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.

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.

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.

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.

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.

Molecular characterization of poxviruses associated with tattoo skin lesions in UK cetaceans.

There is increasing concern for the well-being of cetacean populations around the UK. Tattoo skin disease (characterised by irregular, grey, black or yellowish, stippled cutaneous lesions) caused by poxvirus infection is a potential health indicatora potential health indicator for cetaceans. Limited sequence data indicates that cetacean poxviruses (CPVs) belong to an unassigned genus of the Chordopoxvirinae. To obtain further insight into the phylogenetic relationships between CPV and other Chordopoxvirinae members we partially characterized viral DNA originating from tattoo lesions collected in Delphinidae and Phocoenidae stranded along the UK coastline in 1998-2008. We also evaluated the presence of CPV in skin lesions other than tattoos to examine specificity and sensitivity of visual diagnosis. After DNA extraction, regions of the DNA polymerase and DNA topoisomerase I genes were amplified by PCR, sequenced and compared with other isolates. The presence of CPV DNA was demonstrated in tattoos from one striped dolphin (Stenella coeruleoalba), eight harbour porpoises (Phocoena phocoena) and one short-beaked common dolphin (Delphinus delphis) and in one 'dubious tattoo' lesion detected in one other porpoise. Seventeen of the 18 PCR positive skin lesions had been visually identified as tattoos and one as a dubious tattoo. None of the other skin lesions were PCR positive. Thus, visual identification had a 94.4% sensitivity and 100% specificity. The DNA polymerase PCR was most effective in detecting CPV DNA. Limited sequence phylogeny grouped the UK samples within the odontocete poxviruses (CPV group 1) and indicated that two different poxvirus lineages infect the Phocoenidae and the Delphinidae. The phylogenetic tree had three major branches: one with the UK Phocoenidae viruses, one with the Delphinidae isolates and one for the mysticete poxvirus (CPV group 2). This implies a radiation of poxviruses according to the host suborder and the families within these suborders.

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.

Morphogenesis of salmonid gill poxvirus associated with proliferative gill disease in farmed Atlantic salmon (Salmo salar) in Norway.

Proliferative gill disease (PGD) is an emerging problem in Norwegian culture of Atlantic salmon (Salmo salar). Parasites (Ichthyobodo spp.) and bacteria (Flexibacter/Flavobacterium) may cause PGD, but for most cases of PGD in farmed salmon in Norway, no specific pathogen has been identified as the causative agent. However, Neoparamoeba sp. and several bacteria and viruses have been associated with this disease. In the spring of 2006, a new poxvirus, salmon gill poxvirus (SGPV), was discovered on the gills of salmon suffering from PGD in fresh water in northern Norway. Later the same year, this virus was also found on gills of salmon at two marine sites in western Norway. All farms suffered high losses associated with the presence of this virus. In this study, we describe the entry and morphogenesis of the SGP virus in epithelial gill cells from Atlantic salmon. Intracellular mature virions (IMVs) are the only infective particles that seem to be produced. These are spread by cell lysis and by "budding" of virus packages, containing more that 100 IMVs, from the apical surface of infected cells. Entry of the IMVs appears to occur by attachment to microridges on the cell surface and fusion of the viral and cell membranes, delivering the cores into the cytoplasm. The morphogenesis starts with the emergence of crescents in viroplasm foci in perinuclear areas of infected cells. These crescents consist of two tightly apposed unit membranes (each 5 nm thick) that seem to be derived from membranes of the endoplasmic reticulum. The crescents develop into spheres, immature virions (IVs), that are 350 nm in diameter and surrounded by two unit membranes. The maturation of the IVs occurs by condensation of the core material and a change from spherical to boat-shaped particles, intracellular mature virions (IMVs), that are about 300 nm long. Hence, the IMVs from the SGP virus have a different morphology compared to other vertebrate poxviruses that are members of the subfamily Chordopoxvirinae, and they are more similar to members of subfamily Entomopoxvirinae, genus Alphaentomopoxvirus. However, it is premature to make a taxonomic assignment until the genome of the SGP virus has been sequenced, but morphogenesis clearly shows that this virus is a member of family Poxviridae.

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.

Poxvirus entry and membrane fusion.

