Classic paper: Are the chickenpox virus and the zoster virus identical?: HELMUT RUSKA.

As early as 1943, the German physician Helmut Ruska visualized the virus of varicella and zoster (at that time, he was not completely certain whether the virus was the same) by the newly developed electron microscope; he is regarded as the discoverer of this virus. Here, we present a translation of his classical paper into the English language. In our introduction and commentary to his paper, we discuss the significance of Helmut Ruska's work for the development of virology, his distinction between the varicella, zoster, and herpes virus group on one hand and poxviruses on the other, as well as the development of imaging techniques which have refined or substituted for electron microscopy of viruses and virus-infected cells.

The varicella-zoster virus portal protein is essential for cleavage and packaging of viral DNA.

The varicella-zoster virus (VZV) open reading frame 54 (ORF54) gene encodes an 87-kDa monomer that oligomerizes to form the VZV portal protein, pORF54. pORF54 was hypothesized to perform a function similar to that of a previously described herpes simplex virus 1 (HSV-1) homolog, pUL6. pUL6 and the associated viral terminase are required for processing of concatemeric viral DNA and packaging of individual viral genomes into preformed capsids. In this report, we describe two VZV bacterial artificial chromosome (BAC) constructs with ORF54 gene deletions, Δ54L (full ORF deletion) and Δ54S (partial internal deletion). The full deletion of ORF54 likely disrupted essential adjacent genes (ORF53 and ORF55) and therefore could not be complemented on an ORF54-expressing cell line (ARPE54). In contrast, Δ54S was successfully propagated in ARPE54 cells but failed to replicate in parental, noncomplementing ARPE19 cells. Transmission electron microscopy confirmed the presence of only empty VZV capsids in Δ54S-infected ARPE19 cell nuclei. Similar to the HSV-1 genome, the VZV genome is composed of a unique long region (UL) and a unique short region (US) flanked by inverted repeats. DNA from cells infected with parental VZV (VZVLUC strain) contained the predicted UL and US termini, whereas cells infected with Δ54S contained neither. This result demonstrates that Δ54S is not able to process and package viral DNA, thus making pORF54 an excellent chemotherapeutic target. In addition, the utility of BAC constructs Δ54L and Δ54S as tools for the isolation of site-directed ORF54 mutants was demonstrated by recombineering single-nucleotide changes within ORF54 that conferred resistance to VZV-specific portal protein inhibitors. Importance: Antivirals with novel mechanisms of action would provide additional therapeutic options to treat human herpesvirus infections. Proteins involved in the herpesviral DNA encapsidation process have become promising antiviral targets. Previously, we described a series of N-α-methylbenzyl-N'-aryl thiourea analogs that target the VZV portal protein (pORF54) and prevent viral replication in vitro. To better understand the mechanism of action of these compounds, it is important to define the structural and functional characteristics of the VZV portal protein. In contrast to HSV, no VZV mutants have been described for any of the seven essential DNA encapsidation genes. The VZV ORF54 deletion mutant described in this study represents the first VZV encapsidation mutant reported to date. We demonstrate that the deletion mutant can serve as a platform for the isolation of portal mutants via recombineering and provide a strategy for more in-depth studies of VZV portal structure and function.

Single virus detection by means of atomic force microscopy in combination with advanced image analysis.

In the present contribution virions of five different virus species, namely Varicella-zoster virus, Porcine teschovirus, Tobacco mosaic virus, Coliphage M13 and Enterobacteria phage PsP3, are investigated using atomic force microscopy (AFM). From the resulting height images quantitative features like maximal height, area and volume of the viruses could be extracted and compared to reference values. Subsequently, these features were accompanied by image moments, which quantify the morphology of the virions. Both types of features could be utilized for an automatic discrimination of the five virus species. The accuracy of this classification model was 96.8%. Thus, a virus detection on a single-particle level using AFM images is possible. Due to the application of advanced image analysis the morphology could be quantified and used for further analysis. Here, an automatic recognition by means of a classification model could be achieved in a reliable and objective manner.

Aberrant virion assembly and limited glycoprotein C production in varicella-zoster virus-infected neurons.

