A Patient-Derived iPSC-Based Model Reveals Neural Lineage-Specific Transcription of Endogenous HHV-6B

Abstract

Background

Endogenous human herpesvirus 6 (eHHV-6), in which the entire viral genome is integrated into human chromosomes, is present in approximately 1% of the population and has been associated with various clinical conditions, including neurological disorders. However, its biological significance remains unclear due to the lack of appropriate experimental models.

Methods

We established a patient-derived induced pluripotent stem cell (iPSC)–based tissue culture model using lymphoblastoid cell lines from individuals with eHHV-6B. iPSCs retaining the integrated viral genome were generated and subsequently differentiated into neural stem cells (NSCs). Viral gene expression was evaluated by RT-qPCR under basal conditions and following chemical stimulation.

Results

The integrated HHV-6B genome was transcriptionally silent in iPSCs but exhibited spontaneous low-level expression of the immediate-early gene U90 and the late gene U100 in NSCs. Chemical stimulation further enhanced U90 expression, whereas induction of U100 did not reach statistical significance. These findings indicate preferential activation of early viral transcriptional programs in neural lineage cells.

Conclusions

Neural lineage cells provide a permissive environment for expression of eHHV-6B transcripts. This patient-derived iPSC-based model provides a platform for investigating the biological significance of neural lineage-specific eHHV-6 transcripts and may facilitate interpretation of HHV-6 DNA detection in patients with eHHV-6 by enabling studies of cell type-dependent viral transcription.

Summary:

A patient-derived iPSC model demonstrates that endogenous HHV-6B is transcriptionally silent in pluripotent cells but becomes spontaneously and inducibly active in neural stem cells, providing a platform to study its potential role in human disease.

Introduction

Human herpesviruses 6A and 6B (HHV-6A/B) can integrate into the human germline, enabling vertical transmission of viral DNA [1, 2]. This condition, historically termed inherited chromosomally integrated HHV-6A/B, was officially renamed endogenous HHV-6A/B (eHHV-6A/B) at the 2025 International HHV-6 Conference [3]. eHHV-6A/B is found in approximately 0.4–2.9% of the general population [4–7]. The entire HHV-6A/B genome is integrated into the telomeric regions of human chromosomes via homologous recombination [8, 9], and is transmitted to half of an individual’s offspring in accordance with Mendelian inheritance. Most individuals with eHHV-6A/B harbor a single copy of the viral genome per nucleated cell, whereas rare individuals carry two distinct integration sites on different chromosomes [10]. Because viral DNA is present in every nucleated cell, detection of HHV-6A/B DNA may be misinterpreted as active infection, resulting in diagnostic confusion and unnecessary antiviral treatment [10, 11]. The widespread implementation of the FilmArray Meningitis/Encephalitis panel (BioFire Diagnostics, Salt Lake City, UT, USA) has further increased incidental detection of eHHV-6A/B during evaluation of central nervous system (CNS) infections [10, 12], highlighting the need to better understand the biological and pathological significance of eHHV-6A/B.

Several clinical conditions—including angina pectoris [13], preeclampsia [14], spontaneous abortion [15], and acute graft-versus-host disease [16]—have been reported to be associated with eHHV-6A/B, although these associations remain observational and do not establish a causality. Viral reactivation has been proposed as one possible biological mechanism, but direct experimental evidence is lacking. Accumulating evidence indicates that eHHV-6A/B is transcriptionally active under some conditions. Spontaneous or stimulus-induced viral gene expression has been demonstrated in cells harboring integrated genomes [1, 14, 17], and HHV-6A/B transcripts are enriched in the brain tissue of individuals with eHHV-6A/B compared with other organs [18]. Moreover, several reports have suggested associations between eHHV-6A/B and neurological or autoimmune disorders, including encephalitis and systemic lupus erythematosus [4, 19]. Together, these findings suggest that transcription from integrated eHHV-6A/B may have biological relevance even without productive viral replication.

