SUMMARYChromosomally integrated human herpesvirus 6 (ciHHV-6) is a condition in which the complete HHV-6 genome is integrated into the host germ line genome and is vertically transmitted in a Mendelian manner. The condition is found in less than 1% of controls in the USA and UK, but has been found at a somewhat higher prevalence in transplant recipients and other patient populations in several small studies. HHV-6 levels in whole blood that exceed 5.5 log10 copies/ml are strongly suggestive of ciHHV-6. Monitoring DNA load in plasma and serum is unreliable, both for identifying and for monitoring subjects with ciHHV-6 due to cell lysis and release of cellular DNA. High HHV-6 DNA loads associated with ciHHV-6 can lead to erroneous diagnosis of active infection. Transplant recipients with ciHHV-6 may be at increased risk for bacterial infection and graft rejection. ciHHV-6 can be induced to a state of active viral replication in vitro. It is not known whether ciHHV-6 individuals are put at clinical risk by the use of drugs that have been associated with HHV-6 reactivation in vivo or in vitro. Nonetheless, we urge careful observation when use of such drugs is indicated in individuals known to have ciHHV-6. Little is known about whether individuals with ciHHV-6 develop immune tolerance for viral proteins. Further research is needed to determine the role of ciHHV-6 in disease. Copyright © 2011 John Wiley & Sons, Ltd.

Shortly after the discovery of human herpesvirus 6 (HHV-6), two distinct variants, HHV-6A and HHV-6B, were identified. In 2012, the International Committee on Taxonomy of Viruses (ICTV) classified HHV-6A and HHV-6B as separate viruses. This review outlines several of the documented epidemiological, biological, and immunological distinctions between HHV-6A and HHV-6B, which support the ICTV classification. The utilization of virus-specific clinical and laboratory assays for distinguishing HHV-6A and HHV-6B is now required for further classification. For clarity in biological and clinical distinctions between HHV-6A and HHV-6B, scientists and physicians are herein urged, where possible, to differentiate carefully between HHV-6A and HHV-6B in all future publications.

Characterizing complex viral transcriptomes by conventional RNA sequencing approaches is complicated by high gene density, overlapping reading frames, and complex splicing patterns. Direct RNA sequencing (direct RNA-seq) using nanopore arrays offers an exciting alternative whereby individual polyadenylated RNAs are sequenced directly, without the recoding and amplification biases inherent to other sequencing methodologies. Here we use direct RNA-seq to profile the herpes simplex virus type 1 (HSV-1) transcriptome during productive infection of primary cells. We show how direct RNA-seq data can be used to define transcription initiation and RNA cleavage sites associated with all polyadenylated viral RNAs and demonstrate that low level read-through transcription produces a novel class of chimeric HSV-1 transcripts, including a functional mRNA encoding a fusion of the viral E3 ubiquitin ligase ICP0 and viral membrane glycoprotein L. Thus, direct RNA-seq offers a powerful method to characterize the changing transcriptional landscape of viruses with complex genomes.

Varicella-zoster virus (VZV), an alphaherpesvirus, establishes lifelong latent infection in the neurons of >90% humans worldwide, reactivating in one-third to cause shingles, debilitating pain and stroke. How VZV maintains latency remains unclear. Here, using ultra-deep virus-enriched RNA sequencing of latently infected human trigeminal ganglia (TG), we demonstrate the consistent expression of a spliced VZV mRNA, antisense to VZV open reading frame 61 (ORF61). The spliced VZV latency-associated transcript (VLT) is expressed in human TG neurons and encodes a protein with late kinetics in productively infected cells in vitro and in shingles skin lesions. Whereas multiple alternatively spliced VLT isoforms (VLTly) are expressed during lytic infection, a single unique VLT isoform, which specifically suppresses ORF61 gene expression in co-transfected cells, predominates in latently VZV-infected human TG. The discovery of VLT links VZV with the other better characterized human and animal neurotropic alphaherpesviruses and provides insights into VZV latency.