Highly pure (>95%) terminally differentiated neurons derived from pluripotent stem cells appear healthy at 2 weeks after infection with varicella-zoster virus (VZV), and the cell culture medium contains no infectious virus. Analysis of the healthy-appearing neurons revealed VZV DNA, transcripts, and proteins corresponding to the VZV immediate early, early, and late kinetic phases of replication. Herein, we further characterized virus in these neuronal cells, focusing on (i) transcription and expression of late VZV glycoprotein C (gC) open reading frame 14 (ORF14) and (ii) ultrastructural features of virus particles in neurons. The analysis showed that gC was not expressed in most infected neurons and gC expression was markedly reduced in a minority of VZV-infected neurons. In contrast, expression of the early-late VZV gE glycoprotein (ORF68) was abundant. Transcript analysis also showed decreased gC transcription compared with gE. Examination of viral structure by high-resolution transmission electron microscopy revealed fewer viral particles than typically observed in cells productively infected with VZV. Furthermore, viral particles were more aberrant, in that most capsids in the nuclei lacked a dense core and most enveloped particles in the cytoplasm were light particles (envelopes without capsids). Together, these results suggest a considerable deficiency in late-phase replication and viral assembly during VZV infection of neurons in culture.

ORF9p phosphorylation by ORF47p is crucial for the formation and egress of varicella-zoster virus viral particles.

The role of the tegument during the herpesvirus lytic cycle is still not clearly established, particularly at the late phase of infection, when the newly produced viral particles need to be fully assembled before being released from the infected cell. The varicella-zoster virus (VZV) protein coded by open reading frame (ORF) 9 (ORF9p) is an essential tegument protein, and, even though its mRNA is the most expressed during the productive infection, little is known about its functions. Using a GalK positive/negative selection technique, we modified a bacterial artificial chromosome (BAC) containing the complete VZV genome to create viruses expressing mutant versions of ORF9p. We showed that ORF9p is hyperphosphorylated during the infection, especially through its interaction with the viral Ser/Thr kinase ORF47p; we identified a consensus site within ORF9p recognized by ORF47p and demonstrated its importance for ORF9p phosphorylation. Strikingly, an ultrastructural analysis revealed that the mutation of this consensus site (glutamate 85 to arginine) strongly affects viral assembly and release, reproducing the ORF47 kinase-dead VZV phenotype. It also slightly diminishes the infectivity toward immature dendritic cells. Taken together, our results identify ORF9p as a new viral substrate of ORF47p and suggest a determinant role of this phosphorylation for viral infectivity, especially during the process of viral particle formation and egress.

3D reconstruction of VZV infected cell nuclei and PML nuclear cages by serial section array scanning electron microscopy and electron tomography.

Varicella-zoster virus (VZV) is a human alphaherpesvirus that causes varicella (chickenpox) and herpes zoster (shingles). Like all herpesviruses, the VZV DNA genome is replicated in the nucleus and packaged into nucleocapsids that must egress across the nuclear membrane for incorporation into virus particles in the cytoplasm. Our recent work showed that VZV nucleocapsids are sequestered in nuclear cages formed from promyelocytic leukemia protein (PML) in vitro and in human dorsal root ganglia and skin xenografts in vivo. We sought a method to determine the three-dimensional (3D) distribution of nucleocapsids in the nuclei of herpesvirus-infected cells as well as the 3D shape, volume and ultrastructure of these unique PML subnuclear domains. Here we report the development of a novel 3D imaging and reconstruction strategy that we term Serial Section Array-Scanning Electron Microscopy (SSA-SEM) and its application to the analysis of VZV-infected cells and these nuclear PML cages. We show that SSA-SEM permits large volume imaging and 3D reconstruction at a resolution sufficient to localize, count and distinguish different types of VZV nucleocapsids and to visualize complete PML cages. This method allowed a quantitative determination of how many nucleocapsids can be sequestered within individual PML cages (sequestration capacity), what proportion of nucleocapsids are entrapped in single nuclei (sequestration efficiency) and revealed the ultrastructural detail of the PML cages. More than 98% of all nucleocapsids in reconstructed nuclear volumes were contained in PML cages and single PML cages sequestered up to 2,780 nucleocapsids, which were shown by electron tomography to be embedded and cross-linked by an filamentous electron-dense meshwork within these unique subnuclear domains. This SSA-SEM analysis extends our recent characterization of PML cages and provides a proof of concept for this new strategy to investigate events during virion assembly at the single cell level.

Non-axial view of the varicella-zoster virus portal protein reveals conserved crown, wing and clip architecture.

