Effects of Grapevine Leafroll associated Virus 3 (GLRaV-3) and duration of infection on fruit composition and wine chemical profile of Vitis vinifera L. cv. Sauvignon blanc.

In order to determine the effects of Grapevine Leafroll associated Virus 3 (GLRaV-3) on fruit composition and chemical profile of juice and wine from Vitis vinifera L. cv. Sauvignon blanc grown in New Zealand, composition variables were measured on fruit from vines either infected with GLRaV-3 (established or recent infections) or uninfected vines. Physiological ripeness (20.4°Brix) was the criterion established to determine the harvest date for each of the three treatments. Date of grape ripeness was strongly affected by virus infection. In juice and wine, GLRaV-3 infection prior to 2008 reduced titratable acidity compared with the uninfected control. Differences observed in amino acids from the three infection status groups did not modify basic wine chemical properties. In conclusion, GLRaV-3 infection slowed grape ripening, but at equivalent ripeness to result in minimal effects on the juice and wine chemistry. Time of infection produced differences in specific plant physiological variables.

Insulin-degrading enzyme binds to the nonglycosylated precursor of varicella-zoster virus gE protein found in the endoplasmic reticulum.

Insulin degradation enzyme (IDE) is a 110-kDa zinc metalloprotease found in the cytosol of all cells. IDE degrades insulin and a variety of small proteins including amyloid-beta. Recently, IDE has been proposed as the receptor for varicella-zoster virus (VZV) attachment. During our reassessment, some of the original studies were repeated and expanded in scope. We first confirmed that IDE antibody reduced VZV spread. For additional controls, we repeated the same experiments with herpes simplex virus (HSV)-infected cells as well as uninfected cells. There was a visible reduction in HSV spread but less than seen in the VZV system. Of greater importance, IDE antibody also inhibited the growth of uninfected cells. Second, we repeated the coprecipitation assays. We confirmed that antibodies to VZV gE (open reading frame 68) coprecipitated IDE and that anti-IDE antibody coprecipitated gE. However, the detected gE protein was not the mature 98-kDa form; rather, it was a precursor 73-kDa gE form found in the endoplasmic reticulum. Additional control experiments included VZV-infected cell cultures treated with tunicamycin to block gE glycosylation in the endoplasmic reticulum; again, the anti-IDE antibody coprecipitated a 73-kDa gE product. Finally, Orbitrap mass spectrometry analysis of a chromatographically purified gE sample revealed four cellular proteins associated with the unfolded protein response: BiP (HSPA5), HSPA8, HSPD1, and PPIA (peptidyl-propyl cis-trans isomerase). We conclude that IDE protease binds to the 73-kDa gE precursor and that this event occurs in the cytosol but not as a receptor/ligand interaction.

Genomic cartography of varicella-zoster virus: a complete genome-based analysis of strain variability with implications for attenuation and phenotypic differences.

In order to gain a better perspective on the true variability of varicella-zoster virus (VZV) and to catalogue the location and number of differences, 11 new complete genome sequences were compared with those previously in the public domain (18 complete genomes in total). Three of the newly sequenced genomes were derived from a single strain in order to assess variations that can occur during serial passage in cell culture. The analysis revealed that while VZV is relatively stable genetically it does posses a certain degree of variability. The reiteration regions, origins of replication and intergenic homopolymer regions were all found to be variable between strains as well as within a given strain. In addition, the terminal viral sequences were found to vary within and between strains specifically at the 3' end of the genome. Analysis of single nucleotide polymorphisms (SNPs) identified a total of 557 variable sites, 451 of which were found in coding regions and resulted in 187 different in amino acid substitutions. A comparison of the SNPs present in the two gE mutant strains, VZV-MSP and VZV-BC, suggested that the missense mutation in gE was primarily responsible for the accelerated cell spread phenotype. Some of the variations noted with high passage in cell culture are consistent with variations seen in the IE62 gene of the vaccine strains (S628G, R958G and I1260V) that may help in pinpointing variations essential for attenuation. Although VZV has been considered to be one of the most genetically stable human herpesviruses, this initial assessment of genomic VZV cartography provides insight into ORFs with previously unreported variations.

