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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
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
the effects of post-exposure smallpox vaccination on clinical disease presentation: addressing the data gaps between historical epidemiology and modern surrogate model data.decades after public health interventions - including pre- and post-exposure vaccination - were used to eradicate smallpox, zoonotic orthopoxvirus outbreaks and the potential threat of a release of variola virus remain public health concerns. routine prophylactic smallpox vaccination of the public ceased worldwide in 1980, and the adverse event rate associated with the currently licensed live vaccinia virus vaccine makes reinstatement of policies recommending routine pre-exposure vaccination unl ...201323994378
a rev-independent gag/pol eliminates detectable psi-gag recombination in lentiviral vectors.lentiviral vectors (lvs) are being developed for clinical use in humans for applications including gene therapy and immunotherapy. a safety concern for use of lvs in humans is the generation of replication-competent lentivirus (rcl), which may arise due to recombination between the split genomes of third-generation lvs. although no rcl has been detected to date, design optimizations that minimize recombination events between split genome vectors would provide an added safety benefit that may fur ...201324380052
vaccinia virus entry is followed by core activation and proteasome-mediated release of the immunomodulatory effector vh1 from lateral bodies.host cell entry of vaccinia virus, the prototypic poxvirus, involves a membrane fusion event delivering the viral core and two proteinaceous lateral bodies (lbs) into the cytosol. uncoating of viral cores is poorly characterized, and the composition and function of lbs remains enigmatic. we found that cytosolic cores rapidly dissociated from lbs and expanded in volume, which coincided with reduction of disulfide-bonded core proteins. we identified the abundant phosphoprotein f17, the dual-specif ...201323891003
an oncolytic vaccinia virus expressing the human sodium iodine symporter prolongs survival and facilitates spect/ct imaging in an orthotopic model of malignant pleural mesothelioma.the purpose of this work was to examine the ability of an oncolytic vaccinia virus expressing the human sodium iodine transporter (hnis) to provide real time monitoring of viral therapy and effective treatment of malignant pleural mesothelioma (mpm).201323890748
interactions of the vaccinia virus a19 protein.the a19 protein of vaccinia virus (vacv) is conserved among chordopoxviruses, expressed late in infection, packaged in the virus core, and required for a late step in morphogenesis. multiple-sequence alignments of a19 homologs indicated conservation of a series of lysines and arginines, which could represent a nuclear localization or nucleic acid binding motif, and a pair of cxxc motifs that suggested a zinc finger or redox active sites. the importance of the cxxc motif was confirmed by cysteine ...201323885084
safety mechanism assisted by the repressor of tetracycline (smart) vaccinia virus vectors for vaccines and therapeutics.replication-competent viruses, such as vaccinia virus (vacv), are powerful tools for the development of oncolytic viral therapies and elicit superior immune responses when used as vaccine and immunotherapeutic vectors. however, severe complications from uncontrolled viral replication can occur, particularly in immunocompromised individuals or in those with other predisposing conditions. vacvs constitutively expressing interferon-γ (ifn-γ) replicate in cell culture indistinguishably from control ...201323988330
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
viral infection: propelling vaccinia virus to the neighbours. 201323872944
vaccinia virus f11 promotes viral spread by acting as a pdz-containing scaffolding protein to bind myosin-9a and inhibit rhoa signaling.the vaccinia f11 protein promotes viral spread by modulating the cortical actin cytoskeleton by inhibiting rhoa signaling via an unknown mechanism. pdz domains are widely conserved protein interaction modules whose occurrence in viral proteins is unprecedented. we found that f11 contains a central pdz-like domain that is required to downregulate rhoa signaling and enhance viral spread. the pdz-like domain interacts with the pdz binding motif of the rho gtpase-activating protein (gap) myosin-9a. ...201323870313
a mechanism for induction of a hypoxic response by vaccinia virus.viruses have evolved sophisticated strategies to exploit host cell function for their benefit. here we show that under physiologically normal oxygen levels (normoxia) vaccinia virus (vacv) infection leads to a rapid stabilization of hypoxia-inducible factor (hif)-1α, its translocation into the nucleus and the activation of hif-responsive genes, such as vascular endothelial growth factor (vegf), glucose transporter-1, and pyruvate dehydrogenase kinase-1. hif-1α stabilization is mediated by vacv p ...201323836663
