Vaccinia virus A11 is required for membrane rupture and viral membrane assembly.

Although most enveloped viruses acquire their membrane from the host by budding or by a wrapping process, collective data argue that nucleocytoplasmic large DNA viruses (NCLDVs) may be an exception. The prototype member of NCLDVs, vaccinia virus (VACV) may induce rupture of endoplasmic-reticulum-derived membranes to build an open-membrane sphere that closes after DNA uptake. This unconventional membrane assembly pathway is also used by at least 3 other members of the NCLDVs. In this study, we identify the VACV gene product of A11, as required for membrane rupture, hence for VACV membrane assembly and virion formation. By electron tomography, in the absence of A11, the site of assembly formed by the viral scaffold protein D13 is surrounded by endoplasmic reticulum cisternae that are closed. We use scanning transmission electron microscopy-electron tomography to analyse large volumes of cells and demonstrate that in the absence of A11, no open membranes are detected. Given the pivotal role of D13 in initiating VACV membrane assembly, we also analyse viral membranes in the absence of D13 synthesis and show that this protein is not required for rupture. Finally, consistent with a role in rupture, we show that during wild-type infection, A11 localises predominantly to the small ruptured membranes, the precursors of VACV membrane assembly. These data provide strong evidence in favour of the unusual membrane biogenesis of VACV and are an important step towards understanding its molecular mechanism.

Multi-modal cryo-EM reveals trimers of protein A10 to form the palisade layer in poxvirus cores.

Poxviruses are among the largest double-stranded DNA viruses, with members such as variola virus, monkeypox virus and the vaccination strain vaccinia virus (VACV). Knowledge about the structural proteins that form the viral core has remained sparse. While major core proteins have been annotated via indirect experimental evidence, their structures have remained elusive and they could not be assigned to individual core features. Hence, which proteins constitute which layers of the core, such as the palisade layer and the inner core wall, has remained enigmatic. Here we show, using a multi-modal cryo-electron microscopy (cryo-EM) approach in combination with AlphaFold molecular modeling, that trimers formed by the cleavage product of VACV protein A10 are the key component of the palisade layer. This allows us to place previously obtained descriptions of protein interactions within the core wall into perspective and to provide a detailed model of poxvirus core architecture. Importantly, we show that interactions within A10 trimers are likely generalizable over members of orthopox- and parapoxviruses.

Novel poxvirus in big brown bats, northwestern United States.

A wildlife hospital and rehabilitation center in northwestern United States received several big brown bats with necrosuppurative osteomyelitis in multiple joints. Wing and joint tissues were positive by PCR for poxvirus. Thin-section electron microscopy showed poxvirus particles within A-type inclusions. Phylogenetic comparison supports establishment of a new genus of Poxviridae.

Detection limit of negative staining electron microscopy for the diagnosis of bioterrorism-related micro-organisms.

AIMS: To determine the detection limit of diagnostic negative staining electron microscopy for the diagnosis of pathogens that could be used for bioterrorism. METHODS AND RESULTS: Suspensions of vaccinia poxvirus and endospores of Bacillus subtilis were used at defined concentrations as a model for poxviruses and spores of anthrax (Bacillus anthracis), both of which are pathogens that could be used for bioterrorist attacks. Negative staining electron microscopy was performed directly or after sedimentation of these suspensions on to the sample supports using airfuge ultracentrifugation. For both virus and spores, the detection limit using direct adsorption of a 10-µl sample volume onto the sample support was 10(6) particles per ml. Using airfuge ultracentrifugation with a sample volume of 80 µl, the detection limit could be reduced to 10(5) particles per ml for spores and to 5 × 10(4) particles per ml for poxviruses. The influence on particle detection of incubation time, washing and adsorption procedures was investigated. CONCLUSIONS: The reproducibility and sensitivity of the method were acceptable, particularly considering the small sample volume and low particle number applied onto the sample support. SIGNIFICANCE AND IMPACT OF THE STUDY: Diagnostic negative staining electron microscopy is used for the diagnosis of pathogens in emergency situations because it allows a rapid examination of all particulate matter down to the nanometre scale. This study provides precise detection limit for the method, an important factor for the validation and improvement of the technique.

