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  • A site of varicella-zoster virus vulnerability identified by structural studies of neutralizing antibodies bound to the glycoprotein complex gHgL PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Xing, Y., Oliver, S. L., TuongVi Nguyen, T., Ciferri, C., Nandi, A., Hickman, J., Giovani, C., Yang, E., Palladino, G., Grose, C., Uematsu, Y., Lilja, A. E., Arvin, A. M., Carfi, A. 2015; 112 (19): 6056-6061

    Abstract

    Varicella-zoster virus (VZV), of the family Alphaherpesvirinae, causes varicella in children and young adults, potentially leading to herpes zoster later in life on reactivation from latency. The conserved herpesvirus glycoprotein gB and the heterodimer gHgL mediate virion envelope fusion with cell membranes during virus entry. Naturally occurring neutralizing antibodies against herpesviruses target these entry proteins. To determine the molecular basis for VZV neutralization, crystal structures of gHgL were determined in complex with fragments of antigen binding (Fabs) from two human monoclonal antibodies, IgG-94 and IgG-RC, isolated from seropositive subjects. These structures reveal that the antibodies target the same site, composed of residues from both gH and gL, distinct from two other neutralizing epitopes identified by negative-stain electron microscopy and mutational analysis. Inhibition of gB/gHgL-mediated membrane fusion and structural comparisons with herpesvirus homologs suggest that the IgG-RC/94 epitope is in proximity to the site on VZV gHgL that activates gB. Immunization studies proved that the anti-gHgL IgG-RC/94 epitope is a critical target for antibodies that neutralize VZV. Thus, the gHgL/Fab structures delineate a site of herpesvirus vulnerability targeted by natural immunity.

    View details for DOI 10.1073/pnas.1501176112

    View details for Web of Science ID 000354390600062

    View details for PubMedID 25918416

    View details for PubMedCentralID PMC4434712

  • The cytoplasmic domain of varicella-zoster virus glycoprotein h regulates syncytia formation and skin pathogenesis. PLoS pathogens Yang, E., Arvin, A. M., Oliver, S. L. 2014; 10 (5)

    View details for DOI 10.1371/journal.ppat.1004173

    View details for PubMedID 24874654

  • An immunoreceptor tyrosine-based inhibition motif in varicella-zoster virus glycoprotein B regulates cell fusion and skin pathogenesis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Oliver, S. L., Brady, J. J., Sommer, M. H., Reichelt, M., Sung, P., Blau, H. M., Arvin, A. M. 2013; 110 (5): 1911-1916

    Abstract

    Herpesvirus entry functions of the conserved glycoproteins gB and gH-gL have been delineated, but their role in regulating cell-cell fusion is poorly understood. Varicella-zoster virus (VZV) infection provides a valuable model for investigating cell-cell fusion because of the importance of this process for pathogenesis in human skin and sensory ganglia. The present study identifies a canonical immunoreceptor tyrosine-based inhibition motif (ITIM) in the gB cytoplasmic domain (gBcyt) and demonstrates that the gBcyt is a tyrosine kinase substrate. Orbitrap mass spectrometry confirmed that Y881, central to the ITIM, is phosphorylated. To determine whether the gBcyt ITIM regulates gB/gH-gL-induced cell-cell fusion in vitro, tyrosine residues Y881 and Y920 in the gBcyt were substituted with phenylalanine separately or together. Recombinant viruses with these substitutions were generated to establish their effects on syncytia formation in replication in vitro and in the human skin xenograft model of VZV pathogenesis. The Y881F substitution caused significantly increased cell-cell fusion despite reduced cell-surface gB. Importantly, the Y881F or Y881/920F substitutions in VZV caused aggressive syncytia formation, reducing cell-cell spread. These in vitro effects of aggressive syncytia formation translated to severely impaired skin infection in vivo. In contrast, the Y920F substitution did not affect virus replication in vitro or in vivo. These observations suggest that gB modulates cell-cell fusion via an ITIM-mediated Y881 phosphorylation-dependent mechanism, supporting a unique concept that intracellular signaling through this gBcyt motif regulates VZV syncytia formation and is essential for skin pathogenesis.

    View details for DOI 10.1073/pnas.1216985110

    View details for Web of Science ID 000314558100067

    View details for PubMedID 23322733

    View details for PubMedCentralID PMC3562845

  • HIV-1 inhibitory properties of eCD4-Igmim2 determined using an Env-mediated membrane fusion assay. PloS one Yang, E., Gardner, M. R., Zhou, A. S., Farzan, M., Arvin, A. M., Oliver, S. L. 2018; 13 (10): e0206365

    Abstract

    Human Immunodeficiency Virus-1 (HIV-1) entry is dependent on the envelope glycoprotein (Env) that is present on the virion and facilitates fusion between the envelope and the cellular membrane. The protein consists of two subunits, gp120 and gp41, with the former required for binding the CD4 receptor and either the CXCR4 or CCR5 coreceptor, and the latter for mediating fusion. The requirement of fusion for infection has made Env an attractive target for HIV therapy development and led to the FDA approval of enfuvirtide, a fusion inhibitor. Continued development of entry inhibitors is warranted because enfuvirtide resistant HIV-1 strains have emerged. In this study, a novel HIV-1 fusion assay was validated using neutralizing antibodies and then used to investigate the mechanism of action of eCD4-Igmim2, an HIV-1 inhibitor proposed to cooperatively bind the CD4 binding site and the sulfotyrosine-binding pocket of gp120. Greater reduction in fusion levels was observed with eCD4-Igmim2 in the fusion assay than all of the gp120 antibodies evaluated. Lab adapted isolates, HIV-1HXB2 and HIV-1YU2, were sensitive to eCD4-Igmim2 in the fusion assay, while primary isolates, HIV-1BG505 and HIV-1ZM651 were resistant. These results correlated with greater IC50 values for primary isolates compared to the lab adapted isolates observed in a virus neutralization assay. Analysis of gp120 models identified differences in the V1 and V2 domains that are associated with eCD4-Igmim2 sensitivity. This study highlights the use of a fusion assay to identify key areas for improving the potency of eCD4-Igmim2.

