B R I E F R E P O R T

Human Herpesvirus 6 Latent

Infection in Patients With Glioma
Jing Chi,
1,a
Bin Gu,
1,2,a
Chun Zhang,
1,3
Guangyong Peng,
4
Feng Zhou,
1
Yun Chen,
1
Guofeng Zhang,
1,3
Yidi Guo,
1
Dandan Guo,
1
Jian Qin,
5
Jinfeng Wang,
1
Lingyun Li,
1
Fang Wang,
1,6
Genyan Liu,
1,6
Fangyi Xie,
1
Dongju Feng,
1
Hong Zhou,
1
Xingxu Huang,
7
Shiqiang Lu,
1
Yingxia Liu,
1
Weixing Hu,
3
and Kun Yao
1
1
Department of Microbiology and Immunology, Nanjing Medical University,
2
Department of Neurosurgery, Zhongda hospital, School of Medicine, Southeast
University, and
3
Department of Neurosurgery, First Afliated Hospital of Nanjing
Medical University, China;
4
Division of Infectious Diseases, Allergy, and
Immunology, Department of Internal Medicine, Saint Louis University, Missouri,
5
College of Foreign Languages, Hehai University,
6
Department of Laboratory
Medicine, First Afliated Hospital of Nanjing Medical University, and
7
Model
Animal Research Center, Nanjing University, China
The etiology of glioma remains unclear so far. Human her-
pesvirus 6 (HHV-6) might be associated with glioma, but
there is no direct evidence to support this. High percentages
of HHV-6 DNA and protein were detected in tissue from
gliomas, compared with normal brain tissue. In addition, a
strain of HHV-6Awas isolated from the uid specimens from
glioma cysts. High levels of interleukin 6 (IL-6), interleukin 8
(IL-8), tumor necrosis factor , and transforming growth
factor (TGF-) were detected in the cyst uid specimens
from HHV-6positive patients with glioma. Furthermore,
HHV-6A infection promoted IL-6, IL-8, and TGF- produc-
tion in astrocyte cultures. Our studies strongly suggest the in-
volvement of HHV-6 infection in the pathogenesis of glioma.
Glioma is one of the most common primary brain tumors,
with a high fatality rate [1]. However, the etiology of glioma
has remained unclear until recently [1]. Recent studies have
suggested that viral agents might be an etiological factor for
the development of glioma [2]. An early study reported that
virus-like particles were found in glioma tissues by electron
microscopy [3]. Furthermore, one study demonstrated that
90% of the examined glioblastoma samples were cytomegalo-
virus (CMV) antigen and DNA positive [4]. However, their
studies did not provide any direct evidence showing the exis-
tence of viable CMV in the tumor microenvironment. Besides
CMV detection, more-recent studies have demonstrated that
HHV-6 might be associated with glioma [57].
HHV-6 is a double-stranded DNA virus with both human
lymphocyte tropism and neurotropism. It has 2 variants,
HHV-6A and HHV-6B. Furthermore, HHV-6 infection has
been associated with a number of neurological disorders, in-
cluding encephalitis and seizures [8, 9]. In addition, HHV-6
early and late antigens have been detected in primary and
recurrent central nervous system tumors and, even more
frequently, in glioma [5, 6].
Given that HHV-6 may be a potential etiological agent for
the pathogenesis of glioma, we investigated HHV-6 infection
in glioma tissues, using nest polymerase chain reaction (PCR)
and immunohistochemical analysis. Importantly, we isolated a
HHV-6 strain from cyst uid specimens obtained from a
patient with glioma, strongly suggesting that HHV-6 may be
an important etiological factor for glioma. We further showed
that HHV-6 infection can induce proinammatory cytokines
in cyst uids of HHV-6positive patients with glioma and in
astrocyte cultures.
MATERIALS AND METHODS
Clinical Specimens
Forty glioma tissue specimens were obtained from patients
aged 2273 years. Thirteen normal brain tissue specimens
were obtained from autopsy of individuals with nonneurologi-
cal causes of death (age range, 2570 years). Blood samples
were collected from 40 patients with glioma and 20 healthy
volunteers. Cyst uid specimens were obtained from 4 patients
with glioma. These specimens were provided by the First Afl-
iated Hospital of Nanjing Medical University. Brain tumors
were classied according to World Health Organization crite-
ria. These studies were approved by the local ethics committee
and institutional review board. All samples were obtained with
consent from patients and volunteers.