A novel poxvirus isolated from an Egyptian fruit bat in Israel.

An Egyptian fruit bat (Rousettus aegyptiacus) from the Zoological Gardens, at Tel Aviv, Israel, showed pox-like clinical signs including vesicular and nodular skin lesions on the wings. Cell culture isolation, histopathology, electron microscopy and molecular analysis, revealed the presence of a novel bat poxvirus. Future research is needed to determine whether this virus can affect human health.

First Evidence of Carp Edema Virus Infection of Koi Cyprinus carpio in Chiang Mai Province, Thailand.

The presence of carp edema virus (CEV) was confirmed in imported ornamental koi in Chiang Mai province, Thailand. The koi showed lethargy, loss of swimming activity, were lying at the bottom of the pond, and gasping at the water's surface. Some clinical signs such as skin hemorrhages and ulcers, swelling of the primary gill lamella, and necrosis of gill tissue, presented. Clinical examination showed co-infection by opportunistic pathogens including Dactylogyrus sp., Gyrodactylus sp. and Saprolegnia sp. on the skin and gills. Histopathologically, the gill of infected fish showed severe necrosis of epithelial cells and infiltrating of eosinophilic granular cells. Electron microscope examination detected few numbers of virions were present in the cytoplasm of gill tissue which showed an electron dense core with surface membranes worn by surface globular units. Molecular detection of CEV DNA from gill samples of fish was performed by polymerase chain reaction (PCR) and confirmed by nested-PCR. Phylogenetic analyses revealed that CEV isolate had 99.8% homology with the CEV isolated from South Korea (KY946715) and Germany (KY550420), and was assigned to genogroup IIa. In conclusion, this report confirmed the presence of CEV infection of koi Cyprinus carpio in Chiang Mai province, Thailand using pathological and molecular approaches.

Ecthyma contagiosum (orf)--report of a human case from the United Arab Emirates and review of the literature.

BACKGROUND: Ecthyma contagiosum (orf) is caused by an epitheliotropic parapox virus. It is a zoonosis usually transmitted to humans from affected sheep or goat through direct contact or contaminated fomites. METHODS: We report a 36-year-old patient with multiple skin lesions on his left hand, first observed 5 days before admission followed by red streaks on the forearm and an erythema on the upper arm 1 day prior to admission. The patient reported that he was working on a sheep farm. RESULTS: Histopathologic examination showed evidence of a viral infection. Subsequent transmission electron microscopy showed typical parapox virus particles, predominantly in the scaled-off layers of degenerated keratinocytes and monocytes. The results were verified and specified by two newly established polymerase chain reaction (PCR) assays and subsequent sequencing of the amplicons: one broadly reacting 'general parapox virus PCR', and one assay which allows--following sequencing--discrimination between individual orf virus strains. CONCLUSION: Despite the multiplicity of the lesions, there was a significant improvement after 2 weeks of treatment. Sequencing showed the uniqueness of this virus compared with previously published strains from other countries.

Impact of poxvirus lesions on saltwater crocodile (Crocodylus porosus) skins.

Cutaneous poxvirus infections are common in several crocodilian species and are of importance in crocodile farming due to their potential impact on the tanned hide. To confirm poxvirus infection and understand the impact on saltwater crocodile (Crocodylus porosus) skin, fourteen animals from different age groups (five hatchlings, five yearlings and four grow-outs) were selected based on a criterion of ten poxvirus-like lesions per animal. One lesion on each animal was extruded for genetic analysis and transmission electron microscopy. Both methods confirmed poxvirus so the remainder of lesions were re-examined every six weeks over a 24 week study period. Each lesion went through four distinct phases: early active, active, expulsion and healing. To understand how these lesions impact on the final skin product, one crocodile from each age group was euthanised and the lesions examined. Using standard skin grading techniques (light-table), the early phase (early active - expulsion) lesions were all translucent and would lead to downgrading of the skin or, at worst, rendering them unsaleable. At the later stages of healing, the translucency reduces. Histological examination of the phases confirm that the basement membrane is not breached by the infection further indicating that poxvirus lesions, given enough time, will eventually have no detrimental effect on skin quality. This is obviously dependent upon no more lesions developing in the interim.