The DNA sequences of the Oka varicella vaccine virus (V-Oka) and its parental virus (P-Oka) were completed. Comparison of the sequences revealed 42 base substitutions, which led to 20 amino acid conversions and length differences in tandem repeat regions (R1, R3, and R4) and in an origin of DNA replication. Amino acid substitutions existed in open reading frames (ORFs) 6, 9A, 10, 21, 31, 39, 50, 52, 55, 59, 62, and 64. Of these, 15 base substitutions, leading to eight amino acid substitutions, were in the gene 62 region alone. Further DNA sequence analysis showed that these substitutions were specific for V-Oka and were not present in nine clinical isolates. The immediate-early gene 62 product (IE62) of P-Oka had stronger transactivational activity than the mutant IE62 contained in V-Oka in 293 and CV-1 cells. An infectious center assay of a plaque-purified clone (S7-01) from the V-Oka with 8 amino acid substitutions in ORF 62 showed smaller plaque formation and less-efficient virus-spreading activity than did P-Oka in human embryonic lung cells. Another clone (S-13) with only five substitutions in ORF 62 spread slightly faster than S7-01 but not as effectively as P-Oka. Moreover, transient luciferase assay in 293 cells showed that transactivational activities of IE62s of S7-01 and S7-13 were lower than that of P-Oka. Based on these results, it appears that amino acid substitutions in ORF 62 are responsible for virus growth and spreading from infected to uninfected cells. Furthermore, the Oka vaccine virus was completely distinguishable from P-Oka and 54 clinical isolates by seven restriction-enzyme fragment length polymorphisms that detected differences in the DNA sequence.Varicella-zoster virus (VZV) is a human herpesvirus that causes chickenpox (varicella) and shingles (herpes zoster). A live attenuated varicella vaccine, the Oka strain was originally developed by Takahashi et al. in Japan (56) and is routinely used in children in Japan and other countries, including the United States. Clinical symptoms caused by this live vaccine are very rare in healthy children. Although the Oka vaccine virus (V-Oka) is an avirulent virus, its parental virus (P-Oka), isolated from a patient with typical varicella, is thought to be virulent in vivo. It has not been clarified which gene(s) is involved in the pathogenicity of VZV infection. Thus, comparison of the complete genomes of V-Oka and P-Oka should reveal correlations between DNA sequence and virulence.The complete DNA sequence of the VZV Dumas strain was first determined by Davison and Scott (9). The genome is a linear double-stranded DNA of ca. 125,000 bp and consists of unique long regions (ULs) flanked by terminal repeat long (TRL) and internal repeat long (IRL) inverted repeat regions, and a unique short (US) region flanked by internal repeat short (IRS) and terminal repeat short (TRS) inverted repeat regions. The genome contains ca. 70 open reading frames (ORFs), three of which exist in both IRS and TRS regions; genes 62 through 64 correspond to genes 69 throug...

†These authors contributed equally to this work.The final envelopment of most herpesviruses occurs at Golgi or post-Golgi compartments, such as the trans Golgi network (TGN); however, the final envelopment site of human herpesvirus 6 (HHV-6) is uncertain. In this study, we found novel pathways for HHV-6 assembly and release from T cells that differed, in part, from those of alphaherpesviruses. Electron microscopy showed that late in infection, HHV-6-infected cells were larger than uninfected cells and contained many newly formed multivesicular body (MVB)-like compartments that included small vesicles. These MVBs surrounded the Golgi apparatus. Mature virions were found in the MVBs and MVB fusion with plasma membrane, and the release of mature virions together with small vesicles was observed at the cell surface. Immunoelectron microscopy demonstrated that the MVBs contained CD63, an MVB/ late endosome marker, and HHV-6 envelope glycoproteins. The viral glycoproteins also localized to internal vesicles in the MVBs and to secreted vesicles (exosomes). Furthermore, we found virus budding at TGNassociated membranes, which expressed CD63, adaptor protein (AP-1) and TGN46, and CD63 incorporation into virions. Our findings suggest that mature HHV-6 virions are released together with internal vesicles through MVBs by the cellular exosomal pathway. This scenario has significant implications for understanding HHV-6's maturation pathway.