Varicella zoster virus: out of Africa and into the research laboratory.

This review updates on numerous topics relating to the evolutionary origins of varicella zoster virus (VZV), the replication cycle, virion assembly and the recent genomic analyses. VZV is one of eight human herpesviruses that have existed for at least 400 million years. It has co-evolved with humankind and is present in all nationalities globally. The pathogenesis of varicella (chickenpox) is dependent on viral replication and dispersion through the body in T-lymphocytes. VZV replication is similar to that of herpes simplex virus. A complete analysis of VZV transcripts has identified their relative abundance, with transcripts for the regulatory proteins (open reading frame) ORF62 and ORF63 among the greatest. Studies of virion assembly have shown that endocytosis pathways are involved in the envelopment process by the viral glycoproteins. The complete sequencing of five VZV strains has identified numerous single nucleotide polymorphisms, and in turn, VZV strains have been segregated into European/North American and Asian clades. Furthermore, a small number of mutant VZV strains have been identified. These results suggest more diversity between VZV strains than previously recognized.

Partner's influence on patient preference for treatment in early prostate cancer.

OBJECTIVE: To determine the partner's influence on the patient's choice of treatment for early prostate cancer, and whether partner characteristics and biases predict the preference. PATIENTS, SUBJECTS AND METHODS: Questionnaires for partners to complete retrospectively were sent to consecutive patients recruited in a study comparing treatment options for early prostate cancer. The partners' perceptions about prostate cancer were explored and the partners asked to comment on the suitability of each treatment option. Partners recorded their influence on the patient's choice using a 10-point visual linear analogue scale. RESULTS: Questionnaires were sent to 116 eligible patients and 82 were returned for analysis (mean partner age 63 years). When asked to recall the treatment options initially discussed, all partners recalled radiotherapy (EBRT), all but one radical prostatectomy (RP), 51% brachytherapy, but only 29% watchful waiting (WW); 41% of partners stated RP as their chosen option, 37% EBRT, 12% brachytherapy and 10% no clear favourite. None preferred WW. Employment and education status were not significant predictors of partners' preference but retired partners and those aged > 65 years were 3 times more likely to prefer EBRT than were their employed and younger counterparts, respectively. The partners' mean (median, SD) self-assessed influence factor was 4.8 (5, 3.4). Of the partners, 88% reported active involvement throughout the process, identifying information-gathering and emotional support as their primary roles. Most deliberately chose not to influence the patient's final decision. CONCLUSION: Partner preference is influenced by pre-existing conceptions about cancer and its treatment. While undoubtedly influential throughout the decision-making process, partners deliberately left the final decision to the patient.

Genomic analysis of varicella-zoster virus: primers for individual open reading frames.

The genome of varicella-zoster virus (VZV) contains nearly 125,000 bp. Preliminary genomic analysis has revealed that VZV may be less immutable than once thought. Through the investigation of the VZV genome using specifically designed oligonucleotides, it has been learned that sequence variation within VZV open reading frame 62 can distinguish between vaccine and wild-type virus. Additionally, the presence of single nucleotide polymorphisms within the VZV genome has identified distinct VZV populations originating from circumscribed geographic locations. In order for future studies of VZV genetic diversity to be carried out, amplifying and sequencing primers for individual VZV genes have been catalogued. Additionally, this report will facilitate the selection of VZV primers by which to distinguish clinical VZV isolates from vaccinia virus isolates.

Varicella-zoster Virus gB and gE coexpression, but not gB or gE alone, leads to abundant fusion and syncytium formation equivalent to those from gH and gL coexpression.

