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the canarypox virus vector alvac induces distinct cytokine responses compared to the vaccinia virus-based vectors mva and nyvac in rhesus monkeys.despite the growing use of poxvirus vectors as vaccine candidates for multiple pathogens and cancers, their innate stimulatory properties remain poorly characterized. here we show that the canarypox virus-based vector alvac induced distinct systemic proinflammatory and antiviral cytokine and chemokine levels following the vaccination of rhesus monkeys compared to the vaccinia virus-based vectors mva and nyvac. these data suggest that there are substantial biological differences among leading pox ...201424257612
identification of 10 cowpox virus proteins that are necessary for induction of hemorrhagic lesions (red pocks) on chorioallantoic membranes.cowpox viruses (cpxv) cause hemorrhagic lesions ("red pocks") on infected chorioallantoic membranes (cam) of embryonated chicken eggs, while most other members of the genus orthopoxvirus produce nonhemorrhagic lesions ("white pocks"). cytokine response modifier a (crma) of cpxv strain brighton red (br) is necessary but not sufficient for the induction of red pocks. to identify additional viral proteins involved in the induction of hemorrhagic lesions, a library of single-gene cpxv knockout mutan ...201424850732
a plasmid-based reverse genetics system for mammalian orthoreoviruses driven by a plasmid-encoded t7 rna polymerase.mammalian orthoreoviruses (reoviruses) have served as highly useful models for studies of virus replication and pathogenesis. the development of a plasmid-based reverse genetics system represented a major breakthrough in reovirus research. the current reverse genetics systems for reoviruses rely on the expression of t7 rna polymerase within cells transfected with reovirus gene-segment cdna plasmids. in these systems, the t7 rna polymerase is provided by using a recombinant vaccinia virus express ...201424183920
epithelial immunization induces polyfunctional cd8+ t cells and optimal mousepox protection.we assessed several routes of immunization with vaccinia virus (vacv) in protecting mice against ectromelia virus (ectv). by a wide margin, skin scarification provided the greatest protection. humoral immunity and resident-memory t cells notwithstanding, several approaches revealed that circulating, memory cd8(+) t cells primed via scarification were functionally superior and conferred enhanced virus control. immunization via the epithelial route warrants further investigation, as it may also pr ...201424899206
changes in the mitochondrial network during ectromelia virus infection of permissive l929 cells.mitochondria are extremely important organelles in the life of a cell. recent studies indicate that mitochondria also play a fundamental role in the cellular innate immune mechanisms against viral infections. moreover, mitochondria are able to alter their shape continuously through fusion and fission. these tightly regulated processes are activated or inhibited under physiological or pathological (e.g. viral infection) conditions to help restore homeostasis. however, many types of viruses, such ...201424660173
vaccination directed against the human endogenous retrovirus-k (herv-k) gag protein slows herv-k gag expressing cell growth in a murine model system.human endogenous retroviruses (hervs) are remnants of ancestral infections and chromosomally integrated in all cells of an individual, are transmitted only vertically and are defective in viral replication. however enhanced expression of herv-k accompanied by the emergence of anti-herv-k-directed immune responses has been observed inter-alia in hiv-infected individuals and tumor patients. therefore herv-k might serve as a tumor-specific antigen or even as a constant target for the development of ...201424669861
development and evaluation of single domain antibodies for vaccinia and the l1 antigen.there is ongoing interest to develop high affinity, thermal stable recognition elements to replace conventional antibodies in biothreat detection assays. as part of this effort, single domain antibodies that target vaccinia virus were developed. two llamas were immunized with killed viral particles followed by boosts with the recombinant membrane protein, l1, to stimulate the immune response for envelope and membrane proteins of the virus. the variable domains of the induced heavy chain antibodi ...201425211488
crystal structure of the vaccinia virus dna polymerase holoenzyme subunit d4 in complex with the a20 n-terminal domain.vaccinia virus polymerase holoenzyme is composed of the dna polymerase e9, the uracil-dna glycosylase d4 and a20, a protein with no known enzymatic activity. the d4/a20 heterodimer is the dna polymerase co-factor whose function is essential for processive dna synthesis. genetic and biochemical data have established that residues located in the n-terminus of a20 are critical for binding to d4. however, no information regarding the residues of d4 involved in a20 binding is yet available. we expres ...201424603707
expression of a non-coding rna in ectromelia virus is required for normal plaque formation.poxviruses are dsdna viruses with large genomes. many genes in the genome remain uncharacterized, and recent studies have demonstrated that the poxvirus transcriptome includes numerous so-called anomalous transcripts not associated with open reading frames. here, we characterize the expression and role of an apparently non-coding rna in orthopoxviruses, which we call viral hairpin rna (vhrna). using a bioinformatics approach, we predicted expression of a transcript not associated with an open re ...201424078045
genome sequence of wau86/88-1, a new variant of vaccinia virus lister strain from poland.the poxviruses warsaw agricultural university 86 (wau86) and 88-1 (wau88-1) were isolated in 1986 to 1988 from separate outbreaks in laboratory mice in poland and described as ectromelia virus isolates. the genome sequences of these poxviruses reveal that they are almost identical and represent a novel variant of the vaccinia virus lister strain.201424407630
α4-integrins control viral meningoencephalitis through differential recruitment of t helper cell subsets.natalizumab blocks α4-integrins and is a prototypic agent for a series of anti-inflammatory drugs that impair trafficking of immune cells into the cns. however, modulation of the access of immune cells to the cns is associated with impaired immune surveillance and detrimental viral infections of the cns. here, we explored the potency of cellular immune responses within the cns to protect against viral encephalitis in mice with t cell conditional disruption of vla-4 integrin (α4β1) expression.201424606807
genome segregation and packaging machinery in acanthamoeba polyphaga mimivirus is reminiscent of bacterial apparatus.genome packaging is a critical step in the virion assembly process. the putative atp-driven genome packaging motor of acanthamoeba polyphaga mimivirus (apmv) and other nucleocytoplasmic large dna viruses (ncldvs) is a distant ortholog of prokaryotic chromosome segregation motors, such as ftsk and hera, rather than other viral packaging motors, such as large terminase. intriguingly, apmv also encodes other components, i.e., three putative serine recombinases and a putative type ii topoisomerase, ...201424623441
