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Human herpesvirus 7 (HHV-7), which belongs to the betaherpesvirus subfamily and infects mainly CD4 + T cells in vitro, infects children during infancy. HHV-7 contains two genes, U12 and U51, that encode putative homologs of cellular G-protein-coupled receptors. To analyze the biological function of the U12 and U51 genes, we cloned these genes and expressed the proteins in cells. U12 and U51 encoded functional calcium-mobilizing receptors for β-chemokines, which include thymus and activation-regulated chemokine (TARC), macrophage-derived chemokine (MDC), EBI1-ligand chemokine (ELC), and secondary lymphoid-tissue chemokine (SLC), but not for other chemokines, suggesting that the chemokine selectivities of the U12 and U51 products were distinct from those of the known mammalian chemokine receptors. ELC and SLC induced migration in Jurkat cells stably expressing U12, but TARC and MDC did not. In contrast, none of these chemokines induced migration in Jurkat cells stably expressing U51. Together, these data indicate that the products of U12 and U51 may play important and different roles in the pathogenesis of HHV-7 through transmembrane signaling.

The complete genome of the varicella-zoster virus (VZV) Oka strain has been cloned as a bacterial artificial chromosome (BAC). Following electroporation into Escherichia coli (E. coli) strain DH10B, the VZV BAC was stably propagated over multiple generations of its host. Human embryonic lung (HEL) cells transfected with VZV BAC DNA recovered from DH10B showed cytopathic effect (CPE), and virus spread to neighbouring cells was observed. BAC vector sequences are flanked by loxP sites and, coinfection of the reconstituted virus, with a recombinant adenovirus expressing Cre recombinase removed the bacterial sequences. The resulting recombinant rV02 grew as well as the parental virus in HEL cells. The recombinant VZV will promote VZV research and increase use of the viral genome as an investigative tool.

Human herpesvirus 6 (HHV-6) glycoproteins H and L (gH and gL, respectively) and the 80-kDa form of glycoprotein Q (gQ-80K) form a heterotrimeric complex that is found on the viral envelope and that is a viral ligand for human CD46. Besides gQ-80K, the gQ gene encodes an additional product whose mature molecular mass is 37 kDa (gQ-37K) and which is derived from a different transcript. Therefore, we designated gQ-80K as gQ1 and gQ-37K as gQ2. We show here that gQ2 also interacts with the gH-gL-gQ1 complex in HHV-6-infected cells and in virions. To examine how these components interact in HHV-6-infected cells, we performed pulse-chase studies. The results demonstrated that gQ2-34K, which is endo-β- N -acetylglucosaminidase H sensitive and which is the precursor form of gQ2-37K, associates with gQ1-74K, which is the precursor form of gQ1-80K, within 30 min of the pulse period. After a 1-h chase, these precursor forms had associated with the gH-gL dimer. Interestingly, an anti-gH monoclonal antibody coimmunoprecipitated mainly gQ1-80K and gQ2-37K, with little gQ1-74K or gQ2-34K. These results indicate that although gQ2-34K and gQ1-74K interact in the endoplasmic reticulum, the gH-gL-gQ1-80K-gQ2-37K heterotetrameric complex arises in the post-endoplasmic reticulum compartment. The mature complex is subsequently incorporated into viral particles.

Kaposi's sarcoma-associated herpesvirus (KSHV; also known as human herpesvirus 8, HHV-8) belongs to the γ-herpesvirus subfamily. The KSHV ORF57 gene is thought to be a homolog of posttranscriptional regulators that are conserved in the herpesvirus family and are essential for replication. We generated specific monoclonal antibodies (mAbs) against the ORF57 protein that detected the 51-kDa protein expressed in the nucleus of KSHV-infected cells. We also found that the ORF57 protein interacted with poly(rC)-binding protein 1 (PCBP1), a cellular RNA-binding, posttranscriptional regulator. ORF57's interaction with PCBP1 enhanced the activity of not only poliovirus internal ribosome-entry site (IRES)-dependent translation but also X-linked inhibitor of apoptosis (XIAP) and KSHV vFLIP IRES. Actually, when ORF57 expression was induced by the expression of replication and transcription activator (RTA) in KSHV-infected cells, the expression of XIAP was enhanced. These results suggest that ORF57 binds to PCBP1 as a functional partner for posttranscriptional regulation and is involved in the regulation of the expression of both cellular and viral genes through IRESs.

In the latent infection of Kaposi's sarcoma-associated herpesvirus (KSHV), its 160-kb circularized episomal DNA is replicated and maintained in the host nucleus. KSHV latency-associated nuclear antigen (LANA) is a key factor for maintaining viral latency. LANA binds to the terminal repeat (TR) DNA of the viral genome, leading to its localization to specific dot structures in the nucleus. In such an infected cell, the expression of the viral genes is restricted by a mechanism that is still unclear. Here, we found that LANA interacts with SUV39H1 histone methyltransferase, a key component of heterochromatin formation, as determined by use of a DNA pull-down assay with a biotinylated DNA fragment that contained a LANA-specific binding sequence and a maltose-binding protein pull-down assay. The diffuse localization of LANA on the chromosomes of uninfected cells changed to a punctate one with the introduction of a bacterial artificial chromosome containing most of the TR region, and SUV39H1 clearly colocalized with the LANA-associated dots. Thus, the LANA foci in KSHV-infected cells seemed to include SUV39H1 as well as heterochromatin protein 1. Furthermore, a chromatin immunoprecipitation assay revealed that the TR and the open reading frame (ORF) K1 and ORF50/RTA genes, but not the ORF73/LANA gene, lay within the heterochromatin during KSHV latency. Taken together, these observations indicate that LANA recruits heterochromatin components to the viral genome, which may lead to the establishment of viral latency and govern the transcription program.

Replication and transcription activator (RTA), mostly encoded by Kaposi's sarcoma-associated herpesvirus (KSHV) open reading frame 50, is expressed in the immediate-early phase of reactivation and plays a critical role in inducing the viral lytic cycle in KSHV-infected cells. We established cell clones from BJAB cells and replication-deficient BCBL-1 cells in which KSHV RTA expression was controlled by an inducible promoter of the tetracycline-based Tet-Off expression system. In RTA-inducible BJAB cells, tetracycline removal induced the synthesis of RTA, resulting in cell death. DNA fragmentation, structural changes in the cell membrane, and poly(ADP-ribose) polymerase (PARP) cleavage were observed in the RTA-induced BJAB cells, indicating that RTA expression induced caspase activation and cell death by apoptosis. However, expression of RTA in RTA-inducible BCBL-1 cells did not undergo apoptosis and cell death. These results suggested that KSHV RTA is an apoptosis inducer that is opposed by an antiapoptotic pathway in infected cells.

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.

