Molecular Aspects of Varicella-Zoster Virus Latency

Primary varicella-zoster virus (VZV) infection causes varicella (chickenpox) and the establishment of a lifelong latent infection in ganglionic neurons. VZV reactivates in about one-third of infected individuals to cause herpes zoster, often accompanied by neurological complications. The restricted host range of VZV and, until recently, a lack of suitable in vitro models have seriously hampered molecular studies of VZV latency. Nevertheless, recent technological advances facilitated a series of exciting studies that resulted in the discovery of a VZV latency-associated transcript (VLT) and provide novel insights into our understanding of VZV latency and factors that may initiate reactivation. Deducing the function(s) of VLT and the molecular mechanisms involved should now be considered a priority to improve our understanding of factors that govern VZV latency and reactivation. In this review, we summarize the implications of recent discoveries in the VZV latency field from both a virus and host perspective and provide a roadmap for future studies

Development of 3D Bit-Map-Based CAD and Its Application to Hydraulic Pump Model Fabrication

A novel 3D bit-map-based CAD has been developed. This program, named CellCAD, relies on techniques which provides easy manipulation of huge sized 3D bit-map, poly-triangle and 2D bit-map data. It can be attached to various kinds of additional plug-in processors to extend its functions and to customize it highly for broad applications. CellCAD can be applied in fields using computed tomography digitizers and/or layering fabricators. The authors applied CellCAD to hydraulic pump model fabrication. This paper reports the basic design concept and implementation techniques of CellCAD from the viewpoint of design engineering, and also introduces the methods and results of an actual CellCAD application.Mechanical Engineerin

神経誘導因子Netrin-1は軟骨形成や骨形成においてBMPまたはNogginにより調節される

This is the first report describing neurogenic factor of Netrin-1 related to chondrogenesis or osteogenesis in a human cells. Netrin is a morphogenetic factor that induces a growth cone of an axial filament of the nervous system. However, the roles of Netrin in chondrogenesis or osteogenesis are not yet understood. We analyzed the relationship between Netrin and bone morphogenetic protein-2 (BMP-2) in chondrogenesis or osteogenesis, using a human chondrocyte-like cell line (USAC), which also retains multi-potency to differentiate into osteoblasts and adipocytes. Netrin-1 mRNA was decreased in USAC cells, though the expression was increased during osteogenic differentiation at the stage when osteocalcin mRNA were increased by BMP-2. Furthermore, inhibition of Netrin-1 gene increased Cbfa1 mRNA expression, and decreased Sox9 mRNA expression. We also found that Netrin-1was strongly expressed in immature chondrocytes of cartilage-like tissues that were formed in an exo vivo experiment with diffusion chambers. The se findings indicate that Netrin-1 and BMP-2 regulates in the stage dependent process of mesenchymal cell differentiation to chondrocytes or osteoblasts.骨芽細胞または脂肪細胞への分化多能を保持するヒト軟骨細胞様細胞系(USAC)を用い、軟骨形成または骨形成におけるNetrinと骨形成蛋白質-2(BMP-2)との関係を調べた。Netrin-1 mRNAはUSAC細胞中では減少するが、オステオカルシンmRNA濃度がBMPによって上昇する際の骨芽細胞分化時にNetrin-1 mRNAの発現が増加した。Netrin-1遺伝子を阻害すると、Cbfal mRNA発現は増加しSox9 mRNA発現は減少した。またNetrin-1は軟骨様組織の未成熟軟骨細胞において強く発現した。Netrin-1とBMP-2が、間葉細胞の軟骨細胞または骨芽細胞へ分化プロセスを制御すると考えた

Varicella-zoster virus ORF 58 gene is dispensable for viral replication in cell culture

<p>Abstract</p> <p>Background</p> <p>Open reading frame 58 (ORF58) of varicella-zoster virus (VZV) lies at the 3'end of the Unique long (U_L) region and its functional is unknown. In order to clarify whether ORF58 is essential for the growth of VZV, we constructed a deletion mutant of ORF58 (pOka-BACΔ58) from the Oka parental genome cloned into a bacterial artificial chromosome (pOka-BAC).</p> <p>Results</p> <p>The ORF58-deleted virus (rpOkaΔ58) was reconstituted from the pOka-BACΔ58 genome in MRC-5 cells, indicating that the ORF58 gene is non-essential for virus growth. Comparison of the growth rate of rpOkaΔ58 and recombinant wild-type virus by assessing plaque sizes revealed no significant differences between them both in MRC-5 cells and malignant melanoma cells.</p> <p>Conclusion</p> <p>This study shows that the ORF58 gene is dispensable for viral replication and does not affect the virus' ability to form plaques <it>in vitro</it>.</p

A Variant Allele in Varicella-Zoster Virus Glycoprotein B Selected during Production of the Varicella Vaccine Contributes to Its Attenuation

