Latent, early or late HHV-6B expression in adult mesial temporal lobe epilepsy: Association of virus life cycle with in�ammatory cytokinesin brain tissue and cerebral spinal �uid

Background: Human herpes virus-6B (HHV-6B) was suggested as an important etiologic factor of mesial temporal lobe epilepsy, while the mechanism is still unknown. Here, we aimed to analyze antigens representing latent, early and late HHV-6B infection and the association with in�ammatory cytokines in brain tissue and cerebral spinal �uid (CSF) from MTLE patients with HHV-6B-positivity. Results: Nested polymerase chain reaction (nPCR) in brain tissue revealed HHV-6B DNA in 19 of 49 MTLE patients (39%) and 1 of 19 controls (5%) (P<0.001), but not in CSF. ICH showed HHV-6B early antigen (P41) positivity in 3 patients (6%), late antigen (gp116/54/64) positivity in 5 patients (10%), latent antigen (U94) positivity in 8 patients (16%), and multiple antigen (early and late or/and latent) positivity in 9 patients (18%). None of these HHV-6B related proteins were found positive in control brain tissue. PCR revealed signi�cant up-regulation of IL-1a, IL-2 and IL-7 mRNA levels in brain tissue from MTLE patients with different virus state. Suspension bead array in CSF con�rmed signi�cant up-regulation of IL-1a and IL-7 protein expression. Conclusions: Our �nding suggests HHV-6B is a common etiologic agent of MTLE. Different virus life cycle may play an important modifying role in in�ammatory biology that warrants further investigation. Though virus DNA is di�cult detected in CSF, up-regulation of IL-1a and IL-7 in CSF indicates the two cytokines may be taken as indirect biomarker of HHV-6B infection.

HHV-6B can establish latency thougout life after primay infection and reactivate in the host.It indicates HHV-6B may play an epileptogenitic role in that may affect chronic course of MTLE, particularly associated with HS and history of febric convulsions [9,10].We noted that in several previous studies DNA detection ranged from 9% to 69% while protein detection in 50% to 69% MTLE patients [4-8, 11, 12].
HHV-6B DNA and protein level may respond to different states of the virus such as latency, replication or reactivation, acute or chronic infection.One study revealed high HHV-6 DNA detection (55.6%) but not protein (2.6%) in brain tissue from MTLE patients [13].The viral DNA and protein did not match each other, which indicates that the virus may periodically reactivate.However, It is not known whether these observations represent latent, early or late HHV-6 expression, since there has been no larger scale analysis of HHV-6 latent or active expression published to date.
It may indicate different virus state such as latency, replication or reactivation may be involved in MTLE chronic process.In this study, we try to differentiate the different viral states in MTLE.We also attempted to detect HHV-6B DNA in cerebrospinal uid in MTLE patients which has not been reported previously to our knowledge.
Cytokines are soluble proteins acting as immune mediators in in ammatory process.Previous studies suggest that HHV-6B can infect astrocytes and oligodendrocytes [14]and promote the expression of various pro-in ammatory factors.up-regulation of in ammatory cytokines as IL-6, IL-8, IL-1β, IL-10, IL-11 may be involved in the procession of acute or chronic HHV-6 infection.[15][16][17][18]The data suggest that HHV-6B triggers immune modulatory factors.However, there is not known whether in ammatory cytokines is associated with different viral states.To further elucidate a role of cytokines in HHV-6B infection, PCR and suspension bead array were performed.