The enhancement of immunity against varicella-zoster vaccine (VZV) by subcutaneous injection of a live varicella vaccine was assessed by the VZV skin test for cell-mediated immunity (CMI), and immunoadherence hemagglutination assay (IAHA) and gpELISA antibody assays in the elderly people of 50-79 years of age. A total of 127 subjects were examined: 79 aged 50-59, 25 aged 60-69, and 25 aged 70-79. All were seropositive by the gpELISA assay (one was seronegative in the IAHA antibody assay). In contrast, a notable decline was observed in the VZV skin test with increasing age. Negative reaction was observed in 16/79 (20.2%) of the subjects in their 50s, 12/25 (48.0%) in their 60s and 14/25 (56.0%) in the 70s. After the vaccination, the results of the VZV skin test changed from negative to positive in 15/16 (91.8%) of subjects in their 50s, 11/12 (91.7%) in their 60s and 12/14 (85.7%) in their 70s. The mean antibody titer in the IAHA and the gpELISA increased approximately two-fold after the vaccination in each group. Immunity to VZV in 35 elderly subjects who were vaccinated previously was followed up for 4 years. All were positive by the VZV skin test after the previous vaccination. After 4 years, 31 (88.6%) were positive by the skin test, 4 were negative and became positive after revaccination. Although this study was uncontrolled open study, the results suggest that administering live varicella vaccine to the elderly is effective for enhancing immunity, particularly CMI to VZV.

Human herpesvirus 6 (HHV- 6), which belongs to the betaherpesvirus subfamily and infects mainly T cells in vitro, causes acute and latent infections. HHV- 6 contains two genes (U12 and U51) that encode putative homologs of cellular G-protein-coupled receptors (GCR), while three other betaherpesviruses, human cytomegalovirus, murine cytomegalovirus, and human herpesvirus 7, have three, one, and two GCR-homologous genes, respectively. The U12 gene is expressed late in infection from a spliced mRNA. The U12 gene was cloned, and the protein was expressed in cells and analyzed for its biological characteristics. U12 functionally encoded a calcium-mobilizing receptor for beta-chemokines such as regulated upon activation, normal T expressed and secreted (RANTES), macrophage inflammatory proteins 1alpha and 1beta (MIP-1alpha and MIP-1beta) and monocyte chemoattractant protein 1 but not for the alpha-chemokine interleukin-8, suggesting that the chemokine selectivity of the U12 product was distinct from that of the known mammalian chemokine receptors. These findings suggested that the product of U12 may play an important role in the pathogenesis of HHV- 6 through transmembrane signaling by binding with beta-chemokines.

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.

Open reading frame (ORF) 50 of human herpesvirus 8 (HHV8, Kaposi's sarcoma-associated herpesvirus) is one of the immediate-early gene and a homologue of BRLF1 gene of Epstein-Barr virus. It encodes a key switch protein to trigger viral lytic replication from latency. We have established several hybridoma clones producing monoclonal antibodies (MAbs) to the products of HHV8 ORFs. Using these antibodies, we analyzed antigen expression in a HHV8 infected cell line after treatment with phorbol ester (12-O-tetradecanoylphorbol-13-acetate, TPA). A MAb reacted to 110 kilodalton (kDa) and 62 kDa proteins encoded by ORF50 (ORF50 protein). Kinetic studies of antigen expression by Western blotting revealed that ORF50 protein was induced as early as 6 h after TPA treatment. The proteins encoded by ORFK3, ORFK5, ORFK9, ORF59 and ORFK8.1 were not detected earlier than ORF50 protein. However, when antigen positive cells were counted by immunofluorescent antibody (IFA) test, number of ORFK5 protein positive cells were higher than that of ORF50 protein positive cells at all time after TPA or mock treatment. To confirm the results of IFA test, individual cell was analyzed by reverse transcription polymerase chain reaction. Some cells expressed ORFK5 transcript but not ORF50 transcript. Therefore, we concluded that, although ORF50 protein is a key switch protein of ORFK3, ORFK9, ORF59 and ORFK8.1 expression, it is not essential to trigger ORFK5 gene.

The replication and transcription activator (RTA) of Kaposi's sarcoma-associated herpesvirus (KSHV), or human herpesvirus 8, a homologue of Epstein-Barr virus BRLF1 or Rta, is a strong transactivator and inducer of lytic replication. RTA acting alone can induce lytic replication of KSHV in infected cell lines that originated from primary effusion lymphomas, leading to virus production. During the lytic replication process, RTA activates many kinds of genes, including polyadenylated nuclear RNA, K8, K9 (vIRF), ORF57, and so on. We focused here on the mechanism of how RTA upregulates the K9 (vIRF) promoter and identified two independent cis -acting elements in the K9 (vIRF) promoter that responded to RTA. These elements were finally confined to the sequence 5′-TCTGGGACAGTC-3′ in responsive element (RE) I-2B and the sequence 5′-GTACTTAAAATA-3′ in RE IIC-2, both of which did not share sequence homology. Multiple factors bound specifically with these elements, and their binding was correlated with the RTA-responsive activity. Electrophoretic mobility shift assay with nuclear extract from infected cells and the N-terminal part of RTA expressed in Escherichia coli , however, did not show that RTA interacted directly with these elements, in contrast to the RTA responsive elements in the PAN/K12 promoter region, the ORF57/K8 promoter region. Thus, it was likely that RTA could transactivate several kinds of unique cis elements without directly binding to the responsive elements, probably through cooperation with other DNA-binding factors.

SOCS-1 is a negative regulatory molecule of the JAK-STAT signal cascade. Here, we demonstrate that SOCS-1 is a critical downregulating factor for LPS signal pathways. SOCS-1 expression was promptly induced in macrophages upon LPS stimulation. SOCS-1-deficient mice were highly sensitive to LPS-induced shock and produced increased levels of inflammatory cytokines. Introduction of SOCS-1 inhibited LPS-induced NF-κB and STAT1 activation in macrophages. Furthermore, LPS tolerance, a refractory state to second LPS stimulation, was not observed in SOCS-1-deficient mice. These results suggest SOCS-1 as an essential, negative regulator in LPS responses that protects the host from harmful overresponses to LPS and may provide new insight into the endotoxin-induced fatal syndrome that occasionally occurs following infection.

Human herpesvirus 6 (HHV-6) infection in recipients of cord blood stem cell transplants (CBSCTs) was estimated by semiquantitative and real-time quantitative polymerase chain reaction (PCR) and reverse-transcription PCR. Of the CBSCT recipients, 7 (70%) of 10 had active HHV-6 infection after transplantation, and all 7 were inferred from their age to have already had a primary infection. Because HHV-6 DNA is seldom detected in cord blood, these cases were considered likely to represent reactivation. In contrast, the 3 patients without HHV-6 infection were all believed to be naive regarding HHV-6 primary infection because of their age and the results of PCR assays given before the transplantation procedure. The incidence of HHV-6 infection after transplantation was significantly higher (P

Background: Infants are usually protected from various viral infections, including human herpesvirus-6 (HHV-6) and human herpesvirus-7 (HHV-7) infections, during the early infantile period by antibodies transferred from their mothers. However, rare cases of exanthem subitum (ES) in neonates have been described in published reports. Methods: From the infantile patients of febrile illness, HHV-6 and HHV-7 DNA were examined by the polymerase chain reaction method. Antibodies to HHV-6 and HHV-7 were detected by indirect immuno­fluorescence assay and neutralization test. Viral isolation was attempted from the patient’s peripheral blood mononuclear cells (PBMC) during the acute phase of febrile illness. Results: Human herpesvirus-6 was verified virologically in two neonates who were clinically diagnosed as ES within the first month of life. Although high copies of HHV-6 DNA were detected in their PBMC during the acute phase, the isolation of HHV-6 from their PBMC was not successful. Neutralizing antibodies to HHV-6 were detected in sera of the acute phase, and those antibodies were considered to be transferred from their mothers. Antibody titers showed fourfold elevation in sera of the convalescent phase. The HHV-6 infection occurred despite the presence of pre-existing maternal antibody. Human herpesvirus-7 and HHV-7 DNA were not detected from their clinical samples. Conclusions: This observation suggests that HHV-6 infection could not be protected by only humoral immunity.

