The genome of Yoka poxvirus

Yoka poxvirus was isolated almost four decades ago from a mosquito pool in the Central African Republic. Its classification as a poxvirus is based solely upon the morphology of virions visualized by electron microscopy. Here we describe sequencing of the Yoka poxvirus genome using a combination of Roche/454 and Illumina next-generation sequencing technologies. A single consensus contig of ～175 kb in length that encodes 186 predicted genes was generated. Multiple methods were used to show that Yoka poxvirus is most closely related to viruses in the Orthopoxvirus genus, but it is clearly distinct from previously described poxviruses. Collectively, the phylogenetic and genomic sequence analyses suggest that Yoka poxvirus is the prototype member of a new genus in the family Poxviridae.     

Identification of Novel Cetacean Poxviruses in Cetaceans Stranded in South West England

Poxvirus infections in marine mammals have been mainly reported through their clinical lesions and electron microscopy (EM). Poxvirus particles in association with such lesions have been demonstrated by EM and were previously classified as two new viruses, cetacean poxvirus 1 (CePV-1) and cetacean poxvirus 2 (CePV-2). In this study, epidermal pox lesions in cetaceans stranded in South West England (Cornwall) between 2008 and 2012 were investigated by electron microscopy and molecular analysis. PCR and sequencing of a highly conserved region within the viral DNA polymerase gene ruled out both parapox- and orthopoxviruses. Moreover, phylogenetic analysis of the PCR product clustered the sequences with those previously described as cetacean poxviruses. However, taking the close genetic distance of this gene fragment across the family of poxviridae into account, it is reasonable to postulate further, novel cetacean poxvirus species. The nucleotide similarity within each cluster (tentative species) detected ranged from 98.6% to 100%, whilst the similarity between the clusters was no more than 95%. The detection of several species of poxvirus in different cetacean species confirms the likelihood of a heterogeneous cetacean poxvirus genus, comparable to the heterogeneity observed in other poxvirus genera.     

来源于羊痘病毒的白介素-18结合蛋白基因的表达和活性研究

近来,对白细胞介素18受体(IL-18R)及其转导机制的研究取得了较大进展。已经发现的与IL-18R有关的蛋白质有三种：第-种是IL-1受体相关蛋白(IL-1Rrp),即IL-18Rα,是IL-18R功的能性组分;第二种被称为辅助蛋白(AcPL),也即IL-18Rβ;第三种是IL-18结合蛋白(IL-18BP),其氨基酸序列与已知的细胞因子受体氨基酸序列均不同,可以在体外消除IL-18诱导的IFN-γ和IL-8的生成及对NFkB的激活。尽管IL-18BP与IL-18Rα没有同源性,结构也不相同,但是小鼠和人的IL-18BP均能抑制IL-18与其受体的结合。这表明,IL-18BP具有拮抗IL-18生物学活性的作用,是IL-18的拮抗剂。     

Increasing prevalence of avian poxvirus in Darwin's finches and its effect on male pairing success

Island populations harbour a comparatively species‐poor pathogen community, often resulting in naïve host species that experience compromised immunity when faced with novel diseases. Over 95% of the Galápagos avifauna have survived 400 years of human settlement, yet currently face threats due to introduced diseases such as avian poxvirus. On Hawaii, declining populations of birds and even some extinctions have been attributed to avian poxvirus, and hence, identifying the prevalence and fitness costs of avian poxvirus on the Galápagos is a conservation priority. Surveys of avian poxvirus in Darwin's finches on Santa Cruz Island between 2000 and 2004 found a 33% annual increase in the prevalence of pox lesions in ground finches. Comparisons of pox prevalence on three islands (Santa Cruz, Floreana, and Isabela) were made in 2004, which indicated significant variation in pox prevalence across islands (Isabela>Santa Cruz>Floreana). Darwin's finch species were found to be differentially affected by poxvirus, with a higher prevalence in ground finches than in tree finches. There was a significant effect of habitat, even within species, with higher prevalence in the lowlands than highlands. Pox prevalence was not correlated with sex or body condition. However, male small ground finches Geospiza fuliginosa with evidence of pox were less likely to have a mate (16.6% paired) compared with males without pox (77% paired), indicating fitness costs associated with poxvirus infection.     