development of a novel, guinea pig-specific ifn-γ elispot assay and characterization of guinea pig cytomegalovirus gp83-specific cellular immune responses following immunization with a modified vaccinia virus ankara (mva)-vectored gp83 vaccine.the guinea pig (cavia porcellus) provides a useful animal model for studying the pathogenesis of many infectious diseases, and for preclinical evaluation of vaccines. however, guinea pig models are limited by the lack of immunological reagents required for characterization and quantification of antigen-specific t cell responses. to address this deficiency, an enzyme-linked immunospot (elispot) assay for guinea pig interferon (ifn)-γ was developed to measure antigen/epitope-specific t cell respon ...201424856783
mouse cmv infection delays antibody class switch upon an unrelated virus challenge.poor immune protection upon vaccination is a critical determinant of immunosenescence. latent cytomegalovirus (cmv) infection has been associated with poor antibody responses to vaccination, but a causative role for cmv in the poor immune response requires experimental evidence and thus could not be confirmed in clinical studies. to test the hypothesis that latent cmv infection causes poor antibody responses, we infected young or adult mice with mouse cmv and challenged them with vesicular stoma ...201424462805
prediction of steps in the evolution of variola virus host range.variola virus, the agent of smallpox, has a severely restricted host range (humans) but a devastatingly high mortality rate. although smallpox has been eradicated by a world health organization vaccination program, knowledge of the evolutionary processes by which human super-pathogens such as variola virus arise is important. by analyzing the evolution of variola and other closely related poxviruses at the level of single nucleotide polymorphisms we detected a hotspot of genome variation within ...201424626337
[apoptin enhances the oncolytic activity of vaccinia virus].chicken anemia virus gene encoding apoptin, a selective killer of cancer cells was synthesized and inserted into vaccinia virus (strain l-ivp) genome. the insertion has replaced major part of the viral c11r gene encoding viral growth factor (vgf), which is important for the virulence. the recombinant virus vvdgf-apos24/2 was obtained through the transient dominant selection technique with the use of puromycin resistance gene as the selective marker. the expression apoptin gene from a synthetic e ...201425509357
laboratory-acquired buffalopox virus infection, india. 201424447599
a loss of function analysis of host factors influencing vaccinia virus replication by rna interference.vaccinia virus (vacv) is a large, cytoplasmic, double-stranded dna virus that requires complex interactions with host proteins in order to replicate. to explore these interactions a functional high throughput small interfering rna (sirna) screen targeting 6719 druggable cellular genes was undertaken to identify host factors (hf) influencing the replication and spread of an egfp-tagged vacv. the experimental design incorporated a low multiplicity of infection, thereby enhancing detection of cellu ...201424901222
relationship between poor immunogenicity of hla-a2-restricted peptide epitopes and paucity of naïve cd8(+) t-cell precursors in hla-a2-transgenic mice.we examined the immunogenicity of h-2 class i-restricted and hla-a2-restricted epitopes through peptide immunization of hla-a2-transgenic mice that also express mouse h-2 class i molecules. all four of the tested epitopes restricted by h-2 class i robustly elicited t-cell responses, but four of seven epitopes restricted by hla-a2 did not induce t-cell responses, showing that hla-a2-restricted peptide epitopes tend to be poorly immunogenic in hla-a2-transgenic mice. this finding was confirmed in ...201425177254
a small molecule inhibits virion attachment to heparan sulfate- or sialic acid-containing glycans.primary attachment to cellular glycans is a critical entry step for most human viruses. some viruses, such as herpes simplex virus type 1 (hsv-1) and hepatitis c virus (hcv), bind to heparan sulfate, whereas others, such as influenza a virus (iav), bind to sialic acid. receptor mimetics that interfere with these interactions are active against viruses that bind to either heparan sulfate or to sialic acid. however, no molecule that inhibits the attachment of viruses in both groups has yet been id ...201424789779
a novel mode of poxvirus superinfection exclusion that prevents fusion of the lipid bilayers of viral and cellular membranes.superinfection exclusion is a widespread phenomenon that prevents secondary infections by closely related viruses. the vaccinia virus a56 and k2 proteins in the cell membrane can prevent superinfection by interacting with the entry-fusion complex of subsequent viruses. here, we described another form of exclusion that is established earlier in infection and does not require the a56 or k2 protein. cells infected with one or more infectious virions excluded hundreds of superinfecting vaccinia viru ...201424920806
inactivation of bacterial and viral biothreat agents on metallic copper surfaces.in recent years several studies in laboratory settings and in hospital environments have demonstrated that surfaces of massive metallic copper have intrinsic antibacterial and antiviral properties. microbes are rapidly inactivated by a quick, sharp shock known as contact killing. the underlying mechanism is not yet fully understood; however, in this process the cytoplasmic membrane is severely damaged. pathogenic bacterial and viral high-consequence species able to evade the host immune system a ...201425100640
combination of biobarcode assay with on-chip capillary electrophoresis for ultrasensitive and multiplex biological agent detection.early diagnosis of biological agents is of paramount importance to prevent the casualties and fatal disease in human during bioterrorism or biological warfare. in this study, we reported an efficient and sensitive multiplex biological agent detection method based on the dna biobarcode assay and the micro-capillary electrophoresis (μce) technology. monoplex as well as multiplex pathogen identification was performed using five targets including bacillus anthracis, francisella tularensis, yersinia ...201424878840
novel mode of isg15-mediated protection against influenza a virus and sendai virus in mice.isg15 is a diubiquitin-like modifier and one of the most rapidly induced genes upon type i interferon stimulation. hundreds of host proteins and a number of viral proteins have been shown to be isgylated, and understanding how these modifications affect the interferon response and virus replication has been of considerable interest. isg15(-/-) mice exhibit increased susceptibility to viral infection, and in the case of influenza b virus and vaccinia virus, isg15 conjugation has been shown to res ...201425320315
a single vertebrate dna virus protein disarms invertebrate immunity to rna virus infection.virus-host interactions drive a remarkable diversity of immune responses and countermeasures. we found that two rna viruses with broad host ranges, vesicular stomatitis virus (vsv) and sindbis virus (sinv), are completely restricted in their replication after entry into lepidopteran cells. this restriction is overcome when cells are co-infected with vaccinia virus (vacv), a vertebrate dna virus. using rnai screening, we show that lepidopteran rnai, nuclear factor-κb, and ubiquitin-proteasome pat ...201424966209
