Kazuya Shimada

Identification of Human Herpesvirus 6 Latency-Associated Transcripts

ABSTRACT Four kinds of latency-associated transcripts of human herpesvirus 6 were identified which were detected only in latently infected cells. Although they were oriented in the same direction as the immediate-early 1 and 2 (IE1/IE2) genes and shared their protein-coding region with IE1/IE2, their transcription start sites and exon(s) were latency associated.

Recognition of a Novel Stage of Betaherpesvirus Latency in Human Herpesvirus 6

ABSTRACT Latency-associated transcripts of human herpesvirus 6 (H6LTs) (K. Kondo et al. J. Virol. 76:4145-4151, 2002) were maximally expressed at a fairly stable intermediate stage between latency and reactivation both in vivo and in vitro. H6LTs functioned as sources of immediate-early protein 1 at this stage, which up-regulated the viral reactivation.

Increase in the IgG avidity index due to herpes simplex virus type 1 reactivation and its relationship with cognitive function in amnestic mild cognitive impairment and Alzheimer’s disease

After infection with herpes simplex virus type 1 (HSV-1), latent infection persists for life in the trigeminal ganglion and reactivation results in an outbreak of cold sores around the mouth. Many previous studies have reported HSV-1 reactivation to be a risk factor for Alzheimers disease (AD). This study enrolled subjects with AD (n=85), subjects with amnestic mild cognitive impairment (aMCI; a prodromal stage of AD) (n=34), and healthy controls (n=28). The avidity index of anti-HSV-1 IgG antibodies--a known indicator of HSV-1 reactivation--was measured in order to clarify the relationship between HSV-1 reactivation and symptoms of cognitive function in AD. Cognitive function in AD and aMCI were evaluated using scores from the mini-mental state examination (MMSE) and frontal assessment battery (FAB). The results showed that the subjects with aMCI, for which cerebral function is better preserved than subjects with AD, had a higher anti-HSV-1 IgG antibody avidity index than the AD subjects or healthy controls. Furthermore, the anti-HSV-1 IgG antibody avidity index was even higher in the subjects with high MMSE scores on orientation to time and three-step command subscores. We observed a negative correlation between the anti-HSV-1 IgG antibody avidity index and plasma BDNF concentration, which is an indicator of encephalitis. This suggests that HSV-1 reactivation, as observed through an increase in the anti-HSV-1 IgG avidity index, does not progress to encephalitis. These results suggest that HSV-1 reactivation occurs from the stage of aMCI, which is prodromal to AD, and can affect AD symptoms without an intermediary stage of severe encephalitis. The study demonstrates that the anti-HSV-1 IgG antibody avidity index could be a useful biomarker for the early diagnosis of aMCI as well as AD, and suggests that antiviral medication to treat HSV-1 could play a role in preventing the onset of AD.

Human herpesvirus 6 immediate-early 2 protein interacts with heterogeneous ribonucleoprotein K and casein kinase 2

Human herpesvirus 6 (HHV‐6) immediate‐early (IE) 2 protein (IE2) may play important but incompletely defined roles during infection. We used yeast two‐hybrid screening to detect proteins interacting with HHV‐6 IE2, and found heterogeneous nuclear ribonucleoprotein K (hnRNP K) and the beta subunit of casein kinase 2 (CK2β) specifically interacted with HHV‐6 IE2. The interactions were confirmed by GST pull‐down assay, coimmunoprecipitation, and colocalization studies. These findings indicate that the HHV‐6 IE2 protein interacts with hnRNP K and CK2, and these interactions may affect viral and cellular RNA transcription and translation in viral replication.

Detection of a Gene Cluster That Is Dispensable for Human Herpesvirus 6 Replication and Latency

ABSTRACT The U3-U7 gene cluster of human herpesvirus 6 (HHV-6) was replaced with an enhanced green fluorescent protein-puromycin gene cassette containing the cytomegalovirus major immediate-early promoter. Neither viral replication in T cells nor latency and reactivation in macrophages was impaired. During HHV-6 latency, the cytomegalovirus promoter used the transcription start sites employed in cytomegalovirus latency.

Development of Biomarkers Based on DNA Methylation in the NCAPH2/LMF2 Promoter Region for Diagnosis of Alzheimer’s Disease and Amnesic Mild Cognitive Impairment

From the standpoint of early interventions for dementia, a convenient method of diagnosis using biomarkers is required for Alzheimer’s disease (AD) in the early stage as well as amnesic mild cognitive impairment (aMCI). Focusing on differences in DNA methylation due to AD and aMCI, in the present study, we first conducted genome-wide screening, measuring blood DNA methylation levels by the Illumina Infinium HD Methylation Assay in 3 small age-and gender-matched groups consisting of 4 subjects each: normal controls (NC), aMCI and AD. The genome-wide analysis produced 11 DNA methylation loci that distinguished the 3 groups. For confirmation, we increased group sizes and examined samples by pyrosequencing which revealed that DNA methylation in the NCAPH2/LMF2 promoter region was significantly decreased in the AD (n = 30) and aMCI (n = 28) groups as compared to the NC group (n = 30) (P < 0.0001, ANCOVA). No association was found between methylation levels and APOE genotype. NCAPH2/LMF2 methylation levels were considered to potentially be a convenient and useful biomarker for diagnosis of AD and aMCI.

