Prevalence of varicella-zoster virus DNA in dissociated human trigeminal ganglion neurons and nonneuronal cells.
(9/1300)
Previous analyses using in situ hybridization alone or together with PCR have yielded conflicting results regarding the cell type in which latent varicella-zoster virus (VZV) resides. We separated human trigeminal ganglia (TG) into neuronal and nonneuronal fractions, followed by primary and nested PCR to quantitate VZV DNA at the single cell level. Both TG from each of eight cadavers were dissociated and separated into neuronal and nonneuronal cell suspensions by differential filtration. Analysis of the neuron fraction (5,000 neurons per sample) revealed VZV DNA in 9 of 16 samples, with copy numbers ranging from 1 to 12, whereas only 2 of 16 nonneuronal cell samples were positive for VZV DNA, with 1 copy each. Further analysis of 10 samples of 100 neurons and the corresponding nonneuronal cell fractions from each TG of a single subject revealed VZV DNA in 3 of 10 samples of the left TG (range, 2 to 5 copies) and in 1 of 10 samples of the right TG (2 copies) but in none of the 20 nonneuronal cell fractions. These data indicate that latent VZV DNA is present primarily, if not exclusively, in neurons, at a frequency of two to five copies per latently infected neuron. (+info)
Phenotypic and genetic characterization of thymidine kinase from clinical strains of varicella-zoster virus resistant to acyclovir.
(10/1300)
Varicella-zoster virus (VZV) is a common herpesvirus responsible for disseminated or chronic infections in immunocompromised patients. Effective drugs such as acyclovir (ACV), famciclovir (prodrug of penciclovir), and foscarnet are available to treat these infections. Here we report the phenotypic and genetic characterization of four ACV-resistant VZV strains isolated from AIDS patients and transplant recipients. Sensitivity to six antiviral drugs was determined by an enzyme-linked immunosorbent assay, viral thymidine kinase (TK) activity was measured by comparing [(3)H]thymidine and 1-beta-D-arabinofuranosyl-[(3)H]thymine as substrates, and the TK gene open reading frame was sequenced. Three strains were found to be TK deficient, and the fourth was a mixed population composed of TK-positive and TK-deficient viruses. Each strain presented a unique TK gene mutation that could account for ACV resistance. In one strain, the deletion of two nucleotides at codon 215 induced a premature stop signal at codon 217. In another strain, a single nucleotide addition at codon 167 resulted in a premature stop signal at codon 206. In both other strains, we identified amino acid substitutions already described in other ACV-resistant VZV strains: either Glu-->Gly at residue 48 or Arg-->Gly at residue 143. According to our work and data previously reported on resistant VZV strains, there are three areas in the TK gene where 71% of the mutations described to date are located. These areas are putative candidates for a genotypic diagnosis of ACV resistance. (+info)
Quantitation of latent varicella-zoster virus and herpes simplex virus genomes in human trigeminal ganglia.
(11/1300)
Using real-time fluorescence PCR, we quantitated the numbers of copies of latent varicella-zoster virus (VZV) and herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) genomes in 15 human trigeminal ganglia. Eight (53%) and 1 (7%) of 15 ganglia were PCR positive for HSV-1 or -2 glycoprotein G genes, with means of 2,902 +/- 1,082 (standard error of the mean) or 109 genomes/10(5) cells, respectively. Eleven of 14 (79%) to 13 of 15 (87%) of the ganglia were PCR positive for VZV gene 29, 31, or 62. Pooling of the results for the three VZV genes yielded a mean of 258 +/- 38 genomes/10(5) ganglion cells. These levels of latent viral genome loads have implications for virus distribution in and reactivation from human sensory ganglia. (+info)
The TATA motif specifies the differential activation of minimal promoters by varicella zoster virus immediate-early regulatory protein IE62.
(12/1300)
The immediate-early IE62 protein of varicella zoster virus is an acidic transcriptional activator capable of up-regulating many viral and cellular promoters with varying efficiencies. We demonstrate that, in the context of a minimal promoter, a TATA element is both sufficient and essential for IE62-mediated transcriptional activation. Differential levels of activation by IE62 in this context were conferred by a panel of naturally occurring sequence variations within the TATA motif itself. TATA motif-specific, differential induction was not obtained when the IE62 acidic activation domain was targeted as a GAL4 fusion protein to the same panel. The prototype acidic transactivator, VP16 of herpes simplex virus, failed to discriminate between these different TATA motifs when they were placed into an appropriate responsive promoter context. Nonetheless, a chimeric IE62 polypeptide substituted with the VP16 activation domain retained the ability to differentially modulate minimal promoters with various TATA motifs. Taken together with its binding to TATA box-binding protein (TBP) and transcription factor IIB in vitro, we suggest that IE62 has the unusual ability to achieve differential levels of transcriptional activation through different TATA motifs, which may be accomplished either directly or indirectly by recognizing conformational variations in DNA-bound TBP, TBP-transcription factor IIA/B, or TBP-TATA-associated factor complexes. (+info)
Infection of human T lymphocytes with varicella-zoster virus: an analysis with viral mutants and clinical isolates.