the escherichia coli effector espj blocks src kinase activity via amidation and adp ribosylation.the hallmark of enteropathogenic escherichia coli (epec) infection is the formation of actin-rich pedestal-like structures, which are generated following phosphorylation of the bacterial effector tir by cellular src and abl family tyrosine kinases. this leads to recruitment of the nck-wip-n-wasp complex that triggers arp2/3-dependent actin polymerization in the host cell. the same phosphorylation-mediated signalling network is also assembled downstream of the vaccinia virus protein a36 and the p ...201425523213
characterization of t-cell responses to conserved regions of the hiv-1 proteome in balb/c mice.a likely requirement for a protective vaccine against human immunodeficiency virus type 1 (hiv-1)/aids is, in addition to eliciting antibody responses, induction of effective t cells. to tackle hiv-1 diversity by t-cell vaccines, we designed an immunogen, hivconsv, derived from the most functionally conserved regions of the hiv-1 proteome and demonstrated its high immunogenicity in humans and rhesus macaques when delivered by regimens combining plasmid dna, nonreplicating simian (chimpanzee) ade ...201425230940
t cell antigen discovery using soluble vaccinia proteome reveals recognition of antigens with both virion and nonvirion association.vaccinia virus (vacv) is a useful model system for understanding the immune response to a complex pathogen. proteome-wide ab profiling studies reveal the humoral response to be strongly biased toward virion-associated ags, and several membrane proteins induce ab-mediated protection against vacv challenge in mice. some studies have indicated that the cd4 response is also skewed toward proteins with virion association, whereas the cd8 response is more biased toward proteins with early expression. ...201425024392
cellular sensing of viral dna and viral evasion mechanisms.mammalian cells detect foreign dna introduced as free dna or as a result of microbial infection, leading to the induction of innate immune responses that block microbial replication and the activation of mechanisms that epigenetically silence the genes encoded by the foreign dna. a number of dna sensors localized to a variety of sites within the cell have been identified, and this review focuses on the mechanisms that detect viral dna and how the resulting responses affect viral infections. viru ...201425002095
t-bet and eomes instruct the development of two distinct natural killer cell lineages in the liver and in the bone marrow.trail(+)dx5(-)eomes(-) natural killer (nk) cells arise in the mouse fetal liver and persist in the adult liver. their relationships with trail(-)dx5(+) nk cells remain controversial. we generated a novel eomes-gfp reporter murine model to address this question. we found that eomes(-) nk cells are not precursors of classical eomes(+) nk cells but rather constitute a distinct lineage of innate lymphoid cells. eomes(-) nk cells are strictly dependent on both t-bet and il-15, similarly to nkt cells. ...201424516120
a novel role for the apoptosis inhibitor arc in suppressing tnfα-induced regulated necrosis.tnfα signaling can promote apoptosis or a regulated form of necrosis. arc (apoptosis repressor with card (caspase recruitment domain)) is an endogenous inhibitor of apoptosis that antagonizes both the extrinsic (death receptor) and intrinsic (mitochondrial/er) apoptosis pathways. we discovered that arc blocks not only apoptosis but also necrosis. tnfα-induced necrosis was abrogated by overexpression of wild-type arc but not by a card mutant that is also defective for inhibition of apoptosis. con ...201424440909
sirna screen of early poxvirus genes identifies the aaa+ atpase d5 as the virus genome-uncoating factor.poxvirus genome uncoating is a two-step process. first, cytoplasmic viral cores are activated and early viral genes are expressed. next, cores are disassembled and the genomes released. this second step depends on an early viral factor(s) that has eluded identification for over 40 years. we used a large-scale, high-throughput rnai screen directed against vaccinia virus (vacv) to identify the vacv aaa+ atpase d5 as the poxvirus uncoating factor. we show that the atpase activity of d5 is required ...201424439902
oncolytic vaccinia virotherapy for endometrial cancer.oncolytic virotherapy is a promising modality in endometrial cancer (ec) therapy. in this study, we compared the efficacy of the copenhagen and wyeth strains of oncolytic vaccinia virus (vv) incorporating the human thyroidal sodium iodide symporter (hnis) as a reporter gene (vvnis-c and vvnis-w) in ec.201424434058
tumor vascularization is critical for oncolytic vaccinia virus treatment of peritoneal carcinomatosis.peritoneal carcinomatosis (pc) represents a significant clinical challenge for which there are few treatment options. oncolytic viruses are ideal candidates for pc treatment because of their high tumor specificity, excellent safety profile and suitability for peritoneal delivery. here, we described the use of vvdd-sr-rfp, a recombinant vaccinia virus, in xenograft and syngeneic models of colorectal pc. colorectal cancer cell lines were highly susceptible to vvdd-sr-rfp replication and cytotoxici ...201423893655
synergistic cytotoxicity of radiation and oncolytic lister strain vaccinia in (v600d/e)braf mutant melanoma depends on jnk and tnf-α signaling.melanoma is an aggressive skin cancer that carries an extremely poor prognosis when local invasion, nodal spread or systemic metastasis has occurred. recent advances in melanoma biology have revealed that ras-raf-mek-erk signaling has a pivotal role in governing disease progression and treatment resistance. proof-of-concept clinical studies have shown that direct braf inhibition yields impressive responses in advanced disease but these are short-lived as treatment resistance rapidly emerges. the ...201423624923
vaccinia virus glv-1h153 in combination with 131i shows increased efficiency in treating triple-negative breast cancer.we investigated the therapeutic efficacy of a replication-competent oncolytic vaccinia virus, glv-1h153, carrying human sodium iodide symporter (hnis), in combination with radioiodine in an orthotopic triple-negative breast cancer (tnbc) murine model. in vitro viral infection was confirmed by immunoblotting and radioiodine uptake assays. orthotopic xenografts (mda-mb-231 cells) received intratumoral injection of glv-1h153 or pbs. one week after viral injection, xenografts were randomized into 4 ...201424186964
giving oncolytic vaccinia virus more bite. 201424384909
preventing the return of smallpox: molecular modeling studies on thymidylate kinase from variola virus.smallpox was one of the most devastating diseases in the human history and still represents a serious menace today due to its potential use by bioterrorists. considering this threat and the non-existence of effective chemotherapy, we propose the enzyme thymidylate kinase from variola virus (vartmpk) as a potential target to the drug design against smallpox. we first built a homology model for vartmpk and performed molecular docking studies on it in order to investigate the interactions with inhi ...201423998201