Human herpesvirus 6 and 7 are biomarkers for fatigue, which distinguish between physiological fatigue and pathological fatigue

Fatigue reduces productivity and is a risk factor for lifestyle diseases and mental disorders. Everyone experiences physiological fatigue and recovers with rest. Pathological fatigue, however, greatly reduces quality of life and requires therapeutic interventions. It is therefore necessary to distinguish between the two but there has been no biomarker for this. We report on the measurement of salivary human herpesvirus (HHV-) 6 and HHV-7 as biomarkers for quantifying physiological fatigue. They increased with military training and work and rapidly decreased with rest. Our results suggested that macrophage activation and differentiation were necessary for virus reactivation. However, HHV-6 and HHV-7 did not increase in obstructive sleep apnea syndrome (OSAS), chronic fatigue syndrome (CFS) and major depressive disorder (MDD), which are thought to cause pathological fatigue. Thus, HHV-6 and HHV-7 would be useful biomarkers for distinguishing between physiological and pathological fatigue. Our findings suggest a fundamentally new approach to evaluating fatigue and preventing fatigue-related diseases.

Novel Gene Therapy Viral Vector Using Non-Oncogenic Lymphotropic Herpesvirus

Despite the use of retroviral vectors, efficiently introducing target genes into immunocytes such as T cells is difficult. In addition, retroviral vectors carry risks associated with the oncogenicity of the native virus and the potential for introducing malignancy in recipients due to genetic carryover from immortalized cells used during vector production. To address these issues, we have established a new virus vector that is based on human herpesvirus 6 (HHV-6), a non-oncogenic lymphotropic herpesvirus that infects CD4+ T cells, macrophages, and dendritic cells. In the present study, we have altered the cell specificity of the resulting recombinant HHV-6 by knocking out the U2–U8 genes. The resulting virus proliferated only in activated cord blood cells and not in peripheral blood cells. Umbilical cord blood cells produced replication-defective recombinant virus in sufficiently high titer to omit the use of immortalized cells during vector production. HHV-6 vectors led to high rates (>90%) of gene transduction in both CD4+ and CD8+ T cells. These viruses showed low-level replication of viral DNA that supported greater expression of the induced genes than that of other methods but that was insufficient to support the production of replication-competent virus. Furthermore, HHV-6 vectors containing short hairpin RNAs against CD4 and HIV Gag remarkably inhibited the production of these proteins and HIV particles. Here we demonstrate the utility of HHV-6 as a new non-carcinogenic viral vector for immunologic diseases and immunotherapy.

Usefulness of DNA Methylation Levels in COASY and SPINT1 Gene Promoter Regions as Biomarkers in Diagnosis of Alzheimer’s Disease and Amnestic Mild Cognitive Impairment

In order to conduct early therapeutic interventions for Alzheimer’s disease (AD), convenient, early diagnosis markers are required. We previously reported that changes in DNA methylation levels were associated with amnestic mild cognitive impairment (aMCI) and AD. As the results suggested changes in DNA methylation levels in the COASY and SPINT1 gene promoter regions, in the present study we examined DNA methylation in these regions in normal controls (NCs, n = 30), aMCI subjects (n = 28) and AD subjects (n = 30) using methylation-sensitive high resolution melting (MS-HRM) analysis. The results indicated that DNA methylation in the two regions was significantly increased in AD and aMCI as compared to NCs (P < 0.0001, P < 0.0001, ANOVA). Further analysis suggested that DNA methylation in the COASY gene promoter region in particular could be a high sensitivity, high specificity diagnosis biomarker (COASY: sensitivity 96.6%, specificity 96.7%; SPINT1: sensitivity 63.8%, specificity 83.3%). DNA methylation in the COASY promoter region was associated with CDR Scale Sum of Boxes (CDR-SB), an indicator of dementia severity. In the SPINT1 promoter region, DNA methylation was negatively associated with age in NCs and elevated in aMCI and AD subjects positive for antibodies to Herpes simplex virus type 1 (HSV-1). These findings suggested that changes in DNA methylation in the COASY and SPINT1 promoter regions are influenced by various factors. In conclusion, DNA methylation levels in the COASY and SPINT1 promoter regions were considered to potentially be a convenient and useful biomarker for diagnosis of AD and aMCI.