Herpesvirus Nuclear Egress across the Outer Nuclear Membrane.

Herpesvirus capsids are assembled in the nucleus and undergo a two-step process to cross the nuclear envelope. Capsids bud into the inner nuclear membrane (INM) aided by the nuclear egress complex (NEC) proteins UL31/34. At that stage of egress, enveloped virions are found for a short time in the perinuclear space. In the second step of nuclear egress, perinuclear enveloped virions (PEVs) fuse with the outer nuclear membrane (ONM) delivering capsids into the cytoplasm. Once in the cytoplasm, capsids undergo re-envelopment in the Golgi/trans-Golgi apparatus producing mature virions. This second step of nuclear egress is known as de-envelopment and is the focus of this review. Compared with herpesvirus envelopment at the INM, much less is known about de-envelopment. We propose a model in which de-envelopment involves two phases: (i) fusion of the PEV membrane with the ONM and (ii) expansion of the fusion pore leading to release of the viral capsid into the cytoplasm. The first phase of de-envelopment, membrane fusion, involves four herpes simplex virus (HSV) proteins: gB, gH/gL, gK and UL20. gB is the viral fusion protein and appears to act to perturb membranes and promote fusion. gH/gL may also have similar properties and appears to be able to act in de-envelopment without gB. gK and UL20 negatively regulate these fusion proteins. In the second phase of de-envelopment (pore expansion and capsid release), an alpha-herpesvirus protein kinase, US3, acts to phosphorylate NEC proteins, which normally produce membrane curvature during envelopment. Phosphorylation of NEC proteins reverses tight membrane curvature, causing expansion of the membrane fusion pore and promoting release of capsids into the cytoplasm.

Viruses Infecting the European Catfish (Silurus glanis).

In aquaculture, disease management and pathogen control are key for a successful fish farming industry. In past years, European catfish farming has been flourishing. However, devastating fish pathogens including limiting fish viruses are considered a big threat to further expanding of the industry. Even though mainly the ranavirus (Iridoviridea) and circovirus (Circoviridea) infections are considered well- described in European catfish, more other agents including herpes-, rhabdo or papillomaviruses are also observed in the tissues of catfish with or without any symptoms. The etiological role of these viruses has been unclear until now. Hence, there is a requisite for more detailed information about the latter and the development of preventive and therapeutic approaches to complete them. In this review, we summarize recent knowledge about viruses that affect the European catfish and describe their origin, distribution, molecular characterisation, and phylogenetic classification. We also highlight the knowledge gaps, which need more in-depth investigations in the future.

Characterization of gene deletion mutants of Cyprinid herpesvirus 3 (koi herpesvirus) lacking the immunogenic envelope glycoproteins pORF25, pORF65, pORF148 and pORF149.

Cyprinid herpesvirus 3 (CyHV-3) or koi herpesvirus is a global pathogen causing mass mortality in koi and common carp, against which improved vaccines are urgently needed. In this study we investigated the role of four nonessential, but immunogenic envelope glycoproteins encoded by members of the ORF25 gene family (ORF25, ORF65, ORF148 and ORF149) during CyHV-3 replication. Single deletion of ORF65 did not affect in vitro replication, and deletion of ORF148 even slightly enhanced virus growth on common carp brain (CCB) cells. Deletions of ORF25 or ORF149 led to reduced plaque sizes and virus titers, which was due to delayed entry into host cells. An ORF148/ORF149 double deletion mutant exhibited wild-type like growth indicating opposing functions of the two proteins. Electron microscopy of CCB cells infected with either mutant did not indicate any effects on virion formation and maturation in nucleus or cytoplasm, nor on release of enveloped particles. The ORF148, ORF149 and double deletion mutants were also tested in animal experiments using juvenile carp, and proved to be insufficiently attenuated for use as live virus vaccines. However, surviving fish were protected against challenge with wild-type CyHV-3, demonstrating that these antibody inducing proteins are dispensable for an efficient immune response in vivo.

In Vitro Replication of Chelonid Herpesvirus 5 in Organotypic Skin Cultures from Hawaiian Green Turtles (Chelonia mydas).