    View details for PubMedID 30359435

  • The Glycoprotein B Cytoplasmic Domain Lysine Cluster Is Critical for Varicella-Zoster Virus Cell-Cell Fusion Regulation and Infection JOURNAL OF VIROLOGY Yang, E., Arvin, A. M., Oliver, S. L. 2017; 91 (1)
  • Dysregulated Glycoprotein B-Mediated Cell-Cell Fusion Disrupts Varicella-Zoster Virus and Host Gene Transcription during Infection JOURNAL OF VIROLOGY Oliver, S. L., Yang, E., Arvin, A. M. 2017; 91 (1)
  • Varicella-Zoster Virus Glycoproteins: Entry, Replication, and Pathogenesis. Current clinical microbiology reports Oliver, S. L., Yang, E., Arvin, A. M. 2016; 3 (4): 204-215

    Abstract

    Varicella-zoster virus (VZV), an alphaherpesvirus that causes chicken pox (varicella) and shingles (herpes zoster), is a medically important pathogen that causes considerable morbidity and, on occasion, mortality in immunocompromised patients. Herpes zoster can afflict the elderly with a debilitating condition, postherpetic neuralgia, triggering severe, untreatable pain for months or years. The lipid envelope of VZV, similar to all herpesviruses, contains numerous glycoproteins required for replication and pathogenesis.To summarize the current knowledge about VZV glycoproteins and their roles in cell entry, replication and pathogenesis.The functions for some VZV glycoproteins are known, such as gB, gH and gL in membrane fusion, cell-cell fusion regulation, and receptor binding properties. However, the molecular mechanisms that trigger or mediate VZV glycoproteins remains poorly understood.VZV glycoproteins are central to successful replication but their modus operandi during replication and pathogenesis remain elusive requiring further mechanistic based studies.

    View details for DOI 10.1007/s40588-016-0044-4

    View details for PubMedID 28367398

  • Role for the aV Integrin Subunit in Varicella-Zoster Virus-Mediated Fusion and Infection. Journal of virology Yang, E., Arvin, A. M., Oliver, S. L. 2016; 90 (16): 7567-7578

    Abstract

    Varicella-zoster virus (VZV) is an alphaherpesvirus that causes varicella and herpes zoster. Membrane fusion is essential for VZV entry and the distinctive syncytium formation in VZV-infected skin and neuronal tissue. Herpesvirus fusion is mediated by a complex of glycoproteins gB and gH-gL, which are necessary and sufficient for VZV to induce membrane fusion. However, the cellular requirements of fusion are poorly understood. Integrins have been implicated to facilitate entry of several human herpesviruses, but their role in VZV entry has not yet been explored. To determine the involvement of integrins in VZV fusion, a quantitative cell-cell fusion assay was developed using a VZV-permissive melanoma cell line. The cells constitutively expressed a reporter protein and short hairpin RNAs (shRNAs) to knock down the expression of integrin subunits shown to be expressed in these cells by RNA sequencing. The ?V integrin subunit was identified as mediating VZV gB/gH-gL fusion, as its knockdown by shRNAs reduced fusion levels to 60% of that of control cells. A comparable reduction in fusion levels was observed when an anti-?V antibody specific to its extracellular domain was tested in the fusion assay, confirming that the domain was important for VZV fusion. In addition, reduced spread was observed in ?V knockdown cells infected with the VZV pOka strain relative to that of the control cells. This was demonstrated by reductions in plaque size, replication kinetics, and virion entry in the ?V subunit knockdown cells. Thus, the ?V integrin subunit is important for VZV gB/gH-gL fusion and infection.Varicella-zoster virus (VZV) is a highly infectious pathogen that causes chickenpox and shingles. A common complication of shingles is the excruciating condition called postherpetic neuralgia, which has proven difficult to treat. While a vaccine is now available, it is not recommended for immunocompromised individuals and its efficacy decreases with the recipient's age. These limitations highlight the need for new therapies. This study examines the role of integrins in membrane fusion mediated by VZV glycoproteins gB and gH-gL, a required process for VZV infection. This knowledge will further the understanding of VZV entry and provide insight into the development of better therapies.

    View details for DOI 10.1128/JVI.00792-16

    View details for PubMedID 27279620

    View details for PubMedCentralID PMC4984616

  • Molecular mechanisms of varicella zoster virus pathogenesis NATURE REVIEWS MICROBIOLOGY Zerboni, L., Sen, N., Oliver, S. L., Arvin, A. M. 2014; 12 (3): 197-210

    Abstract

    Varicella zoster virus (VZV) is the causative agent of varicella (chickenpox) and zoster (shingles). Investigating VZV pathogenesis is challenging as VZV is a human-specific virus and infection does not occur, or is highly restricted, in other species. However, the use of human tissue xenografts in mice with severe combined immunodeficiency (SCID) enables the analysis of VZV infection in differentiated human cells in their typical tissue microenvironment. Xenografts of human skin, dorsal root ganglia or foetal thymus that contains T cells can be infected with mutant viruses or in the presence of inhibitors of viral or cellular functions to assess the molecular mechanisms of VZV-host interactions. In this Review, we discuss how these models have improved our understanding of VZV pathogenesis.