triple-negative breast cancer: new perspectives for novel therapies.triple-negative breast cancer (tnbc) represents 10-20 % of all mammary tumors. it is often found in younger women and has been associated with poor prognosis, due to aggressive tumor phenotype(s), early metastasis to visceral organ or brain after chemotherapy and present lack of clinically established targeted therapies. in recent years, a greater understanding of the biology of this disease has led to the development of numerous and varied therapeutic approaches, especially the trials on poly ( ...201323824643
double-deleted vaccinia virus in virotherapy for refractory and metastatic pediatric solid tumors.previous studies have shown successful antitumor effects of systemically delivered double-deleted vaccinia virus (vvdd) against a number of adult tumor models, including glioma, colon and ovarian cancers. the purpose of this study was to investigate the oncolytic potential of vvdd against a panel of cell lines representative of pediatric solid tumors that are currently difficult to cure.201323816608
role of the vaccinia virus o3 protein in cell entry can be fulfilled by its sequence flexible transmembrane domain.the vaccinia virus o3 protein, a component of the entry-fusion complex, is encoded by all chordopoxviruses. we constructed truncation mutants and demonstrated that the transmembrane domain, which comprises two-thirds of this 35 amino acid protein, is necessary and sufficient for interaction with the entry-fusion complex and function in cell entry. nevertheless, neither single amino acid substitutions nor alanine scanning mutagenesis revealed essential amino acids within the transmembrane domain. ...201323816434
environmental persistence of vaccinia virus on materials.smallpox is caused by the variola virus, and ranks as one of the most serious diseases that could originate from a biological weapon. however, limited data exist on the persistence of variola and related viruses on materials (that may act as fomites), under controlled environmental conditions. to fill these data gaps, we determined the persistence of the vaccinia virus (an established surrogate for the variola virus) as a function of temperature, relative humidity and material. experiments were ...201323815079
vaccinia virus expressing bone morphogenetic protein-4 in novel glioblastoma orthotopic models facilitates enhanced tumor regression and long-term survival.glioblastoma multiforme (gbm) is one of the most aggressive forms of cancer with a high rate of recurrence. we propose a novel oncolytic vaccinia virus (vacv)-based therapy using expression of the bone morphogenetic protein (bmp)-4 for treating gbm and preventing recurrence.201323800258
effect of the deletion of genes encoding proteins of the extracellular virion form of vaccinia virus on vaccine immunogenicity and protective effectiveness in the mouse model.antibodies to both infectious forms of vaccinia virus, the mature virion (mv) and the enveloped virion (ev), as well as cell-mediated immune response appear to be important for protection against smallpox. ev virus particles, although more labile and less numerous than mv, are important for dissemination and spread of virus in infected hosts and thus important in virus pathogenesis. the importance of the ev a33 and b5 proteins for vaccine induced immunity and protection in a murine intranasal ch ...201323785523
vaccinia virus-mediated expression of human erythropoietin in tumors enhances virotherapy and alleviates cancer-related anemia in mice.recombinant human erythropoietin (rhepo), a glycoprotein hormone regulating red blood cell (rbc) formation, is used for the treatment of cancer-related anemia. the effect of rhepo on tumor growth, however, remains controversial. here, we report the construction and characterization of the recombinant vaccinia virus (vacv) glv-1h210, expressing hepo. glv-1h210 was shown to replicate in and kill a549 lung cancer cells in culture efficiently. in mice bearing a549 lung cancer xenografts, treatment w ...201323765443
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
specificity profiling of protein phosphatases toward phosphoseryl and phosphothreonyl peptides.a combinatorial library method was developed to systematically profile the substrate specificity of protein phosphatases toward phosphoseryl (ps) and phosphothreonyl (pt) peptides. application of this method and a previously reported phosphotyrosyl (py) library screening technique to dual-specificity phosphatase (dusp) vh1 of vaccinia virus revealed that vh1 is highly active toward both ps/pt and py peptides. vh1 exhibits different and more stringent sequence specificity toward ps/pt than py sub ...201323758517