Fibropapillomatosis (FP) is a tumor disease of marine turtles associated with chelonid herpesvirus 5 (ChHV5), which has historically been refractory to growth in tissue culture. Here we show, for the first time, de novo formation of ChHV5-positive intranuclear inclusions in cultured green turtle cells, which is indicative of active lytic replication of the virus. The minimal requirements to achieve lytic replication in cultured cells included (i) either in vitro cultures of ChHV5-positive tumor biopsy specimens (plugs) or organotypic cultures (rafts) consisting of ChHV5-positive turtle fibroblasts in collagen rafts seeded with turtle keratinocytes and (ii) keratinocyte maturation induced by raising raft or biopsy cultures to the air-liquid interface. Virus growth was confirmed by detailed electron microscopic studies that revealed intranuclear sun-shaped capsid factories, tubules, various stages of capsid formation, nuclear export by budding into the perinuclear space, tegument formation, and envelopment to complete de novo virus production. Membrane synthesis was also observed as a sign of active viral replication. Interestingly, cytoplasmic particles became associated with keratin filaments, a feature not seen in conventional monolayer cell cultures, in which most studies of herpesvirus replication have been performed. Our findings draw a rich and realistic picture of ChHV5 replication in cells derived from its natural host and may be crucial not only to better understand ChHV5 circulation but also to eventually complete Koch's postulates for FP. Moreover, the principles described here may serve as a model for culture of other viruses that are resistant to replication in conventional cell culture.IMPORTANCE A major challenge in virology is the study of viruses that cannot be grown in the laboratory. One example is chelonid herpesvirus 5 (ChHV5), which is associated with fibropapillomatosis, a globally distributed, debilitating, and fatal tumor disease of endangered marine turtles. Pathological examination shows that ChHV5 is shed in skin. Here we show that ChHV5 will grow in vitro if we replicate the complex three-dimensional structure of turtle skin. Moreover, lytic virus growth requires a close interplay between fibroblasts and keratinocytes. Finally, the morphogenesis of herpesviral growth in three-dimensional cultures reveals a far richer, and likely more realistic, array of capsid morphologies than that encountered in traditional monolayer cell cultures. Our findings have applications to other viruses, including those of humans.

Detection of Cyprinid herpesvirus 2 in association with an Aeromonas sobria infection of Carassius carassius (L.), in Italy.

Sixteen specimens of female crucian carp, Carassius carassius (L.), during the breeding season, were investigated for post-mortem and full diagnostic examination during a mortality outbreak in a tributary stream of the Arno River in Tuscany in 2011. Necropsy highlighted the presence of a swollen anus and widespread haemorrhages in the body, fins, gills and eyes. Haemorrhages in internal organs and spleen granulomas were also observed. Bacteria isolated from the brain, kidney and spleen of affected fish were identified as A. sobria. Microscopic lesions observed in gills were characterized by necrosis of the secondary lamellae, congestion and multifocal lamellar fusion. The kidney showed necrosis, oedema, fibrin exudation and areas of haemorrhages, while in the spleen the main lesions were by multifocal necrosis of the lymphoid tissue. In the gills, transmission electron microscopy revealed herpesvirus-like particles, subsequently identified as Cyprinid herpesvirus-2 (CyHV-2) with a nested PCR protocol. Although it was not possible to attribute a pathogenic role to CyHV-2 in this mortality event, the identification of this herpesvirus in crucian carp increases the concern about its potential role in this species.

The way out: what we know and do not know about herpesvirus nuclear egress.

Herpesvirus capsids are assembled in the nucleus of infected cells whereas final maturation occurs in the cytosol. To access the final maturation compartment, intranuclear capsids have to cross the nuclear envelope which represents a formidable barrier. They do so by budding at the inner nuclear membrane, thereby forming a primary enveloped particle residing in the perinuclear cleft. Formation of primary envelopes is driven by a heterodimeric complex of two conserved herpesviral proteins, designated in the herpes simplex virus nomenclature as pUL34, a tail-anchored transmembrane protein located in the nuclear envelope, and pUL31. This nuclear egress complex recruits viral and cellular kinases to soften the nuclear lamina and allowing access of capsids to the inner nuclear membrane. How capsids are recruited to the budding site and into the primary virus particle is still not completely understood, nor is the composition of the primary enveloped virion in the perinuclear cleft. Fusion of the primary envelope with the outer nuclear membrane then results in translocation of the capsid to the cytosol. This fusion event is clearly different from fusion during infectious entry of free virions into target cells in that it does not require the conserved essential core herpesvirus fusion machinery. Nuclear egress can thus be viewed as a vesicle (primary envelope)-mediated transport of cargo (capsids) through thenuclear envelope, a process which had been unique in cell biology. Only recently has a similar process been identified in Drosophila for nuclear egress of large ribonucleoprotein complexes. Thus, herpesviruses appear to subvert a hitherto cryptic cellular pathway for translocation of capsids from the nucleus to the cytosol.