    View details for DOI 10.1038/nrmicro3215

    View details for Web of Science ID 000331623900006

    View details for PubMedID 24509782

    View details for PubMedCentralID PMC4066823

  • ORF11 Protein Interacts with the ORF9 Essential Tegument Protein in Varicella-Zoster Virus Infection JOURNAL OF VIROLOGY Che, X., Oliver, S. L., Reichelt, M., Sommer, M. H., Haas, J., Rovis, T. L., Arvin, A. M. 2013; 87 (9): 5106-5117

    Abstract

    The tegument proteins encoded by ORF11 and ORF9 of varicella-zoster virus (VZV) are conserved among all alphaherpesvirus. We previously demonstrated that the ORF9 gene is essential, whereas ORF11 is dispensable in vitro but its deletion severely impairs VZV infection of skin xenografts in the SCID mouse model in vivo. Here we report that ORF11 protein interacts with ORF9 protein in infected cells as well as in the absence of other viral proteins, and we have mapped the ORF11 protein domain involved in their interaction. Although ORF11 is an RNA binding protein, the interaction between ORF11 and ORF9 proteins was not mediated by RNA or DNA bridging. VZV recombinants with mutations preventing ORF11 protein binding to ORF9 protein had no effect on 6-day growth kinetics based on plaque numbers, but plaque sizes were reduced in vitro. However, disruption of the ORF11 and ORF9 protein interaction was associated with failure to replicate in skin xenografts in vivo. Further, we demonstrate that in the absence of their interaction, the ORF9 protein displays an identical cellular localization, accumulating in the trans-Golgi region, whereas the ORF11 protein exhibits aberrant localization, dispersing throughout the cytoplasm. Overall, our observations suggest that while complete tegument assembly may not be necessary for VZV replication in vitro, the interaction between the ORF11 and ORF9 proteins appears to be critical for the proper localization of ORF11 protein to the assembly complex and for production of infectious virus during VZV pathogenesis in skin.

    View details for DOI 10.1128/JVI.00102-13

    View details for Web of Science ID 000317416400029

    View details for PubMedID 23427162

    View details for PubMedCentralID PMC3624291

  • Structure-function analysis of varicella-zoster virus glycoprotein H identifies domain-specific roles for fusion and skin tropism PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Vleck, S. E., Oliver, S. L., Brady, J. J., Blau, H. M., Rajamani, J., Sommer, M. H., Arvin, A. M. 2011; 108 (45): 18412-18417

    Abstract

    Enveloped viruses require membrane fusion for cell entry and replication. For herpesviruses, this event is governed by the multiprotein core complex of conserved glycoproteins (g)B and gH/gL. The recent crystal structures of gH/gL from herpes simplex virus 2, pseudorabies virus, and Epstein-Barr virus revealed distinct domains that, surprisingly, do not resemble known viral fusogens. Varicella-zoster virus (VZV) causes chicken pox and shingles. VZV is an ?-herpesvirus closely related to herpes simplex virus 2, enabling prediction of the VZV gH structure by homology modeling. We have defined specific roles for each gH domain in VZV replication and pathogenesis using structure-based site-directed mutagenesis of gH. The distal tip of domain (D)I was important for skin tropism, entry, and fusion. DII helices and a conserved disulfide bond were essential for gH structure and VZV replication. An essential (724)CXXC(727) motif was critical for DIII structural stability and membrane fusion. This assignment of domain-dependent mechanisms to VZV gH links elements of the glycoprotein structure to function in herpesvirus replication and virulence.

    View details for DOI 10.1073/pnas.1111333108

    View details for Web of Science ID 000296700000053

    View details for PubMedID 22025718

    View details for PubMedCentralID PMC3215059

  • Disruption of PML Nuclear Bodies Is Mediated by ORF61 SUMO-Interacting Motifs and Required for Varicella-Zoster Virus Pathogenesis in Skin PLOS PATHOGENS Wang, L., Oliver, S. L., Sommer, M., Rajamani, J., Reichelt, M., Arvin, A. M. 2011; 7 (8)

    Abstract

    Promyelocytic leukemia protein (PML) has antiviral functions and many viruses encode gene products that disrupt PML nuclear bodies (PML NBs). However, evidence of the relevance of PML NB modification for viral pathogenesis is limited and little is known about viral gene functions required for PML NB disruption in infected cells in vivo. Varicella-zoster virus (VZV) is a human alphaherpesvirus that causes cutaneous lesions during primary and recurrent infection. Here we show that VZV disrupts PML NBs in infected cells in human skin xenografts in SCID mice and that the disruption is achieved by open reading frame 61 (ORF61) protein via its SUMO-interacting motifs (SIMs). Three conserved SIMs mediated ORF61 binding to SUMO1 and were required for ORF61 association with and disruption of PML NBs. Mutation of the ORF61 SIMs in the VZV genome showed that these motifs were necessary for PML NB dispersal in VZV-infected cells in vitro. In vivo, PML NBs were highly abundant, especially in basal layer cells of uninfected skin, whereas their frequency was significantly decreased in VZV-infected cells. In contrast, mutation of the ORF61 SIMs reduced ORF61 association with PML NBs, most PML NBs remained intact and importantly, viral replication in skin was severely impaired. The ORF61 SIM mutant virus failed to cause the typical VZV lesions that penetrate across the basement membrane into the dermis and viral spread in the epidermis was limited. These experiments indicate that VZV pathogenesis in skin depends upon the ORF61-mediated disruption of PML NBs and that the ORF61 SUMO-binding function is necessary for this effect. More broadly, our study elucidates the importance of PML NBs for the innate control of a viral pathogen during infection of differentiated cells within their tissue microenvironment in vivo and the requirement for a viral protein with SUMO-binding capacity to counteract this intrinsic barrier.