Human herpesvirus-6B (HHV-6B) is a T lymphotropic β-herpesvirus that is clearly distinct from human herpesvirus-6A (HHV-6A) according to molecular biological features. The International Committee on Taxonomy of Viruses recently classified HHV-6B as a separate species. The primary HHV-6B infection causes exanthem subitum and is sometimes associated with severe encephalopathy. More than 90% of the general population is infected with HHV-6B during childhood, and the virus remains throughout life as a latent infection. HHV-6B reactivation causes encephalitis in immunosuppressed patients. The cellular receptor for HHV-6A entry was identified as human CD46, but the receptor for HHV-6B has not been clear. Here we found that CD134, a member of the TNF receptor superfamily, functions as a specific entry receptor for HHV-6B. A T-cell line that is normally nonpermissive for HHV-6B infection became highly susceptible to infection when CD134 was overexpressed. CD134 was down-regulated in HHV-6B-infected T cells. Soluble CD134 interacted with the HHV-6B glycoprotein complex that serves as a viral ligand for cellular receptor, which inhibited HHV-6B but not HHV-6A infection in target cells. The identification of CD134 as an HHV-6B specific entry receptor provides important insight into understanding HHV-6B entry and its pathogenesis.H uman herpesvirus-6B (HHV-6B) is a T lymphotropic β-herpesvirus (1) and is clearly distinct from human herpesvirus-6A (HHV-6A) according to their genetic and antigenic differences and their cell tropism (2-5). Recently the International Committee on Taxonomy of Viruses classified HHV-6B as a separate species.The primary HHV-6B infection causes exanthem subitum (6) and is sometimes associated with severe encephalopathy, whereas the diseases caused by HHV-6A are still unknown. More than 90% of the general population is infected with HHV-6B during childhood, and the virus remains throughout life as a latent infection (7). HHV-6B reactivation causes encephalitis in immunosuppressed patients. HHV-6B reactivation is also associated with drug-induced hypersensitivity syndrome, and recent studies have suggested that it could be related to the severity of this disease (8, 9).HHV-6A can infect a broader variety of human cells than HHV-6B (10), although the homology between HHV-6A and -6B is almost 90% over their entire genome (11-13). Human CD46 has been shown to be a cellular receptor of , and its viral ligand is a glycoprotein (g) complex made up of viral glycoprotein H (gH)/glycoprotein L (gL)/glycoprotein Q1 (gQ1)/ glycoprotein Q2 (gQ2) (15). However, the HHV-6A gH/gL/gQ1/ gQ2 complex binds to its human cellular receptor, CD46, whereas the corresponding complex of HHV-6B does not bind to it (10, 15). Moreover, anti-CD46 antibody does not block HHV-6B infection into the cells, whereas it does HHV-6A infection, indicating that the cellular receptor exists specific for HHV-6B infection. Because HHV-6B remains as a lifelong latent infection in more than 90% of the population and causes severe disease, it is...

Varicella-zoster virus (VZV) and herpes simplex virus (HSV) are prevalent neurotropic herpesviruses that cause various nervous system diseases. Similar to other enveloped viruses, membrane fusion is an essential process for viral entry. Therefore, identification of host molecules that mediate membrane fusion is important to understand the mechanism of viral infection. Here, we demonstrate that myelin-associated glycoprotein (MAG), mainly distributed in neural tissues, associates with VZV glycoprotein B (gB) and promotes cell-cell fusion when coexpressed with VZV gB and gH/gL. VZV preferentially infected MAG-transfected oligodendroglial cells. MAG also associated with HSV-1 gB and enhanced HSV-1 infection of promyelocytes. These findings suggested that MAG is involved in VZV and HSV infection of neural tissues.herpes simplex virus | neurotropism | membrane fusion | Varicella-zoster virus | virus entry V aricella-zoster virus (VZV) mainly causes two human diseases, varicella (chickenpox) in children and zoster (shingles) in immune-compromised or elderly individuals (1); VZV also causes diseases of the nervous system, such as meningitis and encephalitis (2). Herpes simplex virus (HSV) also causes neurological disease. A unique characteristic of these viruses is establishment of latency in sensory ganglia (1, 3). Both VZV and HSV are enveloped viruses of the alphaherpesvirus family, whose interactions between its envelope proteins and cell-surface molecules are crucial events for the entry of enveloped viruses into cells (4).Glycoproteins gB, gD, gH, and gL are essential envelope protein for membrane fusion during HSV infection. gD associates with several cell-surface proteins, such as herpesvirus entry mediator (HVEM) and nectin, and gB associates with paired Iglike type-2 receptor α (PILRα) (5). These interactions can play important roles in HSV-1 infection, depending upon cell types (5). On the other hand, glycoproteins gB, gE, gH, and gL have been suggested to participate in membrane fusion during VZV infection (6). Because mannose 6-phosphate (M6P) inhibits cell-free VZV infection, a M6P receptor has been suggested to be involved in cell-free VZV infection by the interaction with VZV glycoproteins that contain M6P (7, 8). Indeed, Chen et al. showed that cation-independent M6P receptor (MPR ci ) is involved in cell-free VZV infection (9). MPR ci is ubiquitously expressed on various tissues and mainly functions as a molecular chaperone that transports proteins modified with N-linked oligosaccharides from the trans-Golgi network to early endosomes (10, 11).VZV-gE is an essential glycoprotein for VZV infection and has been suggested to be required, in concert with its heterodimer partner, gI, for viral replication and for virion assembly in the transGolgi network (12-15). In addition, gE also seems to be involved in membrane fusion between the viral envelope and cellular membrane, although gE alone does not induce membrane fusion (6, 16). Recently, insulin-degrading enzyme (IDE), ubiquitously expressed on various c...