Role of a reducing environment in disassembly of the herpesvirus tegument.

Initiation of infection by herpes family viruses involves a step in which most of the virus tegument becomes detached from the capsid. Detachment takes place in the host cell cytosol near the virus entry site and it is followed by dispersal of tegument proteins and disappearance of the tegument as a distinct entity. Here we describe the results of experiments designed to test the idea that the reducing environment of the cytosol may contribute to tegument detachment and disassembly. Non-ionic detergent was used to remove the membrane of purified herpes simplex virus under control and reducing conditions. The effects on the tegument were then examined by SDS-PAGE and electron microscopy. Protein analysis demonstrated that most major tegument proteins were removed under both oxidizing and reducing conditions except for UL49 which required a reducing environment. It is proposed therefore that the reducing conditions in the cytosol are involved in removal of UL49 protein. Electron microscopic analysis revealed that capsids produced under oxidizing conditions contained a coating of protein that was absent in reduced virions and which correlated uniquely with the presence of UL49. This capsid-associated layer is suggested to be the location of UL49 in the extracted virion.

Viral infection of cells in culture--approaches for electron microscopy.

In this article we present and discuss three electron microscopical approaches to investigate the pathway of human cytomegalovirus (Herpesviridae) infection in cultivated human macrophages and fibroblasts. These methods include surface scanning electron microscopy and high-pressure freezing combined with freeze-substitution and electron tomography. With this set of methods, basically all steps of the viral cycle can be investigated and documented. These methods are especially helpful to investigate membrane structures, since viral particles of a diameter of 150 nm fit well into a semi-thin section of about 500 nm that can be imaged in three dimensions with STEM tomography.

A TEM/SEM study of the microbial plaque overlying the necrotic gingival papillae of HIV-seropositive, necrotizing ulcerative periodontitis.

The purpose of this study was to examine by transmission (TEM) and scanning electron microscopy (SEM) the supragingival microbial plaque overlying the ulcerated gingival papillae of necrotizing ulcerative periodontitis (NUP) lesions in HIV-seropositive patients. The microbiota of NUP and HIV-seropositive patients with periodontitis has been reported to be similar to that of conventional periodontitis in non-infected subjects, although several investigators have also reported high recovery rates of microbes not generally associated with the indigenous oral microbial flora. Light and electron microscopic observations and microbial culture studies indicate a similar high prevalence of spirochetes in both necrotizing ulcerative gingivitis (NUG) and NUP. In addition, several studies have reported more frequent isolation of Candida albicans from diseased periodontal sites in HIV-seropositive patients than from non-diseased sites. Ten male and six female patients, each HIV-seropositive and exhibiting NUP, constituted the study population. Two biopsies of involved gingival papillae from between posterior teeth were obtained from each patient and processed for examination by both TEM and SEM. Microscopic examination revealed a surface biofilm comprised of a mixed microbial flora of various morphotypes in 81.3% of biopsy specimens. The subsurface flora featured dense aggregations of spirochetes in 87.5% of specimens. Zones of aggregated polymorphonuclear leukocytes and necrotic cells were also noted. Yeasts were observed in 65.6% of specimens and herpes-like viruses in 56.5% of the specimens. Collectively, except for the presence of yeast and viruses, the results suggest that the microbial flora and possibly the soft tissue lesions of NUP and necrotizing ulcerative gingivitis are very similar.

Isolation and molecular characterization of herpesvirus from cultured European eels Anguilla anguilla in Taiwan.

A herpesvirus has been isolated for the first time from a population of European eels Anguilla anguilla cultured in a recirculated system in Taiwan. Syncytia formation was detected in EP-1 (eel epidermis) cell cultures inoculated with cell-free homogenates prepared from both integument and visceral organs of moribund fish. Inoculation of homogenates onto EK (eel kidney) cell cultures induced giant cell formation. Subsequent passages produced a consistent and progressive cytopathic effect (CPE) in cell cultures. In this study, EP-1 cell cultures infected with EEHV (European eel herpesvirus) were examined using an electron microscope. Numerous nucleocapsids of about 100 nm in diameter were found within the nucleus of infected cells, whereas enveloped particles were observed within the cytoplasm. The mature viral particle, about 235 nm in diameter, had an electron-dense core with a hexagonal nucleocapsid surrounded by a coarse capsule. Histopathological examination of moribund fish showed epithelial hyperplasia with intracytoplasmic metabolic inclusions in the skin. Macrophage aggregates were found in liver, spleen, and kidney. A pair of primers designed from channel catfish virus and salmonid herpesvirus 1 was used in a polymerase chain reaction. A 402 bp fragment was amplified and cloned from genomic DNA of EEHV. The nucleotide homology was 99% (298 of 300) with DNA polymerase of eel herpesvirus (anguillid herpesvirus). EEHV nucleic acids were detected within melanomacrophages in the skin, liver, spleen and kidney by in situ hybridization (ISH).