Materials And Methods
Subjects 49 patients were from the Department of Neurosurgery, West China Hospital.Pre-surgery evaluation of each case included video electronencephalography (VEEG) monitoring, brain magnetic resonance imaging (MRI), positron emission tomography(PET), and/or Depth electrodes.All surgical samples were sliced into small pieces and preserved in nitrogen and 4% formalin respectively.MTLE was diagnosed according to 2011 International League Against Epilepsy (ILAE) Classi cation [19].Lumbar puncture was done before surgery in all patients.
Non-epilepsy control samples were obtained from 19 patients who had serious brain trauma or a vascular event and underwent a neurosurgical procedure.All samples (controls and cases) comprised neocortical tissue and/or hippocampal tissue.CSF of controls was obtained from patients with chronic headache whose diagnosis ruled out CNS infection and tumor.The study was approved by the Ethics Committee of the West China Hospital of Sichuan University.Informed consents were obtained from patients or their direct relatives for the use of brain tissue and CSF.Control brain samples were only included if histological examination was normal.
Nested PCR and RT-qPCR for virus DNA in brain tissue DNA was extracted from every sample using a DNA puri cation kit (Qiagen, Germany).DNA ampli cation was performed using nested primers speci c for highly conserved sequences in viral genome.The total reaction volume of external ampli cation was comprised with 4 µL of 5×PCR buffer, 2ul of 20% Glycerol, 2ul of 10×dNTP 2mM , 0.5ul of forward primer (10uM) , 0.5ul of reverse primer (10uM), 1 ul of DNA sample, 0.5ul of HS-Taq (5 U/μL), 9.5ul of ddH 2 O.DNA was ampli ed followed by 20 cycles of PCR under the following conditions: 94 °C for 5 min, 95 °C for 30 s, 58 °C for 30 s, 72 °C for 45 s , and 72 °C for 2 min.Then a total of 2 μL of PCR products was ampli ed using theinternal primers according above process.A total of 10 µL of the nal PCR product was subjected to 1.5% agarose gel electrophoresis to determine the molecular weight for distinguishing HHV-6B positive samples from negative samples.

Real-time quantitative PCR for In ammatory cytokines in brain tissue
RNA in patient samples was puri ed with RNA puri cation kit (QIAGEN) and spectroscopy con rmed that the RNA 260/280 ration was >1.8.RNA was then reverse transcribed using a Bio-Rad iScript Kit.The cDNA was quanti ed by qPCR using SYBR Green (Bio-Rad).Each reaction was run in triplicate and analyzed following the ΔΔCt method as previously described using actin as a normalization control.
Nested PCR for virus DNA in CSF CSF samples were completely thawed and centrifuged at 3000r/min for 10min before testing.DNA extraction and ampli cation were performed according method described above.

Suspension bead array for cytokines in csf
CSF cytokine protein level was detected by using MILLIPLEX MAP (EMD Millipore Corp, Missouri, USA).
Twelve cytokines including TGF-α, IL-10, IL-1RA, IL-9, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, TNF-α and VEGF were detected.The procedure was performed according to the manufacture's protocol for Cytokine kit, assayed in triplicate, and read on a Molecular microplate reader at 450nm (Menlo Park, CA).Standard curves and individual well concentrations were determined using the Softmax 2.34 software (Molecular Devices, Menlo Park, CA).Data analysis was performed using the MasterPlex QT 1.0 system (MiraiBio, Alameda, CA).A ve-parameter regression formula was used to calculate the sample concentrations from the standard curves.All 96 samples were analyzed with the LINCOplex kit (Linco Research Inc).
Statistical analyses SPSS 19.0 software for Windows was used (Chicago, USA).The Independent-sample t-test was used to compare differences among continuous variables of MTLE patients and control individuals.The chisquare (χ 2 ) test with Fisher's exact was used to compare categorical parameters among groups.MLTE cases were categorized into subgroups according to IHC results for HHV-6B.Univariate analysis was used for measuring the association of HHV-6 viral phase and clinical manifestations.Two-tailed tests were used and a P < 0.05 was considered statistically signi cant