(13/1300)
Varicella-zoster virus (VZV) disseminates in the body in peripheral blood mononuclear cells during chickenpox. Up to 1 in 10,000 mononuclear cells are infected during the viremic phase of the disease. We developed an in vitro system to infect human mononuclear cells with VZV by using umbilical cord blood. In this system, 3 to 4% of T cells were infected with VZV. VZV mutants unable to express certain genes, such as open reading frame 47 (ORF47) or ORF66, were impaired for growth in T cells, while other mutants showed little difference from parental virus. VZV unable to express ORF47 was even more impaired for spread from umbilical cord blood cells to melanoma cells in vitro. Early-passage clinical isolates of VZV infected T cells at a similar rate to the Oka vaccine strain; however, the clinical isolates were more efficient in spreading from infected T cells to melanoma cells. This in vitro system for infecting human T cells with VZV should be useful for identifying cellular and viral proteins that are important for virus replication in T cells and for the spread of virus from T cells to other cells. (+info)
Modulation of major histocompatibility class II protein expression by varicella-zoster virus.
(14/1300)
We sought to investigate the effects of varicella-zoster virus (VZV) infection on gamma interferon (IFN-gamma)-stimulated expression of cell surface major histocompatibility complex (MHC) class II molecules on human fibroblasts. IFN-gamma treatment induced cell surface MHC class II expression on 60 to 86% of uninfected cells, compared to 20 to 30% of cells which had been infected with VZV prior to the addition of IFN-gamma. In contrast, cells that were treated with IFN-gamma before VZV infection had profiles of MHC class II expression similar to those of uninfected cell populations. Neither IFN-gamma treatment nor VZV infection affected the expression of transferrin receptor (CD71). In situ and Northern blot hybridization of MHC II (MHC class II DR-alpha) RNA expression in response to IFN-gamma stimulation revealed that MHC class II DR-alpha mRNA accumulated in uninfected cells but not in cells infected with VZV. When skin biopsies of varicella lesions were analyzed by in situ hybridization, MHC class II transcripts were detected in areas around lesions but not in cells that were infected with VZV. VZV infection inhibited the expression of Stat 1alpha and Jak2 proteins but had little effect on Jak1. Analysis of regulatory events in the IFN-gamma signaling pathway showed that VZV infection inhibited transcription of interferon regulatory factor 1 and the MHC class II transactivator. This is the first report that VZV encodes an immunomodulatory function which directly interferes with the IFN-gamma signal transduction via the Jak/Stat pathway and enables the virus to inhibit IFN-gamma induction of cell surface MHC class II expression. This inhibition of MHC class II expression on VZV-infected cells in vivo may transiently protect cells from CD4(+) T-cell immune surveillance, facilitating local virus replication and transmission during the first few days of cutaneous lesion formation. (+info)
Varicella-zoster virus proteins in skin lesions: implications for a novel role of ORF29p in chickenpox.
(15/1300)
Skin biopsy samples from varicella-zoster virus (VZV)-infected patients examined by immunohistochemistry demonstrated VZV replication in nonepithelial cell types. ORF29p, a nonstructural nuclear protein, was found in nerves of two of six patients with chickenpox. In tissue culture, ORF29p was secreted by VZV-infected fibroblasts. Extracellular ORF29p can be taken up through endocytosis by human neurons, implying a novel role for this protein in pathogenesis. (+info)
Immunisation against varicella in end stage and pre-end stage renal failure. Trans-Pennine Paediatric Nephrology Study Group.
(16/1300)
OBJECTIVES: To investigate the seroconversion rate and duration of persistence of protective antibody titres after varicella immunisation in children with renal failure. DESIGN: 32 children (25 end stage and 7 pre-end stage renal failure) were immunised using 2 x 2,000 plaque forming unit doses of varicella vaccine 3 months apart. Varicella antibody titres were measured by enzyme linked immunosorbent assay. RESULTS: All children initially seroconverted after immunisation. At a mean follow up of 20.3 months, 23 of 28 had protective antibody titres, 4 children having died of unrelated causes. Two children required a third booster dose. 11 children underwent renal transplantation; 10 had protective titres at the time of transplantation and, at a mean of 23.4 months after immunisation, 6 currently have protective titres. Minor side effects occurred after 11 vaccine doses in 9 children. No child developed varicella, despite 10 clear episodes of exposure to the wild-type virus. CONCLUSIONS: Varicella immunisation in children with end stage and pre-end stage renal failure results in a high rate of seroconversion and persistence of protective antibody titres. More widespread use of the vaccine before renal transplantation is recommended. (+info)