Increasing prevalence of avian poxvirus in Darwin's finches and its effect on male pairing success

Island populations harbour a comparatively species-poor pathogen community, often resulting in naïve host species that experience compromised immunity when faced with novel diseases. Over 95% of the Galápagos avifauna have survived 400 years of human settlement, yet currently face threats due to introduced diseases such as avian poxvirus. On Hawaii, declining populations of birds and even some extinctions have been attributed to avian poxvirus, and hence, identifying the prevalence and fitness costs of avian poxvirus on the Galápagos is a conservation priority. Surveys of avian poxvirus in Darwin's finches on Santa Cruz Island between 2000 and 2004 found a 33% annual increase in the prevalence of pox lesions in ground finches. Comparisons of pox prevalence on three islands (Santa Cruz, Floreana, and Isabela) were made in 2004, which indicated significant variation in pox prevalence across islands (Isabela>Santa Cruz>Floreana). Darwin's finch species were found to be differentially affected by poxvirus, with a higher prevalence in ground finches than in tree finches. There was a significant effect of habitat, even within species, with higher prevalence in the lowlands than highlands. Pox prevalence was not correlated with sex or body condition. However, male small ground finches Geospiza fuliginosa with evidence of pox were less likely to have a mate (16.6% paired) compared with males without pox (77% paired), indicating fitness costs associated with poxvirus infection.     

Modulation of the host immune response by cowpox virus

Cowpox virus, a zoonotic poxvirus endemic to Eurasia, infects a large number of host species which makes its eradication impossible. The elimination of world-wide smallpox vaccination programs renders the human population increasingly susceptible to infection by orthopoxviruses resulting in a growing number of zoonotic infections including CPXV transmitted from domestic animals to humans. The ability of CPXV to infect a wide range of mammalian host is likely due to the fact that, among the orthopoxviruses, CPXV encodes the most complete set of open reading frames expected to encode immunomodulatory proteins. This renders CPXV particularly interesting for studying poxviral strategies to evade and counteract the host immune responses.     

Characterization of the promoter activity of a poxvirus conserved element

The conserved sequence element (CSE) is a highly conserved 42-bp poxvirus sequence that can function as a poxvirus promoter element. The CSE is composed of 2 repeats, each containing the highly conserved late poxvirus promoter sequence TAAAT. To define the location of the nucleotides critical for promoter function, polymerase chain reaction was carried out using primers that inserted modified versions of the CSE upstream of the green fluorescent protein (GFP), and the constructs were transiently transfected into cells by using GFP levels as a measure of promoter function. The results of this analysis revealed that the second TAAAT sequence, but not the first TAAAT sequence, is critical for promoter function of the CSE. Furthermore, deletion of half of the intervening sequence, i.e., from 10 to 5 nt, increases the promoter strength of the CSE as compared with the wild-type CSE. These results indicate the potential of this novel poxvirus promoter for driving high levels of gene expression.     

Poxvirus infection in a colony of common marmosets (Callithrix jacchus)

An epizootic poxvirus infection occurred in a colony of 80 common marmosets (Callithrix jacchus) recently introduced to a laboratory facility. Over an 18-week period, 29 of the monkeys exhibited skin lesions that persisted for 4-6 weeks. Although eight marmosets died during the outbreak, their deaths were not attributed directly to the poxvirus infection. The skin lesions developed over the entire body surface including the soles and palms. Initially characterized as erythematous papules, they quickly changed to elevated coalescing lesions with extensive scab formation. Histopathologically, the lesions revealed moderate to marked acanthosis, and they progressed to full-thickness epidermal necrosis and ulceration. Intracytoplasmic inclusion bodies were observed occasionally within degenerate keratinocytes. These inclusions most probably constituted the intracytoplasmic aggregates of viral particles observed ultrastructurally and confirmed as members of the poxvirus group by negative staining of direct skin scrapings.     