the d10 decapping enzyme of vaccinia virus contributes to decay of cellular and viral mrnas and to virulence in mice.posttranscriptional mechanisms are important for regulation of cellular and viral gene expression. the presence of the 5' cap structure m(7)g(5')ppp(5')nm is a general feature of mrnas that provides protection from exoribonuclease digestion and enhances translation. vaccinia virus and other poxviruses encode enzymes for both cap synthesis and decapping. decapping is mediated by two related enzymes, d9 and d10, which are synthesized before and after viral dna replication, respectively. the timing ...201424155373
t-cell engager-armed oncolytic vaccinia virus significantly enhances antitumor therapy.oncolytic vaccinia virus (vv) therapy has shown promise in preclinical models and in clinical studies. however, complete responses have rarely been observed. this lack of efficacy is most likely due to suboptimal virus spread through the tumor resulting in limited tumor cell destruction. we reasoned that redirecting t cells to the tumor has the potential to improve the antitumor activity of oncolytic vvs. we, therefore, constructed a vv encoding a secretory bispecific t-cell engager consisting o ...201424135899
langerin+ dermal dc, but not langerhans cells, are required for effective cd8-mediated immune responses after skin scarification with vaccinia virus.skin scarification (s.s.) with vaccinia virus (vacv) is essential for generation of an optimal protective t-cell memory immune response. dendritic cells (dcs), which are professional antigen-presenting cells, are required for naive t-cell priming and activation. at least three subsets of skin-resident dc have been identified: langerhans cells (lcs), dermal langerin+ dc (lang+ ddc), and dermal langerin- dc (lang- ddc). using langerin-diphtheria toxin receptor mice and established mouse model of v ...201424126845
t-cell help dependence of memory cd8+ t-cell expansion upon vaccinia virus challenge relies on cd40 signaling.due to their capacity to differentiate into long-lived memory cells, cd8(+) t cells are able to resolve subsequent infections faster than during the primary response. among other factors, cd4(+) t cells play a crucial role during primary and secondary cd8(+) t-cell responses. however, the timing and mechanisms by which they influence cd8(+) t cells may differ in primary and secondary responses. here, we demonstrate that during both primary and secondary vaccinia virus infection, cd4(+) t cells a ...201424108445
profile of natural killer cells after a previous natural vaccinia virus infection in an in vitro viral re-exposure.the present study compares the profile of nk cells in an in vitro re-exposure by vaccinia virus (vacv), in groups that have had a previous vaccination or natural infection. our data suggests that stimulation with vacv triggers a cytotoxic response by nk cells marked by an increase of ncrs: nkp30, nkp44, and nkp46 in infected (vaccinated and unvaccinated) subjects and in non-infected vaccinated patients, when compared with non-infected unvaccinated individuals. however, the degranulation and secr ...201424530576
the master regulator of the cellular stress response (hsf1) is critical for orthopoxvirus infection.the genus orthopoxviridae contains a diverse group of human pathogens including monkeypox, smallpox and vaccinia. these viruses are presumed to be less dependent on host functions than other dna viruses because they have large genomes and replicate in the cytoplasm, but a detailed understanding of the host factors required by orthopoxviruses is lacking. to address this topic, we performed an unbiased, genome-wide pooled rnai screen targeting over 17,000 human genes to identify the host factors t ...201424516381
critical role of endoplasmic reticulum aminopeptidase 1 in determining the length and sequence of peptides bound and presented by hla-b27.hla-b27 and endoplasmic reticulum aminopeptidase 1 (erap1) are the two strongest genetic factors predisposing to ankylosing spondylitis (as). a key aminopeptidase in class i major histocompatibility complex presentation, erap1 potentially contributes to the pathogenesis of as by altering hla-b27 peptide presentation. the aim of this study was to analyze the effects of erap1 on the hla-b27 peptide repertoire and peptide presentation to cytotoxic t lymphocytes (ctls).201424504800
[effect of antibiotic virusin 1609 on vaccinia virus and taylor's virus]. 201424544539
detection of vaccinia virus in blood and faeces of experimentally infected cows.bovine vaccinia (bv), a zoonosis caused by vaccinia virus (vacv), affects dairy cattle and milkers, causing economic, veterinary and human health impacts. despite such impacts, there are no experimental studies about the pathogenesis of bv in cows to assess whether there is a systemic spread of the virus and whether there are different ways of vacv shedding. trying to answer some of these questions, a study was proposed using experimental inoculation of vacv in cows. all experimentally infected ...201322909142
inactivation of vaccinia virus by natural sunlight and by artificial uvb radiation.this study determined the sensitivity of vaccinia virus, an orthopox virus commonly used as a surrogate for variola virus (etiological agent of smallpox), exposed to uvb radiation emitted by a solar simulator, or to direct natural sunlight. the data obtained indicate that: (1) the virucidal effect of natural sunlight can be mimicked adequately by an artificial light source with similar spectral characteristics in the uvb, (2) viral sensitivity to uvb or to solar radiation can be correlated with ...201322816993
airway cd8(+) t cells induced by pulmonary dna immunization mediate protective anti-viral immunity.vaccination strategies for protection against a number of respiratory pathogens must induce t-cell populations in both the pulmonary airways and peripheral lymphoid organs. in this study, we show that pulmonary immunization using plasmid dna formulated with the polymer polyethyleneimine (pei-dna) induced antigen-specific cd8(+) t cells in the airways that persisted long after antigen local clearance. the persistence of the cells was not mediated by local lymphocyte proliferation or persistent an ...201322806099
targeting the antigen encoded by adenoviral vectors to the dec205 receptor modulates the cellular and humoral immune response.replication-defective adenoviral vectors have emerged as promising vaccine candidates for diseases relying on strong cd8(+) t-cell responses for protection. in this study, we modified a non-replicative adenoviral vector to selectively deliver, in situ, an encoded ovalbumin (ova) model antigen to dendritic cells (dcs). efficient uptake and presentation of ova was achieved through fusion of the antigen to a single-chain antibody directed against dec205, an endocytic receptor expressed on dcs. the ...201323184617
bovine vaccinia, a systemic infection: evidence of fecal shedding, viremia and detection in lymphoid organs.bovine vaccinia (bv) is a zoonosis caused by vaccinia virus (vacv) that affects dairy cattle and milkers, causing economic losses and impacting animal and human health. based on the clinical presentation, bv appears to be a localized disease, with lesions restricted to the skin of affected individuals. however, there are no studies on the pathogenesis of the disease in cows to determine if there is a systemic spread of the virus and if there are different ways of vacv shedding. the objective of ...201323021861