Due to the lack of experimental models that accurately recapitulate eHHV-6A/B biology, the mechanisms regulating eHHV-6A/B gene expression remain poorly understood. Most previous studies have relied on genomic cohort analyses [7, 13, 19] or transcriptomic profiling of clinical specimens, providing limited mechanistic insight [14]. Peddu et al. reported that HHV-6A/B transcripts are more frequently detected in brain tissue than in other organs in individuals with eHHV-6A/B [18], suggesting tissue-specific permissiveness for viral gene expression. To investigate whether neural lineage cells permit eHHV-6B transcription, we established an iPSC-based model using patient-derived lymphoblastoid cell lines (LCLs) from our previously described eHHV-6B cohort [6, 8, 15, 20–22]. The iPSCs were differentiated into neural stem cells (NSCs), a tractable neural lineage model. Using this platform, we examined whether the integrated eHHV-6B genome was maintained during reprogramming and neural differentiation, whether spontaneous viral gene expression occurred, and whether transcription could be pharmacologically induced. This model provides a platform for investigating the regulation of eHHV-6B transcription in neural lineage cells.

Methods

Patient samples and establishment of iPSCs

eHHV-6A/B was diagnosed as previously described [6]. Chromosomal integration sites were determined using FISH, and viral species were identified by restriction fragment length polymorphism analysis [8]. Peripheral blood mononuclear cells (PBMCs) from eHHV-6B–positive individuals were transformed with EBV to generate LCLs [23]. iPSCs were established from LCLs using episomal plasmids encoding OCT3/4, SOX2, KLF4, L-MYC, LIN28, and shRNA against p53 [24] and maintained on laminin-coated dishes in StemFit AK02N medium (Ajinomoto Co., Inc., Tokyo, Japan). All iPSC-like colonies from each donor were pooled and expanded, yielding polyclonal rather than single-cell-derived cultures. G-banded karyotyping was performed by NIHON GENE RESEARCH LABORATORIES, INC. (Sendai, Japan), with 20 metaphase cells analyzed per iPSC line. FISH was performed on metaphase chromosome preparations using an HHV-6-specific probe and the corresponding chromosome-specific reference probe [25].Written informed consent was obtained from all participants under protocols approved by the Ethics Committee of Fujita Health University (HM23–497, CI25–694).

Neural induction and expansion

NSCs were induced from patient-derived iPSCs using PSC Neural Induction Medium (Thermo Fisher Scientific, Waltham, MA, USA) according to instructions. iPSCs were plated at 15–25% confluency and cultured in Neural Induction Medium for 7 days. Neuroepithelial-like cells were detached using Accutase^® (Nacalai Tesque, Kyoto, Japan) and expanded as NSCs in Neural Expansion Medium on Geltrex^®-coated dishes (Thermo Fisher Scientific).

Verification of eHHV-6B genome maintenance in iPSCs and NSCs

Genomic DNA was extracted from LCLs, iPSCs, and NSCs using the QIAamp DNA Blood Mini Kit (QIAGEN, Venlo, The Netherlands). HHV-6B DNA load was quantified by qPCR targeting the U31 gene [26]. EBV DNA was quantified by qPCR targeting the EBNA-1 gene [27]. Viral DNA copy numbers were normalized to β-actin genomic DNA. These analyses assessed eHHV-6B genome maintenance and EBV episome loss during reprogramming and differentiation.

Validation of pluripotency and neural lineage identity

Pluripotency and neural lineage identity were validated by RT-qPCR, immunoblotting, and immunofluorescence staining using OCT4, NANOG, and SOX2 as pluripotency markers and SOX2 and NESTIN as neural stem cell markers.

RT-qPCR

Total RNA was isolated using the AllPrep DNA/RNA Mini Kit (QIAGEN), and cDNA was synthesized using a high-capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Marker expression was quantified by qPCR using gene-specific primers (Supplemental Table 1) and normalized to 18S ribosomal RNA.

Immunoblotting

Cells were lysed with protein lysis buffer, and proteins were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes (MilliporeSigma, Burlington, MA, USA). Membranes were incubated with the primary antibodies against NANOG, OCT4A, SOX2, NESTIN, and β-actin, followed by HRP-conjugated secondary antibodies (all Cell Signaling Technology, Danvers, MA, USA). Signals were detected by chemiluminescence.