    View details for DOI 10.1371/journal.ppat.1002157

    View details for Web of Science ID 000294298100008

    View details for PubMedID 21901090

    View details for PubMedCentralID PMC3161977

  • Mutagenesis of Varicella-Zoster Virus Glycoprotein I (gI) Identifies a Cysteine Residue Critical for gE/gI Heterodimer Formation, gI Structure, and Virulence in Skin Cells JOURNAL OF VIROLOGY Oliver, S. L., Sommer, M. H., Reichelt, M., Rajamani, J., Vlaycheva-Beisheim, L., Stamatis, S., Cheng, J., Jones, C., Zehnder, J., Arvin, A. M. 2011; 85 (9): 4095-4110

    Abstract

    Varicella-zoster virus (VZV) is the alphaherpesvirus that causes chicken pox (varicella) and shingles (zoster). The two VZV glycoproteins gE and gI form a heterodimer that mediates efficient cell-to-cell spread. Deletion of gI yields a small-plaque-phenotype virus, ?gI virus, which is avirulent in human skin using the xenograft model of VZV pathogenesis. In the present study, 10 mutant viruses were generated to determine which residues were required for the typical function of gI. Three phosphorylation sites in the cytoplasmic domain of gI were not required for VZV virulence in vivo. Two deletion mutants mapped a gE binding region in gI to residues 105 to 125. A glycosylation site, N116, in this region did not affect virulence. Substitution of four cysteine residues highly conserved in the Alphaherpesvirinae established that C95 is required for gE/gI heterodimer formation. The C95A and ?105-125 (with residues 105 to 125 deleted) viruses had small-plaque phenotypes with reduced replication kinetics in vitro similar to those of the ?gI virus. The ?105-125 virus was avirulent for human skin in vivo. In contrast, the C95A mutant replicated in vivo but with significantly reduced kinetics compared to those of the wild-type virus. In addition to abolished gE/gI heterodimer formation, gI from the C95A or the ?105-125 mutant was not recognized by monoclonal antibodies that detect the canonical conformation of gI, demonstrating structural disruption of gI in these viruses. This alteration prevented gI incorporation into virus particles. Thus, residues C95 and 105 to 125 are critical for gI structure required for gE/gI heterodimer formation, virion incorporation, and ultimately, effective viral spread in human skin.

    View details for DOI 10.1128/JVI.02596-10

    View details for Web of Science ID 000289618600005

    View details for PubMedID 21345964

    View details for PubMedCentralID PMC3126246

  • Identification and functional characterization of the Varicella zoster virus ORF11 gene product VIROLOGY Che, X., Oliver, S. L., Sommer, M. H., Rajamani, J., Reichelt, M., Arvin, A. M. 2011; 412 (1): 156-166

    Abstract

    The deletion of ORF11 severely impaired VZV infection of human skin xenografts. Here, we investigate the characteristics and functions of the ORF11 gene product. ORF11 is expressed as a 118kDa polypeptide in VZV-infected cells; the protein is present in the nucleus and cytoplasm and is incorporated into VZ virions. Although ORF11 had little effect in transactivating VZV gene promoters in transfection assays, deleting ORF11 from the virus was associated with reduced expression of immediate early proteins IE4, IE62 and IE63, and the major glycoprotein, gE. ORF11 was identified as an RNA binding protein and its RNA binding domain was defined. However, disrupting the ORF11 RNA binding domain did not affect skin infection, indicating that RNA binding capacity, conserved among the alphaherpesviruses homologues, is not essential while the contribution of ORF11 to the expression of the IE proteins and gE may be required for VZV pathogenesis in skin in vivo.

    View details for DOI 10.1016/j.virol.2010.12.055

    View details for Web of Science ID 000288778200018

    View details for PubMedID 21276599

    View details for PubMedCentralID PMC3068617

  • Varicella-Zoster Virus T Cell Tropism and the Pathogenesis of Skin Infection VARICELLA-ZOSTER VIRUS Arvin, A. M., Moffat, J. F., Sommer, M., Oliver, S., Che, X., Vleck, S., Zerboni, L., Ku, C. 2010; 342: 189-209

    Abstract

    Varicella-zoster virus (VZV) is a medically important human alphaherpesvirus that causes varicella and zoster. VZV initiates primary infection by inoculation of the respiratory mucosa. In the course of primary infection, VZV establishes a life-long persistence in sensory ganglia; VZV reactivation from latency may result in zoster in healthy and immunocompromised patients. The VZV genome has at least 70 known or predicted open reading frames (ORFs), but understanding how these gene products function in virulence is difficult because VZV is a highly human-specific pathogen. We have addressed this obstacle by investigating VZV infection of human tissue xenografts in the severe combined immunodeficiency mouse model. In studies relevant to the pathogenesis of primary VZV infection, we have examined VZV infection of human T cell (thymus/liver) and skin xenografts. This work supports a new paradigm for VZV pathogenesis in which VZV T cell tropism provides a mechanism for delivering the virus to skin. We have also shown that VZV-infected T cells transfer VZV to neurons in sensory ganglia. The construction of infectious VZV recombinants that have deletions or targeted mutations of viral genes or their promoters and the evaluation of VZV mutants in T cell and skin xenografts has revealed determinants of VZV virulence that are important for T cell and skin tropism in vivo.