gfp co-expression reduces the a33r gene expression driven by a fowlpox vector in replication permissive and non-permissive cell lines.the development of an effective prophylactic vaccine is still necessary to improve the safety of the conventional although-discontinued smallpox vaccine, and to protect from the threat of deliberate release of variola virus. this need also arises from the number of new cases of animal orthopoxvirus infections each year, and to reduce the risk to animal handlers. fowlpox (fp) recombinants only replicate in avian species and have been developed against human infectious diseases, as they can elicit ...201323000750
oncolytic vaccinia virus and radiotherapy in head and neck cancer.oncolytic forms of attenuated vaccinia virus are now in clinical development, assessing the compatibility of this novel treatment with radiotherapy may reveal exploitable synergistic relationships.201322925693
forensic proteomics of poxvirus production.the field of microbial forensics has recently sought to develop methods to discern biological signatures to indicate production methods for biological agents. viral agents have received less attention to date. their obligate propagation in living cells makes purification from cellular material a challenge. this leads to potential carryover of protein-rich signatures of their production system. here we have explored a proteomic analysis of vaccinia virus as a model poxvirus system in which to com ...201323979794
poxviral protein a52 stimulates p38 mitogen-activated protein kinase (mapk) activation by causing tumor necrosis factor receptor-associated factor 6 (traf6) self-association leading to transforming growth factor β-activated kinase 1 (tak1) recruitment.vaccinia virus encodes a number of proteins that inhibit and manipulate innate immune signaling pathways that also have a role in virulence. these include a52, a protein shown to inhibit il-1- and toll-like receptor-stimulated nfκb activation, via interaction with interleukin-1 receptor-associated kinase 2 (irak2). interestingly, a52 was also found to activate p38 mapk and thus enhance toll-like receptor-dependent il-10 induction, which was traf6-dependent, but the manner in which a52 manipulate ...201324114841
new vaccinia virus promoter as a potential candidate for future vaccines.here we describe the design and strength of a new synthetic late-early optimized (leo) vaccinia virus (vacv) promoter used as a transcriptional regulator of gfp expression during modified vaccinia ankara infection. in contrast to the described synthetic vacv promoter (ps), leo induced significantly higher levels of gfp expression in vitro within the first hour after infection, which correlated with an enhancement in the gfp-specific cd8 t-cell response detected in vivo, demonstrating its potenti ...201324077296
the formin fhod1 and the small gtpase rac1 promote vaccinia virus actin-based motility.vaccinia virus dissemination relies on the n-wasp-arp2/3 pathway, which mediates actin tail formation underneath cell-associated extracellular viruses (cevs). here, we uncover a previously unappreciated role for the formin fhod1 and the small gtpase rac1 in vaccinia actin tail formation. fhod1 depletion decreased the number of cevs forming actin tails and impaired the elongation rate of the formed actin tails. recruitment of fhod1 to actin tails relied on its gtpase binding domain in addition to ...201324062339
direct formation of vaccinia virus membranes from the endoplasmic reticulum in the absence of the newly characterized l2-interacting protein a30.5.crescents consisting of a single lipoprotein membrane with an external protein scaffold comprise the initial structural elements of poxvirus morphogenesis. crescents enlarge to form spherical immature virions, which enclose viroplasm consisting of proteins destined to form the cores of mature virions. previous studies suggest that the l2 protein participates in the recruitment of endoplasmic reticulum (er)-derived membranes to form immature virions within assembly sites of cytoplasmic factories. ...201324027302
tracking viral genomes in host cells at single-molecule resolution.viral dna trafficking in cells has large impacts on physiology and disease development. current methods lack the resolution and accuracy to visualize and quantify viral dna trafficking at single-molecule resolution. we developed a noninvasive protocol for accurate quantification of viral dna-genome (vdna) trafficking in single cells. ethynyl-modified nucleosides were used to metabolically label newly synthesized adenovirus, herpes virus, and vaccinia virus vdna, without affecting infectivity. su ...201324139403
optical detection and virotherapy of live metastatic tumor cells in body fluids with vaccinia strains.metastatic tumor cells in body fluids are important targets for treatment, and critical surrogate markers for evaluating cancer prognosis and therapeutic response. here we report, for the first time, that live metastatic tumor cells in blood samples from mice bearing human tumor xenografts and in blood and cerebrospinal fluid samples from patients with cancer were successfully detected using a tumor cell-specific recombinant vaccinia virus (vacv). in contrast to the fda-approved cellsearch syste ...201324019862
neutralizing antibodies associated with exposure factors to orthopoxvirus in laboratory workers.extensive use of vaccinia virus (vacv) in research has led to associated accidental human exposure in laboratories worldwide. in spite of the social and economic relevance of bovine vaccinia outbreaks in brazil, national data concerning laboratory workers handling these infectious agents are relatively scarce. therefore, a serological survey was conducted in a brazilian laboratory to evaluate staff exposure to orthopoxviruses (opvs). information concerning direct work with opvs, vaccination stat ...201323973323
a leader sequence capable of enhancing rna expression and protein synthesis in mammalian cells.many applications in biotechnology require human proteins generated from human cells. stable cell lines commonly used for this purpose are difficult to develop, and scaling to large numbers of proteins can be problematic. transient expression can circumvent this problem, but protein yields are generally too low for most applications. here we report a novel 37-nucleotide leader sequence that promotes rapid and high transgene expression in mammalian cells. this sequence was identified by in vitro ...201323908110
genetic variation in il18r1 and il18 genes and inteferon γ elispot response to smallpox vaccination: an unexpected relationship.genetic association studies demonstrated a role for cytokine proteins and cytokine or cytokine receptor gene polymorphisms in smallpox vaccine-induced adaptive immunity.201323901078
vaccinia virus immune evasion: mechanisms, virulence and immunogenicity.virus infection of mammalian cells is sensed by pattern recognition receptors and leads to an innate immune response that restricts virus replication and induces adaptive immunity. in response, viruses have evolved many countermeasures that enable them to replicate and be transmitted to new hosts, despite the host innate immune response. poxviruses, such as vaccinia virus (vacv), have large dna genomes and encode many proteins that are dedicated to host immune evasion. some of these proteins are ...201323999164