open membranes are the precursors for assembly of large dna viruses.nucleo cytoplasmic large dna viruses (ncldvs) are a group of double-stranded dna viruses that replicate their dna partly or entirely in the cytoplasm in association with viral factories (vfs). they share about 50 genes suggesting that they are derived from a common ancestor. using transmission electron microscopy (tem) and electron tomography (et) we showed that the ncldv vaccinia virus (vacv) acquires its membrane from open membrane intermediates, derived from the er. these open membranes contr ...201323751082
clinical, hematological and biochemical parameters of dairy cows experimentally infected with vaccinia virus.vaccinia virus (vacv) is the etiological agent of bovine vaccinia (bv), an important zoonosis that affects dairy cattle. there are many aspects of the disease that remain unknown, and aiming to answer some of these questions, the clinical, hematological, and biochemical parameters of vacv experimentally infected cows were evaluated. in the first part of the study, lactating cows were infected with vacv-gp2 strain. in the second part, animals previously infected with vacv-gp2 were divided into tw ...201323747141
barrier to autointegration factor (baf) inhibits vaccinia virus intermediate transcription in the absence of the viral b1 kinase.barrier to autointegration factor (baf/banf1) is a cellular dna-binding protein found in the nucleus and cytoplasm. cytoplasmic baf binds to foreign dna and can act as a defense against vaccinia dna replication. to evade baf, vaccinia expresses the b1 kinase, which phosphorylates baf and blocks its ability to bind dna. interestingly, b1 is also needed for viral intermediate gene expression via an unknown mechanism. therefore, we evaluated the impact of b1-baf signaling on vaccinia transcription. ...201323891157
group 1 vaccinia virus zoonotic outbreak in maranhao state, brazil.in brazil, several exanthematic autochthone vaccinia virus (vacv) outbreaks affecting dairy cattle and rural workers have been reported since 1999. although outbreaks had been first described in the brazilian southeast, vacv outbreaks were notified in all brazilian regions in < 10 years. however, in this context, vacv outbreaks had not been described in some brazilian states, likely because of a lack of notification, or yet unknown epidemiological reasons. here, we describe the first vacv outbre ...201324166043
transmission of vaccinia virus, possibly through sexual contact, to a woman at high risk for adverse complications.severe adverse events, including eczema vaccinatum (ev), can result after smallpox vaccination. persons at risk for ev include those with underlying dermatologic conditions, such as atopic dermatitis. we investigated a case of vaccinia infection, possibly acquired during sexual contact with a recently vaccinated military service member, in a female maryland resident with atopic dermatitis. the u.s. department of defense's vaccine healthcare centers network (vhcn) and the centers for disease cont ...201324306023
cd8 t cell memory to a viral pathogen requires trans cosignaling between hvem and btla.defining the molecular interactions required to program activated cd8 t cells to survive and become memory cells may allow us to understand how to augment anti-viral immunity. hvem (herpes virus entry mediator) is a member of the tumor necrosis factor receptor (tnfr) family that interacts with ligands in the tnf family, light and lymphotoxin-α, and in the ig family, b and t lymphocyte attenuator (btla) and cd160. the ig family members initiate inhibitory signaling when engaged with hvem, but may ...201324205056
vaccinia virus in household environment during bovine vaccinia outbreak, brazil. 201324274787
reemergence of vaccinia virus during zoonotic outbreak, pará state, brazil.in 2010, vaccinia virus caused an outbreak of bovine vaccinia that affected dairy cattle and rural workers in pará state, brazil. genetic analyses identified the virus as distinct from bean58058 vaccinia virus (identified in 1960s) and from smallpox vaccine virus strains. these findings suggest spread of autochthonous group 1 vaccinia virus in this region.201324274374
proteomic, cellular, and network analyses reveal new dusp3 interactions with nucleolar proteins in hela cells.dusp3 (or vaccinia virus phosphatase vh1-related; vhr) is a small dual-specificity phosphatase known to dephosphorylate c-jun n-terminal kinases and extracellular signal-regulated kinases. in human cervical cancer cells, dusp3 is overexpressed, localizes preferentially to the nucleus, and plays a key role in cellular proliferation and senescence triggering. other dusp3 functions are still unknown, as illustrated by recent and unpublished results from our group showing that this enzyme mediates d ...201324245651