BACKGROUND: Herpesviridae encode a family of protein homologues that function as the 'port of entry' for insertion of the viral DNA into preformed capsids during encapsidation. METHODS: Transmission electron microscopy (TEM) of recombinant varicella-zoster virus pORF54 was performed. RESULTS: Results suggest that pORF54 forms higher-order structures with itself. Enriched fractions analyzed by TEM revealed non-axial oriented portals with defined central channels and distinguishable crown, wing and clip regions. CONCLUSION: These morphological features are consistent with those previously reported for other herpesvirus and bacteriophage portal proteins.

VZV infection of keratinocytes: production of cell-free infectious virions in vivo.

Varicella-zoster virus (VZV) is the cause of varicella (chickenpox) and zoster (shingles). Varicella is a primary infection that spreads rapidly in epidemics while zoster is a secondary infection that occurs sporadically as a result of the reactivation of previously acquired VZV. Reactivation is made possible by the establishment of latency during the initial episode of varicella. The signature lesions of both varicella and zoster are cutaneous vesicles, which are filled with a clear fluid that is rich in infectious viral particles. It has been postulated that the skin is the critical organ in which both host-to-host transmission of VZV and the infection of neurons to establish latency occur. This hypothesis is built on evidence that the large cation-independent mannose 6-phosphate receptor (MPR(ci)) interacts with VZV in virtually all infected cells, except those of the suprabasal epidermis, in a way that prevents the release of infectious viral particles. Specifically, the virus is diverted in an MPR(ci)-dependent manner from the secretory pathway to late endosomes where VZV is degraded. Because nonepidermal cells are thus prevented from releasing infectious VZV, a slow process, possibly involving fusion of infected cells with their neighbors, becomes the means by which VZV is disseminated. In the epidermis, however, the maturation of keratinocytes to give rise to corneocytes in the suprabasal epidermis is associated uniquely with a downregulation of the MPR(ci). As a result, the diversion of VZV to late endosomes does not occur in the suprabasal epidermis where vesicular lesions occur. The formation of the waterproof, chemically resistant barrier of the epidermis, however, requires that constitutive secretion outlast the downregulation of the endosomal pathway. Infectious VZV is therefore secreted by default, accounting for the presence of infectious virions in vesicular fluid. Sloughing of corneocytes, aided by scratching, then aerosolizes the virus, which can float with dust to be inhaled by susceptible hosts. Infectious virions also bathe the terminals of those sensory neurons that innervate the epidermis. These terminals become infected with VZV and provide a route, retrograde transport, which can conduct VZV to cranial nerve (CNG), dorsal root ganglia (DRG), and enteric ganglia (EG) to establish latency. Reactivation returns VZV to the skin, now via anterograde transport in axons, to cause the lesions of zoster. Evidence in support of these hypotheses includes observations of the VZV-infected human epidermis and studies of guinea pig neurons in an in vitro model system.

Overview of varicella-zoster virus glycoproteins gC, gH and gL.

The VZV genome is smaller than the HSV genome and only encodes nine glycoproteins. This chapter provides an overview of three VZV glycoproteins: gH (ORF37), gL (ORF60), and gC (ORF14). All three glycoproteins are highly conserved among the alpha herpesviruses. However, VZV gC exhibits unexpected differences from its HSV counterpart gC. In particular, both VZV gC transcription and protein expression are markedly delayed in cultured cells. These delays occur regardless of the virus strain or the cell type, and may account in part for the aberrant assembly of VZV particles. In contrast to VZV gC, the general properties of gH and gL more closely resemble their HSV homologs. VZV gL behaves as a chaperone protein to facilitate the maturation of the gH protein. The mature gH protein in turn is a potent fusogen. Its fusogenic activity can be abrogated when infected cultures are treated with monoclonal anti-gH antibodies.

Varicella zoster virus is not a disease-relevant antigen in multiple sclerosis.

Herpesvirions and varicella zoster virus (VZV) DNA were recently reported in all 15 cerebrospinal fluid (CSF) samples from patients with relapsing-remitting multiple sclerosis (MS) obtained within 1 week of exacerbation. Using identical electron microscopic and polymerase chain reaction techniques, including additional primer sets representing different regions of the VZV genome, we found no herpesvirions or VZV DNA in MS CSF or acute MS plaques. Although enzyme-linked immunosorbent assay analysis demonstrated a higher titer of VZV antibody in MS CSF than in inflammatory control samples, recombinant antibodies prepared from clonally expanded MS CSF plasma cells did not bind to VZV. VZV is not a disease-relevant antigen in MS.

Near-atomic cryo-electron microscopy structures of varicella-zoster virus capsids.