High resolution structural studies of complex icosahedral viruses: a brief overview.

Structural descriptions of viral particles are key to our understanding of their assembly mechanisms and properties. We will describe the application of X-ray crystallography and electron cryomicroscopy to the structural determination of the bluetongue virus core and the herpesvirus capsid. These represent the highest resolution structural studies carried out by these techniques on such complex and large icosahedral virus particles. The bluetongue virus core consists of two layers of distinct proteins with different protein packing symmetries, while the herpes virus capsid is made up of four types of proteins with 3.3 MDa per asymmetric unit. The structural results reveal that each of these proteins has distinct folds and they are packed uniquely to form stable particles.

Inactivation of frog virus 3 and channel catfish virus by esculentin-2P and ranatuerin-2P, two antimicrobial peptides isolated from frog skin.

While it is clear that some amphibian populations have recently experienced precipitous declines, the causes of those die-offs are complex and likely involve multiple variables. One theory suggests that environmental factors may trigger events that result in depressed immune function and increased susceptibility to infectious disease. Here we examine one aspect of innate immunity in amphibians and show that esculentin-2P (E2P) and ranatuerin-2P (R2P), two antimicrobial peptides isolated from Rana pipiens, inactivate frog virus 3, a potentially pathogenic iridovirus infecting anurans, and channel catfish herpesvirus. In contrast to mammalian antimicrobial peptides, E2P and R2P act within minutes, at temperatures as low as 0 degrees C, to inhibit viral infectivity. Moreover, these compounds appear to inactivate the virus directly and do not act by inhibiting replication in infected cells. This is the first report linking amphibian antimicrobial peptides with protection from an amphibian viral pathogen and suggests that these compounds may play a role in maintaining amphibian health.

Genetic and ultrastructural characterization of a European isolate of the fatal endotheliotropic elephant herpesvirus.

A male Asian elephant (Elephas maximus) died at the Berlin zoological gardens in August 1998 of systemic infection with the novel endotheliotropic elephant herpesvirus (ElHV-1). This virus causes a fatal haemorrhagic disease in Asian elephants, the so-called endothelial inclusion body disease, as reported from North American zoological gardens. In the present work, ElHV-1 was visualized ultrastructurally in affected organ material. Furthermore, a gene block comprising the complete glycoprotein B (gB) and DNA polymerase (DPOL) genes as well as two partial genes was amplified by PCR-based genome walking and sequenced. The gene content and arrangement were similar to those of members of the Betaherpesvirinae. However, phylogenetic analysis with gB and DPOL consistently revealed a very distant relationship to the betaherpesviruses. Therefore, ElHV-1 may be a member of a new genus or even a new herpesvirus subfamily. The sequence information generated was used to set up a nested-PCR assay for diagnosis of suspected cases of endothelial inclusion body disease. Furthermore, it will aid in the development of antibody-based detection methods and of vaccination strategies against this fatal herpesvirus infection in the endangered Asian elephant.

Oral hairy leukoplakia: an ultrastructural study and review of the literature.

A 39-year-old, homosexual, Caucasian man with a 9-month history of acquired immunodeficiency syndrome by reduced CD4 lymphocyte count alone was found to have extensive, asymptomatic, nonremovable, corrugated, white patches on the lateral borders and ventral aspects of the tongue typical of oral hairy leukoplakia (OHL). Histologically, irregular hyperparakeratosis, acanthosis, and clusters of ballooned keratinocytes in the stratum spinosum were present in the biopsied lateral tongue. Some of the superficial ballooned keratinocytes had peripherally beaded nuclei, whereas others had ground glass intranuclear inclusions. Ultrastructurally, the ballooned keratinocytes had three important findings of diagnostic significance. First, frequent herpesvirus nucleocapsids were largely confined to superficial ballooned keratinocytes having marginated and condensed chromatin. In searching for herpesvirus nucleocapsids, the marginated and condensed chromatin was an invaluable marker for cells harboring the virions. Second, the marginated and condensed chromatin frequently had a distinctive punched-out or cribriform appearance. Third, the ground glass intranuclear inclusion bodies consisted of central, medium electron-dense, finely granular material containing frequent herpesvirus nucleocapsids and partially surrounded or capped by prominent, clumped chromatin. The patient died with progressive multifocal leukoencephalopathy 24 months after OHL was diagnosed.