Clinical isolates of human herpesvirus 7 (HHV-7) from the saliva of healthy individual were investigated for genetic variations in the regions of two immediate-early (IE) genes, the glycoprotein B (gB) and glycoprotein H (gH) genes, and in R2-repeat. The genomic DNA of 24 isolates from citizens of Thailand, Japan, and the United States was amplified to detect size variations in the IE-1 and IE-2 loci, but none was observed, suggesting that there was no deletion or insertion in these genes, in contrast with an IE gene of human herpesvirus 6 (HHV-6). The sequences of the gB gene from isolates acquired from 5 Japanese and 8 Thai subject were then compared with those of American strains JI and RK with respect to codons that are known to differentiate gB alleles. All the isolates were found to have gB allele C except for the JI strain, which has allele F. Variability was also observed in five specific gH codons, resulting in 6 different groups. The HHV-7 isolates might be classified into two major genetic variants by combining their gB and gH allelic groupings. In the present study, only JI belonged to variant 1, while the rest of the isolates appeared to belong to variant 2. In the R2-repeat region, size heterogeneities were observed among the 24 isolates, due to different repeat numbers (17, 15, 14, 13, or 12 repeats). Therefore, we used the R2-repeat to identify the origins of isolates in a study of HHV-7 transmission, and found HHV-7 to be transmitted within a family from both mothers and fathers to their children.

Whole-virus (WV) vaccines from influenza A/duck/Hokkaido/77 (H3N2), and its reassortant strains H3N4, H3N5 and H3N7, which have the same haemagglutinin (HA) gene but different neuraminidase (NA) genes, were prepared from our influenza virus library. Mice were intranasally immunized with equivalent doses of each vaccine (1–0.01 µg per mouse). All of the mice that received the highest dose of each vaccine (1 µg per mouse) showed equivalent high HA-inhibiting (HI) antibody titres and survived the H3N2 challenge viruses. However, mice that received lower doses of vaccine (0.1 or 0.01 µg per mouse) containing a heterologous NA had lower survival rates than those given the H3N2-based vaccine. The lungs of mice challenged with H3N2 virus showed a significantly higher virus clearance rate when the vaccine contained the homologous NA (N2) versus a heterologous NA, suggesting that NA contributed to the protection, especially when the HI antibody level was low. These results suggested that, even if vaccines prepared for a possible upcoming pandemic do not induce sufficient HI antibodies, WV vaccines can still be effective through other matched proteins such as NA.

Herpes simplex virus-1 (HSV) or varicella zoster virus (VZV) DNA was detected by nested polymerase chain reaction in peripheral blood mononuclear cells of patients with Meniere's disease (one of 28 patients for HSV-1, 2 of 28 patients for VZV) during acute illness (within 5 days after onset). On the other hand, neither HSV-1 DNA or VZV DNA was detected in PBMCs of 50 age- and sex-matched healthy individuals and 50 pregnant women. These findings may imply that reactivation of HSV- 1 or VZV may be associated with the development of some cases of Meniere's disease.

Background: Human herpesvirus 6 (HHV-6), a causative virus of exanthem subitum, may occasionally present with a severe clinical form in immunosuppressed patients after transplantation. In this study, HHV-6 DNA was sequentially measured with a polymerase chain reaction (PCR) method, a quick and sensitive modality in pediatric living-related liver transplantation (LTx). Methods: Subjects consisted of 5 post-operative biliary atresia patients undergoing living-related LTx at ages from 8 months to 4 yr. Immunosuppression was performed with Tacrolimus (blood trough level 8-18 within 1 month and 5-10 ng/mL thereafter) and low-dose steroid. Specimens were peripheral blood mononuclear cells (PBMC), plasma, and liver biopsy tissue. The amount of HHV-6 DNA was semi-quantified as follows: 1 +, 1-10; 2 +, 10-100; 3 +, 100-1000; 4 +, over 1000 copies/10 5 PBMCs. Results: A total of 69 blood samples and three liver biopsies were provided for the examination. HHV-6 DNA in PBMC was positive in 2 donors and 3 recipients before LTx. Two patients with negative DNA were converted to 3 + at 2-3 wk after LTx and 3 with positive DNA remained 2 + 10 3 + throughout the post-LTx period. Only 1 patient developed clinical symptoms, such as fever, liver dysfunction, petechiae, idiopathic thrombocytopenic purpura, and finally bone marrow suppression. HHV-6 DNA in the liver biopsy tissue and plasma in this patient were 4 + and 2 +, respectively. Conclusion: HHV-6 DNA in PBMC measured by the PCR method may be persistently high in pediatric recipients after living-related LTx. Although HHV-6 DNA in PBMC may be positive in case of evident infection, positivity in PBMC may not be always associated with the clinical symptoms.