Immunofluorescence staining analysis and imaging analysis

Cells were fixed formaldehyde and permeabilized with Triton X-100. LCLs and iPSCs were stained with antibodies against OCT4A and NANOG. iPSCs and NSCs were stained with antibodies against NESTIN and SOX2 (All primary antibody from Cell Signaling Technology). After incubation with fluorescent secondary antibodies (Thermo Fisher Scientific), nuclei were counterstained with Hoechst 33342 (Thermo Fisher Scientific). Fluorescence images were acquired using a Keyence BZ-X800 fluorescence microscope (Keyence, Osaka, Japan). Exposure settings were kept constant within each experiment.

eHHV-6B gene expression by TaqMan-based RT-qPCR

Transcripts of immediate-early gene U90 and late gene U100 were quantified. These genes were selected because previous studies identified them as among the most highly expressed HHV-6 genes in the brains of individuals with eHHV-6 [18], and validated TaqMan RT-qPCR assays are available in our laboratory. Viral transcription was evaluated in iPSCs and NSCs derived from three donors (CI19–2, CI19–4, and CI20–2). Cells were plated at a density of 2–4 × 10⁵ per well on day 1 and harvested on days 4, 6, and 8 (Figure 2A). RT-qPCR was performed as previously described [28]. Gene expression was normalized to 18S ribosomal RNA. Supplemental table 1 provides primer and probe sequences. To assess genomic DNA contamination, RT-negative control reactions were performed using RNA samples without reverse transcriptase. Raw Ct values and RT-negative control data are provided in Supplementary Table S2.

Chemical stimulation of eHHV-6B transcription

Etoposide was selected to induce DNA damage, trichostatin A (TSA) to promote chromatin relaxation through histone deacetylase inhibition, and 12-O-tetradecanoyl-13 acetate (TPA) as a protein kinase C activator previously reported to induce herpesvirus gene expression. iOX2 plus hydrocortisone (HC) was included based on a recent study demonstrating enhanced HHV-6A replication and reconstitution through HIF-1α stabilization [29]. NSCs were treated with 5 μM etoposide (Funakoshi Co., Ltd., Tokyo, Japan) or 100 ng/mL TSA (FUJIFILM) plus 10 ng/mL TPA (Funakoshi) for 72 h [1, 30]. In parallel, NSCs were treated with 10 μM iOX2 (Sigma) plus 9 μg/mL hydrocortisone (Sigma-Aldrich, St. Louis, MO, USA) for 120 h [29]. Control cells were treated with vehicle alone (100% ethanol) (Figure 3A). Viral transcription was assessed by TaqMan-based RT-qPCR for U90 and U100, as described above.

Cell growth assay

Cells cultured in 24-well plates were dissociated by gentle pipetting. A 50 μL aliquot of the cell suspension was transferred to a white opaque-bottom 96-well plate, followed by the addition of an equal volume of CellTiter-Glo reagent (Promega Corporation, Madison, WI, USA). Luminescence was measured using a GloMax^® Explorer. Relative cell growth was expressed as a percentage of the untreated control, which was set to 100%.

Statistical analysis

Experiments were performed as three independent biological replicates using the same donor-derived iPSC and NSC cultures. RT-qPCR and qPCR assays were conducted in duplicate, and mean Ct values of the duplicate measurements were used for analyses. Statistical analyses were performed separately for each donor-derived cell line, without pooling data across donors. Data are presented as box-and-whisker plots showing the median and interquartile range. Statistical comparisons were performed using Dunn’s multiple comparison test in JMP version 13.2.1 (SAS Institute, Cary, NC, USA). P-value < 0.05 was considered significant.

