herpes virus 6 gliomas

B R I E F R E P O R T

Human Herpesvirus 6 LatentInfection 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 Yao1

1Department of Microbiology and Immunology, Nanjing Medical University,2Department of Neurosurgery, Zhongda hospital, School of Medicine, SoutheastUniversity, and 3Department of Neurosurgery, First Afliated Hospital of NanjingMedical University, China; 4Division of Infectious Diseases, Allergy, andImmunology, Department of Internal Medicine, Saint Louis University, Missouri,5College of Foreign Languages, Hehai University, 6Department of LaboratoryMedicine, First Afliated Hospital of Nanjing Medical University, and 7ModelAnimal Research Center, Nanjing University, China

The etiology of glioma remains unclear so far. Human her-pesvirus 6 (HHV-6) might be associated with glioma, butthere is no direct evidence to support this. High percentagesof HHV-6 DNA and protein were detected in tissue fromgliomas, compared with normal brain tissue. In addition, astrain of HHV-6Awas isolated from the uid specimens fromglioma cysts. High levels of interleukin 6 (IL-6), interleukin 8(IL-8), tumor necrosis factor , and transforming growthfactor (TGF-) were detected in the cyst uid specimensfrom 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 gliomahas remained unclear until recently [1]. Recent studies havesuggested that viral agents might be an etiological factor for

the development of glioma [2]. An early study reported thatvirus-like particles were found in glioma tissues by electronmicroscopy [3]. Furthermore, one study demonstrated that90% of the examined glioblastoma samples were cytomegalo-virus (CMV) antigen and DNA positive [4]. However, theirstudies did not provide any direct evidence showing the exis-tence of viable CMV in the tumor microenvironment. BesidesCMV detection, more-recent studies have demonstrated thatHHV-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 hasbeen associated with a number of neurological disorders, in-cluding encephalitis and seizures [8, 9]. In addition, HHV-6early and late antigens have been detected in primary andrecurrent central nervous system tumors and, even morefrequently, in glioma [5, 6].Given that HHV-6 may be a potential etiological agent for

the pathogenesis of glioma, we investigated HHV-6 infectionin glioma tissues, using nest polymerase chain reaction (PCR)and immunohistochemical analysis. Importantly, we isolated aHHV-6 strain from cyst uid specimens obtained from apatient with glioma, strongly suggesting that HHV-6 may bean important etiological factor for glioma. We further showedthat HHV-6 infection can induce proinammatory cytokinesin cyst uids of HHV-6positive patients with glioma and inastrocyte cultures.

MATERIALS AND METHODS

Clinical SpecimensForty glioma tissue specimens were obtained from patientsaged 2273 years. Thirteen normal brain tissue specimenswere obtained from autopsy of individuals with nonneurologi-cal causes of death (age range, 2570 years). Blood sampleswere collected from 40 patients with glioma and 20 healthyvolunteers. Cyst uid specimens were obtained from 4 patientswith glioma. These specimens were provided by the First Afl-iated Hospital of Nanjing Medical University. Brain tumorswere classied according to World Health Organization crite-ria. These studies were approved by the local ethics committeeand institutional review board. All samples were obtained withconsent from patients and volunteers.

CellsCord blood mononuclear cells (CBMCs) were puried from thecord blood samples obtained from the Afliated Women and

Received 9 February 2012; accepted 4 June 2012; electronically published 7 September2012.

aJ. 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 ([email protected]). And Weixing Hu, MD, Department of Neurosurgery, First Afliated Hospital ofNanjing Medical University, 300 Guangzhou Rd, Nanjing 210029, Jiangsu Province, China([email protected]).

The Journal of Infectious Diseases 2012;206:13948 The Author 2012. Published by Oxford University Press on behalf of the Infectious DiseasesSociety of America. All rights reserved. For Permissions, please e-mail: [email protected]: 10.1093/infdis/jis513

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Children Hospital of Nanjing Medical University. HSB-2 cell linewas cultured in Roswell Park Memorial Institute 1640 mediumcontaining 10% fetal calf serum (FCS). Primary human fetalastrocytes (PHFAs) were purchased from the Sciencell companyand cultured in DEME/F12 supplemented with 10% FCS.

Virus Isolation and IdenticationThe cyst uid specimens and peripheral blood mononuclearcells (PBMCs) obtained from patients with glioma were cocul-tured with CBMCs. HHV-6 infection was rst identied onthe basis of the cytopathic effects (CPEs) in CBMCs. Virusparticles were further observed by electron microscopy. Oncethe viruses were identied from the CBMCs, they were main-tained and amplied in HSB-2 cells. Virus titers of mediantissue culture infective doses (TCID50) per milliliter weredetermined on the basis of CPEs in HSB-2 cells.