Herpes simplex viruses (HSVs) are members of the neurotropic subfamily (alphaherpesviruses) of the herpesvirus family. Infection with HSV type 1 (HSV-1) is prevalent. HSV-1 infects cells through initial attachment to the plasma membrane and subsequent fusion of the viral envelope with the plasma membrane or through contiguous cell-cell spread. The entry pathway of HSV-1 is divided into three major processes: binding, fusion, and capsid penetration. These processes require several viral envelope glycoproteins (49–51). The initial attachment is mediated through glycoprotein C (gC) and/or gB to cell surface heparan sulfate proteoglycans (17, 18, 45), but this attachment is not sufficient for virus penetration (3, 11, 22, 27). The fusion of the viral envelope with the plasma membrane requires gD, gB, gH, and gL (49–51). These four viral glycoproteins also participate in cell-cell spread (3, 11, 20, 28, 36, 40). Cell-cell spread furthermore requires gE and gI (1, 8–10), but these glycoproteins are not required for entry (8). Thus, entry and cell-cell spread of HSV-1 share similar processes, but these pathways also differ in some significant aspects. Recently, expression cloning has led to the identification and isolation of HSV entry mediators (5, 12, 33, 46, 51). The human receptors identified include a lymphotoxin receptor (31), designated HVEM (33, 65) or HSV entry mediator A (HveA), which belongs to the tumor necrosis factor receptor family; two members of the immunoglobulin (Ig) superfamily (5, 12, 48, 62); and 3-O-sulfated heparan sulfate (46). The two members of the Ig superfamily are closely related to the poliovirus receptor and named poliovirus receptor-related protein 1 (PRR1) and PRR2 (12, 62). Based on their ability to promote entry into cells, PRR1 and PRR2 are also called HveC and HveB, respectively (12, 62). PRR1/HveC is active as an entry mediator for all alphaherpesviruses tested so far (HSV-1, HSV-2, pseudorabies virus, and bovine herpesvirus 1) (12). PRR2/HveB enhances entry of a restricted number of mutant strains of HSV-1 (those carrying mutations in gD, such as rid1, rid2, and ANG), some HSV-2 strains, and pseudorabies virus (62). We recently found that PRR1/HveC and PRR2/HveB are components of a novel cell-cell adhesion system at cadherin-based adherens junctions (AJs) (53). We therefore renamed PRR1/HveC and PRR2/HveB nectin-1 and -2, respectively. Nectin-1 consists of two splicing variants, named nectin-1α and -1β/HIgR (5, 53), and nectin-2 also consists of two splicing variants, named nectin-2α and -2δ (53). Thus, nectin constitutes a family. We furthermore isolated a third member, named nectin-3, consisting of three splicing variants, nectin-3α, -3β, and -3γ (44). Nectin-3 may also serve as a receptor for some virus(es), but it has not been identified. Each member of the nectin family consists of three extracellular Ig-like domains, one transmembrane segment, and one cytoplasmic region. Nectin-1α, -2α, -2δ, and -3α have been shown to be Ca2+-independent homophilic cell-cell adhesion molecules at AJs that are directly associated with afadin, an actin filament (F-actin)-binding protein that connects nectins to the actin cytoskeleton (29, 30, 32, 44, 53). These members form cis homodimers where the monomers are aligned in a parallel orientation. They show cell-cell adhesion activities through trans homointeractions where the monomers interact with each other from opposing cell surfaces in an antiparallel orientation. Nectin-3α furthermore shows trans heterointeraction with nectin-1α or -2α, whereas nectin-1α does not show trans heterointeraction with nectin-2α (44). AJs constitute a junctional complex with tight junctions and desmosomes in polarized epithelial cells. Cadherin is a key cell-cell adhesion molecule at cell-cell AJs (14, 55, 56). The cytoplasmic region of cadherin is associated with the actin cytoskeleton through three F-actin-binding proteins: α-catenin, α-actinin, and vinculin (23, 39, 64). α-Catenin indirectly interacts with cadherin through β-catenin (35, 38), whereas α-actinin and vinculin indirectly interact with cadherin through α-catenin (23, 63, 64). The cadherin-catenin system plays essential roles in the formation and maintenance of cell-cell AJs (14, 55, 56) and is also required for the organization of tight junctions (15, 16, 63). Our recent studies of the role of afadin using afadin−/− mice and embryoid bodies have shown that afadin plays a key role in the proper organization of AJs and tight junctions (21). We have furthermore shown that nectin and cadherin interact through afadin and α-catenin and that the nectin-afadin and cadherin-catenin systems cooperatively organize AJs (52). The interaction of nectin with afadin is necessary for their clustering at cell-cell contact sites (32). Thus, evidence is accumulating that the nectin-afadin and cadherin-catenin systems interact and cooperatively play key roles at cell-cell AJs. Evidence is accumulating that nectin-1 mediates entry of HSV-1 by interaction with gD (4, 24, 25, 41). gD contains a binding domain specific for the first Ig-like domain, called the V domain, of nectin-1 (4, 25). The interaction of nectin-1 with gD has been proposed to activate the membrane-fusing activity of gB or gH-gL that, in addition to gD, are known to be required for this fusion (51). However, the V domain is sufficient to mediate entry of HSV-1, and the second and third Ig-like domains, called the C2 domains, increase the efficiency of HSV-1 entry (4). The cytoplasmic region of nectin-1 is not required for entry of HSV-1 (4, 5), suggesting that the interaction of nectin-1α with afadin does not affect entry. Recently, nectin-1 and -2 have been shown to mediate cell-cell spread of HSV-1 (6). However, it remains to be determined whether the interaction of nectin-1α with afadin is involved in cell-cell spread of HSV-1. Little is known, either, about the role of the cadherin-catenin system in entry and cell-cell spread of HSV-1. In this paper, we examined the role of afadin in entry and/or cell-cell spread of HSV-1 and found that the interaction of nectin-1α with afadin is required for efficient cell-cell spread, but not entry, of this virus. We have also found that the cadherin-catenin system is involved in both entry and cell-cell spread of HSV-1.

The expression of major histocompatibility complex (MHC) class I, class II, CD1a, and CD 83 in dendritic cells (DCs) after infection with human herpesvirus 6 (HHV-6) was examined. Whereas there was no significant change in the expression of CD1a, CD83, and MHC class II in infected DCs, MHC class I expression was downregulated after infection with HHV-6 variant A but not HHV-6B. The expression of HHV-6 immediate-early or early genes was required for the downregulation of MHC class I. The de novo synthesis of MHC class I was greatly suppressed by infection with HHV-6A in DCs, while its rate of degradation was only slightly elevated. These results suggest that HHV-6A may escape from the host immune system in DCs by causing the downregulation of MHC class I synthesis.

We present 3 cases of immunocompetent infants with CMV infection who showed prolonged liver dysfunction. In all cases the CMV genome was detectable in hepatocytes using the in situ hybridization method. Combination therapy with ganciclovir (GCV) and hyperimmune gammaglobulin (HGG) was instituted in 2 cases and successfully suppressed the replication of CMV, with sustained improvement in liver function. In 1 of these cases, signals for CMV DNA were undetectable in the liver 12 months after termination of combination therapy. These results help to confirm the etiology of CMV for persistent hepatitis in immunocompetent infants using the in situ hybridization method and also show the efficacy of combination therapy with a virostatic agent, GCV, and an immune-modulating agent, HGG.

Background Pityriasis rosea (PR) is a common papulosquamous skin disorder that is suspected to have an infectious aetiology. Objectives We aimed to study the role of human herpesvirus (HHV)-7 and HHV-6 in the pathogenesis of PR. Methods We performed seroepidemiological studies (indirect immunofluorescence test) and polymerase chain reaction (PCR) analysis for HHV-6 and HHV-7 in patients with PR. Seventy-two serum samples and 37 samples of peripheral blood mononuclear cells (PBMC) from 44 patients with PR were obtained. Twenty-five patients with other skin disorders such as drug eruption, urticaria or herpes zoster were studied as controls in the PCR analysis. Results HHV-7 DNA was detected in 13 of 30 (43%) samples of PBMC of the patients with PR and 14 of 25 (56%) samples of PBMC of controls. HHV-6 DNA was detected in six of 29 (21%) patients with PR and nine of 23 (39%) controls. Thus there was no difference in the prevalence of HHV-6 or HHV-7 in PBMC between patients with PR and those with other skin disorders. In the seroepidemiological study, two cases of at least a fourfold rise in titre and five cases of a fourfold decrease in titre to HHV-7 antibody, and two cases of a fourfold rise in titre and two cases of a fourfold decrease in titre to HHV-6 antibody, were observed in 24 patients with PR. This seroepidemiological study revealed antibody responses consistent with active infection in several PR patients, but the greater proportion of the patients had no definite increase in the antibody titres. Conclusions We conclude that HHV-7 and HHV-6 may play a part in some patients with PR, but that other causative agents may exist. Further analyses are needed to determine the causative agents of PR.