Results

Establishment of iPSCs and differentiation into NSCs from eHHV-6B–positive donors

To archive limited numbers of cells obtained from patients with eHHV-6A/B, we routinely generated LCLs from PBMCs. LCLs from six individuals diagnosed with eHHV-6B (CI15–263, CI15–274, CI15–275, CI19–2, CI19–4, and CI20–2) were used in this study. Integration of the HHV-6B genome was confirmed by FISH (Supplemental Figure 1), and donor characteristics are summarized in Table 1. Notably, CI19–2 and CI20–2 harbored two independent HHV-6B integration genome sites on chromosomes 20 and 22, respectively. Polyclonal iPSC populations were established from these LCLs and subsequently differentiated into NSCs. Representative morphology is shown in Figure 1B. Pluripotency of iPSCs was confirmed by RT-qPCR, immunoblotting, and immunofluorescence for NANOG, OCT4A, and SOX2 (Figure 1C–E). Following neural differentiation, NSCs expressed SOX2 and NESTIN with concomitant downregulation of pluripotency markers. Representative G-banded karyotype analysis of iPSC lines derived from three donors (CI19–2, CI19–4, and CI20–2) demonstrated normal karyotypes without detectable numerical or structural chromosomal abnormalities in analysis of 20 metaphase cells per line (Figure 1F). Representative FISH analysis confirmed retention of the integrated HHV-6 genome following reprogramming (Figure 1G). qPCR demonstrated stable maintenance of HHV-6B genome copy numbers during reprogramming and neural differentiation, consistent with the one or two integrated copies identified by FISH in the parental LCLs (Figure 1H). In contrast, EBV genome was detectable only in parental LCLs but became undetectable following reprogramming, remaining absent following neural differentiation.

Table 1. Clinical and genetic characteristics of individuals with eHHV-6 used for iPSC generation.

Sex, viral species, and chromosomal integration sites of eHHV-6–positive donors included in this study.

Patient #	Sex	eHHV-6 species	Integration site
CI15–263	F	HHV-6B	22q
CI15–274	M	HHV-6B	Yp
CI15–275	M	HHV-6B	Xp
CI19–2	F	HHV-6B	20p & 22q
CI19–4	M	HHV-6B	20p
CI20–2	M	HHV-6B	20p & 22q

F, female; M, male; eHHV-6, endogenous human herpesvirus 6; HHV-6B, human herpesvirus 6B; iPSC, induced pluripotent stem cells.

Figure 1. Establishment and validation of eHHV-6B iPSCs–derived neural model.

(A) Schematic overview of the experimental workflow showing establishment of lymphoblastoid cell lines (LCLs) from PBMCs of individuals with endogenous HHV-6B (eHHV-6B), followed by reprogramming into induced pluripotent stem cells (iPSCs) and differentiation into neural stem cells (NSCs). (B) Representative phase-contrast images of iPSCs and NSCs. Scale bar = 100 μm. (C) Gene expression of pluripotency markers (NANOG, OCT4 and SOX2) and neural stem cell markers (SOX2 and NESTIN) in each cell line confirmed by RT-qPCR. Box plots and dots show the relative gene expression normalized to each patient’s iPSCs. Boxes indicate the interquartile range and the center line indicates the median. Colors indicate each individual with eHHV-6B (CI15–263, blue; CI15–274, red; CI15–275, green; CI19–2, purple; CI19–4, brown; CI20–2, light green). *p < 0.05, **p < 0.01, ***p < 0.001 versus iPSCs. (D) Immunoblot validation of pluripotency and neural stem cell marker expression. (E) Immunofluorescence staining confirming expression of pluripotency markers in iPSCs and neural stem cell markers in NSCs. Scale bar = 20 μm. (F) Representative G-banded karyotypes of iPSC lines derived from three individuals with eHHV-6B. (G) Representative fluorescence in situ hybridization (FISH) image of the iPSC line derived from CI19–4, demonstrating maintenance of the integrated HHV-6 genome following reprogramming. Metaphase chromosomes were co-hybridized with an HHV-6-specific probe (yellow arrow) and a chromosome 20q13.33 subtelomeric probe (white arrows), confirming retention of the viral integration site. (H) Quantitative PCR analysis demonstrating maintenance of HHV-6 DNA copy number during reprogramming and neural differentiation, and loss of EBV DNA following reprogramming. Box plots and dots show relative DNA load normalized to CI15–263 LCL. Boxes indicate the interquartile range and the center line indicates the median. Colors indicate each individual with eHHV-6B (CI15–263, blue; CI15–274, red; CI15–275, green; CI19–2, purple; CI19–4, brown; CI20–2, light green). Triangles indicate samples from CI19–2 and CI20–2, which harbor two integrated copies of HHV-6B. ***p < 0.001 versus LCLs.