Debilitating cutaneous poxvirus infection in a Hodgson's grandala (Grandala coelicolor).

A case of cutaneous avian pox infection in a Hodgson's grandala (Grandala coelicolor) is described. The bird was emaciated and had nodules on the eyelids, bill, neck, legs, and toes. Eosinophilic intracytoplasmic inclusion bodies were visualized by light microscopy in epithelial cells of the cutaneous nodules. Electron microscopy revealed numerous pox virions in the inclusion bodies. This is the first report of cutaneous poxvirus infection in a Hodgson's grandala.

Debilitating cutaneous poxvirus lesions on two captive houbara bustards (Chlamydotis undulata).

Poxvirus was isolated from cutaneous nodules on two young Houbara bustards (Chlamydotis undulata) bred in captivity in Saudi Arabia. Birds were emaciated and presented nodules on tibiotarso-tarsometatarsal joints, toes, and the carpal joint. Diagnosis was confirmed by histopathology, virus isolation on inoculated chorioallantoic membranes of embryonated chicken's eggs, and electron microscopy. Progressive leg lesions were extensive and interfered with walking, significantly debilitating the birds. Successful excisions of these lesions were performed.

Natural pox and herpes as a dual virus infection in chickens.

A natural dual viral infection was confirmed in chickens of University of Illinois Poultry Farm by histopathologic, immunologic, and electron-microscopic examination of formalin-fixed tracheal tissue. Histopathologic examination of the tracheal mucosa revealed eosinophilic cytoplasmic inclusions characteristic of fowlpox and intranuclear inclusions suggestive of Marek's disease. Treatment of the formalin-fixed tracheal tissue with peroxidase and fluorescent-labeled antibody against Marek's disease virus revealed specificity of the reaction. Electron microscopy showed viral particles of two morphologic forms, i.e., pox and herpes. A single cell having dual infection with herpes virus in the nucleus and pox virus in the cytoplasm was also encountered on electron-microscopic examination.

Isolation of surface tubules of fowlpox virus.

Surface tubules of fowlpox virus were isolated using chemical and physical methods. Suspensions of lipid cytoplasmic inclusion bodies were obtained by treating infected chorioallantoic membranes with 1% trypsin. Inclusions were treated with ultrasonic sound, detergents, and enzymes and were examined by electron microscopy. Although lipase treatment altered the morphology of lipid inclusions, no viral surface tubules were recovered. Treatment with the detergent Nonidet-P40 followed by 2-mercaptoethanol disrupted virions without allowing surface tubules to be recovered. Disruption of lipid inclusions by ultrasonic sound or manual grinding of chorioallantoic membranes produced free virions but only small numbers of tubules. These results indicate that surface tubules can be recovered, but that the lipid nature of cytoplasmic inclusions interferes with procedures commonly used in tubule purification.

Cutaneous tumour-like lesions due to poxvirus infection in Chilean flamingos.

Cutaneous tumour-like growths were observed on the face and other areas of the body surface of young Chilean flamingos. In the cells of these lesions, avian pox-specific cytoplasmic inclusion bodies were observed by light microscopy and virus particles were detected under an electron-microscope. It was diagnosed as avian pox.

Comparison of the external dimensions of capripoxvirus isolates.

No significant difference was found between the external dimensions of the M forms of isolates of sheep pox virus from Nigeria, sheep and goat pox virus from Kenya and lumpy skin disease virus from South Africa. Earlier reports that isolates of capripoxvirus can be distinguished by their relative sizes could not be substantiated.