Cells
Cord blood mononuclear cells (CBMCs) were puried from the
cord blood samples obtained from the Afliated Women and
Received 9 February 2012; accepted 4 June 2012; electronically published 7 September
2012.
a
J. C. and B. G. contributed equally to the study.
Correspondence: Kun Yao, MD, Department of Microbiology and Immunology, Nanjing
Medical University,140 Hanzhong Rd, Nanjing 210029, Jiangsu Province, China (yaokun@
njmu.edu.cn). And Weixing Hu, MD, Department of Neurosurgery, First Afliated Hospital of
Nanjing Medical University, 300 Guangzhou Rd, Nanjing 210029, Jiangsu Province, China
(hwx66@126.com).
The Journal of Infectious Diseases 2012;206:13948
The Author 2012. Published by Oxford University Press on behalf of the Infectious Diseases
Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@
oup.com.
DOI: 10.1093/infdis/jis513
1394 JID 2012:206 (1 November) BRIEF REPORT

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Children Hospital of Nanjing Medical University. HSB-2 cell line
was cultured in Roswell Park Memorial Institute 1640 medium
containing 10% fetal calf serum (FCS). Primary human fetal
astrocytes (PHFAs) were purchased from the Sciencell company
and cultured in DEME/F12 supplemented with 10% FCS.
Virus Isolation and Identication
The cyst uid specimens and peripheral blood mononuclear
cells (PBMCs) obtained from patients with glioma were cocul-
tured with CBMCs. HHV-6 infection was rst identied on
the basis of the cytopathic effects (CPEs) in CBMCs. Virus
particles were further observed by electron microscopy. Once
the viruses were identied from the CBMCs, they were main-
tained and amplied in HSB-2 cells. Virus titers of median
tissue culture infective doses (TCID
50
) per milliliter were
determined on the basis of CPEs in HSB-2 cells.
DNA Extraction and Nested PCR
DNA from tissues and cells was isolated using a DNA extrac-
tion kit (Biomiga). The DNA samples were then subjected to
nested PCR to determine virus infection. Five microliters of
DNA from each sample was amplied in a 50-L reaction
mixture containing PCR buffer and each outer primer. Two
microliters of the rst-round PCR product were amplied in a
second-round PCR, using each inner primer. Ten microliters
of the second-round PCR product were electrophoresed on a
1% agarose gel and visualized by ethidium bromide staining.
The second-round products also were sequenced and then
subjected to a BLAST search against the GenBank database.
The sequences of the nested PCR primers of HHVs are shown
in Supplementary Table 1.
Immunouorescence and Immunohistochemical Analyses
The CBMCs with typical CPEs of virus infection were harvest-
ed for smearing. Indirect immunouorescence analysis was
performed using a HHV-6-specic monoclonal antibody
(Santa Cruz Biotechnology) and a secondary antibody labeled
with FITC ( Jackson). Immunohistochemical staining was per-
formed on parafn-embedded and frozen sections, using En-
vision plus Peroxidase Mouse detection kits (Gene Tech) with
an antiHHV-6 monoclonal antibody (Santa Cruz Biotechnol-
ogy). Isotype antibody (Sigma) served as a negative control.
Infection of Astrocytes by the Isolated HHV-6
PHFAs (2 10
5
/well) were cultured in 6-well plates and then
infected with the isolated HHV-6 at a multiplicity of infection
of 100. The culture supernatants were collected for cytokine
detection at 0, 24, 48, 72, and 96 hours after infection.
Cytokine Detection by Enzyme-Linked Immunosorbent Assay
(ELISA)
The presence of tumor necrosis factor (TNF-), interferon
(IFN-), transforming growth factor (TGF-), interleukin 6
(IL-6), interleukin 8 (IL-8), and interleukin 10 (IL-10) in the
astrocyte culture supernatants infected by HHV-6 or in cyst
uid specimens from patients with glioma was determined
using ELISA kits (Bender). All cytokine assays were performed
in triplicate.