Varicella-zoster virus (VZV) is distinguished from herpes simplex virus type 1 (HSV-1) by the fact that cell-to-cell fusion and syncytium formation require only gH and gL within a transient-expression system. In the HSV system, four glycoproteins, namely, gH, gL, gB, and gD, are required to induce a similar fusogenic event. VZV lacks a gD homologous protein. In this report, the role of VZV gB as a fusogen was investigated and compared to the gH-gL complex. First of all, the VZV gH-gL experiment was repeated under a different set of conditions; namely, gH and gL were cloned into the same vaccinia virus (VV) genome. Surprisingly, the new expression system demonstrated that a recombinant VV-gH+gL construct was even more fusogenic than seen in the prior experiment with two individual expression plasmids containing gH and gL (K. M. Duus and C. Grose, J. Virol. 70:8961-8971, 1996). Recombinant VV expressing VZV gB by itself, however, effected the formation of only small syncytia. When VZV gE and gB genes were cloned into one recombinant VV genome and another fusion assay was performed, extensive syncytium formation was observed. The degree of fusion with VZV gE-gB coexpression was comparable to that observed with VZV gH-gL: in both cases, >80% of the cells in a monolayer were fused. Thus, these studies established that VZV gE-gB coexpression greatly enhanced the fusogenic properties of gB. Control experiments documented that the fusion assay required a balance between the fusogenic potential of the VZV glycoproteins and the fusion-inhibitory effect of the VV infection itself.

Varicella-zoster virus ORF47 protein serine kinase: characterization of a cloned, biologically active phosphotransferase and two viral substrates, ORF62 and ORF63.

Varicella-zoster virus (VZV) codes for a protein serine kinase called ORF47; the herpes simplex virus (HSV) homolog is UL13. No recombinant alphaherpesvirus serine kinase has been biologically active in vitro. We discovered that preservation of the intrinsic kinase activity of recombinant VZV ORF47 required unusually stringent in vitro conditions, including physiological concentrations of polyamines. In this assay, ORF47 phosphorylated two VZV regulatory proteins: the ORF62 protein (homolog of HSV ICP4) and the ORF63 protein (homolog of HSV ICP22). Of interest, ORF47 kinase also coprecipitated ORF63 protein from the kinase assay supernatant.

Mutational analysis of the repeated open reading frames, ORFs 63 and 70 and ORFs 64 and 69, of varicella-zoster virus.

Varicella-zoster virus (VZV) open reading frame 63 (ORF63), located between nucleotides 110581 and 111417 in the internal repeat region, encodes a nuclear phosphoprotein which is homologous to herpes simplex virus type 1 (HSV-1) ICP22 and is duplicated in the terminal repeat region as ORF70 (nucleotides 118480 to 119316). We evaluated the role of ORFs 63 and 70 in VZV replication, using recombinant VZV cosmids and PCR-based mutagenesis to make single and dual deletions of these ORFs. VZV was recovered within 8 to 10 days when cosmids with single deletions were transfected into melanoma cells along with the three intact VZV cosmids. In contrast, VZV was not detected in transfections carried out with a dual deletion cosmid. Infectious virus was recovered when ORF63 was cloned into a nonnative AvrII site in this cosmid, confirming that failure to generate virus was due to the dual ORF63/70 deletion and that replication required at least one gene copy. This requirement may be related to our observation that ORF63 interacts directly with ORF62, the major immediate-early transactivating protein of VZV. ORF64 is located within the inverted repeat region between nucleotides 111565 and 112107; it has some homology to the HSV-1 Us10 gene and is duplicated as ORF69 (nucleotides 117790 to 118332). ORF64 and ORF69 were deleted individually or simultaneously using the VZV cosmid system. Single deletions of ORF64 or ORF69 yielded viral plaques with the same kinetics and morphology as viruses generated with the parental cosmids. The dual deletion of ORF64 and ORF69 was associated with an abnormal plaque phenotype characterized by very large, multinucleated syncytia. Finally, all of the deletion mutants that yielded recombinants retained infectivity for human T cells in vitro and replicated efficiently in human skin in the SCIDhu mouse model of VZV pathogenesis.

Multimeric humanized varicella-zoster virus antibody fragments to gH neutralize virus while monomeric fragments do not.