    View details for DOI 10.1007/82_2010_29

    View details for Web of Science ID 000282104800012

    View details for PubMedID 20397071

  • Analysis of the Functions of Glycoproteins E and I and Their Promoters During VZV Replication In Vitro and in Skin and T-Cell Xenografts in the SCID Mouse Model of VZV Pathogenesis VARICELLA-ZOSTER VIRUS Arvin, A. M., Oliver, S., Reichelt, M., Moffat, J. F., Sommer, M., Zerboni, L., Berarducci, B. 2010; 342: 129-146

    Abstract

    The two VZV glycoproteins, gE and gI, are encoded by genes that are designated open reading frames, ORF67 and ORF68, located in the short unique region of the VZV genome. These proteins have homologs in the other alphaherpesviruses. Like their homologues, VZV gE and gI exhibit prominent co-localization in infected cells and form heterodimers. However, VZV gE is much larger than its homologues because it has a unique N-terminal domain, consisting of 188 amino acids that are not present in these other gene products. VZV gE also differs from the related gE proteins, in that it is essential for viral replication. Targeted mutations of gE that are compatible with VZV replication in cultured cells have varying phenotypes in skin and T-cell xenografts in the SCID mouse model of VZV pathogenesis in vivo. While gI is dispensable for growth in cultured cells in vitro, this glycoprotein is essential for VZV infection of differentiated human skin and T cells in vivo. The promoter regions of gE and gI are regulated by the cellular transactivator, specificity protein factor 1 (Sp1) in combination with the major VZV transactivator in reporter construct experiments and some Sp1 promoter elements are important for VZV virulence in vivo. Further analysis of VZV gE and gI functions and their interactions with other viral and host cell proteins are important areas for studies of VZV replication and pathogenesis.

    View details for DOI 10.1007/82_2009_1

    View details for Web of Science ID 000282104800008

    View details for PubMedID 20186616

  • Anti-Glycoprotein H Antibody Impairs the Pathogenicity of Varicella-Zoster Virus in Skin Xenografts in the SCID Mouse Model JOURNAL OF VIROLOGY Vleck, S. E., Oliver, S. L., Reichelt, M., Rajamani, J., Zerboni, L., Jones, C., Zehnder, J., Grose, C., Arvin, A. M. 2010; 84 (1): 141-152

    Abstract

    Varicella-zoster virus (VZV) infection is usually mild in healthy individuals but can cause severe disease in immunocompromised patients. Prophylaxis with varicella-zoster immunoglobulin can reduce the severity of VZV if given shortly after exposure. Glycoprotein H (gH) is a highly conserved herpesvirus protein with functions in virus entry and cell-cell spread and is a target of neutralizing antibodies. The anti-gH monoclonal antibody (MAb) 206 neutralizes VZV in vitro. To determine the requirement for gH in VZV pathogenesis in vivo, MAb 206 was administered to SCID mice with human skin xenografts inoculated with VZV. Anti-gH antibody given at 6 h postinfection significantly reduced the frequency of skin xenograft infection by 42%. Virus titers, genome copies, and lesion size were decreased in xenografts that became infected. In contrast, administering anti-gH antibody at 4 days postinfection suppressed VZV replication but did not reduce the frequency of infection. The neutralizing anti-gH MAb 206 blocked virus entry, cell fusion, or both in skin in vivo. In vitro, MAb 206 bound to plasma membranes and to surface virus particles. Antibody was internalized into vacuoles within infected cells, associated with intracellular virus particles, and colocalized with markers for early endosomes and multivesicular bodies but not the trans-Golgi network. MAb 206 blocked spread, altered intracellular trafficking of gH, and bound to surface VZV particles, which might facilitate their uptake and targeting for degradation. As a consequence, antibody interference with gH function would likely prevent or significantly reduce VZV replication in skin during primary or recurrent infection.

    View details for DOI 10.1128/JVI.01338-09

    View details for Web of Science ID 000272564300013

    View details for PubMedID 19828615

    View details for PubMedCentralID PMC2798403

  • Mutagenesis of Varicella-Zoster Virus Glycoprotein B: Putative Fusion Loop Residues Are Essential for Viral Replication, and the Furin Cleavage Motif Contributes to Pathogenesis in Skin Tissue In Vivo JOURNAL OF VIROLOGY Oliver, S. L., Sommer, M., Zerboni, L., Rajamani, J., Grose, C., Arvin, A. M. 2009; 83 (15): 7495-7506

    Abstract

    Glycoprotein B (gB), the most conserved protein in the family Herpesviridae, is essential for the fusion of viral and cellular membranes. Information about varicella-zoster virus (VZV) gB is limited, but homology modeling showed that the structure of VZV gB was similar to that of herpes simplex virus (HSV) gB, including the putative fusion loops. In contrast to HSV gB, VZV gB had a furin recognition motif ([R]-X-[KR]-R-|-X, where | indicates the position at which the polypeptide is cleaved) at residues 491 to 494, thought to be required for gB cleavage into two polypeptides. To investigate their contribution, the putative primary fusion loop or the furin recognition motif was mutated in expression constructs and in the context of the VZV genome. Substitutions in the primary loop, W180G and Y185G, plus the deletion mutation Delta491RSRR494 and point mutation 491GSGG494 in the furin recognition motif did not affect gB expression or cellular localization in transfected cells. Infectious VZV was recovered from parental Oka (pOka)-bacterial artificial chromosomes that had either the Delta491RSRR494 or 491GSGG494 mutation but not the point mutations W180G and Y185G, demonstrating that residues in the primary loop of gB were essential but gB cleavage was not required for VZV replication in vitro. Virion morphology, protein localization, plaque size, and replication were unaffected for the pOka-gBDelta491RSRR494 or pOka-gB491GSGG494 virus compared to pOka in vitro. However, deletion of the furin recognition motif caused attenuation of VZV replication in human skin xenografts in vivo. This is the first evidence that cleavage of a herpesvirus fusion protein contributes to viral pathogenesis in vivo, as seen for fusion proteins in other virus families.

