deficiency. Consistent with this, astrocytes
from the patient’s temporal lobe made no glutamate transporter mRNA
(mRNA is an essential precursor for protein to be produced). Finally,
infection of astrocytes isolated from a patient without MTLE with HHV-6B
greatly reduced expression of glutamate transporter in these astrocytes.
What Do These Findings Mean? These findings, together with those
from the previous study, reveal that nearly two-thirds of patients with
MTLE (but no patients with other forms of epilepsy) have an active HHV-
6B infection in the brain region where their epilepsy originates. Overall,
they provide strong support for the idea that HHV-6B infections might
cause MTLE, particularly given the results obtained from the patient
whose condition only improved after multiple brain operations had
removed all the virally infected material. Furthermore, the demonstration
that HHV-6B infection reduces glutamate transporter expression in
astrocytes suggests that HHV-6B infection might cause astrocyte
dysfunction. This dysfunction could lead to injury of the sensitive
neurons in the hippocampus and trigger MTLE. Additional patients now
need to be studied both to confirm the association between HHV-6B
infection and MTLE and to discover exactly how this virus triggers
Additional Information. Please access these Web sites via the online
version of this summary at http://dx.doi.org/10.1371/journal.pmed.
? MedlinePlus encyclopedia page on epilepsy (in English and Spanish)
? World Health Organization fact sheet on epilepsy (in English, French,
Spanish, Russian, Arabic, and Chinese)
? US National Institute for Neurological Disorders and Stroke epilepsy
information page (in English and Spanish)
? UK National Health Service Direct information for patients on epilepsy
(in several languages)
? Neuroscience for kids, an educational Web site prepared by Eric
Chudler (University of Washington, Seattle, Washington, United
States), who also has a site that includes information on epilepsy
and a list of links to epilepsy organizations (mainly in English but some
sections in other languages as well)
? A short scientific article on human herpes virus 6 in the journal
Emerging Infectious Diseases
PLoS Medicine | www.plosmedicine.orgMay 2007 | Volume 4 | Issue 5 | e1800857
HHV-6 and Epilepsy