Murine monoclonal antibody 206 (MAb mu206) binds to gH, the varicella-zoster virus (VZV) fusogen, neutralizing the virus in vitro in the absence of complement and inhibiting cell-to-cell spread and egress of VZV in cultured cells. We have humanized this antibody to generate MAb hu206 by complementarity determining region grafting. MAb hu206 retained binding and in vitro neutralizing activity, as well as cross-reactivity with ten different VZV strains. Single-chain antibody fragments (scAb) derived from MAb hu206 were produced in Escherichia coli. These scAb retained the binding properties of the whole antibody. However, monomeric scAb exhibited markedly reduced neutralizing activity compared to the bivalent parental MAb hu206. Shortening the peptide linker joining the V(H) to the V(kappa) domain from 14 to 5 or even 0 residues encouraged multimerization and increased neutralizing efficacy. The fact that Fab fragments enzymatically generated from whole MAb hu206 lost their neutralizing potency lent support to the proposal that valency is important for VZV neutralization at this epitope.

Varicella-zoster virus with a lost gE epitope: evidence for immunological pressure by the human antibody response.

The varicella-zoster virus (VZV) genome contains about 70 open reading frames (ORF). ORF 68 codes for glycoprotein gE, formerly called gpl, which is the predominant VZV glycoprotein; gE is a typical type 1 transmembrane protein with 623 amino acids. Recently, a variant virus was discovered which has a mutation in gE codon 150; this mutation converts an aspartic acid into an asparagine residue.

Interactions among structural proteins of varicella zoster virus.

Varicella zoster virus tegument components include the regulatory proteins IE4, IE62, IE63 and the ORFI0 protein, a protein kinase (ORF47) and an abundant protein encoded in ORF9 which is the homolog of HSV VP22. The kinase is able to phosphorylate IE62 and the ORF9 protein specifically in viral particles. We show that interactions among these proteins are, at least in part, dependent on the presence or absence of phosphate groups and we suggest models for tegument formation and for its dissolution in the infected cell.

Varicella-zoster virus gE escape mutant VZV-MSP exhibits an accelerated cell-to-cell spread phenotype in both infected cell cultures and SCID-hu mice.

Varicella-zoster virus is considered to have one of the most stable genomes of all human herpesviruses. In 1998, we reported the unanticipated discovery of a wild-type virus that had lost an immunodominant B-cell epitope on the gE ectodomain (VZV-MSP); the gE escape mutant virus exhibited an unusual pattern of egress. Further studies have now documented a markedly enhanced cell-to-cell spread by the mutant virus in cell culture. This property was investigated by laser scanning confocal microscopy combined with a software program that allows the measurement of pixel intensity of the fluorescent signal. For this new application of imaging technology, the VZV immediate early protein 62 (IE 62) was selected as the fluoresceinated marker. By 48 h postinfection, the number of IE 62-positive pixels in the VZV-MSP-infected culture was nearly fourfold greater than the number of pixels in a culture infected with a low-passage laboratory strain. Titrations by infectious center assays supported the above image analysis data. Confirmatory studies in the SCID-hu mouse documented that VZV-MSP spread more rapidly than other VZV strains in human fetal skin implants. Generally, the cytopathology and vesicle formation produced by other strains at 21 days postinfection were demonstrable with VZV-MSP at 14 days. To assess whether additional genes were contributing to the unusual VZV-MSP phenotype, approximately 20 kb of the VZV-MSP genome was sequenced, including ORFs 31 (gB), 37 (gH), 47, 60 (gL), 61, 62 (IE 62), 66, 67 (gI), and 68 (gE). Except for a few polymorphisms, as well as the previously discovered mutation within gE, the nucleotide sequences within most open reading frames were identical to the prototype VZV-Dumas strain. In short, VZV-MSP represents a novel variant virus with a distinguishable phenotype demonstrable in both infected cell cultures and SCID-hu mice.

Subacute sclerosing panencephalitis, a measles complication, in an internationally adopted child.