deletion of immunomodulator c6 from vaccinia virus strain western reserve enhances virus immunogenicity and vaccine efficacy.vectors based on vaccinia virus (vacv), the vaccine used to eradicate smallpox, are currently popular candidates for the vaccination against numerous infectious diseases including malaria and aids. although vacv induces robust cellular and humoral responses, enhancing the safety and efficacy of these vectors remains an important area of research. here, we describe the enhanced immunogenicity of a recombinant vacv western reserve (wr) strain lacking the immunomodulatory protein c6 (vδc6). intrade ...201323288427
[oncolytic properties of some orthopoxviruses, adenoviruses and parvoviruses in human glioma cells].currently one of the most promising approaches in development of cancer virotherapy is based on the ability of oncolytic viruses to selective infection and lysis of tumor cells.201324741936
cd4 and cd8 t cells participate in the immune memory response against vaccinia virus after a previous natural infection.the present study evaluates the immune response of memory cd4(+) and cd8(+) t cells from patients following a natural vaccinia virus (vacv) infection. a total of 42 individuals were involved in the study being: 22 previously infected individuals (vaccinated or not against smallpox) and 20 non-infected individuals (vaccinated or not). a short-term in vitro stimulation with uv-inactivated vacv of whole blood cells was performed. our study showed that previously infected individuals have a lower pe ...201324600565
transport and stability of the vaccinia virus a34 protein is affected by the a33 protein.vaccinia virus (vacv) has two infectious forms called intracellular mature virus and extracellular enveloped virus (eev). two of the seven viral proteins in the eev outer envelope, a33 and a34, are type ii membrane glycoproteins that each interact with another eev protein called b5; however, evidence for direct a33-a34 interaction is lacking. the localization and stability of a34 is affected by b5 and here data are presented showing that a34 is also affected by a33. in the absence of a33, just a ...201323255618
biogenesis of the vaccinia virus membrane: genetic and ultrastructural analysis of the contributions of the a14 and a17 proteins.vaccinia virus membrane biogenesis requires the a14 and a17 proteins. we show here that both proteins can associate with membranes co- but not posttranslationally, and we perform a structure function analysis of a14 and a17 using inducible recombinants. in the absence of a14, electron-dense virosomes and distinct clusters of small vesicles accumulate; in the absence of a17, small vesicles form a corona around the virosomes. when the proteins are induced at 12 h postinfection (hpi), crescents app ...201323135725
viral antigen density and confinement time regulate the reactivity pattern of cd4 t-cell responses to vaccinia virus infection.t-cell recognition of ligands is polyspecific. this translates into antiviral t-cell responses having a range of potency and specificity for viral ligands. how these ligand recognition patterns are established is not fully understood. here, we show that an activation threshold regulates whether robust cd4 t-cell activation occurs following viral infection. the activation threshold was variable because of its dependence on the density of the viral peptide (p)mhc displayed on infected cells. furth ...201323248307
crosstalk between immune cell and oncolytic vaccinia therapy enhances tumor trafficking and antitumor effects.the combination of an oncolytic virus, that directly destroys tumor cells and mediates an acute immune response, with an immune cell therapy, capable of further enlisting and enhancing the host immune response, has the potential to create a potent therapeutic effect. we have previously developed several strategies for optimizing the delivery of oncolytic vaccinia virus vectors to their tumor targets, including the use of immune cell-based carrier vehicles and the incorporation of mutations that ...201323229093
poxvirus membrane biogenesis: rupture not disruption.enveloped viruses acquire their membrane from the host by budding at, or wrapping by, cellular membranes. transmission electron microscopy (tem) images, however, suggested that the prototype member of the poxviridae, vaccinia virus (vacv), may create its membrane 'de novo' with free open ends exposed in the cytosol. within the frame of the german-wide priority programme we re-addressed the biogenesis and origin of the vacv membrane using electron tomography (et), cryo-em and lipid analysis of pu ...201323168015
recombinant dna vaccine against neurite outgrowth inhibitors attenuates behavioral deficits and decreases abeta in an alzheimer's disease mouse model.alzheimer's disease (ad) is a chronic neurodegenerative disease that causes a progressive loss in learning and memory capabilities and eventually results in dementia. the non-renewable nature of neurons in the central nervous system leads to the basic pathological changes that are related to the various behavioral and psychological symptoms of ad. oligodendrocyte- and myelin-related neurite outgrowth inhibitors (nois) tend to hinder the regeneration of neurons. we designed a recombinant dna vacc ...201323201352
analysis of viral membranes formed in cells infected by a vaccinia virus l2-deletion mutant suggests their origin from the endoplasmic reticulum.assembly of the poxvirus immature virion (iv) membrane is a poorly understood event that occurs within the cytoplasm. at least eight viral proteins participate in formation of the viral membrane. of these, a14, a17, and d13 are structural components whereas a6, a11, f10, h7, and l2 participate in membrane biogenesis. l2, the object of this study, is conserved in all chordopoxviruses, expressed early in infection, and associated with the endoplasmic reticulum (er) throughout the cell and at the e ...201323192873
preventing postoperative metastatic disease by inhibiting surgery-induced dysfunction in natural killer cells.natural killer (nk) cell clearance of tumor cell emboli following surgery is thought to be vital in preventing postoperative metastases. using a mouse model of surgical stress, we transferred surgically stressed nk cells into nk-deficient mice and observed enhanced lung metastases in tumor-bearing mice as compared with mice that received untreated nk cells. these results establish that nk cells play a crucial role in mediating tumor clearance following surgery. surgery markedly reduced nk cell t ...201323090117
oncolytic vaccinia virus safely and effectively treats skin tumors in mouse models of xeroderma pigmentosum.xeroderma pigmentosum (xp) is an orphan autosomal recessive disorder of dna repair. when exposed to genotoxic stress, xp patients have reduced capacity to remove bulky adducts by nucleotide excision repair and are thus greatly predisposed to cancer. unfortunately, given the nature of their underlying genetic defect, tumor-bearing xp patients cannot be treated with conventional dna damaging therapies. engineered strains of the poxvirus vaccinia have been shown to cure cancer in numerous preclinic ...201322733395