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.     

Poxvirus infection of the white-tailed tropicbird (Phaethon lepturus) in Bermuda

Lesions caused by an avian poxvirus were identified on the face and nares of fledgling white-tailed tropicbirds (Phaethon lepturus catesbyi) in the natural environment on Bermuda. Between 1958 and 1978, 6 of 81 fledglings found off the nest and unable to fly at departure time had lesions suggestive of poxvirus infection. More detailed nest-site surveys from 1974 to 1978 indicated an overall prevalence of less than 0.5%, involving the fledgling population only.     

Pathology and preliminary characterization of a parapoxvirus isolated from a California sea lion (Zalophus californianus)

Cutaneous pox-like lesions are a common complication in the rehabilitation of pinnipeds. However, the exact identity, taxonomy, and host range of pinniped parapoxviruses remain unknown. During a poxvirus outbreak in May 2003 in California sea lions (Zalophus californianus) at a marine mammal rehabilitation facility, multiple raised, firm, 1-3-cm skin nodules from the head, neck, and thorax of one sea lion weanling pup that spontaneously died were collected. Histologically, the nodules were characterized by inflammation and necrosis of the dermis and epidermis, acanthosis, and ballooning degeneration of the stratum spinosum. Large, coalescing eosinophilic cytoplasmic inclusions were observed in the ballooned cells. A parapoxvirus (sea lion poxvirus 1, SLPV-1) was isolated on early passage California sea lion kidney cells inoculated with a tissue homogenate of a skin nodule. The morphology of the virions on electron microscopy was consistent with that of parapoxviruses. Partial sequencing of the genomic region encoding the putative major virion envelope antigen p42K confirmed the assignment of the sea lion poxvirus to the genus Parapoxvirus. Although SLPV-1 is most closely related to the poxvirus of harbor seals of the European North Sea, it is significantly different from orf virus, bovine papular stomatitis virus, pseudocowpox virus and the parapoxvirus of New Zealand red deer.     

Poxvirus tropism

Despite the success of the WHO-led smallpox eradication programme a quarter of a century ago, there remains considerable fear that variola virus, or other related pathogenic poxviruses such as monkeypox, could re-emerge and spread disease in the human population. Even today, we are still mostly ignorant about why most poxvirus infections of vertebrate hosts show strict species specificity, or how zoonotic poxvirus infections occur when poxviruses occasionally leap into novel host species. Poxvirus tropism at the cellular level seems to be regulated by intracellular events downstream of virus binding and entry, rather than at the level of specific host receptors as is the case for many other viruses. This review summarizes our current understanding of poxvirus tropism and host range, and discusses the prospects of exploiting host-restricted poxvirus vectors for vaccines, gene therapy or tissue-targeted oncolytic viral therapies for the treatment of human cancers.     

Inverted terminal repeats in rabbit poxvirus and vaccinia virus DNA.

In both rabbit poxvirus and vaccinia virus DNA have demonstrated an identical distribution of eight HinfI. The length of the terminal repeats was found to be 3.4 to 3.6 megadaltons (Mdaltons) for rabbit poxvirus DNA and 7.4 to 8.0 Mdaltons for vaccinia virus DNA. Maps of the HinfI restriction sites within isolated EcoRI end fragments of rabbit poxvirus and vaccinia virus DNA PHAVE DEMONSTRATED AN IDENTICAL DISTRIBUTION OF EIGHT HinfI sites in an internal part (approximately 2 Mdaltons) of the EcoRI end fragments of the two genomes.     