Clinical data and pathological ndings
Of the 49 patients with epilepsy, 20 (41%) were male.Mean age was 26.7 years (standard deviation [SD] 11.1, range 15 to 55).Epilepsy duration ranged from 3 to 32 years (mean 10.4,SD 6.5).Clinical and pathological ndings of the 49 cases are provided in Table 1. 9 of the 19 controls were male (47%).Mean age was 32.52 years (SD 8.1, range 15 to 61). 12 cases had traumatic injuries and the remaining 7 had cerebral hemorrhage or infarction.Clinical and pathological ndings of the 19 cases are provided in Table 2.The resected tissues included temporal neocortex (11 cases) and hippocampus (8 cases).CSF was obtained from 15 patients with headache.Nine of them were male (60%).Mean age was 45.1 years (SD Dissussion HHV-6B is considered as an etiologic agent involved in etiopathogenesis of MTLE [10,21,22].Our nding con rmed that HHV-6B is a potential etiologic agent involved in etiopathogenesis of MTLE.The data showed higher HHV-6B DNA (19 cases, 39%) and protein (25 cases, 51%) expression.Previous studies that showed 9-69% DNA and 50-69% protein positivity in MTLE patients.A meta analysis which mergers 10 researchs in recent years about MTLE and HHV-6b infection shows that the HHV-6 DNA detection ratio in MTLE patients (126/645,19.5%) is higher than the control group (14/136,10.3%)(p 0.05) [9].No HHV-6A has been detected [21].The discrepancy of detection in the study is potentially not only due to sample size, race and region etc, but also involve viral life cycle and technology sensitivity.
HHV-6 like other herpesviruses causes both productive and latent infections.In productive infection, the infected cells express most if not all viral antigens.P41, an early antigen yielding proteins of 41Da [15,23,24],is a major DNA-binding protein and processivity factor stimulating viral replication.It frequently appears 12 to 18 hours after infection before viral DNA synthesis [25,26].The expression of P41 plays an important role in viral propagation and active replication.The late membrane antigen complex Gp116/64/54 against glycoprotein B is a late protein which is useful in identifying reactivation of HHV-6B [27].In our nding, the regulatory immediate early antigen (P41) and late antigens (GP116) are expressed alone or together, and the later is more common (P<0.05).The detection of both P41 and late gp116/64/54 antigens are suggestive of active virus infection.However, we did not found lymphocytic in ltration in brain samples.It suggests neuro-immune defense system is not activated and HHV-6 may be involved in the pathogenic process of MTLE through in ammatory mediators such as chemokinesorcytokines.It may permit viral reactivation without severe in ammatory response.
HHV-6 is the only herpesvirus encoding a rep-like function, while latency is a general feature of this family of viruses.HHV-6 integrates the genome into the telomere sequence of host chromosomes, and U94 may play a role in this process.Previous research showed that U94 is expressed in the incubation period, and has the activities of DNA binding protein, adenosine triphosphate enzyme, exonuclease and helicase, and regulates viral gene expression and enables the establishment and/or maintenance of latent infection in vitro [28,29].HHV-6 is the only human beta herpes virus endoding U94 protein.In our study, U94 antigen, which is speci c for HHV-6B, was detected in 11 (22%) cases, with alone 8 (16%) and co-expressed with productive antigens (6%).The data demonstrate that the latent state of HHV-6B infection is common in MTLE.U94-positivity was found in temporal lobe tissue and hippocampus from six MTLE patients who had only with U94-immunostaining-positivity without viral DNA detection.This suggests that the levels of viral DNA remain below the limit of detection.These ndings are consistent with the hypothesis that U94 antigen may regulate the viral life cycle by restricting beta herpes virus replication [30].It also suggests that unlike the state of persistent viral replication, only small-amount viral genes are expressed in the latent state.In previous studies, available evidence showed only latency-associated transcripts are detectable in the case of HSV during viral latency [28].It indicates that U94 may be considered as a potential hallmark of HHV-6B latency.But more research is needed to discover U94's role in HHV integration and latency process.Previous study showed that.We observed that U94 co-expressed with productive antigens in three cases, which suggests that the virus may be reactivated during latent infection or in the state of latency and reactivation at the same time [31].It may be associated with different viral life cycle of virues distributed in different regions of hippocampus.However, there have been recent ndings that U94 is not required for HHV-6A integration [32].Further studies are needed to explore the role of U94 and the mechanisms of HHV-6 reactivation in these processes.
HHV-6 is a low copy number virus compared to HSV-1, and is highly cell associated.Encephalitis patients can have very high HHV-6 viral load in the brain tissue with negligible levels in the CSF and plasma [33].HHV-6 DNA can be detected inthe CSF in patients with the acute phase of HHV-6 encephalopathy (7/13,53.8%),but the level is very low compared to in serum [16].In HHV-6A was be detected in the CSF which was collected in a subset of MS patients, but only during relapse and not during remission [34][35][36][37][38].As expected, we did not detect viral DNA in any CSF samples.It may be that HHV-6 DNA can be immediately after a seizure, and our patients were not acutely ill.Since the virus spreads cell-to-cell and since HHV-6B does not cause cell death during active latency, HHV-6B DNA from lysed cells, if any,fell below our limit of detection or technique sensitivity.It suggests that the DNA level of HHV-6B in CSF from MTLE patients with HHV-6B-positivity is different with that of in acute HHV-6 encephalitis.We were not able to use HHV-6B DNA in CSF as biomarker of HHV-6B infection in brain tissues, others may be more successful using ultra-sensitive methods such as high speed centrifugeto concentrate a large volume of material.
We study cytokine changes in HHV-6B infection in MTLE patients at different stages .There are few study on cytokines in MTLE patients with HHV-6B-positivity.No typical in ammatory in ltration changes were found in the pathological specimens of TLE patients, and searching for in ammatory immune mediators may help to nd the relationship between HHV infection and the pathogenesis of MTLE.Our data showed up-regulated mRNA of IL-1a, IL-2 and IL-7 in brain tissue and up-regulation of IL-1a and IL-7 protein in CSF from MTLE patients with latent, early, late and multiple antigen positivity for HHV-6.The distribution of these cytokines differed between brain and CSF.The up-regulation of cytokines is not completely equivalent to the results of previous studies on HHV-6 encephalopathy that showed upregulated IL-8 and IL-6 [24,36].It suggests different immune-mediated mechanism between HHV-6 encephalopathy and MTLE with HHV-6 infection.IL-1α and IL-7 was upregulated signi cantly in early antigen expression compared to other samples of latency, late or multiple forms of HHV-6B infection.It indicates that the two cytokines may be associated with the pathogenesis of the MTLE with HHV-6B infection and could be considered as indirectly biomarkers of HHV-6 re-activation.
A major shortcoming of our study is that we could not certain whether these antigens mark the same infected glial cells because all HHV-6 antibodies are mouse origin.The preferential expression of both early and late viral antigens may be related to the productive state of the virus and the differentiation state of the transformed cell.We found that the distribution of early, latancy and late antigens was preferentially localized in different cells, however, we could not speculate whether it was related to cellspeci c infections in different viral cycle.Larger studies or basic animal studies are needed to help clarify whether cytokine expression is affected by different HHV infection states time of seizure onset other viral infection factorsand etc.These studies may provide insights for early antiviral and future development and application of anti-in ammatory drugs.