We analyzed the U79-80 gene of human herpesvirus 6 (HHV-6), which is predicted to be a positional homolog of the UL112-113 gene of human cytomegalovirus (HCMV). The U79-80 gene encoded a family of nuclear proteins of 36, 41, 44, and 59 kDa. These proteins had common amino termini and were generated by complex alternative splicing. Transcripts from U79-80 appeared as early as 3 h postinfection and could be detected in the presence of phosphonoformate. U79-80 proteins were seen as early as 8 h postinfection and could be detected in the presence of phosphonoformate but not in the presence of cycloheximide combined with actinomycin D treatment. The U79-80 proteins were localized to the nucleus of infected cells, where they were detected as a speckled or punctuate pattern. Moreover, the U79-80 proteins colocalized with the components of the viral DNA replication machinery and appeared to distribute adjacent to or touching nuclear domain 10, where viral DNA replication occurs. From the sequence analysis of genomic DNA, the predicted amino acid similarity between U79-80 and UL112-113 was lower than between other genes, but the characteristics of the transcripts and proteins encoded by U79-80 were similar to those of UL112-113. These results suggest that the U79-80 proteins have a role in viral DNA replication and are functional homologues of the UL112-113 proteins.

Kaposi's sarcoma-associated herpesvirus (KSHV), or human herpesvirus 8 (HHV-8), belongs to the gammaherpesvirus subfamily and encodes ∼80 open reading frames (ORFs). Among them are a few candidates for immediate-early genes (e.g., K5). We developed a monoclonal antibody (MAb), 328C7, against the K5 antigen. This MAb reacted with the K5 gene product by immunoscreening of a cDNA library from BCBL-1 cells, and this result was confirmed by transfection of the K5 ORF into Cos-7 cells. After induction of lytic infection by treatment with 12- O -tetradecanoylphorbol-13-acetate, MAb 328C7 reacted with an antigen in the cytoplasm of BCBL-1 and BC-3 cells as early as after 4 h of induction. Immunoelectron microscopy showed that the K5 antigen was situated mainly in the endoplasmic reticulum but was not present on the virion or in the nucleus. Northern blotting with a K5-specific probe revealed a single transcript of 1.2 kb, while Western blotting showed the antigen to be a 36-kDa polypeptide. The 5′ and 3′ ends were then determined by rapid amplification of cDNA, followed by sequencing of RACE products, and a splice was revealed upstream of the K5 ORF. K5 expression was unaffected by the respective DNA and protein synthesis inhibitors phosphonoformic acid and cycloheximide plus actinomycin D, confirming its immediate-early nature. Transient-transfection assays showed that the K5 promoter was transactivated by ORF 50 (KSHV Rta), a homolog of Epstein-Barr virus Rta, but the K5 gene product exhibited no transregulation of its own promoter or those of DNA polymerase and the human immunodeficiency virus type 1 long terminal repeat. This is the first such analysis of an immediate-early gene product; determination of its specific biological function requires further investigation.

We have attempted to reactivate human herpesvirus 6 (HHV-6) by infection with HHV-7 using childhood exanthem subitum patients in vitro. Peripheral blood mononuclear cells (PBMCs) were collected from children who had a history of exanthem subitum(ES) by HHV-6 and were infected by human herpesvirus 7 (HHV-7) in vitro. The antigen positive rate to HHV-6 started to increase 7 days after the infection and reached a maximum by Day 15 using an immunofluorescence antibody test. The copy number of HHV-6 DNA also increased in the samples in 10 days after infection in vitro. No antigen or increase in DNA was detected in PBMCs, that were mock-infected or infected with supernatant of stock virus after ultracentrifugation, suggesting that an infection by HHV-7 is necessary to reactivate HHV-6. In the paired sera samples during the acute and the convalescent phases of ES, seven to ten bands, that were specific for HHV-6, were recognized in samples from the acute phase, and at least 5 dominant polypeptides were found more intensively after HHV-7 infection.

When the nucleotide sequences of the Oka vaccine and its parental varicella-zoster virus (VZV) were compared in 6 open reading frames (ORFs), glycoprotein C (gC) and 5 transactivator genes, mutations were detected only in the immediate-early gene 62. The vaccine virus contained a mixture of different sequences that had variations at 15 nucleotide positions, but only one sequence was found for the Oka parental virus gene 62. The Oka vaccine virus gene 62 could be distinguished from the parental virus gene using a simplified restriction-enzyme fragment length polymorphism analysis, using NaeI and BssHII. This analysis was based on the sequence data obtained in this study. Studies of the regulatory activities of the ORF62 gene product (IE62) in a transient transfection assay indicated that IE62 of the parental virus had a stronger transactivational activity than that of the vaccine virus in activating immediate-early, early, and late gene promoters. These data suggest that IE62 might play an important role in the attenuation of VZV.

OBJECTIVE—The aim of the present study was to evaluate the regional cerebral blood flow (rCBF) in patients with postinfectious acute cerebellar ataxia using single photon emission computed tomography (SPECT). METHODS—Five children with postinfectious acute cerebellar ataxia and five control subjects were examined. The distribution of rCBF was measured by SPECT imaging after intravenous administration of 123I-IMP (111 MBq). The rCBF ratio—defined as the ratio of rCBF in the region of interest (ROI) to that in the occipital cortex—was calculated for each cortical and subcortical ROI. The mean rCBF ratio of each region was then compared between the ataxic and control subjects. These patients and all control subjects were also evaluated using MRI. RESULTS—The rCBF ratio was significantly lower in the cerebellum of the ataxic patients than in the cerebellum of the control subjects (p

The anti-herpesvirus activity of (1'S,2'R)-9-[[1',2'-bis(hydroxymethyl)cycloprop-1'-yl]methyl]guani ne (A-5021) was evaluated in murine cells and in several murine models of herpes simplex virus (HSV) infection. Against HSV type 1 (HSV-1), A-5021 was 15-30- and 30-60-fold more active, and against HSV type 2 (HSV-2), it was 2- and 8-fold more active than acyclovir and penciclovir in Balb/3T3 cells, respectively. When antiviral compounds were administered orally (once daily) to mice infected intraperitoneally with HSV-1 (Tomioka), A-5021 was more active than acyclovir or famciclovir in spite of its relatively low oral bioavailability. A-5021 was as active as penciclovir when the antiviral compounds were given intravenously (three times daily) to mice infected intraperitoneally with HSV-2 (186). In mice with a cutaneous HSV-1 (KOS) infection, three times daily oral therapy with A-5021 at 25 mg/kg per day produced more significant reduction in severity of skin lesions than equivalent treatment with acyclovir or famciclovir. In mice infected intracerebrally with HSV-1 (Tomioka), complete survival was observed in the group treated intravenously with A-5021 at 25 mg/kg per day (three times daily), while more than 50% of mice died in the groups treated intravenously with acyclovir of up to 100 mg/kg per day (three times daily). Moreover, A-5021 was more effective than acyclovir in clearing infectious virus from the brain. These findings demonstrate that A-5021 has potent anti-HSV activity in several murine models.