The study of poxvirus entry and membrane fusion has been invigorated by new biochemical and microscopic findings that lead to the following conclusions: (1) the surface of the mature virion (MV), whether isolated from an infected cell or by disruption of the membrane wrapper of an extracellular virion, is comprised of a single lipid membrane embedded with non-glycosylated viral proteins; (2) the MV membrane fuses with the cell membrane, allowing the core to enter the cytoplasm and initiate gene expression; (3) fusion is mediated by a newly recognized group of viral protein components of the MV membrane, which are conserved in all members of the poxvirus family; (4) the latter MV entry/fusion proteins are required for cell to cell spread necessitating the disruption of the membrane wrapper of extracellular virions prior to fusion; and furthermore (5) the same group of MV entry/fusion proteins are required for virus-induced cell-cell fusion. Future research priorities include delineation of the roles of individual entry/fusion proteins and identification of cell receptors.

Clinical presentation and antiviral therapy for poxvirus infection in pudu (Pudu puda).

A severe poxvirus infection occurred in three pudu (Pudu puda), resulting in two fatalities. Cutaneous ulcers with mucopurulent exudate were present around the eyes and nose, at the lip margins, coronary bands, and teats. Mucosal ulcers were present in the oral cavity, esophagus, and forestomachs. In the two fatalities, a secondary disseminated fungal infection also occurred. Affected animals were leukopenic, hypocalcemic, and hyperphosphatemic and had elevated serum alkaline phosphatase, alanine aminotransferase, and aspartate aminotransferase levels. Electron microscopic examination of affected skin confirmed the presence of a poxvirus. Neutralizing antibody titers to this virus were present in the two pudu tested. One case was treated with cidofovir, 5 mg/kg i.v. q7d for four treatments. Complete recovery occurred in the treated animal. This is the second report of poxvirus infection in pudu and the first report describing clinical presentation, presence of secondary disseminated fungal infection, and successful treatment.

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.

Poxvirus virions: their surface ultrastructure and interaction with the surface membrane of host cells.

Virions of vaccinia and orf viruses were examined by ultrahigh-resolution scanning electron microscopy using a non-coating method. Intracellular mature particles of vaccinia virus appeared to be covered with a net and ultrastructurally their surface consists of many fine ridges and globules, while the surfaces of orf virus mature particles recovered from infected cells consist of spirally running protrusions. The ridge-like structures of vaccinia virus were presumed to correspond to surface tubules shown by negative staining of this virus, while the spiral protrusions of orf virus were presumed to correspond to spiral threads having a criss-cross appearance by the same staining. Using scanning electron microscopy in which the samples were prepared by the conventional method, we observed: (i) many virions, i.e. one or two hundreds, or occasionally more reaching about one thousand particles, of the IHD strain of vaccinia virus, (ii) many or a moderate number of virions, i.e. about one hundred or fewer particles, of the 58 strain of cowpox virus and (iii) rather few virions, i.e. several tens or fewer particles, of the Iwate strain of orf virus on the free surface of each cell infected with these viruses. It must be noted that the number of virions detected considerably differed in respective cells examined. Virus budding was frequently observed at the cell surface of monolayer cells infected with vaccinia virus but it was never detected with cowpox or orf virus, indicating a difference in the mechanism of virus release between vaccinia and the other two viruses. When whole cells infected with vaccinia virus were examined by a combination of high-voltage and scanning electron microscopies, virions on the cell surface and those inside the cells were clearly differentiated. All virions on the cell surface had an envelope, and some of the envelopes had a slack and/or one or more bulges.

Morphological and molecular characterization of the poxvirus BeAn 58058.

BeAn 58058 virus (BAV) was isolated from an Oryzomis rodent in Brazil. BAV was shown to be antigenically related to another poxvirus also isolated in Brazil, the Cotia virus, but it remained ungrouped. Electron microscopy revealed that BAV has a typical poxvirus morphology. The Hind III DNA profile of BAV genome was similar with that of VV WR and Lister, but some differences in the profile were detected. We have also detected the presence of genes homologous to vaccinia virus (VV WR) genes in the genome of BAV. Genes related to vaccinia thymidine kinase (TK) gene and vaccinia growth factor (VGF) gene were found. The patterns of TK and VGF mRNA transcripts described for vaccinia virus infected cells were observed in BAV infected cells. Nucleotide sequence of BAV VGF homologous gene was similar to VV WR VGF sequences. This similarity was further seen when cross-hybridization of total genomes of BAV and VV was done. Polypeptide synthesis of BAV and vaccinia in infected cells also showed similar profiles. The genetic data was used to construct a phylogenetic tree where BAV and VV were placed at the same cluster. Based on our findings we propose that BAV is a vaccinia virus variant.