Attenuation of the live varicella Oka vaccine (vOka) has been attributed to mutations in the genome acquired during cell culture passage of pOka (parent strain); however, the precise mechanisms of attenuation remain unknown. Comparative sequence analyses of several vaccine batches showed that over 100 single-nucleotide polymorphisms (SNPs) are conserved across all vaccine batches; 6 SNPs are nearly fixed, suggesting that these SNPs are responsible for attenuation. By contrast, prior analysis of chimeric vOka and pOka recombinants indicates that loci other than these six SNPs contribute to attenuation. Here, we report that pOka consists of a heterogenous population of virus sequences with two nearly equally represented bases, guanine (G) or adenine (A), at nucleotide 2096 of the ORF31 coding sequence, which encodes glycoprotein B (gB) resulting in arginine (R) or glutamine (Q), respectively, at amino acid 699 of gB. By contrast, 2096A/699Q is dominant in vOka (.99.98%). gB699Q/gH/gL showed significantly less fusion activity than gB699R/gH/gL in a cell-based fusion assay. Recombinant pOka with gB669Q (rpOka_gB699Q) had a similar growth phenotype as vOka during lytic infection in cell culture including human primary skin cells; however, rpOka_gB699R showed a growth phenotype similar to pOka. rpOka_gB699R entered neurons from axonal terminals more efficiently than rpOka_gB699Q in the presence of cell membrane-derived vesicles containing gB. Strikingly, when a mixture of pOka with both alleles equally represented was used to infect human neurons from axon terminals, pOka with gB699R was dominant for virus entry. These results identify a variant allele in gB that contributes to attenuation of vOka. IMPORTANCE The live-attenuated varicella vaccine has reduced the burden of chickenpox. Despite its development in 1974, the molecular basis for its attenuation is still not well understood. Since the live-attenuated varicella vaccine is the only licensed human herpesvirus vaccine that prevents primary disease, it is important to understand the mechanism for its attenuation. Here we identify that a variant allele in glycoprotein B (gB) selected during generation of the varicella vaccine contributes to its attenuation. This variant is impaired for fusion, virus entry into neurons from nerve terminals, and replication in human skin cells. Identification of a variant allele in gB, one of the essential herpesvirus core genes, that contributes to its attenuation may provide insights that assist in the development of other herpesvirus vaccines

Characterization of the human herpesvirus 6A U23 gene

AbstractHuman herpesvirus 6 (HHV-6), which replicates abundantly in T cells, belongs to the Roseolovirus genus within the betaherpesvirus subfamily. Members of the Roseolovirus genus encode seven unique genes, U20, U21, U23, U24, U24A, U26, and U100. The present study focused on one of these, U23, by analyzing the characteristics of its gene product in HHV-6A-infected cells. The results indicated that the U23 protein was expressed at the late phase of infection as a glycoprotein, but was not incorporated into virions, and mostly stayed within the trans Golgi network (TGN) in HHV-6A-infected cells. Furthermore, analysis using a U23-defective mutant virus showed that the gene is nonessential for viral replication in vitro

In vitro system using human neurons demonstrates that varicella-zoster vaccine virus is impaired for reactivation, but not latency

Varicella-zoster virus (VZV) establishes latency in human sensory and cranial nerve ganglia during primary infection (varicella), and the virus can reactivate and cause zoster after primary infection. The mechanism of how the virus establishes and maintains latency and how it reactivates is poorly understood, largely due to the lack of robust models. We found that axonal infection of neurons derived from hESCs in a microfluidic device with cell-free parental Oka (POka) VZV resulted in latent infection with inability to detect several viral mRNAs by reverse transcriptase-quantitative PCR, no production of infectious virus, and maintenance of the viral DNA genome in endless configuration, consistent with an episome configuration. With deep sequencing, however, multiple viral mRNAs were detected. Treatment of the latently infected neurons with Ab to NGF resulted in production of infectious virus in about 25% of the latently infected cultures. Axonal infection of neurons with vaccine Oka (VOka) VZV resulted in a latent infection similar to infection with POka; however, in contrast to POka, VOka-infected neurons were markedly impaired for reactivation after treatment with Ab to NGF. In addition, viral transcription was markedly reduced in neurons latently infected with VOka compared with POka. Our in vitro system recapitulates both VZV latency and reactivation in vivo and may be used to study viral vaccines for their ability to establish latency and reactivate

A spliced latency-associated VZV transcript maps antisense to the viral transactivator gene

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

Atelocollagen enhanced osteogenesis in a geometricstructured beta–TCP scaffold by VEGF induction.

In order to establish the convertibility of a host for bone augmentation, we herein developed a new honeycombshaped β-tricalcium phosphate (37H) using atelocollagen as a scaffold, which exhibited unique geometric properties for in vitro and in vivo studies.Human mesenchymal stem cells (MSC) were cultured with 37H or atelocollagen-coated honeycomb-shaped β-tricalcium phosphate (Col37H), and their osteoblastic differentiation was then analyzed. Atelocollagen promoted cell adhesion and formation of vessel-like structures in the tunnels of scaffolds of cultured MSC. The mRNA expression levels of type I collagen, osteocalcin, and VEGF were greater in MSC cultured with Col37H than with 37H. Bone generation with Col37H in the rat calvaria was greater than with 37H, and this was attributed to early vessel construction. A large number of blood vessels invaded tunnels from the periosteum and existing bone surface. A strong VEGF signal was detected immediately before the new bone surface in the tunnels of Col37H. These results indicate that the addition of atelocollagen to Col37H has potential in the construction of functional artificial bone