Varicella-zoster virus (VZV) is a medically important human herpesvirus that causes chickenpox and shingles, but its cell-associated nature has hindered structure studies. Here we report the cryo-electron microscopy structures of purified VZV A-capsid and C-capsid, as well as of the DNA-containing capsid inside the virion. Atomic models derived from these structures show that, despite enclosing a genome that is substantially smaller than those of other human herpesviruses, VZV has a similarly sized capsid, consisting of 955 major capsid protein (MCP), 900 small capsid protein (SCP), 640 triplex dimer (Tri2) and 320 triplex monomer (Tri1) subunits. The VZV capsid has high thermal stability, although with relatively fewer intra- and inter-capsid protein interactions and less stably associated tegument proteins compared with other human herpesviruses. Analysis with antibodies targeting the N and C termini of the VZV SCP indicates that the hexon-capping SCP-the largest among human herpesviruses-uses its N-terminal half to bridge hexon MCP subunits and possesses a C-terminal flexible half emanating from the inner rim of the upper hexon channel into the tegument layer. Correlation of these structural features and functional observations provide insights into VZV assembly and pathogenesis and should help efforts to engineer gene delivery and anticancer vectors based on the currently available VZV vaccine.

Varicella-zoster virus in cerebrospinal fluid at relapses of multiple sclerosis.

OBJECTIVE: Recent studies in peripheral blood mononuclear cells (PBMCs) have indicated that exacerbations of multiple sclerosis (MS) could be associated with the reactivation of latent varicella-zoster virus (VZV). METHODS: Ultrastructural observations for viral particles were made by electron microscopy in cerebrospinal fluid (CSF) from 15 MS patients during relapse, 19 MS patients during remission, and 28 control subjects. Initial findings were reproduced in a confirmation cohort. In addition, DNA from VZV was quantified by real-time polymerase chain reaction in PBMCs and CSF from a large number of MS patients (n = 78). RESULTS: We found by electron microscopy the presence of abundant viral particles identical to VZV in CSF obtained from MS patients within the first few days of an acute relapse. In contrast, viral particles were not seen in CSF samples from MS patients in remission or from neurological control subjects. Also, DNA from VZV was present in CSF and in PBMCs during relapse, disappearing in most patients during remission. The mean viral load was 542 times greater in CSF at relapse than in CSF at remission and 328 times greater in CSF at relapse than in PBMCs at relapse. INTERPRETATION: The ultrastructural finding of viral particles identical to VZV, together with the simultaneous presence of large quantities of DNA from VZV in the subarachnoid space, almost restricted to the periods of exacerbation, as well as its steady diminution and eventual disappearance from clinical relapse to clinical remission are surprising and constitute the strongest evidence to support the participation of VZV in the pathogenesis of MS.

Morphologic changes in fibroadenoma of breast due to chickenpox: a case report with suspicious cytology in fine needle aspiration smears.

BACKGROUND: Fibroadenomas with stromal giant cell reaction have been described in the literature, but cytologic atypia including giant cell reaction due to chickenpox giving rise to suspicious cytology has not been reported. CASE REPORT: A 25-year-old woman, recovering from chickenpox, presented with a 1.5 x 1.5-cm mass in the lower outer quadrant of her right breast. Fine needle aspiration smears showed sheets of benign ductal cells with overlapping myoepithelial cells and many bipolar bare nuclei. Cells showing nuclear enlargement, prominent nucleoli and multilobated or multinucleated giant cell formation occurred in separate sheets or dispersed among groups of benign ductal cells. Cytodiagnosis was suspicion for malignancy; excision biopsy was advised. Histopathologic examination showed fibroadenoma with evidence of epithelial hyperplasia, nuclear enlargement and multilobated giant cell formation. Atypical ductal cells, including the giant cells, were immunohistochemically positive for epithelial membrane antigen, estrogen receptor and progesterone receptor and negative for smooth muscle actin, indicating epithelial origin. Both cytologic and histologic specimens showed focal positive reaction with HSV-1 and HSV-2 antibodies. Ultrastructural examination of aspirated material revealed cytoplasmic viral particles with characteristic surface projections. CONCLUSION: Herpes zoster virus can produce morphologic alteration mimicking a malignancy. Pathologists should be aware of these changes to avoid a false positive diagnosis.

Pathogenesis of varicella-zoster angiitis in the CNS.

A 20-year-old man with Hodgkin's disease experienced ophthalmic zoster with dissemination and CNS involvement. At autopsy, he was found to have granulomatous angiitis involving the basilar artery, and electron microscopy revealed virus-like particles in the outer layers of the vessel walls, but not the endothelium. This suggests that granulomatous angiitis of the CNS in varicella-zoster infections results from direct viral invasion of blood vessels, perhaps by contiguous spread from cranial nerves.