    View details for DOI 10.1128/JVI.00400-09

    View details for Web of Science ID 000267747400015

    View details for PubMedID 19474103

    View details for PubMedCentralID PMC2708640

  • Comparison of the efficacy of rotavirus VLP vaccines to a live homologous rotavirus vaccine in a pig model of rotavirus disease VACCINE El-Attar, L., Oliver, S. L., Mackie, A., Charpilienne, A., Poncet, D., Cohen, J., Bridger, J. C. 2009; 27 (24): 3201-3208

    Abstract

    Rotavirus-like particles (VLPs) have shown promise as rotavirus vaccine candidates in mice, rabbits and pigs. In pigs, VLP vaccines reduced rotavirus shedding and disease but only when used in conjunction with live attenuated human rotavirus. Using a porcine rotavirus pig model, rotavirus antigen shedding was reduced by up to 40% after vaccination with VLPs including the neutralizing antigens VP7 and VP8* when used in combination with the adjuvant polyphosphazene poly[di(carbozylatophenoxy)phoshazene] (PCPP). In contrast, complete protection from rotavirus antigen shedding and disease was induced by vaccination with the virulent porcine rotavirus PRV 4F. This is the first study to demonstrate some post-challenge reductions in rotavirus antigen shedding in a pig model of rotavirus disease after vaccination with VLPs without combining with infectious rotavirus.

    View details for DOI 10.1016/j.vaccine.2009.03.043

    View details for Web of Science ID 000266698200013

    View details for PubMedID 19446192

  • Heterogeneity in the capsid protein of bovine enteric caliciviruses belonging to a new genus VIROLOGY D'Mello, F., Jervis, S. M., Edwards, P. M., Oliver, S. L., Bridger, J. C. 2009; 387 (1): 109-116

    Abstract

    Some bovine enteric caliciviruses form a new genus in the family Caliciviridae. In this study, Bayesian phylogenetic analysis of 31 full length capsid sequences from Europe, North America and Asia revealed that this new genus had four currently circulating lineages that showed both temporal and geographical distribution. These groupings were supported by the distribution of the frequency of pair-wise distances. However, the nucleotide and amino acid heterogeneity was low, with a maximum nucleotide and amino acid divergence of 16.7% and 8.4%, respectively. Most variability was found between amino acid residues 288 and 420 of the capsid protein and the sequence motifs observed in this region supported the division of the four lineages. Homology modelling using the structure of the San Miguel sea lion capsid indicated that most variation occurred in the predicted P2 domain and thus, may affect antigenic sites on the surface of the capsid of this newly described genus.

    View details for DOI 10.1016/j.virol.2009.01.035

    View details for Web of Science ID 000265561800013

    View details for PubMedID 19254803

  • Development of recombinant varicella-zoster viruses expressing luciferase fusion proteins for live in vivo imaging in human skin and dorsal root ganglia xenografts JOURNAL OF VIROLOGICAL METHODS Oliver, S. L., Zerboni, L., Sommer, M., Rajamani, J., Arvin, A. M. 2008; 154 (1-2): 182-193

    Abstract

    Varicella-zoster virus (VZV) is a host specific human pathogen that has been studied using human xenografts in SCID mice. Live whole-animal imaging is an emerging technique to measure protein expression in vivo using luminescence. Currently, it has only been possible to determine VZV protein expression in xenografts postmortem. Therefore, to measure immediate early (IE63) and late (glycoprotein E [gE]) protein expression in vivo viruses expressing IE63 or gE as luciferase fusion proteins were generated. Viable recombinant viruses pOka-63-luciferase and pOka-63/70-luciferase, which had luciferase genes fused to ORF63 and its duplicate ORF70, or pOka-gE-CBR were recovered that expressed IE63 or gE as fusion proteins and generated luminescent plaques. In contrast to pOka-63/70-luciferase viruses, the luciferase gene was rapidly lost in vitro when fused to a single copy of ORF63 or ORF68. IE63 expression was successfully measured in human skin and dorsal root ganglia xenografts infected with the genomically stable pOka-63/70-luciferase viruses. The progress of VZV infection in dorsal root ganglia xenografts was delayed in valacyclovir treated mice but followed a similar trend in untreated mice when the antiviral was withdrawn 28 days post-inoculation. Thus, IE63-luciferase fusion proteins were effective for investigating VZV infection and antiviral activity in human xenografts.

    View details for DOI 10.1016/j.jviromet.2008.07.033

    View details for Web of Science ID 000261838800026

    View details for PubMedID 18761377

    View details for PubMedCentralID PMC2657092

  • Envelope protein variability among HBV-Infected asymptomatic carriers and immunized children with breakthrough infections JOURNAL OF MEDICAL VIROLOGY Mendy, M., D'Mello, F., Kanellos, T., Oliver, S., Whittle, H., Howard, C. R. 2008; 80 (9): 1537-1546

    Abstract

    A detailed study of hepatitis B virus (HBV) surface variants and their role in breakthrough infections has been conducted in The Gambia, West Africa. Samples from 1856 vaccinated subjects were tested for hepatitis B surface antigen (HBsAg). Evidence of infection was found in 11% (22/192) of subjects with breakthrough infections and 18 (81.8%) were also positive for HBV DNA following PCR analysis. A cohort of 58 unvaccinated carriers which also included 11 patients with hepatocellular carcinoma was also investigated in order to establish the prevalence of surface variants in the unvaccinated population. Analysis of the S gene from HBV PCR-positive subjects (n = 64) revealed little variation in the S gene of these subjects. Twenty-four S protein sequences (37.5%) were identical and a further 22 sequences differed by only a single amino acid. The K141E variant found in previous work was not detected and little variation was observed in the immunodominant "a" determinant; a single change was found in one vaccinated patient (Q129H) and nine changes detected among six unvaccinated carriers. This study showed that breakthrough HBV infection in vaccinated Gambians is mainly caused by the wild type genoytype E strain and that immune escape mutants are uncommon. However, HBV mutants may play a role in establishing infection later in life when anti-HBs antibodies have begun to decline. Further investigation is required to determine the cause of these breakthrough infections and whether they contribute to the establishment of the carrier state.