Yaba tumor poxvirus synthesis in vitro. II. Adsorption, inactivation, and assay studies

Yohn, David S. (Roswell Park Memorial Institute, Buffalo, N.Y.), Fanny R. Marmol, Victoria A. Haendiges, and James T. Grace, Jr. Yaba tumor poxvirus synthesis in vitro. II. Adsorption, inactivation, and assay studies. J. Bacteriol. 91:1953-1958. 1966.-Means to increase the efficiency of the Yaba tumor poxvirus assay in BSC-1 cell cultures were sought. A method was devised wherein 0.4 ml of each virus dilution was layered onto BSC-1 cells in 80-mm Leighton tubes and permitted to adsorb at 25 C for 18 hr prior to incubation at 35 C. The diluent found most reliable was medium 199 containing 50% bovine amniotic fluid adjusted to 2.0 mm calcium and 1.0 mm magnesium at pH 7.0. These modifications yielded highly reproducible titrations with a greater than twofold increase in assay sensitivity. Irreversible adsorption of Yaba virus by BSC-1 cells proceeds comparatively slowly at 25 to 33 C. Although the process is more rapid at 35 or 37 C, the increased thermal lability of the virus at these temperatures results in lower titers than with virus adsorbed at 25 C for longer periods of time. Yaba poxvirus appears to be 5 to 10 times more sensitive to thermal inactivation than vaccinia poxvirus.     

Poxvirus infection of the Manx shearwater (Puffinus puffinus)

During studies on the etiology of puffinosis, a disease of the Manx shearwater, 1 to 4% of full-grown birds were found to have dry, non-pigmented lesions on the webs of the feet. Poxvirus infection was detected in six of seven full-grown birds with such lesions. The lesions contained large encapsulated inclusions which were packed with mature and immature poxvirus particles. Poxvirus infection was not apparent in shearwater fledglings during puffinosis epizootics, and its spatial distribution was not related to that of puffinosis. The results indicate that poxvirus infection produces a mild, self-limiting disease in shearwaters and is not the cause of puffinosis.     

Molecular weight of DNA from four entomopoxviruses determined by electron microscopy.

DNA was isolated from entomopoxviruses infected Amsacta moorei and Euxoa auxiliaris (Lepidoptera), Goeldichironomus holoprasinus (Diptera), and Othnonius batesi (Coleoptera) and compared with vertebrate virus DNA (vaccinia). After incubation in Pronase, sodium lauryl sulfate, and deoxycholate, poxvirus preparations shadowed with platinum and palladium revealed subcore particles 45 to 60 nm in diameter. Continued incubation in Pronase resulted in the gradual release of DNA from the particles. Metal-shadowed DNA molecules were photographed in the electron microscope and measured, and the average molecular weights were calculated. Lepidopteran poxvirus DNA (135 X 10(6)) was approximately equal to vaccinia DNA (131.7 X 10(6)) in molecular weight. The molecular weight of dipteran and coleopteran poxvirus DNA (200 X 10(6) to 251 X 10(6)) was approximately 50% greater than vaccinia DNA. Based on the concentration of DNA and protein per virion, Amsacta entomopoxvirus contained 5.7 to 7.7% DNA.     

Intracellular location of rabbit poxvirus nucleic acid within infected cells as determined by in situ hybridization.

The intracellular location of rabbit poxvirus DNA within cells during the course of infection has been determined by the hybridization in situ of labeled viral DNA probes to uninfected and infected cells under various conditions. Extensive control experiments were performed to demonstrate that DNA could be detected selectively and accurately within the cell. Our results suggest that rabbit poxvirus DNA is located only within the cytoplasm during the reproductive cycle, and we found no evidence that viral DNA enters the cell nucleus. The pattern of hybridization of viral DNA at early times (1 and 2 h postinfection) and in the presence of inhibitors of viral DNA synthesis suggests that there may be an association between the input viral DNA and some structural component of the host cell. A number of observations support the hypothesis that the host cell nucleus is required for a productive poxvirus infection. Our results are discussed in terms of the possible role of the nucleus in the replication of poxviruses.     