Herpesvirus-associated papillomas in koi carp (Cyprinus carpio).

From January through November 1994, 32% (7/22) of koi carp (Cyprinus carpio) maintained in indoor aquariums developed proliferative cutaneous lesions that consisted of single to multiple 2-10-mm whitish to pink fleshy masses usually associated with fin rays. Although scaleless koi were more commonly affected (3/6) than were normally scaled koi (4/16), the difference in incidence rates was not significant (chi2 text, P > 0.05). Lesions typically resolved spontaneously in 1-3 wk, occasionally persisted for >3 mo, and recurred in several fish after 2-5 mo. Fish were otherwise asymptomatic. Wet mount preparations from lesions were densely cellular and consisted of hyperplastic epidermal cells of normal morphology without parasites or inflammatory cells. Histologically, biopsies were consistent with papillomas and were characterized by a marked benign epidermal hyperplasia without inclusion bodies or inflammatory infiltrate. Transmission electron microscopic examination revealed intranuclear and intracytoplasmic herpesvirus virions. Virus isolation attempts were unsuccessful.

Kaposi's sarcoma: breeding ground of herpesviridae - A tour de force over viral evolution (review).

After reviewing the molecular biological basis of prominent theories for the integration of viruses into the earliest forms of living matter, an account is given on the immunoevasive strategies viruses have had to acquire in order to secure their existence against the most sophisticated anti-viral defensive mechanisms evolving in their hosts. Herpes-viridae and Kaposi's sarcoma illustrate the complexity of host-virus relationship. In following the evolutionary steps of simians and hominoids to Homo, it becomes evident that: a) Epstein-Barr virus evolved in Africa and its ancestral viruses are present in cercopithecines and hominoids; b) human herpes-virus-8-related viruses are present in macaques, in S. American primates and in Homo but such isolates from the great apes are missing. Thus interspecies transfer occurred from lower monkeys to Homo but when and at what geographical location? The human retrolentiviruses also jumped species barriers: this occurred recently in Africa, from great apes (chimpanzee and bonobo) to Homo sapiens (except when HIV-2 was transferred to mankind from sooty mangabeys). The matter is further complicated by the long coevolutionary cooperative interactions between herpes- and retrolentiviruses. Of pathological entities suspected to be etiologically affected by such complex viral cooperation, the origin of Reed-Sternberg cells of Hodgkin's disease is singled out for critical analysis. In this article the senior author summarizes his own 52 years of studentship in virology.

Malignant seminoma with metastasis and herpesvirus infection in a free-living sea otter (Enhydra lutris).

In winter 1990, an adult male sea otter (Enhydra lutris) was found dead along the eastern shore of Prince William Sound, Alaska. Necropsy findings included an enlarged retained left testicle with a twisted spermatic cord, enlarged left sublumbar lymph node, emaciation, dental attrition, oral papules and ulcers, and luminal intestinal hemorrhage associated with numerous acanthocephalids. A malignant seminoma was present in the left testicle and left sublumbar lymph node. Additionally, herpesvirus like intranuclear inclusion bodies were found in oral, esophageal, and corneal epithelial cells. Virions consistent with a herpesvirus were found in esophageal epithelium. Dental attrition, severe intestinal acanthocephaliasis, the malignant seminoma, and emaciation were considered contributing factors in causing death. The herpesviral disease was probably secondary to the debilitation and stress. This is the first report of malignant seminoma with metastasis in a sea otter.

Epidermal tumors of rainbow smelt with associated virus.

Epithelial tumors of the skin occurred in landlocked populations of rainbow smelt (Osmerus mordax) in several lakes in New Hampshire (USA) during the spawning runs. Histologically, these were noninvasive epithelial cell lesions. Herpesvirus-like particles could be seen in the nucleus and cytoplasm. The lesions occurred in both males and females. Prevalence which varied annually, was as high as 30%.