Project description:The highly conserved herpesvirus glycoprotein complex, gB/gH-gL, mediates membrane fusion during virion entry and cell-cell fusion. Varicella-zoster virus (VZV) characteristically forms multi-nucleated cells, or syncytia, during the infection of human tissues but little is known about this process. The cytoplasmic domain of VZV gB (gBcyt) has been implicated in cell-cell fusion regulation because a gB[Y881F] substitution causes hyperfusion. The gBcyt regulation is necessary for VZV pathogenesis as the hyperfusogenic mutant gB[Y881F] is severely attenuated in human skin xenografts. In this study, gBcyt regulated fusion was investigated by comparing melanoma cells infected with wild type-like VZV or hyperfusogenic mutants. The gB[Y881F] mutant exhibited dramatically accelerated syncytia formation in melanoma cells caused by fusion of infected cells with many uninfected cells, increased cytoskeleton reorganization and rapid displacement of nuclei to dense central structures when compared to pOka using live cell confocal microscopy. VZV and human transcriptomes were concurrently investigated using RNA-seq to identify viral and cellular responses induced when the gBcyt regulation was disrupted by the gB[Y881F] substitution. The expression of four vital VZV genes, ORF61 and glycoproteins, gC, gE and gI, was significantly reduced at 36 hours post infection for the hyperfusogenic mutants. Importantly, hierarchical clustering demonstrated an association of differential gene expression with dysregulated gBcyt-mediated fusion. A subset of Ras GTPase genes linked to membrane remodeling were upregulated in cells infected with the hyperfusogenic mutants. These data implicate the gBcyt in the regulation gB fusion function that, if unmodulated, triggers cellular processes leading to hyperfusion that attenuates VZV infection.
Project description:Enteric fever is a major public health problem and causes numerous deaths annually. Ty21a is the only efficacious oral, live attenuated typhoid vaccine currently licensed for use, however, its mechanism of protection is poorly understood. To address this knowledge gap, we interrogated transcriptional profiles following vaccination with Ty21a and an immunogenic experimental oral live attenuated vaccine, M01ZH09, and related these findings to immunogenicity, and incubation period and disease severity following challenge with Salmonella Typhi four weeks after vaccination. Despite originating from the same parent strain (Ty2), we detected marked differences in the gene expression between both vaccines. Analysis of the transcriptome 7 days after M01ZH09 vaccination implicated transcriptional patterns associated with the cell cycle correlated significantly with humoral immunogenicity 28 days after vaccination. In contrast, significantly induced T and NK cell responses were associated with Ty21a vaccination, and integrative analysis indicated signatures reflecting amino acid metabolism with delayed onset of disease. Stimulation of PBMCs collected from participants prior to and following vaccination with the two vaccine strains in vitro confirmed the superior capacity of Ty21a to induce NK cells, validating gene expression results. These data provide insight into the effects of oral live attenuated typhoid vaccines on the human molecular immune response and underline the involvement of T cell response signatures with protection following challenge.
Project description:Our aim was to investigate the interaction between epidermal differentiation and VZV infection. By means of a calcium-induced keratinocyte differentiation model and RNA-seq we show VZV infection has a profound effect on differentiating keratinocytes and hijacks the normal process of epidermal gene expression to generate a signature resembling patterns of gene expression seen in both heritable and acquired skin-blistering disorders. Analysis of the viral transcriptome provides evidence that VZV replication in skin is tightly linked to differentiation and critically, that late viral gene expression is associated with cellular differentiation. The experiment was performed on human primary keratinocytes under four conditions: undifferentiated/uninfected, uninfected/differentiated, VZV-infected/undifferentiated and VZV-infected/differentiated.
Project description:During primary infection, varicella-zoster virus (VZV) is spread via lymphocytes to skin, where it induces a rash and establishes latency in sensory ganglia. A live, attenuated varicella vaccine (vOka) was generated by using the VZV Oka strain (pOka), but the molecular basis for vOka attenuation remains unknown. Little is known concerning the effects of wild-type or attenuated VZV on cellular gene regulation in the host cells that are critical for pathogenesis. In this study, transcriptional profiles of primary human T cells and fibroblasts infected with VZV in cell culture were determined by using 40,000-spot human cDNA microarrays. Cellular gene transcription in human skin xenografts in SCID mice that were infected with VZV in vivo was also evaluated. The profiles of cellular gene transcripts that were induced or inhibited in infected human foreskin fibroblasts (HFFs), T cells, and skin in response to pOka and vOka infection were similar. However, significant alterations in cellular gene regulation were observed among the three differentiated human cell types that were examined, suggesting specific differences in the biological consequences of VZV infection related to the target cell. Changes in cellular gene transcription detected by microarray analysis were confirmed for selected genes by quantitative real-time reverse transcription-PCR analysis of VZV-infected cells. Interestingly, the transcription of caspase 8 was found to be decreased in infected T cells but not in HFFs or skin, which may signify a tissue-specific antiapoptosis mechanism. The use of microarrays to demonstrate differences in effects on host cell genes in primary, biologically relevant cell types provides background information for experiments to link these various response phenotypes with mechanisms of VZV pathogenesis that are important for the natural course of human infection.