Poxvirus orthologous clusters: toward defining the minimum essential poxvirus genome

Increasingly complex bioinformatic analysis is necessitated by the plethora of sequence information currently available. A total of 21 poxvirus genomes have now been completely sequenced and annotated, and many more genomes will be available in the next few years. First, we describe the creation of a database of continuously corrected and updated genome sequences and an easy-to-use and extremely powerful suite of software tools for the analysis of genomes, genes, and proteins. These tools are available free to all researchers and, in most cases, alleviate the need for using multiple Internet sites for analysis. Further, we describe the use of these programs to identify conserved families of genes (poxvirus orthologous clusters) and have named the software suite POCs, which is available at www.poxvirus.org. Using POCs, we have identified a set of 49 absolutely conserved gene families-those which are conserved between the highly diverged families of insect-infecting entomopoxviruses and vertebrate-infecting chordopoxviruses. An additional set of 41 gene families conserved in chordopoxviruses was also identified. Thus, 90 genes are completely conserved in chordopoxviruses and comprise the minimum essential genome, and these will make excellent drug, antibody, vaccine, and detection targets. Finally, we describe the use of these tools to identify necessary annotation and sequencing updates in poxvirus genomes. For example, using POCs, we identified 19 genes that were widely conserved in poxviruses but missing from the vaccinia virus strain Tian Tan 1998 GenBank file. We have reannotated and resequenced fragments of this genome and verified that these genes are conserved in Tian Tan. The results for poxvirus genes and genomes are discussed in light of evolutionary processes.     

Deep-etch EM reveals that the early poxvirus envelope is a single membrane bilayer stabilized by a geodetic "honeycomb" surface coat

Three-dimensional "deep-etch" electron microscopy (DEEM) resolves a longstanding controversy concerning poxvirus morphogenesis. By avoiding fixative-induced membrane distortions that confounded earlier studies, DEEM shows that the primary poxvirus envelope is a single membrane bilayer coated on its external surface by a continuous honeycomb lattice. Freeze fracture of quick-frozen poxvirus-infected cells further shows that there is only one fracture plane through this primary envelope, confirming that it consists of a single lipid bilayer. DEEM also illustrates that the honeycomb coating on this envelope is completely replaced by a different paracrystalline coat as the poxvirus matures. Correlative thin section images of infected cells freeze substituted after quick-freezing, plus DEEM imaging of Tokuyasu-type cryo-thin sections of infected cells (a new application introduced here) all indicate that the honeycomb network on immature poxvirus virions is sufficiently continuous and organized, and tightly associated with the envelope throughout development, to explain how its single lipid bilayer could remain stable in the cytoplasm even before it closes into a complete sphere.     

The role of cell signaling in poxvirus tropism: the case of the M-T5 host range protein of myxoma virus

Poxviruses demonstrate strict species specificity in vivo that range from narrow to broad, however the fundamental factors that mediate the basis of poxvirus tropism remain poorly understood. It is generally believed that most, if not all, poxviruses can efficiently bind and enter a wide range of mammalian cells and all of the known host anti-viral pathways that block viral replication in nonpremissive cells operate downstream of virus entry. A productive poxvirus infection is heavily dependent upon the production of a vast array of host modulatory products that specifically target and manipulate both extracellular immune response pathways of the host, as well as intracellular signal transduction pathways of the individually infected cells. The unique pathogenesis and host tropism of specific poxviruses can be attributed to the broad diversity of host modulatory proteins they express. Myxoma virus (MV) is a rabbit-specific poxviruses that encodes multiple host range factors, including an ankyrin-repeat protein M-T5, which functions to regulate tropism of MV for rabbit lymphocytes and some human cancer cells. At the molecular level, M-T5 binds and alters at least two distinct cellular proteins: Akt and cullin-1. The direct interaction between M-T5 and Akt was shown to be a key restriction determinant for MV tropism in a spectrum of human cancer cells making MV an excellent oncolytic candidate. Thus, the intricate relationship between viral encoded proteins and components of the host cell signaling networks can have profound impact on poxvirus tropism. The lessons we continue to learn from poxvirus host range factors like M-T5 will provide further insights into the factors that regulate poxvirus tropism and the mechanisms by which poxviruses micromanipulate the signaling pathways of the infected cell.