A healthy 13-year-old boy who had spent the first 4.5 years of his life in an orphanage in Thailand before adoption by an American couple became ill with subacute sclerosing panencephalitis and died several months later. The boy had most likely contracted wild-type measles in Thailand. Measles complications are a risk in international adoptions.

Inputs to combination-sensitive neurons of the inferior colliculus.

In the mustached bat, combination-sensitive neurons display integrative responses to combinations of acoustic elements in biosonar or social vocalizations. One type of combination-sensitive neuron responds to multiple harmonics of the frequency-modulated (FM) components in the sonar pulse and echo of the bat. These neurons, termed FM-FM neurons, are sensitive to the pulse-echo delay and may encode the distance of sonar targets. FM-FM neurons are common in high-frequency regions of the central nucleus of the inferior colliculus (ICC) and may be created there. If so, they must receive low-frequency inputs in addition to the expected high-frequency inputs. We placed single deposits of a tracer at FM-FM recording sites in the ICC and then analyzed retrograde labeling in the brainstem and midbrain. We were particularly interested in labeling patterns suggestive of low-frequency input to these FM-FM neurons. In most nuclei containing labeled cells, there was a single focus of labeling in regions thought to be responsive to high-frequency sounds. More complex labeling patterns were observed in three nuclei. In the anteroventral cochlear nucleus, labeling in the anterior and marginal cell divisions occurred in regions thought to respond to low-frequency sounds. This labeling comprised 6% of total brainstem labeled cells. Labeling in the intermediate nucleus of the lateral lemniscus and the magnocellular part of the ventral nucleus of the lateral lemniscus together comprised nearly 40% of all labeled cells. In both nuclei, multiple foci of labeling occurred. These different foci may represent groups of cells tuned to different frequency bands. Thus, one or more of these three nuclei may provide low-frequency input to high-frequency-sensitive cells in the ICC, creating FM-FM responses. We also examined whether ICC neurons responsive to lower frequencies project to high-frequency-sensitive ICC regions; only 0.15% of labeling originated from these lower frequency representations. If the spectral integration of FM-FM neurons is created at the level of the ICC, these results suggest that neurons of the anteroventral cochlear nucleus or monaural nuclei of the lateral lemniscus may provide the essential low-frequency input. In contrast, there is little evidence that the low-frequency representation of the ICC contributes to these integrative responses.

Varicella-zoster virus Fc receptor component gI is phosphorylated on its endodomain by a cyclin-dependent kinase.

Varicella-zoster virus (VZV) glycoprotein gI is a type 1 transmembrane glycoprotein which is one component of the heterodimeric gE:gI Fc receptor complex. Like VZV gE, VZV gI was phosphorylated in both VZV-infected cells and gI-transfected cells. Preliminary studies demonstrated that a serine 343-proline 344 sequence located within the gI cytoplasmic tail was the most likely phosphorylation site. To determine which protein kinase catalyzed the gI phosphorylation event, we constructed a fusion protein, consisting of glutathione-S-transferase (GST) and the gI cytoplasmic tail, called GST-gI-wt. When this fusion protein was used as a substrate for gI phosphorylation in vitro, the results demonstrated that GST-gI-wt fusion protein was phosphorylated by a representative cyclin-dependent kinase (CDK) called P-TEFb, a homologue of CDK1 (cdc2). When serine 343 within the serine-proline phosphorylation site was replaced with an alanine residue, the level of phosphorylation of the gI fusion protein was greatly reduced. Subsequent experiments with individually immunoprecipitated mammalian CDKs revealed that the VZV gI fusion protein was phosphorylated best by CDK1, to a lesser degree by CDK2, and not at all by CDK6. Transient-transfection assays carried out in the presence of the specific CDK inhibitor roscovitine strongly supported the prior results by demonstrating a marked decrease in gI phosphorylation while gI protein expression was unaffected. Finally, the possibility that VZV gI contained a CDK phosphorylation site in its endodomain was of further interest because its partner, gE, contains a casein kinase II phosphorylation site in its endodomain; prior studies have established that CDK1 can phosphorylate casein kinase II.