RESULTS
Isolation and Identication of Viruses From Patients With
Glioma
PBMCs obtained from 27 patients and 4 fresh glioma cyst
uid samples were mixed with PHA-stimulated CBMCs. After
11 days of growth, the cyst uid sample from patient 1 showed
CPEs characterized by swelling of scattered cells, increased
transparency, and refraction and fusion of infected cells. At
approximately 15 days of coculture, CPEs were detected >50%
in the cocultured cells. Following the subculture, we found
that the average time to CPE onset was shortened to 57 days,
and the percentage of the cells showing CPEs reached 50%
(Figure 1A). However, the other 3 cyst uid samples and 27
PBMC samples did not show the CPEs after 3 weeks of cocul-
ture. In addition, the herpesvirus-like particles (120300 nm
in diameter) were visible in the cytoplasm of the infected
CBMCs, using electron microscopy (Figure 1B). The isolated
virus can infect HSB-2 cells with a titer of 5 10
3
TCID
50
/mL.
The isolated virus in infected CBMCs was conrmed by per-
forming immunouorescence analysis with a HHV-6specic
monoclonal antibody (Figure 1C). Furthermore, by using
HHV-6 specic primers, we detected a bright band at 287 base
pairs after the rst round of PCR and at 163 base pairs after the
second round of PCR (Figure 1D). To determine whether the
isolate was the HHV-6A or HHV-6B variant, the inner
segment amplied by PCR was sequenced. DNA sequencing
showed 98% homology between this 163base pair fragment
and the U1102 gene in HHV-6A (Figure 1E), suggesting that
this isolate belongs to the HHV-6A variant.
Prevalence of HHV-6 in Tumor Tissues and PBMCs From
Patients With Glioma
To determine whether HHV nucleic acids were present in
glioma tissues, nested PCR for detection of herpes simplex
virus 1, Epstein-Barr virus, CMV, HHV-6, and HHV-7 was
performed on 40 glioma and 13 normal brain specimens.
HHV-6 was the most frequently identied virus among the
HHV family, existing in 17 of 40 glioma tissues (42.5%). Fur-
thermore, only 1 of 13 normal brain tissues (7.7%) yielded
HHV-6 DNA. There was a signicant difference in the HHV-
6 detection rate between the glioma tissues and the normal
brain tissues (P = .03). Notably, HHV-6 positivity varied
among the different types of glioma. A total of 41.2% of astro-
cytomas, 33.3% of oligodendrogliomas, and 50% of glioblasto-
mas were positive for HHV-6 (Table 1).
BRIEF REPORT JID 2012:206 (1 November) 1395

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Figure 1. Isolation and identication of virus from cyst uid samples. A, Virus infectioninduced cytopathic effect (CPE) in cord blood mononuclear
cells (CBMCs). After culture for 11 days, the CBMCs inoculated with the cyst uid sample presented the ballooning and polykaryotic CPE. B, Detection
of viral particles in virus-infected CBMCs by electron microscopy. C, Detection of human herpesvirus 6 (HHV-6) antigen in CBMCs inoculated with cyst
uid. Virus-infected CBMCs showed bright expression of HHV-6specic antigen (green) in the cytosol with an antiHHV-6 (20) monoclonal antibody. D,
Detection of HHV-6specic DNA in infected CBMCs by polymerase chain reaction (PCR). DNA of herpes simplex virus 1 (HSV-1), Epstein-Barr virus
(EBV), cytomegalovirus (CMV), HHV-6, and HHV-7 was detected in virus-infected CBMCs by nested PCR. A bright band at 287 base pairs after the rst-
round PCR and at 163 base pairs after the second-round PCR is shown. E, DNA sequencing showed 98% homology between the sequences of the 163
base pair fragment and the HHV-6A U1102 gene. Furthermore, the sequence of HHV-6B (Z29) contained a HindIII digestion site. However, HHV-6A
(U1102) and the isolated virus did not have a HindIII digestion site in the DNA sequences.