    View details for DOI 10.1002/jmv.21221

    View details for Web of Science ID 000258011500005

    View details for PubMedID 18649345

  • Serotype 1 and 2 bovine noroviruses are endemic in cattle in the United Kingdom and Germany JOURNAL OF CLINICAL MICROBIOLOGY Oliver, S. L., Wood, E., Asobayire, E., Wathes, D. C., Brickell, J. S., Elschner, M., Otto, P., Lambden, P. R., Clarke, I. N., Bridger, J. C. 2007; 45 (9): 3050-3052

    Abstract

    The genomically and antigenically distinct bovine noroviruses Bo/Jena/1980/DE and Bo/Newbury2/1976/UK have been associated with calf diarrhea. In the present seroprevalence study, both were found to be endemic in cattle from Germany and the United Kingdom, a finding in contrast to previous virus prevalence studies. They were less common than group A rotaviruses, particularly in calves, suggesting a different epidemiology.

    View details for DOI 10.1128/JCM.02015-06

    View details for Web of Science ID 000249506900044

    View details for PubMedID 17596356

  • Retrospective study of noroviruses in samples of diarrhoea from cattle, using the Veterinary Laboratories Agency's Farmfile database VETERINARY RECORD Milnes, A. S., Binns, S. H., Oliver, S. L., Bridger, J. C. 2007; 160 (10): 326-330

    Abstract

    A collaborative study was undertaken by the Veterinary Laboratories Agency (vla) and the Royal Veterinary College (rvc) to determine the prevalence of bovine noroviruses in cattle with diarrhoea. Samples of bovine diarrhoea were provided by the vla from routine diagnostic submissions and a reverse transcription-pcr was used by the rvc to detect the viruses. Epidemiological information about the samples was provided retrospectively by the Farmfile database. Noroviruses were detected in 44 (11 per cent) of the 398 samples tested, and Farmfile data were used to investigate the differences between the positive and negative animals.

    View details for Web of Science ID 000245113800010

    View details for PubMedID 17351173

  • Complete genomic characterization and antigenic relatedness of genogroup III, genotype 2 bovine noroviruses ARCHIVES OF VIROLOGY Oliver, S. L., Asobayire, E., Charpilienne, A., Cohen, J., Bridger, J. C. 2007; 152 (2): 257-272

    Abstract

    Bovine enteric noroviruses form a genogroup, III, distinct from the 2 human norovirus genogroups, I and II. Two genogroup III genotypes were suggested by partial genomic analyses. In the present study, analysis of the full-length genome sequence of Bo/Newbury2/76/UK and the more contemporary Newbury2-like virus, Bo/Dumfries/1994/UK, showed that both were 7311 nucleotides in length and had three open reading frames (ORFs), amino acids motifs typical of noroviruses, and 95% or greater amino acid identities to each other in all regions of their genome. Apart from the ORF1 NTPase region, their ORF1 regions had less than 90% identity to the genogroup III genotype 1 Bo/Jena/80/DE virus, confirming two genogroup III genotypes. A close antigenic relationship was demonstrated by ELISA between the genotype 2 viruses, which will allow their serological diagnosis.

    View details for DOI 10.1007/s00705-006-0856-2

    View details for Web of Science ID 000243631200003

    View details for PubMedID 17066248

  • Characterization of a cross-reactive linear epitope in human genogroup I and bovine genogroup III norovirus capsid proteins VIROLOGY Batten, C. A., Clarke, I. N., Kempster, S. L., Oliver, S. L., Bridger, J. C., Lambden, P. R. 2006; 356 (1-2): 179-187

    Abstract

    The Southampton norovirus (SV) capsid protein was expressed as VLPs by recombinant baculoviruses in insect cells and was used to immunize mice for the production of monoclonal antibodies (mAbs). One mAb, CM54, showed broad cross-reactivity to genogroup I (GI) noroviruses, but was not reactive to GII capsid proteins. Interestingly mAb CM54 reacted to a bovine norovirus capsid protein. Immunoblot analysis indicated the binding site for CM54 was located in the shell domain between amino acid residues 102-225 of the SV capsid protein. The epitope was mapped to high resolution using a peptide array and was located to the sequence LEDVRN at amino acid residues 162-167. Alignment of norovirus capsid protein sequences confirmed the epitope sequence was common to particular groups of human and bovine noroviruses. Modeling of the epitope onto the recombinant NV capsid protein revealed it was located to the inner surface of the shell domain.

    View details for DOI 10.1016/j.virol.2006.07.034

    View details for Web of Science ID 000242424800020

    View details for PubMedID 16934306

  • Genomic characterization of the unclassified bovine enteric virus Newbury agent-1 (Newbury1) endorses a new genus in the family Caliciviridae VIROLOGY Oliver, S. L., Asobayire, E., Dastjerdi, A. M., Bridger, J. C. 2006; 350 (1): 240-250

    Abstract

    The pathogenic bovine enteric virus, Newbury agent-1 (Bo//Newbury1/1976/UK), first identified in 1976, was characterized as a possible calicivirus by morphology, buoyant density in CsCl and the presence of a single capsid protein but genomic sequence could not be obtained. In the present study, the complete genome sequence of Newbury1 was determined and classified Newbury1 in a new genus of the Caliciviridae. The Newbury1 genome, of 7454 nucleotides, had two predicted open reading frames (ORFs). ORF1 encoded the non-structural and contiguous capsid proteins. ORF2 encoded a basic protein characteristic of the family Caliciviridae. Compared to the 4 recognized Caliciviridae genera, Norovirus, Sapovirus, Lagovirus and Vesivirus, Newbury1 had less than 39% amino acid (47% nucleotide) identity in the complete 2C-helicase, 3C-protease, 3D-polymerase and capsid regions but had 89% to 98% amino acid (78% to 92% nucleotide) identity to the recently characterized NB virus in these regions. By phylogenetic analyses, Newbury1 and NB viruses formed a distinct clade independent of the 4 recognized genera. However, amino acid identities showed that Newbury1 and the NB virus were distinct polymerase types (90% amino acid identity), but their complete capsid proteins were almost identical (98% amino acid identity). Analyses of contemporary viruses showed that the two polymerase genotypes, Newbury1 and NB, were circulating in UK cattle and antibody to Newbury1-like viruses was common in cattle sera. The present study defined the existence of a new genus in the Caliciviridae that we propose be named Becovirus or Nabovirus to distinguish the new clade from bovine noroviruses.