Spontaneous eHHV-6B gene expression in iPSCs and NSCs

Next, we examined whether neural cell differentiation permitted spontaneous eHHV-6B gene expression. Expression of the immediate-early gene U90 and the late gene U100 was assessed in iPSCs and NSCs derived from three eHHV-6B donors (CI19–2, CI19–4, and CI20–2) (Figure 2A). U90 and U100 transcripts were undetectable or near the lower limit of detection in iPSCs, regardless of integrated HHV-6B copy numbers (Figure 2B). In contrast, both U90 and U100 transcripts were reproducibly detected in NSCs, although transcript levels remained low (Figure 2B). This increased transcription was consistent across all three donor-derived lines, including those harboring dual HHV-6B integrations.

Figure 2. Spontaneous activation of eHHV-6 transcription in neural stem cells.

(A) Experimental design for assessment of spontaneous viral gene expression. iPSCs and NSCs derived from CI19–2, CI19–4, and CI20–2 were collected at days 4, 6, and 8 after culture initiation, and expression of U90 and U100 was analyzed by RT-qPCR. (B) RT-qPCR analysis showing spontaneous expression of U90 and U100 in NSCs. Each dot represents one independent biological replicate (mean of technical duplicates). Box plots and dots show the relative gene expression normalized to NSC on day4. Boxes indicate the interquartile range and the center line indicates the median. For each donor-derived cell line, iPSCs and NSCs were analyzed in three independent experiments (n = 3), with RT-qPCR performed in technical duplicate. Statistical comparisons were performed between iPSCs and NSCs within each donor. *p < 0.05. Colors indicate sampling time points (day 4, blue; day 6, red; day 8, green).

Chemical stimulation enhances eHHV-6B gene expression in NSCs

To determine whether eHHV-6B transcription could be chemically induced, NSCs derived from eHHV-6B donors were treated with the indicated agents. U90 expression was significantly increased following etoposide and histone deacetylases (HDAC) inhibitor treatment compared with untreated controls (P = 0.0097 and 0.0005, respectively) (Figure 3B). In contrast, iOX2 treatment did not significantly induce U90 expression. U100 expression showed a modest increase following etoposide treatment, but the induction did not reach statistical significance (Figure 3B). Cell viability analysis demonstrated that etoposide and iOX2+HC maintained substantially higher viability than TSA+TPA, indicating that etoposide-induced U90 expression was unlikely to result solely from nonspecific cytotoxicity (Figure 3C).

Figure 3. Chemical stimulation enhances eHHV-6 transcription in NSCs.

(A) Experimental design for chemical stimulation experiments. NSCs derived from CI19–2, CI19–4, and CI20–2 were treated with vehicle control, etoposide (72 h), TSA+TPA (72 h), or iOX2+hydrocortisone (120 h), followed by RT-qPCR analysis of U90 and U100 expression. (B) RT-qPCR results showing induction of U90 expression following chemical stimulation with etoposide, TSA+TPA, and iOX2+hydrocortisone. Each dot represents the mean of technical duplicates from three independent biological experiments (n=3) for each donor-derived cell line. Box plots show relative gene expression normalized to untreated controls; boxes indicate the interquartile range and the center line indicates the median. Colors indicate donor lines (CI19–2, purple; CI19–4, brown; CI20–2, light green). Statistical comparisons were performed between untreated and chemically treated NSCs within each donor.**p < 0.01, ***p < 0.001 versus untreated controls. (C) Cell viability following treatment with etoposide, TSA+TPA, or iOX2+HC. Cell viability was normalized to the untreated control. Box plots and dots show relative cell viability. Boxes indicate the interquartile range and the center line indicates the median. Colors indicate donor lines (CI19–2, purple; CI19–4, brown; CI20–2, light green).