Evidence for incomplete replication of a penguin poxvirus in cells of mammalian origin.

The recent discovery of a novel poxvirus [penguin-pox virus (PPV)] from Jackass penguins offers the potential of a unique candidate vaccine vector for use in mammals. Infectivity studies were therefore undertaken using a number of mammalian cell lines and chick embryo fibroblasts (CEF). It was shown that the simian CV-1 cell line was able to support replication of the PPV DNA, but no infectious progeny virus could be recovered from the infected cells. Electron microscopy was used to establish the extent of virus morphogenesis in CV-1 cells as compared to that in both chorio-allantoic membranes (CAMs) of hens' eggs and CEF cells. It appears that CV-1 cells are able to support partial maturation of PPV, but that morphogenesis does not proceed to the stage of mature infectious particles. Vaccinia virus promoters were successful in achieving transient gene expression in PPV-infected cells.

AFM review study on pox viruses and living cells.

Single living cells were studied in growth medium by atomic force microscopy at a high--down to one image frame per second--imaging rate over time periods of many hours, stably producing hundreds of consecutive scans with a lateral resolution of approximately 30-40 nm. The cell was held by a micropipette mounted onto the scanner-piezo as shown in Häberle, W., J. K. H. Hörber, and G. Binnig. 1991. Force microscopy on living cells. J. Vac. Sci. Technol. B9:1210-0000. To initiate specific processes on the cell surface the cells had been infected with pox viruses as reported earlier and, most likely, the liberation of a progeny virion by the still-living cell was observed, hence confirming and supporting earlier results (Häberle, W., J. K. H. Hörber, F. Ohnesorge, D. P. E. Smith, and G. Binnig. 1992. In situ investigations of single living cells infected by viruses. Ultramicroscopy. 42-44:1161-0000; Hörber, J. K. H., W. Häberle, F. Ohnesorge, G. Binnig, H. G. Liebich, C. P. Czerny, H. Mahnel, and A. Mayr. 1992. Investigation of living cells in the nanometer regime with the atomic force microscope. Scanning Microscopy. 6:919-930). Furthermore, the pox viruses used were characterized separately by AFM in an aqueous environment down to the molecular level. Quasi-ordered structural details were resolved on a scale of a few nm where, however, image distortions and artifacts due to multiple tip effects are probably involved--just as in very high resolution (<15-20 nm) images on the cells. Although in a very preliminary manner, initial studies on the mechanical resonance properties of a single living (noninfected) cell, held by the micropipette, have been performed. In particular, frequency response spectra were recorded that indicate elastic properties and enough stiffness of these cells to make the demonstrated rapid scanning of the imaging tip plausible. Measurements of this kind, especially if they can be proven to be cell-type specific, may perhaps have a large potential for biomedical applications. Images of these living cells were also recorded in the widely known (e.g., Radmacher, M., R. W. Tillmann, and H. E. Gaub. 1993. Imaging viscoelasticity by force modulation with the atomic force microscope. Biophys. J. 64:735-742) force modulation mode, yet at one low modulation frequency of approximately 2 kHz. (Note: After the cells were attached to the pipette by suction, they first deformed significantly and then reassumed their original spherical shape, which they also acquire when freely suspended in solution, to a great extent with the exception of the portion adjusting to the pipette edge geometry after approximately 0.5-1 h, which occurred in almost the same manner with uninfected cells, and those that had been infected several hours earlier. This seems to be a process which is at least actively supported by the cellular cytoskeleton, rather than a mere osmotic pressure effect induced by electrolyte transport through the membrane. Furthermore, several hours postinfection (p.i.) infected cells developed many optically visible refraction effects, which appeared as small dark spots in the light microscope, that we believed to be the regions in the cell plasma where viruses are assembled; this is known from the literature on electron microscopy on pox-infected cells and referred to there as "virus factories" (e.g., Moss, B. 1986. Replication of pox viruses. In Fundamental Virology, B. N. Fields and D. M. Knape, editors. Raven Press, New York. 637-655). Therefore, we assume that the cells stay alive during imaging, in our experience for approximately 30-45 h p.i.).

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.