Conclusion
In summary, our nding suggests HHV-6B is a common etiologic agent of MTLE.Cytokines like IL-1a, IL-2 and IL-7 are activated in brain tissue.Up-regulation of IL-1a and IL7 in CSF indicates the two cytokines may be taken as indirect biomarker of viral infection.The role of HHV-6 in regulating the expression of cytokines may be important to the understanding of the pathogenesis and progression of chronic MTLE.Further work is needed to elucidate the function of HHV-6 in MTLE.

Yao
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range 23 to 54).HHV-6B DNA load HHV-6B DNA was detected in brain tissue from 19 MTLE patients and 1 controls by nested PCR (p=0.049).HHV-6B sequence was not detected in any csf samples.The PCR ampli cation products from HHV-6B-positive DNA samples are shown in Fig 1.Pathological nding and detection of HHV-6B latent, early and late antigens by IHC.IHC revealed multiple antibodies immunoreactivity including P41, Gp116/65/64 and/or U94 in 9 MTLE patients.Single early, late and latent antibody positivity was shown in 3, 5 and 8 cases, respectively.HHV-6B latent antibody positivity was presented in 3 control cases.Staining for HHV-6B antibodies were observed to be distributed mainly around or in the nucleus of cells that resembled astrocytes.Antibody staining in sample of the control group was relatively faint.Fig 2 and Fig 3 shows the pathological nding and immunohisitochemical results.The DNA load in the groups of early, late multiple antibodies immunoreactivitywas higher than that of the group with latent antigen.Fig 4 shows the results of the relationship of HHV-6 immunoreactivity and viral DNA load.Analysis of cytokinem RNA level in brain tissue and protein level in csf In brain tissue, detection of cytokine mRNA levels by PCR revealed signi cant up-regulation of IL-1α, IL-1β, IL-2 and IL-7 mRNA levels in MTLE patients with HHV-6B-positivity compared to MTLE patients with HHV-6B-negativity orthe control tissues (P<0.01).The post hoc tests result of cytokines mRNA levels in brain tissuewas showed in Fig 5.In CSF, detection of cytokine protein levels bysuspension bead arrayrevealed that signi cantly upregulation of IL-1αandIL-7 in MTLE patients with HHV-6B-positivity compared to HHV-6B-negativity and the control (P<0.01).The post hoc tests result of cytokines mRNA levels in CSF was showed in Fig 6.
presence of HHV-6B DNA in brain tissue from MTLE patients: Agarosegel electrophoresis of representative PCR ampli cation products from brain tissue.The HHV-6B ampli cation product was 119bp.

Table 2 :
Clinical features of the control casesTotalNumber or mean value ± SD (range) TN, Temporal Neocortex, H, hippocampus, L, left, r, right, NAF, No abnormal ndings.