vaccinia virus protein n2 is a nuclear irf3 inhibitor that promotes virulence.vaccinia virus (vacv) expresses many proteins that are non-essential for virus replication but promote virulence by inhibiting components of the host immune response to infection. these immunomodulators include a family of proteins that have, or are predicted to have, a structure related to the b-cell lymphoma (bcl)-2 protein. five members of the vacv bcl-2 family (n1, b14, a52, f1 and k7) have had their crystal structure solved, others have been characterized and a function assigned (c6, a46), ...201323761407
serological study of vaccinia virus reservoirs in areas with and without official reports of outbreaks in cattle and humans in são paulo, brazil.vaccinia virus (vacv), the etiological agent of an exanthematic disease, has been associated with several bovine outbreaks in brazil since the end of the global vaccination campaign against smallpox. it was previously believed that the vaccine virus used for the who global campaign had adapted to an unknown wild reservoir and was sporadically re-emerging in outbreaks in cattle and milkers. at present, it is known that brazilian vacv is phylogenetically different from the vaccinia virus vaccinal ...201323760628
genomic analysis of vaccinia virus strain tiantan provides new insights into the evolution and evolutionary relationships between orthopoxviruses.vaccinia virus (vacv) strain tiantan was used for much of china's modern history to vaccinate against smallpox, however the only genome sequence contains errors. we have sequenced additional examples of tiantan to obtain a better picture of this important virus. we detected two different subclones. one (tp03) encodes large deletions in the terminal repeats that extend into both vegf genes and create a small plaque variant. the second clone (tp05) encodes a nearly intact complement of genes in th ...201323608359
vaccinia virus f1l protein promotes virulence by inhibiting inflammasome activation.host innate immune responses to dna viruses involve members of the nucleotide-binding domain, leucine-rich repeat and pyrin domain containing protein (nlrp) family, which form "inflammasomes" that activate caspase-1, resulting in proteolytic activation of cytokines interleukin (il)-1β and il-18. we hypothesized that dna viruses would target inflammasomes to overcome host defense. a vaccinia virus (vacv) b-cell cll/lymphoma 2 (bcl-2) homolog, f1l, was demonstrated to bind and inhibit the nlr fami ...201323603272
extracts from rabbit skin inflamed by the vaccinia virus attenuate bupivacaine-induced spinal neurotoxicity in pregnant rats.extracts from rabbit skin inflamed by the vaccinia virus can relieve pain and promote repair of nerve injury. the present study intraperitoneally injected extracts from rabbit skin inflamed by the vaccinia virus for 3 and 4 days prior to and following intrathecal injection of bupivacaine into pregnant rats. the pain threshold test after bupivacaine injection showed that the maximum possible effect of tail-flick latency peaked 1 day after intrathecal injection of bupivacaine in the extract-pretre ...201325206391
vaccinia virus protein k7 is a virulence factor that alters the acute immune response to infection.vaccinia virus (vacv) encodes many proteins that antagonize the innate immune system including a family of intracellular proteins with a b-cell lymphoma (bcl)-2-like structure. one of these bcl-2 proteins called k7 binds toll-like receptor-adaptor proteins and the dead-box rna helicase ddx3 and thereby inhibits the activation of nf-κb and interferon regulatory factor 3. however, the contribution of k7 to virus virulence is not known. here a vacv lacking the k7r gene (vδk7) was constructed and co ...201323580427
unintentional transfer of vaccinia virus associated with smallpox vaccines: acam2000(®) compared with dryvax(®).routine vaccination against smallpox (variola) ceased in the us in 1976. however, in 2002 limited coverage for military personnel and some healthcare workers was reinstituted. in march 2008, acam2000® replaced dryvax® as the vaccine used in the united states against smallpox. unintentional transfer of vaccinia virus from a vaccination site by autoinoculation or contact transmission, can have significant public health implications. we summarize unintentional virus transfer aes associated with aca ...201323571177
anatomically restricted synergistic antiviral activities of innate and adaptive immune cells in the skin.despite extensive ex vivo investigation, the spatiotemporal organization of immune cells interacting with virus-infected cells in tissues remains uncertain. to address this, we used intravital multiphoton microscopy to visualize immune cell interactions with virus-infected cells following epicutaneous vaccinia virus (vv) infection of mice. vv infects keratinocytes in epidermal foci and numerous migratory dermal inflammatory monocytes that outlie the foci. we observed ly6g(+) innate immune cells ...201323414756
getting in front and behind the enemy lines to counter virus infection.how immune cells collaborate to clear a virus from the skin is not well understood. in this issue of cell host & microbe,hickman et al. (2013) show that spatially segregated ly6g(+) monocytes and cd8(+) t cells act synergistically to clear vaccinia virus from a primary skin infection.201323414751
vaccinia virus-mediated melanin production allows mr and optoacoustic deep tissue imaging and laser-induced thermotherapy of cancer.we reported earlier the delivery of antiangiogenic single chain antibodies by using oncolytic vaccinia virus strains to enhance their therapeutic efficacy. here, we provide evidence that gene-evoked production of melanin can be used as a therapeutic and diagnostic mediator, as exemplified by insertion of only one or two genes into the genome of an oncolytic vaccinia virus strain. we found that produced melanin is an excellent reporter for optical imaging without addition of substrate. melanin pr ...201323401518
randomized dose-finding clinical trial of oncolytic immunotherapeutic vaccinia jx-594 in liver cancer.oncolytic viruses and active immunotherapeutics have complementary mechanisms of action (moa) that are both self amplifying in tumors, yet the impact of dose on subject outcome is unclear. jx-594 (pexa-vec) is an oncolytic and immunotherapeutic vaccinia virus. to determine the optimal jx-594 dose in subjects with advanced hepatocellular carcinoma (hcc), we conducted a randomized phase 2 dose-finding trial (n=30). radiologists infused low- or high-dose jx-594 into liver tumors (days 1, 15 and 29) ...201323396206
oncolytic vaccinia virus disrupts tumor-associated vasculature in humans.efforts to selectively target and disrupt established tumor vasculature have largely failed to date. we hypothesized that a vaccinia virus engineered to target cells with activation of the ras/mapk signaling pathway (jx-594) could specifically infect and express transgenes (hgm-csf, β-galactosidase) in tumor-associated vascular endothelial cells in humans. efficient replication and transgene expression in normal human endothelial cells in vitro required either vegf or fgf-2 stimulation. intraven ...201323393196