Discussion

Generating patient-derived iPSC–based tissue culture models

Approximately 1% of the global population—corresponding to an estimated 80 million individuals—harbors an endogenous copy of the HHV-6A/B genome integrated into their chromosomes. Despite this prevalence, its biological consequences remain largely unexplored due to the lack of appropriate experimental models. Here, we established a patient-derived iPSC–based tissue culture model as a versatile and tractable platform for studying the biology of eHHV-6A/B. Leveraging a 20-year collection of LCLs derived from PBMCs of eHHV-6A/B–positive individuals, we generated iPSCs that stably retained the integrated viral genome and could be differentiated into multiple human cell types. Using this system, we focused on the neurotropic potential of HHV-6B [31–35] and demonstrated that the eHHV-6B genome is transcriptionally silent in iPSCs but becomes spontaneously active in NSCs, with expression further enhanced by chemical stimulation. These findings indicate cell type-dependent transcriptional states of eHHV-6B, warranting further mechanistic studies. Beyond HHV-6A/B, this iPSC-based framework offers a generalizable approach for investigating the regulation of transcription from potential of chromosomally integrated viral genomes, providing new opportunities to understand how endogenous viral elements are regulated in different cellular contexts.

We further demonstrated that while the eHHV-6B genome was stably maintained throughout reprogramming and differentiation into NSCs, EBV genomes were lost during the transition from LCLs into iPSCs. These findings indicate that the eHHV-6A/B genome behaves as a stable component of the host genome across distinct cellular states. This contrasting phenotype likely reflects differences in the mechanisms of latent maintenance: EBV persists as an episome, and its maintenance in infected cells requires latent viral protein expression to tether and segregate the episomes into daughter cells [36]. In contrast, eHHV-6A/B is integrated into human telomeric regions and is therefore replicated and inherited as part of the host chromosome, a principle that is well established for integrated viral genomes, including retroviruses and endogenous retroviral elements [37]. From a methodological perspective, the loss of EBV while preserving the eHHV-6A/B genome represents a major advantage of this system. By removing the confounding effects of EBV, this platform provides a clean, disease-relevant model for investigating the transcriptional regulation of eHHV-6A/B in human neural lineage cells.

Spontaneous eHHV-6B gene expression in neural lineage cells

Previous transcriptomic analyses have shown that HHV-6A/B RNA is frequently detected in brain tissue from eHHV-6A/B individuals, with preferential expression of U90 and U100 [18]. Accordingly, we assessed spontaneous eHHV-6B gene expression in iPSCs and NSCs using RT-qPCR. We observed low-level spontaneous expression of eHHV-6B genes in NSCs compared with iPSCs. Specifically, transcription of the immediate-early gene U90 and the late gene U100 was detectable in NSCs under basal conditions, whereas expression of these genes was minimal or undetectable in iPSCs. These findings indicate that neural lineage cells provide a more permissive cellular environment for transcription from the eHHV-6B genome and suggest that neural differentiation is associated with activation of eHHV-6B transcriptional potential. Our results are consistent with previous reports demonstrating the presence of HHV-6A/B transcripts in individuals with eHHV-6A/B [1, 14, 17, 18], including the preferential expression of U90 and U100 observed in brain tissue [20]. Several clinical conditions—including angina pectoris [13], preeclampsia [14], spontaneous abortion [15], acute graft-versus-host disease [16], systemic lupus erythematosus [19], and severe encephalitis [4]—have been associated with eHHV-6A/B, and viral reactivation has been proposed as a potential contributing mechanism. In this context, the patient-derived iPSC-based system established in this study provides a unique platform to differentiate eHHV-6B–positive cells into diverse lineages, enabling future investigations of cell type-specific regulation of eHHV-6B transcription and whether such lineage-specific viral transcription contributes to these clinically associated conditions.