In the diagnosis of human cytomegalovirus (HCMV) infection, it is very important to distinguish symptomatic from asymptomatic infection. The nucleic acid sequence-based amplification (NASBA) technique was compared with single and nested polymerase chainreaction (PCR) methods. For NASBA detection, the beta2.7 transcript was chosen as a target because of its abundant active HCMV-specific expression. Of 20 pediatric bone marrow transplant (BMT) recipients, 8 developed HCMV-related clinical symptoms. The clinical sensitivities and specificities were 50% and 100% for single PCR, 100% and 67% for nested PCR, and 100% and 83% for NASBA, respectively. Follow-up of HCMV infections in pediatric BMT recipients showed that NASBA could both detect viral transcript prior to the onset of clinical symptoms and reflect clinical improvement due to antiviral therapy. These data suggest that NASBA should be useful for both predicting HCMV disease development and monitoring the effect of antiviral therapy.

The seroprevalence to Kaposi's sarcoma-associated herpesvirus (KSHV) or human herpesvirus type 8 (HHV-8) was surveyed in human immunodeficiency virus type 1 (HIV-1) carriers with or without skin diseases, and also in HIV-1 negative individuals in Thailand. Using an immunofluorescence assay, the seropositive rates to lytic antigens of HHV-8 in HIV-1 carriers with or without skin diseases were 25% and 7.4%, respectively, but none of HIV-1 negative individuals had antibody. The seroprevalence to HHV-8 antigens was high in HIV positive individuals with low CD4/CD8 ratio, suggesting that HHV-8 is reactivated during the immunosuppressive state. Several polypeptides with apparent molecular weights of 34-38,000 and 40,000, which were specific to HHV-8, were identified by the immunoprecipitation test using the seropositive sera. Our results suggested that HHV-8 co-existed with HIV in HIV-1 carriers and the existence of HHV-8 may be associated with clinical features in the skin.

The US22 gene family was first discovered in human cytomegalovirus (HCMV) and contains several conserved amino acid motifs. Human herpesvirus 6 (HHV-6) also encodes several genes belonging to the US22 family, including the U3 gene (a positional homolog of HCMV UL24). Because the gene products of the US22 gene family function as gene regulators in general, we analyzed the HHV-6A U3 gene. Six transcripts with different molecular weights of 7.5–1.8 kb were detected by Northern blot analysis using a U3-specific probe. Sequence analyses of the respective cDNA clones and primer extension experiments revealed that the U3 gene encoded the 2.0- and 3.5-kb transcripts and had no splicing within the U3 gene region. By immunofluorescence testing using antibodies raised to a fusion protein of MBP (maltose-binding protein) and U3, the U3 viral antigen was detected as early as 24 h p.i. in HHV-6A-infected U373 cells. The antigens were found in cytoplasmic granules, preferentially in the endoplasmic reticulum. Moreover, cotransfection assay using a luciferase gene expression system revealed that the U3 gene product was capable of activating the human immunodeficiency virus long terminal repeat promoter in CV1 cells.

A method was developed to distinguish the Oka vaccine strain of varicella-zoster virus (VZV) from other clinical isolates. The molecular characteristics of 52 clinical isolates from varicella or zoster patients with no history of VZV vaccination and the Oka strain, including vaccine and parental viruses, were analyzed by PstI cleavage of the PstI site-less (PSL) region. This was followed by single-strand conformational polymorphism (SSCP) after polymerase chain reaction amplification of repeating region 2 (R2). Most of the clinical isolates tested, especially recent isolates, had a PstI site in the PSL region, but the Oka strain did not. The SSCP patterns of R2 in Oka strain virus differed from those of other viruses. These results suggest that analysis of the PstI site followed by SSCP of R2 will be useful for identifying the Oka vaccine virus in isolates.

Neutralizing monoclonal antibody (MAb) escape mutants of Hantaan virus were generated using MAbs to envelope protein G1 (16D2) and G2 (11E10). The mutant viruses (mu16D2 and mu11E10), lacked reactivity only to the selecting MAb, or a MAb belonging to the same antigenic site. Both mutants had a single amino acid (a.a.) substitution. The a.a. substitution, found in mu16D2, was different from that found in another mutant selected with the same MAb (16D2). Although MAb 11E10 immunoprecipitated G2 protein, a deduced a.a. substitution was located in the G1 region. These results suggest that antigenic sites defined by neutralizing MAbs are composed of discontinuous epitopes over the G1 and G2 proteins. Mutant 11E10 showed a significant decrease in virulence in suckling mice. A virulence revertant of mu11E10, selected through passages in suckling mice brain, showed exactly the same deduced a.a. sequence as mu11E10 and still was not neutralized by MAb 11E10. Since mutant 16D2 was virulent for suckling mice, neutralization related epitopes found with MAbs 11E10 and 16D2 were independent of pathogenicity in BALB/c mice.

The adjuvant effect of two lipophilic derivatives of muramyl dipeptide (MDP), B30-MDP and MDP-Lys(L18), on the ability of an inactivated vaccine of B-1 virus (B-1 vaccine) to induce immune response against Hantavirus causing hemorrhagic fever with renal syndrome (HFRS) was examined. When mice were immunized subcutaneously (s.c.) twice at 2-week intervals with B-1 vaccine admixed with or without 100 micrograms mouse-1 of B30-MDP (B-1/B30-MDP) or MDP-Lys(L18) [B-1/MDP-Lys(L18)], mice immunized with B-1/B30-MDP as well as B-1/MDP-Lys(L18) showed significantly higher indirect fluorescent antibody (IFA) titers against HFRS virus than mice immunized with B-1 vaccine alone. Both mice treated with B-1/B30-MDP and B-1/MDP-Lys(L18) also exhibited significantly higher neutralizing antibody titers against HFRS virus than mice immunized with B-1 vaccine alone during 3-9 weeks after the primary immunization. The evaluation of antibody-producing cells by enzyme-linked immunospot (ELISPOT) assay on week 4 revealed that both MDP derivatives enhanced the number of HFRS virus-specific IgG1 and IgM antibody-producing cells. Furthermore, mice treated with B-1/B30-MDP as well as B-1/MDP-Lys(L18) showed a higher level of Th-2 type cytokines, IL-4 and IL-6, in sera than mice treated with B-1 alone. In an in-vitro analysis of T lymphocyte proliferation to baculovirus-expressed recombinant nucleocapsid protein (rNP) of Hantaan 76-118 strain, the splenocytes of mice treated with B-1/B30-MDP and B-1/MDP-Lys(L18) on week 4 showed a significantly higher proliferating activity than those treated with B-1 vaccine alone. In addition, when mice were immunized once with B-1 vaccine admixed with or without B30-MDP and MDP-Lys(L18) and followed by intrafootpad (i.f.) injection of B-1 vaccine on day 7, mice immunized with B-1/B30-MDP and B-1/MDP-Lys(L18) induced a higher delayed-type hypersensitivity (DTH) reaction than mice immunized with B-1 vaccine alone. These results suggest that B30-MDP and MDP-Lys(L18) are useful immunoadjuvants to enhance the ability of inactivated B-1 vaccine to induce a humoral and cellular response to HFRS virus.