DNA Extraction and Nested PCRDNA from tissues and cells was isolated using a DNA extrac-tion kit (Biomiga). The DNA samples were then subjected tonested PCR to determine virus infection. Five microliters ofDNA from each sample was amplied in a 50-L reactionmixture containing PCR buffer and each outer primer. Twomicroliters of the rst-round PCR product were amplied in asecond-round PCR, using each inner primer. Ten microlitersof the second-round PCR product were electrophoresed on a1% agarose gel and visualized by ethidium bromide staining.The second-round products also were sequenced and thensubjected to a BLAST search against the GenBank database.The sequences of the nested PCR primers of HHVs are shownin Supplementary Table 1.

Immunouorescence and Immunohistochemical AnalysesThe CBMCs with typical CPEs of virus infection were harvest-ed for smearing. Indirect immunouorescence analysis wasperformed using a HHV-6-specic monoclonal antibody(Santa Cruz Biotechnology) and a secondary antibody labeledwith FITC ( Jackson). Immunohistochemical staining was per-formed on parafn-embedded and frozen sections, using En-vision plus Peroxidase Mouse detection kits (Gene Tech) withan antiHHV-6 monoclonal antibody (Santa Cruz Biotechnol-ogy). Isotype antibody (Sigma) served as a negative control.

Infection of Astrocytes by the Isolated HHV-6PHFAs (2 105/well) were cultured in 6-well plates and theninfected with the isolated HHV-6 at a multiplicity of infectionof 100. The culture supernatants were collected for cytokinedetection 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 theastrocyte culture supernatants infected by HHV-6 or in cystuid specimens from patients with glioma was determinedusing ELISA kits (Bender). All cytokine assays were performedin triplicate.

RESULTS

Isolation and Identication of Viruses From Patients WithGliomaPBMCs obtained from 27 patients and 4 fresh glioma cystuid samples were mixed with PHA-stimulated CBMCs. After11 days of growth, the cyst uid sample from patient 1 showedCPEs characterized by swelling of scattered cells, increasedtransparency, and refraction and fusion of infected cells. Atapproximately 15 days of coculture, CPEs were detected >50%in the cocultured cells. Following the subculture, we foundthat 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 27PBMC samples did not show the CPEs after 3 weeks of cocul-ture. In addition, the herpesvirus-like particles (120300 nmin diameter) were visible in the cytoplasm of the infectedCBMCs, using electron microscopy (Figure 1B). The isolatedvirus can infect HSB-2 cells with a titer of 5 103 TCID50/mL.The isolated virus in infected CBMCs was conrmed by per-

forming immunouorescence analysis with a HHV-6specicmonoclonal antibody (Figure 1C). Furthermore, by usingHHV-6 specic primers, we detected a bright band at 287 basepairs after the rst round of PCR and at 163 base pairs after thesecond round of PCR (Figure 1D). To determine whether theisolate was the HHV-6A or HHV-6B variant, the innersegment amplied by PCR was sequenced. DNA sequencingshowed 98% homology between this 163base pair fragmentand the U1102 gene in HHV-6A (Figure 1E), suggesting thatthis isolate belongs to the HHV-6A variant.

Prevalence of HHV-6 in Tumor Tissues and PBMCs FromPatients With GliomaTo determine whether HHV nucleic acids were present inglioma tissues, nested PCR for detection of herpes simplexvirus 1, Epstein-Barr virus, CMV, HHV-6, and HHV-7 wasperformed on 40 glioma and 13 normal brain specimens.HHV-6 was the most frequently identied virus among theHHV family, existing in 17 of 40 glioma tissues (42.5%). Fur-thermore, only 1 of 13 normal brain tissues (7.7%) yieldedHHV-6 DNA. There was a signicant difference in the HHV-6 detection rate between the glioma tissues and the normalbrain tissues (P = .03). Notably, HHV-6 positivity variedamong 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).

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Figure 1. Isolation and identication of virus from cyst uid samples. A, Virus infectioninduced cytopathic effect (CPE) in cord blood mononuclearcells (CBMCs). After culture for 11 days, the CBMCs inoculated with the cyst uid sample presented the ballooning and polykaryotic CPE. B, Detectionof viral particles in virus-infected CBMCs by electron microscopy. C, Detection of human herpesvirus 6 (HHV-6) antigen in CBMCs inoculated with cystuid. 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 163base 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 .03a .501 .014a

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.