Table 1. Nested Polymerase Chain Reaction (PCR) Analyses of Human Herpesvirus (HHV) DNA and Immunohistochemical (IHC)
Analyses of HHV-6 Antigen in Glioma and Normal Brain Tissue Samples
Tissue Sample
Nested PCR Analysis IHC Analysis
HSV-1 EBV CMV HHV-6 HHV-7 HHV-6
Glioma (n = 40) 7/40 (17.5) 8/40 (20) 8/40 (20) 17/40 (42.5) 3/40 (7.5) 13/40 (32.5)
Astrocytoma (n =17) 2/17 (11.8) 2/17 (11.8) 3/17 (17.6) 7/17 (41.2) 2/17 (11.8) 5/17 (29.4)
Glioblastoma (n =14) 3/14 (21.4) 5/14 (35.7) 3/14 (21.4) 7/14 (50) 1/14 (7.1) 5/14 (35.7)
Oligodendroglioma (n =9) 2/9 (22.2) 1/9 (11.1) 2/9 (22.2) 3/9 (33.3) 0/9 (0) 3/9 (33.3)
Normal brain (n = 13) 0/13 (0) 0/13 (0) 0/13 (0) 1/13 (7.7) 0/13 (0) 0/13 (0)
P, Fisher exact test .121 .074 .074 .03
a
.501 .014
a
Data are no. of samples with positive results/no. tested (%), unless otherwise indicated.
Abbreviations: CMV, cytomegalovirus; EBV, EpsteinBarr virus; HSV-1, herpes simplex virus 1.
a
P < .05, glioma vs normal brain tissues.
1396 JID 2012:206 (1 November) BRIEF REPORT

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We also analyzed HHV-6 DNA in PBMCs obtained from
patients with glioma and from normal volunteers, using
nested PCR. We found that 22 of 40 PBMCs (55%) were posi-
tive for HHV-6 DNA in patients with glioma. However, only
5 of 20 PBMCs from healthy controls (25%) were positive for
HHV-6 DNA, further demonstrating a signicant difference
in the HHV-6 infection rate between patients with glioma and
healthy controls (P = .026). These results suggested that HHV-
6 presented more commonly in patients with glioma.
We next investigated whether HHV-6 is also locally reacti-
vated in glioma tissues. We performed immunohistochemical
analysis of 40 glioma specimens and 13 normal brain tissue
specimens. Among the glioma specimens, 13 samples (32.5%)
were positive for HHV-6, with both strong nuclear and cyto-
plasmic staining (Supplementary Figure 1). However, we did
not detect any HHV-6 immunoreactivity in normal brain
tissues (P = .014) (Table 1). Collectively, these results clearly
indicate that, compared with other herpersviruses, HHV-6 in-
fection and reactivation is a common feature in patients with
glioma.
Proinammatory and Suppressive Cytokines Induced by HHV-6
Infection
Recent studies have demonstrated that HHV-6 infection in
vitro can change the characteristics of the infected cells and
release inammatory cytokines [10, 11]. Thus, we reasoned
that HHV-6 may reactivate in the brain and create a local in-
ammatory microenvironment that facilitates the development
of glioma. To address these possibilities, we rst determined
cytokine production in cyst uid specimens obtained from pa-
tients with glioma. As shown in Supplementary Figure 2A,
high levels of IL-6, IL-8, TNF-, and TGF- were detected in
cyst uid specimens from 4 patients with glioma. To further
investigate the potential origins of these cytokines, we deter-
mined cytokine production in astrocytes after infection with
the isolated HHV-6. As expected, we found that, in infected
astrocytes, HHV-6 infection signicantly increased the release
of IL-6, IL-8, and TGF-; slightly induced IL-10; and did not
induce TNF- or IFN- (Supplementary Figure 2B). Notably,
cyst uid specimens from patients with glioma and superna-
tants from HHV-6infected astrocytes contained high
amounts of TGF-, suggesting that HHV-6 infection may
induce a suppressive microenvironment in patients with
glioma.
DISCUSSION
In this study, we showed that HHV-6 antigen and DNA were
abundant in glioma specimens. Most importantly, we isolated
a HHV-6A strain from a glioma cyst uid sample. The current
study was the rst report on virus isolation from glioma
specimens and conrmed the importance of HHV-6 infection
in glioma pathogenesis.