Dextran sulfate (DS) is a negatively charged sulfated polysaccharide that suppresses the replication of influenza A viruses. The suppression was thought to be associated with inhibition of the hemagglutinin-dependent fusion activity. However, we previously showed that suppression by DS was observed not only at the initial stage of viral infection, but also later when virus is released from infected cells due to inhibition of neuraminidase (NA) activity. In the present study, we isolated DS-resistant A/Puerto Rico/8/34 (PR8) influenza viruses and analyzed the inhibition by DS. We found six mutations in NA genes of five independent resistant PR8 viruses and each resistant NA gene had two mutations. All mutations were from basic to acidic or neutral amino acids. In addition, R430L, K432E or K435E in the 430-435 region was a common mutation in all resistant NA genes. To determine which amino acid(s) are responsible for this resistance, a panel of recombinant viruses containing a PR8 and A/WSN/33(WSN) chimeric NA gene or an NA gene with different mutation(s) was generated using reverse genetics. Using recombinant viruses containing a PR8/WSN chimeric NA, we showed that one third of the C-terminal region of PR8 NA was responsible for DS-sensitivity. Recombinant viruses with a single mutation in NA replicated better than wild-type PR8 in the presence of DS, but were still DS-sensitive. However, replication of recombinant viruses with double mutations from the resistant viruses was not affected by the presence or absence of DS. In addition, resistant recombinant viruses were found to be sensitive to the NA inhibitor, oseltamivir and the oseltamivir-resistant recombinant virus was sensitive to DS. These results suggested that DS is an NA inhibitor with a different mechanism of action from the currently used NA inhibitors and that DS could be used in combination with these inhibitors to treat influenza virus infections.

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.

Human herpesvirus 6 (HHV-6) is now classified into two distinct variants such as HHV-6 variant A(HHV-6A) and B(HHV-6B) (Ablashi et al., Arch. Virol. 129, 1993, 1–4) and the DNA of HHV-6A strain U1102 was completely sequenced (Gompels et al., Virology 209, 1995, 29–51). We have sequenced a 30-kilobase pair (kbp) (genomic positions around 111–141 kb) of HHV-6B strain HST, and a sequence of this region was compared with that of HHV-6A strain U1102. Dodecameric repeats, G/T and Kpn repeat elements, putative major immediate early 1 (MIE1) and major immediate early 2 (MIE2) genes were found in this region. The DNA sequences of HHV-6A (U1102) and HHV-6B (HSI) were markedly different in the MIE1 region, Kpn repeat elements and the putative MIE2 region. Dodecameric repeat element was located in the putative MIE2 locus of HHV-6. When primers covering dodecameric repeat region were used to amplify HHV-6 DNA of clinical isolates from patients with exanthem subitum (ES) by polymerase chain reaction (PCR), variations in size of PCR products in each isolate were found, indicating strain-specific features. Furthermore, the results of molecular biological analysis by PCR using DNA samples in a family suggest that HHV-6 infects within a family.

Using polyclonal and monoclonal (MAbs) antibodies to human herpesvirus 7 (HHV-7), we have studied HHV-7-specific polypeptides. Human sera were obtained during the convalescent phase from patients with exanthem subitum due to HHV-7, and at least 16 HHV-7-specific polypeptides with apparent molecular masses of 26-210 kDa were immuno-precipitated. Sera prepared in mice also precipitated at least 17 HHV-7-specific polypeptides with molecular masses of 26-210 kDa. Among them, the most commonly observed antigenic protein had an apparent molecular mass of 52 kDa. Forty-two clones secreting MAbs against HHV-7-specific proteins, as determined by immunofluorescence assays, were established from BALB/c mice immunized with HHV-7-infected cell extracts. Seven MAbs which immunoprecipitated HHV-7-specific polypeptides were further characterized. Two of these, MAbs 5E12 and 5F12, reacted predominantly with glyco-proteins of 78 kDa and 85 kDa, respectively, and possessed neutralizing activity. This suggests that there are at least two neutralization-inducing proteins in HHV-7. MAb 16B4 reacted with the major immunogenic protein of 52 kDa. Five of the 42 MAbs also reacted in immunofluorescence assays with HHV-6 antigens to the same degree as to HHV-7. Two other MAbs, 7C10 and 10F1, recognized an HHV-7 protein of 40 kDa, and only 7C10 cross-reacted with an HHV-6 protein of 45 kDa.

We have developed a new sensitive enzyme immunoassay (EIA) and MTT (tetrazolium salt) assay for screening compounds against two variants of human herpesvirus 6 (HHV-6A, HHV-6B) and human herpesvirus 7 (HHV-7) and evaluated the anti-HHV-6 and HHV-7 activity of a series of anti-herpesvirus compounds and acyclic nucleoside phosphonate analogues. The results indicate that the pattern of activity of these compounds against these betaherpesviruses is similar to that for human cytomegalovirus (HCMV). The highest potency and selectivity against the two variants of HHV-6 and HHV-7 was demonstrated by S2242 (N7-isomer of 6-deoxy-ganciclovir). Also, ganciclovir (GCV), foscarnet, (phosphonoformic acid; PFA) and the acyclic nucleoside phosphonate analogues such as cidofovir (HPMPC) exhibited selective inhibitory activity against these viruses. Thymidine kinase (TK)-dependent drugs (acyclovir, ACV; brivudin, BVDU; and sorivudine, BVaraU) showed little, if any, activity. These results suggest a structural homology of the DNA polymerase and a lack of TK gene among these three betaherpesviruses (HHV-6, HHV-7 and HCMV). The finding that HHV-7 was highly sensitive to GCV also suggests that HHV-7 may have an HCMV-UL97-homologue gene for the phosphorylation of GCV. The present EIA method is more rapid and sensitive than the previously reported procedures and could be useful for the large-scale screening of compounds against HHV-6 and HHV-7.