    View details for DOI 10.1016/j.virol.2006.02.027

    View details for Web of Science ID 000238561900023

    View details for PubMedID 16574184

  • Genotype 1 and genotype 2 bovine noroviruses are antigenically distinct but share a cross-reactive epitope with human noroviruses JOURNAL OF CLINICAL MICROBIOLOGY Oliver, S. L., Batten, C. A., Deng, Y., Elschner, M., Otto, P., Charpilienne, A., Clarke, I. N., Bridger, J. C., Lambden, P. R. 2006; 44 (3): 992-998

    Abstract

    The bovine enteric caliciviruses Bo/Jena/1980/DE and Bo/Newbury2/1976/UK represent two distinct genotypes within a new genogroup, genogroup III, in the genus Norovirus of the family Caliciviridae. In the present study, the antigenic relatedness of these two genotypes was determined for the first time to enable the development of tests to detect and differentiate between both genotypes. Two approaches were used. First, cross-reactivity was examined by enzyme-linked immunosorbent assay (ELISA) using recombinant virus-like particles (VLPs) and convalescent-phase sera from calves infected with either Jena (genotype 1) or Newbury2 (genotype 2). Second, cross-reactivity was examined between the two genotypes with a monoclonal antibody, CM39, derived using Jena VLPs. The two genotypes, Jena and Newbury2, were antigenically distinct with little or no cross-reactivity by ELISA to the heterologous VLPs using convalescent calf sera that had homologous immunoglobulin G titers of log10 3.1 to 3.3. CM39 reacted with both Jena and heterologous Newbury2 VLPs. The CM39 epitope was mapped to nine amino acids (31PTAGAQIAA39) in the Jena capsid protein, which was not fully conserved for Newbury2 (31PTAGAPVAA39). Molecular modeling showed that the CM39 epitope was located within the NH2-terminal arm inside the virus capsid. Surprisingly, CM39 also reacted with VLPs from two genogroup II/3 human noroviruses by ELISA and Western blotting. Thus, although the bovine noroviruses Jena and Newbury2 corresponded to two distinct antigenic types or serotypes, they shared at least one cross-reactive epitope. These findings have relevance for epidemiological studies to determine the prevalence of bovine norovirus serotypes and to develop vaccines to bovine noroviruses.

    View details for DOI 10.1128/JCM.44.3.992-998.2006

    View details for Web of Science ID 000236095000050

    View details for PubMedID 16517888

  • A chimeric bovine enteric calicivirus: evidence for genomic recombination in genogroup III of the Norovirus genus of the Caliciviridae VIROLOGY Oliver, S. L., Brown, D. W., Green, J., Bridger, J. C. 2004; 326 (2): 231-239

    Abstract

    The Norovirus genus of the Caliciviridae encompasses viruses that cause outbreaks of gastroenteritis in human and viruses that have been associated with diarrhea in cattle. The two bovine noroviruses, Bo/Newbury2/76/UK and Bo/Jena/80/DE, represent two distinct genetic clusters in the newly described genogroup III. In the present study, Jena-like polymerase sequences were identified for the first time in the UK, but one of these, Bo/Thirsk10/00/UK, was a chimeric virus. Bo/Thirsk10/00/UK had a Jena-like polymerase gene but Newbury2-like capsid and ORF3 genes by comparison of their genome organization, nucleotide, and amino acid identities and phylogenetic analyses. The present study is one of few studies to clearly demonstrate the existence of chimeric genomes in the Norovirus genus and the first, to our knowledge, to identify a chimeric genome in genogroup III. It provides additional support that genomic recombination is part of the natural evolution of noroviruses and is relevant to the diagnosis and immunological control of norovirus diarrhea outbreaks.

    View details for DOI 10.1016/j.virol.2004.06.010

    View details for Web of Science ID 000223454500003

    View details for PubMedID 15302209

  • Molecular characterization of bovine enteric caliciviruses: a distinct third genogroup of noroviruses (Norwalk-like viruses) unlikely to be of risk to humans JOURNAL OF VIROLOGY Oliver, S. L., Dastjerdi, A. M., Wong, S., El-Attar, L., Gallimore, C., Brown, D. W., Green, J., Bridger, J. C. 2003; 77 (4): 2789-2798

    Abstract

    Bovine enteric caliciviruses (BoCVs) have been classified in the Norovirus (Norwalk-like virus) genus of the Caliciviridae, raising questions about zoonotic transmission and an animal reservoir for the human Norwalk-like viruses (NLVs), an important cause of nonbacterial gastroenteritis in humans. We examined the genetic relationship of human NLVs to BoCVs that were identified by using reverse transcription-PCR with primer pairs originally designed to detect human NLVs. Polymerase, capsid, and open reading frame 3 (ORF3) gene sequence analyses of BoCVs that were identified from 1976 to 2000 from throughout the United Kingdom showed that BoCVs formed a distinct third genogroup of closely related viruses distinct from the human genogroup I and II NLVs. Evidence was not obtained to support the concept that BoCVs are circulating in humans and pose a threat to human health.

    View details for DOI 10.1128/JVI.77.4.2789-2798.2003

    View details for Web of Science ID 000180712700057

    View details for PubMedID 12552024

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