An eruptive moderate form of camelpox infection in dromedary camels (Camelus dromedarius) in Saudi Arabia.

An eruptive moderate form of camelpox infection is reported in camels aged three to four years from the Al-Ahsa region of Saudi Arabia. The clinical signs were moderate in nature (between the 'mild' and the 'severe' form). The morbidity rate was 100% while the case fatality rate was 0%. Camelpox virus was isolated and identified using electron microscopy and serological analysis.

An investigation of camelpox outbreaks in two principal camel (Camelus dromedarius) rearing areas of Kenya.

In 1992, during an investigation into camelpox in two principal camel-rearing areas of Kenya, the disease was found in 1,100 camels at a prevalence of 6% in Turkana and 27% in Samburu. In Turkana, outbreaks were detected in two herds of young animals, while in Samburu, outbreaks were found in two herds of adult animals, as well as in two herds of young camels. In all cases, there was 100% morbidity in the affected herds. When young camels were involved, the main lesions were confined to the mouth, nose and muzzle as distinct pustular lesions. In adults, there was also extensive oedema of the head and neck. Direct electron microscopy and virus isolation on tissue culture were used to confirm the orthopoxvirus infection. The outbreaks appeared related to the stress of weaning and, in the case of the adults, to recent long-distance travel.

Reaction of convalescent bovine antisera with strain-specific antigens of parapoxviruses.

Two strains of papular stomatitis (PS) virus, 1 of milker's nodules (MN) virus and 1 of contagious ecthyma (CE) virus possessed 2 distinct external structures when examined by electron microscopy. The innermost, designated coat was closely apposed to the tubular surface, whereas the outer envelope loosely surrounded the virion. When convalescent sera from cattle infected with PS virus were used for immunoelectron microscopy, antibody reacted with coats and envelopes of the PS virus strains, but only with coats of MN and CE viruses. Convalescent sera from cattle infected with PS or MN virus contained complement-dependent antibodies cytolytic to cells infected with the homologous virus. In an indirect immunofluorescence test, the sera reacted with homologous strains to higher titers than with heterologous strains.

Morphological studies of seal poxvirus.

The fine structure of the M and C forms of seal poxvirus as observed in negative stained preparations in the electron microscope are described. The M form has a beaded surface and a serrated outline and particles averaged 312.3 mmicro, in length and 193.8 microns in width. The C form was not beaded, had a serrated periphery and averaged 352.8 mmicro in length and 196 mmicro in width. A short discussion of the morphology of seal poxvirus in relation to other poxviruses is presented.

Poxvirus infection in the white-backed magpie (Gymnorhina hypoleuca) and pox-like conditions in other birds in Australia.

Lesions, grossly and histologically typical of pox infection, occurred in a white-backed magpie from Melbourne, Australia. Electron microscopic examination revealed typical poxvirus particles in lesion material. The disease was experimentally transmitted to other magpies, but chickens, turkeys, pigeons, and canaries were refractory to experimental infection with magpie poxvirus. The epidemiology of magpie pox and the probable occurrence of pox-like disease in other native Australian birds are discussed.

Chlamydial and poxvirus infections of circulating monocytes of a flap-necked chameleon (Chamaeleo dilepis).

Of blood films examined from 170 specimens of 15 Chamaeleo spp. in Tanzania, three C. dilepis had an intracytoplasmic inclusion within monocytes. One of the lizards was maintained in captivity and was sequentially bled over a 55 day period. At 46 days, a second type of inclusion was occasionally seen within monocytes. The lizard became ill and was euthanatized on day 55. All circulating monocytes were found to have either one or both types of inclusions. Histologic examination of multiple tissues demonstrated similar inclusions within macrophages in the spleen and liver. Transmission electron microscopic examination of monocytes revealed the presence of a chlamydia-like organism and pox-like virus. These pathogens have not been reported previously in chameleons, nor has a combined infection of circulating monocytes with these two pathogens been reported for any animal.