The ORF49 tegument protein of varicella-zoster virus (VZV) is one of the core gene products that is conserved among herpesvirus family members. Although ORF49 is known to be a cell-tropic factor, its detailed functions remain elusive. ORF44 is another core gene product reported to be essential, although its characterization and detailed functional analysis have not been reported. These two core gene products form a complex in other herpesviruses beyond the host species and herpesvirus subfamilies. Here, we show that complex formation between ORF44 and ORF49 is conserved in VZV. We serendipitously found that binding is eliminated by an amino acid substitution at position 129 (phenylalanine 129), and four amino acids in the carboxyl-terminal half of the acidic cluster in ORF49 (i.e., aspartate-phenylalanine-aspartate-glutamate from positions 41 to 44 [41DFDE44]) were identified as its binding motif. Alanine substitutions in each domain rendered the ORF44F129A mutation lethal for VZV, similar to deletion of the entire ORF44. The phenotype of the ORF49-41AAAA44 mutation was comparable to that of the ORF49-defective virus, including small-plaque formation, impaired growth, and low infectious virus production. These results suggest that the interaction between ORF44 and ORF49 is essential for their role in VZV infection and that ORF49 is required for the efficient production of infectious progeny virus mediated by the conserved interaction between the two proteins.

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.

The gene for the human herpes virus 7 (HHV-7) glycoprotein B (gB) has been identified by sequencing a molecularly cloned HHV-7 DNA fragment. A 2.5-kb open reading frame (ORF) encoded a protein of 822 amino acids with characteristics of a transmembrane glycoprotein, and showed the strongest similarity (56.5%) with the human herpesvirus 6 (HHV-6) gB. The genes for the transport/capsid assembly protein (tp/cap) and the DNA polymerase (pol) existed upstream and downstream of the gB gene, respectively. This arrangement was the same as that of HHV-6. Antisera were generated by immunizing mice with a glutathione S-transferase-carboxy terminal gB fusion protein. Immunofluorescent tests demonstrated that the antisera reacted specifically with HHV-7 antigens in cytoplasm of infected cells. The antisera immunoprecipitated proteins with apparent molecular masses of 51, 63 and 112 kDa from HHV-7 infected cells by pulse-chase analysis. In the presence of tunicamycin, the protein with a molecular mass of 112 kDa was replaced by a protein with a molecular mass of 88 kDa, and this size was consistent with the predicted size of the primary translation product of the HHV-7 gB gene. These results suggested that the protein with a molecular mass of 112 kDa was a glycoprotein synthesized by addition of N-linked oligosaccharides to a non-glycosylated precursor of the protein with a molecular mass of 88 kDa and then cleaved into the proteins with molecular masses of 51 and 63 kDa in HHV-7 infected cells.

The novel herpesvirus-like DNA sequences, which were identified in AIDS-related Kaposi's sarcoma (KS) and designated KS associated herpesvirus (KSHV) or human herpesvirus 8 (HHV-8), have been reported to be associated with various forms of KS. Here, we searched for the presence of KSHV sequences in various other skin lesions including premalignant or malignant skin tumors of a total of 69 clinical cases without immunodeficiency due to AIDS or following organ transplantation. Strikingly high rates of detection were obtained for premalignant Bowen's disease and malignant squamous cell carcinoma, accounting for 71.4% and 50%, respectively. A less frequent but as yet high incidence (33.3%) was scored for actinic keratosis, a premalignant epidermal disorder. In contrast, the frequency remained low (16.7%) for another type of proliferative skin lesions of extramammary Paget's disease and non-proliferative skin lesions (dermatitis, morphea, epidermal cyst and scar). These results suggest a close association of KSHV with at least some non-KS malignant and premalignant skin lesions in non-immunocompromised patients.

The clinical features of infection with human herpesvirus 7 (HHV-7) are not well described. Exanthem subitum is the only illness that is confirmed to be caused by HHV-7. We report two children who had exanthem subitum associated with central nervous system manifestations. Two strains of HHV-7 were isolated sequentially from peripheral blood mononuclear cells and saliva of the some child who had exanthem subitum complicated with acute hemiplegia in childhood. Two strains were confirmed to be HHV-7 by means of monoclonal antibodies to human herpesvirus 6 (HHV-6) and HHV-7, polymerase chain reaction, and DNA analysis. During the convalescent period, the antibody titer to HHV-7 rose from less than 1:10 to 1:320, whereas the antibody titer to HHV-6 remained less than 1:10. Another child with exanthem subitum complicated by acute hemiplegia had serologic evidence of primary HHV-7 infection. These two cases demonstrate a new relationship between HHV-7 and central nervous system symptoms.

The infectivity of human herpesvirus 6 (HHV-6) in a human hepatoma cell line, Hep G2 cells, and the effect of HHV-6 on production of inflammatory cytokines in these cells were examined to analyze pathogenesis of HHV-6 in the liver. We demonstrated that Hep G2 cells were susceptible to infection with HHV-6, and produced infectious virus. Moreover, infection of Hep G2 cells by HHV-6 induced the expression of IL-8 mRNA, but not IL-1 beta. The effect on induction of IL-8 gene expression was observed only in Hep G2 cells infected with infectious virus, whereas both heat-inactivated HHV-6 and UV-irradiated HHV-6 did not change the IL-8 mRNA level in these cells. These data suggest that HHV-6 may induce the cytokine-mediated inflammatory response by infecting liver cells, which could result in liver dysfunction in vivo.

Segments of an immediate-early (IE) protein (IE03; 958 amino acids (aa)), encoded by clone pSTY03, of human herpesvirus 6 (HHV-6) variant B strain HST were expressed as β-galactosidase fusion proteins in Escherichia coli . Using Western blot analysis, and the serum of a patient having high titer anti-HHV-6 antibodies, an antigenic region of the IE03 protein was mapped between residues 340 and 505 (pUE03IE-M). The fusion protein expresded in E. coli harboring plasmid pUE03IE-M was purified after electrophoresis in SDS-PAGE, and then immunized in mice to obtain a monospecific antibody. Monospecific antibody raised against the fusion protein reacted with IE03 protein species with apparent molecular weights of 155 and 170 kDa, and was detected as granular fluorescence in nuclei of infected cells by an immunofluorescence antibody test. Furthermore, this antibody reacted only with HHV-6 variant B, but did not react with HHV-6 variant A. The IE03 protein was confirmed to be an IE protein, since the synthesis of this protein was observed in infected cells that were first treated with cycloheximide, which was then replaced with actinomycin D. Further, it was also detected as early as 4 h after infection.

CFS, a recently named heterogeneous disorder, is an illness of unknown etiology. The association of CFS with viral infections has been suggested. A common association between CFS and several viruses examined has not been confirmed. Here, we centered on the possible link between CFS and BDV infection. By nested RT-PCR followed by hybridization, BDV RNA was demonstrated as a clear signal in PBMCs in 3 out of 25 CFS patients. The amplified cDNA fragments were cloned and sequenced. A total of 16 clones were studied. Intra-patients divergencies of the p24 were 2–9%, 3–20%, and 3–11% in the deduced amino acids. Inter-patient divergencies among the 16 clones were 3–24%. Antibodies to recombinant BDV p24 protein were detected in 6 CFS patients including one carrying BDV RNA. Overall, these gave the prevalence of 32% (8/25) in Japanese CFS patients, suggesting that Japanese CFS is highly associated with active infection of BDV, or a related agent.