Project description:During primary infection, varicella-zoster virus (VZV) is spread via lymphocytes to skin, where it induces a rash and establishes latency in sensory ganglia. A live, attenuated varicella vaccine (vOka) was generated by using the VZV Oka strain (pOka), but the molecular basis for vOka attenuation remains unknown. Little is known concerning the effects of wild-type or attenuated VZV on cellular gene regulation in the host cells that are critical for pathogenesis. In this study, transcriptional profiles of primary human T cells and fibroblasts infected with VZV in cell culture were determined by using 40,000-spot human cDNA microarrays. Cellular gene transcription in human skin xenografts in SCID mice that were infected with VZV in vivo was also evaluated. The profiles of cellular gene transcripts that were induced or inhibited in infected human foreskin fibroblasts (HFFs), T cells, and skin in response to pOka and vOka infection were similar. However, significant alterations in cellular gene regulation were observed among the three differentiated human cell types that were examined, suggesting specific differences in the biological consequences of VZV infection related to the target cell. Changes in cellular gene transcription detected by microarray analysis were confirmed for selected genes by quantitative real-time reverse transcription-PCR analysis of VZV-infected cells. Interestingly, the transcription of caspase 8 was found to be decreased in infected T cells but not in HFFs or skin, which may signify a tissue-specific antiapoptosis mechanism. The use of microarrays to demonstrate differences in effects on host cell genes in primary, biologically relevant cell types provides background information for experiments to link these various response phenotypes with mechanisms of VZV pathogenesis that are important for the natural course of human infection. A pathogenicity experiment design type is where an infective agent such as a bacterium, virus, protozoan, fungus etc. infects a host organism(s) and the infective agent is assayed. Computed
Project description:Live-attenuated viral vaccines have been successfully used to combat infectious disease for decades but due to their empirical derivation, little is known about their mechanisms of attenuation. This lack of understanding makes the development of next generation live attenuated vaccines difficult. The success of the 17D vaccine and availability of the parent virus, Asibi, makes it an excellent model to understand the molecular basis of attenuation of a live attenuated vaccine and the effects of viral diversity on vaccine function. Due to the differences in genetic diversity between WT Asibi virus and its 17D vaccine derivative, we investigated the changes in genetic diversity of 17D and Asibi viruses following treatment with ribavirin.
Project description:Sequence analyses shown that most exons of VZV circVLTs were overlapped with ORFs, except for exon5, which was an ideal mutation position. Using a galK positive/counter selection VZV bacteria artificial chromosome (BAC) system, we replaced upstream (104,765-104,785 Mutation2, pOKA-M2) or downstream (104,785-104,805, Mutation1, pOKA-M1) of circVLTs BSJ exon5 with 20 bp random sequences in VZV genomic DNA level. The code used in this study and extended data are available from the GitHub repository (https://github.com/ShaominYang/VZV_circRNA)
Project description:Varicella Zoster Virus (VZV) is a skin-tropic virus that infects epidermal keratinocytes and causes chickenpox. Although common, VZV infection can be life-threatening particularly in the immunocompromised. Therefore, understanding VZV-keratinocyte interactions is important to find new treatments beyond vaccination and anti-viral drugs. In VZV- infected skin, Kallikrein 6 (KLK6), and the ubiquitin-ligase MDM2 are up-regulated concomitant with Keratin 10 (K10) down-regulation. MDM2 binds to K10 targeting it for degradation via the ubiquitin-proteasome pathway. Preventing K10 degradation reduced VZV propagation in culture and prevented epidermal disruption in skin explants. K10 knockdown induced expression of the nuclear receptor subfamily 4, group A, member 1 (NR4A1) and enhanced viral propagation in culture. NR4A1 knockdown prevented viral propagation in culture, reduced LC3 levels and increased LAMP2 expression. We therefore describe a novel drug-able pathway whereby MDM2 ubiquitinates and degrades K10 increasing NR4A1 expression allowing VZV replication and propagation.