HHV-6 has also been reported by other groups to be
present in 8%47% of adult brain tumors by PCR analysis,
which accords with the prevalence of 42.5% among glioma
tissue specimens in our current study [5, 7]. The discrepancy
of the detection rates in both diseased and normal tissues
among research groups could potentially be due to differences
in PCR methods and sample preparation. Like HHV-6, DNA
and protein of CMV, another member of the beta herpesvirus
family, has been detected in malignant glioma specimens by
PCR and immunohistochemical analysis [4]. In our experi-
ments, mixed infection with CMV, HHV-6, HHV-7, and EBV
was detected by PCR in some brain tumor tissues (unpub-
lished data). However, the rate of HHV-6 positivity was much
higher than that of any of the other types of HHVs.
HHV-6 primary infection occurs during early childhood.
After primary infection, HHV-6 establishes latency and
remains present throughout the hosts life. HHV-6 can be re-
activated in immunosuppressed individuals. Furthermore, the
distribution of HHV-6 nucleic acid and viral antigen in
the tumor tissues itself already suggested a CNS reservoir for
HHV-6. Our current studies have shown that HHV-6A can
preferentially infect human astrocytes in vitro. In addition,
one trans-activating gene (ORF-1) in HHV-6 has been shown
to exhibit transforming activity and directly crosstalk with the
oncogene p53 [12]. Besides directly altering the infected cell
properties, HHV-6 infection can induce proinammatory
cytokine secretion and create an inammatory microenviron-
ment that facilitates the pathogenesis of glioma. Yoshikawa
et al demonstrated that a latent HHV-6 infection in neuroglial
cells could alter proinammatory cytokines synthesis [11], and
this might be involved in the development of glioma. In the
current study, we found that high levels of IL-6, IL-8, TGF-,
and TNF- were detected in cyst uid specimens from pa-
tients with glioma. Furthermore, we showed that infection
with the HHV-6 isolate promoted IL-6, IL-8, and TGF- pro-
duction in astrocytes cultures. In addition, HHV-6 can disturb
the key immune activation pathways and cytokine networks,
including an upregulation of TNF-, RANTES, interleukin 1,
and IL-10 [10]. Importantly, recent evidence suggested that
HHV-6 is an important immunosuppressive virus. HHV-6
(especially HHV-6A) could cause selective immunosuppres-
sion in otherwise immunocompetent adults [10, 11]. We
showed that HHV-6 infection can induce IL-10secreting
CD4
+
T-regulatory 1 cells [13]. Furthermore, HHV-6 infection
can directly deplete the infected CD4
+
T lymphocytes via the
induction of apoptosis [14]. More recently, we have demon-
strated that HHV-6 suppressed T-cell proliferation through
the induction of cell cycle arrest in the G2/M phase [15].
In summary, our data provide convincing evidence that
HHV-6 is highly present in glioma tissues. We were the rst
BRIEF REPORT JID 2012:206 (1 November) 1397

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to directly isolate HHV-6 from the glioma tumor microenvi-
ronment, suggesting the critical role of HHV-6 in the develop-
ment of glioma. In addition, proinammatory cytokines
induced by HHV-6 infection might provide a chronic inam-
matory environment that facilitates the development of
glioma. These results strongly suggest that HHV-6 is an im-
portant etiological factor in the pathogenesis of glioma.
Supplementary Data
Supplementary materials are available at The Journal of Infectious Diseases
online (http://jid.oxfordjournals.org/). Supplementary materials consist of
data provided by the author that are published to benet the reader. The
posted materials are not copyedited. The contents of all supplementary
data are the sole responsibility of the authors. Questions or messages
regarding errors should be addressed to the author.
Notes
Financial support. This work was supported by the National Natural
Science Foundation of China (grants 30972784 & 81273235 to K. Y.).
Potential conicts of interest. All authors: No reported conicts.
All authors have submitted the ICMJE Form for Disclosure of Potential
Conicts of Interest. Conicts that the editors consider relevant to the
content of the manuscript have been disclosed.
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