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We also analyzed HHV-6 DNA in PBMCs obtained frompatients with glioma and from normal volunteers, usingnested PCR. We found that 22 of 40 PBMCs (55%) were posi-tive for HHV-6 DNA in patients with glioma. However, only5 of 20 PBMCs from healthy controls (25%) were positive forHHV-6 DNA, further demonstrating a signicant differencein the HHV-6 infection rate between patients with glioma andhealthy 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 immunohistochemicalanalysis of 40 glioma specimens and 13 normal brain tissuespecimens. 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 didnot detect any HHV-6 immunoreactivity in normal braintissues (P = .014) (Table 1). Collectively, these results clearlyindicate that, compared with other herpersviruses, HHV-6 in-fection and reactivation is a common feature in patients withglioma.

Proinammatory and Suppressive Cytokines Induced by HHV-6InfectionRecent studies have demonstrated that HHV-6 infection invitro can change the characteristics of the infected cells andrelease inammatory cytokines [10, 11]. Thus, we reasonedthat HHV-6 may reactivate in the brain and create a local in-ammatory microenvironment that facilitates the developmentof glioma. To address these possibilities, we rst determinedcytokine 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 incyst uid specimens from 4 patients with glioma. To furtherinvestigate the potential origins of these cytokines, we deter-mined cytokine production in astrocytes after infection withthe isolated HHV-6. As expected, we found that, in infectedastrocytes, HHV-6 infection signicantly increased the releaseof IL-6, IL-8, and TGF-; slightly induced IL-10; and did notinduce TNF- or IFN- (Supplementary Figure 2B). Notably,cyst uid specimens from patients with glioma and superna-tants from HHV-6infected astrocytes contained highamounts of TGF-, suggesting that HHV-6 infection mayinduce a suppressive microenvironment in patients withglioma.

DISCUSSION

In this study, we showed that HHV-6 antigen and DNA wereabundant in glioma specimens. Most importantly, we isolateda HHV-6A strain from a glioma cyst uid sample. The currentstudy was the rst report on virus isolation from glioma

specimens and conrmed the importance of HHV-6 infectionin 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 gliomatissue specimens in our current study [5, 7]. The discrepancyof the detection rates in both diseased and normal tissuesamong research groups could potentially be due to differencesin PCR methods and sample preparation. Like HHV-6, DNAand protein of CMV, another member of the beta herpesvirusfamily, has been detected in malignant glioma specimens byPCR and immunohistochemical analysis [4]. In our experi-ments, mixed infection with CMV, HHV-6, HHV-7, and EBVwas detected by PCR in some brain tumor tissues (unpub-lished data). However, the rate of HHV-6 positivity was muchhigher 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 andremains present throughout the hosts life. HHV-6 can be re-activated in immunosuppressed individuals. Furthermore, thedistribution of HHV-6 nucleic acid and viral antigen inthe tumor tissues itself already suggested a CNS reservoir forHHV-6. Our current studies have shown that HHV-6A canpreferentially infect human astrocytes in vitro. In addition,one trans-activating gene (ORF-1) in HHV-6 has been shownto exhibit transforming activity and directly crosstalk with theoncogene p53 [12]. Besides directly altering the infected cellproperties, HHV-6 infection can induce proinammatorycytokine secretion and create an inammatory microenviron-ment that facilitates the pathogenesis of glioma. Yoshikawaet al demonstrated that a latent HHV-6 infection in neuroglialcells could alter proinammatory cytokines synthesis [11], andthis might be involved in the development of glioma. In thecurrent 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 infectionwith the HHV-6 isolate promoted IL-6, IL-8, and TGF- pro-duction in astrocytes cultures. In addition, HHV-6 can disturbthe key immune activation pathways and cytokine networks,including an upregulation of TNF-, RANTES, interleukin 1,and IL-10 [10]. Importantly, recent evidence suggested thatHHV-6 is an important immunosuppressive virus. HHV-6(especially HHV-6A) could cause selective immunosuppres-sion in otherwise immunocompetent adults [10, 11]. Weshowed that HHV-6 infection can induce IL-10secretingCD4+ T-regulatory 1 cells [13]. Furthermore, HHV-6 infectioncan directly deplete the infected CD4+ T lymphocytes via theinduction of apoptosis [14]. More recently, we have demon-strated that HHV-6 suppressed T-cell proliferation throughthe 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

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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 cytokinesinduced by HHV-6 infection might provide a chronic inam-matory environment that facilitates the development ofglioma. 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 Diseasesonline (http://jid.oxfordjournals.org/). Supplementary materials consist ofdata provided by the author that are published to benet the reader. Theposted materials are not copyedited. The contents of all supplementarydata are the sole responsibility of the authors. Questions or messagesregarding errors should be addressed to the author.

Notes

Financial support. This work was supported by the National NaturalScience 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 thecontent of the manuscript have been disclosed.

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