Chemical stimulation–induced enhancement of eHHV-6B gene expression

In this study, chemical stimulation selectively enhanced transcription of the eHHV-6B genome in NSCs. Treatment with etoposide and HDAC inhibitors significantly induced expression of the immediate-early gene U90, whereas induction of the late gene U100 was limited, indicating preferential activation of early transcriptional programs rather than full progression to productive viral replication. The rationale for selecting etoposide was the hypothesis that reactivation and replication of eHHV-6A/B require excision of the viral genome from telomeric regions [38, 39]. Telomeres harboring integrated HHV-6A/B are structurally unstable and prone to partial viral genome excision [7, 39], suggesting that DNA damage–inducing agents such as etoposide [40] promote transcriptional activation by destabilizing telomeric chromatin. Consistent with this model, etoposide induced U90 expression without increasing viral DNA copy number. Similarly, HDAC inhibition enhanced U90 expression, supporting the role of epigenetic repression in maintaining eHHV-6B transcriptional silencing. Notably, both TSA plus TPA treatment and etoposide treatment induced significant U90 expression despite marked differences in their effects on cell viability. Whereas TSA plus TPA treatment substantially reduced cell viability, etoposide maintained cell viability at more than 50% of untreated controls, suggesting that the observed transcriptional induction cannot be explained solely by nonspecific cytotoxicity. While HDAC inhibitors have previously been shown to induce eHHV-6A/B transcription in T cells and LCLs [1, 2], our study extends these observations to neural lineage cells, using a stimulation strategy informed by the integrated HHV-6A/B model of Gravel et al. [30]. The absence of significant U100 induction, despite significant induction of U90, suggests that chemical stimulation preferentially promotes immediate-early transcriptional activation of eHHV-6B without progression to late-stage viral gene expression. However, this interpretation remains indirect because only two viral transcripts were examined, and broader viral gene expression and protein production are required to confirm the extent of viral activation. This finding aligns with clinical evidence that productive replication of eHHV-6A/B is rare and largely restricted to conditions of profound immunosuppression [41]. Collectively, these results indicate that eHHV-6B transcription can be experimentally enhanced in neural lineage cells while remaining confined to the early phases of the viral transcriptional cascade. Importantly, our conclusions are limited to viral transcription, as productive viral replication was not assessed in the present study.

Limitations and Future Directions

This study has several limitations. First, although we demonstrated spontaneous and chemically inducible transcription from the eHHV-6B genome, viral protein expression could not be directly assessed due to the lack of well-validated monoclonal antibodies against HHV-6B proteins. Consequently, the extent to which transcriptional activation leads to viral protein production remains unclear and will require the development of reliable reagents or alternative proteomic approaches. Second, transcriptional analyses were performed using NSCs derived from a limited number of donors, and NSCs represent only a limited neural cell population in vivo. Future studies including additional donors and mature neural cell types—including neurons, astrocytes, and oligodendrocytes—will be necessary to determine whether these transcriptional patterns are broadly applicable across neural lineages and to further evaluate cell type-specific regulation of eHHV-6B. Additionally, future studies leveraging this platform should investigate downstream host responses to eHHV-6A/B transcription, including innate immune activation and epigenetic changes, as well as define the cellular heterogeneity of eHHV-6A/B transcriptional activity using single-cell approaches. Extending this system to mature neural lineages will be essential for elucidating the mechanisms by which eHHV-6A/B gene expression contributes to disease susceptibility and pathogenesis. From a clinical perspective, these findings have important implications for the interpretation of HHV-6 detection in diagnostic settings. Because eHHV-6B is present in all nucleated cells, detection of HHV-6 DNA in clinical specimens—particularly in cerebrospinal fluid—does not necessarily indicate active infection. Our results suggest that transcriptional activation of the integrated viral genome may occur in a cell type–dependent manner, particularly in neural lineage cells, even in the absence of productive viral replication. This highlights the need for caution in distinguishing between incidental detection and biologically relevant viral activity in patients with eHHV-6B.

Data availability

The data underlying this article are available in the article and in its online supplementary material.