a36-dependent actin filament nucleation promotes release of vaccinia virus.cell-to-cell transmission of vaccinia virus can be mediated by enveloped virions that remain attached to the outer surface of the cell or those released into the medium. during egress, the outer membrane of the double-enveloped virus fuses with the plasma membrane leaving extracellular virus attached to the cell surface via viral envelope proteins. here we report that f-actin nucleation by the viral protein a36 promotes the disengagement of virus attachment and release of enveloped virus. cells ...201323555252
rational engineering of recombinant picornavirus capsids to produce safe, protective vaccine antigen.foot-and-mouth disease remains a major plague of livestock and outbreaks are often economically catastrophic. current inactivated virus vaccines require expensive high containment facilities for their production and maintenance of a cold-chain for their activity. we have addressed both of these major drawbacks. firstly we have developed methods to efficiently express recombinant empty capsids. expression constructs aimed at lowering the levels and activity of the viral protease required for the ...201323544011
discovering naturally processed antigenic determinants that confer protective t cell immunity.cd8+ t cells (tcd8) confer protective immunity against many infectious diseases, suggesting that microbial tcd8 determinants are promising vaccine targets. nevertheless, current t cell antigen identification approaches do not discern which epitopes drive protective immunity during active infection - information that is critical for the rational design of tcd8-targeted vaccines. we employed a proteomics-based approach for large-scale discovery of naturally processed determinants derived from a co ...201323543059
design of potent poxvirus inhibitors of the heterodimeric processivity factor required for viral replication.smallpox constitutes a major bioterrorism threat, which underscores the need to develop antiviral drugs for rapid response in the event of an attack. viral processivity factors are attractive drug targets in being both specific and essential for their cognate dna polymerases to synthesize extended strands of dna. an in silico model of the vacinnia virus processivity factor, comprised of the a20 and d4 heterocomplex, was constructed and used for lead optimization of an indole-based scaffold ident ...201323527789
crystallization and preliminary x-ray diffraction analysis of three recombinant mutants of vaccinia virus uracil dna glycosylase.amino-acid residues located at a highly flexible area in the uracil dna glycosylase of vaccinia virus were mutated. in the crystal structure of wild-type d4 these residues lie at the dimer interface. specifically, three mutants were generated: (i) residue arg167 was replaced with an alanine (r167ad4), (ii) residues glu171, ser172 and pro173 were substituted with three glycine residues (3gd4) and (iii) residues glu171 and ser172 were deleted (δ171-172d4). mutant proteins were expressed, purified ...201323519808
domain-level rocking motion within a polymerase that translocates on single-stranded nucleic acid.vaccinia virus poly(a) polymerase (vp55) is the only known polymerase that can translocate independently with respect to single-stranded nucleic acid (ssna). previously, its structure has only been solved in the context of the vp39 processivity factor. here, a crystal structure of unliganded monomeric vp55 has been solved to 2.86 å resolution, showing the first backbone structural isoforms among either vp55 or its processivity factor (vp39). backbone differences between the two molecules of vp55 ...201323519670
protective effect of surfactant protein d in pulmonary vaccinia virus infection: implication of a27 viral protein.vaccinia virus (vacv) was used as a surrogate of variola virus (varv) (genus orthopoxvirus), the causative agent of smallpox, to study orthopoxvirus infection. varv is principally transmitted between humans by aerosol droplets. once inhaled, varv first infects the respiratory tract where it could encounter surfactant components, such as soluble pattern recognition receptors. surfactant protein d (sp-d), constitutively present in the lining fluids of the respiratory tract, plays important roles i ...201323518578
targeting cxcl12/cxcr4 signaling with oncolytic virotherapy disrupts tumor vasculature and inhibits breast cancer metastases.oncolytic viruses hold promise for the treatment of cancer, but their interaction with the tumor microenvironment needs to be elucidated for optimal tumor cell killing. because the cxcr4 receptor for the stromal cell-derived factor-1 (sdf-1/cxcl12) chemokine is one of the key stimuli involved in signaling interactions between tumor cells and their stromal microenvironment, we used oncolytic virotherapy with a cxcr4 antagonist to target the cxcl12/cxcr4 signaling axis in a triple-negative 4t1 bre ...201323509246
vaccinia virus glv-1h153 is a novel agent for detection and effective local control of positive surgical margins for breast cancer.surgery is currently the definitive treatment for early-stage breast cancer. however, the rate of positive surgical margins remains unacceptably high. the human sodium iodide symporter (hnis) is a naturally occurring protein in human thyroid tissue, which enables cells to concentrate radionuclides. the hnis has been exploited to image and treat thyroid cancer. we therefore investigated the potential of a novel oncolytic vaccinia virus glv1h-153 engineered to express the hnis gene for identifying ...201323506710
biochemical and biophysical properties of a putative hub protein expressed by vaccinia virus.h5 is a constitutively expressed, phosphorylated vaccinia virus protein that has been implicated in viral dna replication, post-replicative gene expression, and virus assembly. for the purpose of understanding the role of h5 in vaccinia biology, we have characterized its biochemical and biophysical properties. previously, we have demonstrated that h5 is associated with an endoribonucleolytic activity. in this study, we have shown that this cleavage results in a 3'-oh end suitable for polyadenyla ...201323476017
secondary and tertiary transmission of vaccinia virus after sexual contact with a smallpox vaccinee--san diego, california, 2012.on june 24, 2012, cdc notified public health services, county of san diego health and human services agency, of a suspected case of vaccinia virus infection transmitted by sexual contact. the case had been reported to cdc by an infectious disease specialist who had requested vaccinia immune globulin intravenous (vigiv) (cangene corporation, berwyn, pennsylvania) for a patient with lesions suspicious for vaccinia. the patient reported two recent sexual contacts: one with a partner who recently ha ...201323446513
imaging of intratumoral inflammation during oncolytic virotherapy of tumors by 19f-magnetic resonance imaging (mri).oncolytic virotherapy of tumors is an up-coming, promising therapeutic modality of cancer therapy. unfortunately, non-invasive techniques to evaluate the inflammatory host response to treatment are rare. here, we evaluate (19)f magnetic resonance imaging (mri) which enables the non-invasive visualization of inflammatory processes in pathological conditions by the use of perfluorocarbon nanoemulsions (pfc) for monitoring of oncolytic virotherapy.201323441176
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