Varicella-zoster viral glycoprotein envelopment: ultrastructural cytochemical localization.

The periodate-thiocarbohydrazide silver proteinate (PA-TCH-SP) method was used to study the envelopment process in varicella-zoster virus-infected human melanoma cells. Viral envelopment could be seen at two sites, the nuclear membrane and at virus-induced intracytoplasmic vacuoles. Virus-associated glycoconjugates were detected by the PA-TCH-SP method at the plasmalemma and on the inner membrane of the intracytoplasmic vacuoles. Virion envelopes acquired at the nuclear membrane were PA-TCH-SP negative, whereas those acquired at intracytoplasmic vacuoles were PA-TCH-SP positive. All virions found inside these vacuoles contained periodate-reactive envelopes. Release of virions into the extracellular space, where virtually all virions were PA-TCH-SP positive, appeared to be via exocytosis. Thus, the PA-TCH-SP method identifies glycoprotein incorporation at specific cytoplasmic vacuoles distinct from nuclear envelope, endoplasmic reticulum, and Golgi lamellae. These results suggest that envelopment within the cytoplasm is a stage in the assembly of the varicella-zoster virion.

Isomerization of the UL region of varicella-zoster virus DNA.

Varicella-zoster virus (VZV) DNA exists principally as two isomers. Despite the presence of inverted repeats bounding the long sequence region, the unique long sequence, UL, is found in one (prototype) orientation in 95-98% of VZV DNA molecules and in the inverted orientation in only 2-5% of the molecules. In searching for an explanation for this disparity, we superinfected VZV-infected cells with herpes simplex virus type 1 or pseudorabies virus. Neither superinfecting virus produced a measurable change in the frequency of isomerization of the VZV DNA long sequence region.

Replication of varicella zoster virus in Raji cells.

This report provides evidence for the replication of varicella zoster virus (VZV) in Raji cells. Infection was achieved by co-cultivation of Raji cells with VZV-infected human fibroblasts. Replication of VZV, as assessed by immunofluorescence using monoclonal antibodies against VZV-glycoproteins, ranged from 18 to 24% of the cells. Electron microscopy detected complete virions within the membrane-bound cytoplasmic vesicles and free viral particles in the nuclear matrix as late as 12 days post-infection. Western blot analysis of infected Raji cells demonstrated VZV-specific glycoproteins. The availability of a VZV-susceptible cell line growing in suspension culture provides a useful model for future studies.

Effectiveness of live varicella vaccine.

The disease burden of chickenpox to children has been described, and a lower force of neutralising antibody to varicella-zoster virus (VZV) than against measles, either after natural infection or after vaccination, has been reported. In the case of VZV, strong cell-mediated immunity may work efficiently to prevent the spread of the virus. The lower force of humoral antibody to VZV might be related to the occurrence of "breakthrough" varicella cases in a small portion of the vaccine recipients. Safety and high effectiveness of the varicella vaccine--approximately 85% effective for all diseases and 95-100% effective for moderate-to-severe diseases--have been reported. Vaccine-induced immunity persists for 10-20 years. However, concerns have been raised that universal immunisation in children may shift the susceptibility from children to adults, whose symptoms are usually moderate-to-severe. In addition, other concerns have been expressed that, due to lack of exposure to varicella in children, the elderly may develop zoster infections more frequently than before. A clear answer is difficult to give at present, although, for several reasons, such situations may be unlikely to occur.

Ocular varicella-zoster virus infection in the guinea pig. A new in vivo model.

Corneal intrastromal inoculation of guinea pigs with approximately 10(4) plaque-forming units of live, adapted varicella-zoster virus (VZV) resulted in reproducible, acute, superficial corneal disease in all animals. The culture-positive VZV ocular infection progressed to involve 30% to 40% of the corneal surface in a diffuse punctate keratitis and 10% to 15% of this surface with microdendrites, characteristic of VZV-induced ocular disease. Retrograde dissemination of VZV to the trigeminal ganglia, midbrain, cerebellum, and superior cervical ganglia was demonstrated by whole-cell coculture VZV recovery. Central nervous system VZV dissemination, manifested by transient neurologic symptoms and pneumonitis, was evident in 60% of the animals. Varicella-zoster virus spread to the trigeminal ganglion during acute and early-latent infection was evident by electron microscopy.