reliefseq: a gene-wise adaptive-k nearest-neighbor feature selection tool for finding gene-gene interactions and main effects in mrna-seq gene expression data.relief-f is a nonparametric, nearest-neighbor machine learning method that has been successfully used to identify relevant variables that may interact in complex multivariate models to explain phenotypic variation. while several tools have been developed for assessing differential expression in sequence-based transcriptomics, the detection of statistical interactions between transcripts has received less attention in the area of rna-seq analysis. we describe a new extension and assessment of rel ...201324339943
combination of virotherapy and t-cell therapy: arming oncolytic virus with t-cell engagers.while cure rates for several cancers have significantly improved, the outcome for patients with advanced solid tumors remains grimly unchanged over the last decades. thus, there is a need for new therapies that could improve outcome for patients who fail current therapies. oncolytic vaccinia virus (vv) would be an appealing addition to the current therapies of cancers because of its ability to infect, replicate in, and lyse tumor cells, and spread to other tumor cells in successive rounds of rep ...201324333405
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
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
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
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
cascade regulation of vaccinia virus gene expression is modulated by multistage promoters.vaccinia virus contains ~200 genes classified temporally as early, intermediate or late. we analyzed 53 intermediate promoters to determine whether any have dual late promoter activity. our strategy involved (i) construction of a cell line that stably expressed the three late transcription factors, (ii) infection with a vaccinia virus mutant that expresses rna polymerase but neither intermediate nor late transcription factors, and (iii) transfection with plasmids containing a luciferase reporter ...201324210117
follow-up analysis of a randomized phase iii immunotherapeutic clinical trial on melanoma.development of a melanoma-specific vaccine is of clinical necessity. therefore, a phase iii, randomized, double-blind trial was performed (june 1988-june 1991) to assess the clinical effectiveness of our vaccinia melanoma oncolysate (vmo) vaccine in stage iii melanoma patients. patient data were collected from 11 institutions, as well as from the social security death index and were analyzed from april through august 2008 for disease-free interval (dfi) and overall survival (os). the median os f ...201324649193
inconclusive evidence for or against positive antigen selection in the shaping of human immunoglobulin e repertoires: a call for new approaches.the mechanisms driving the development of immunoglobulin e (ige) antibody repertoires are a matter of debate. alternatives to the classical view on antibody development, involving somatic mutation and antigen-driven selection of high-affinity variants in germinal centers, have been proposed.201323343692
critical role for all-trans retinoic acid for optimal effector and effector memory cd8 t cell differentiation.a plethora of work implicates important effects of the vitamin a derivative retinoic acid (ra) in myeloid differentiation, whereas fewer studies explore the role of ra in lymphoid cells. most work on lymphoid cells has focused on the influence of ra on cd4 t cells. little information about the role of ra in cd8 t cell differentiation is available, and even less on cell-intrinsic effects in the cd8 t cell. this study explores the role of ra in effector and memory differentiation in a cell-intrins ...201323338237
protein kinase c overexpression does not enhance immune-stimulatory surface markers of vaccinia-infected dendritic cells and dc cell lines.one of the shortcomings of vaccinia virus (vacv) as immunization vector is the down-regulation of hla and costimulatory molecules in antigen presenting cells. to overcome this problem we investigated the use of protein kinase c (pkc) as immune stimulatory agent. thus several classical and atypical pkcs were inserted into wild-type or attenuated vacv using recombination into the hemagglutinin gene and the expression driven by the vacv 7,5k-ie gene promoter. recombinant constructs expressing pkc-a ...201323323525
atomic structure of dual-specificity phosphatase 26, a novel p53 phosphatase.regulation of p53 phosphorylation is critical to control its stability and biological activity. dual-specificity phosphatase 26 (dusp26) is a brain phosphatase highly overexpressed in neuroblastoma, which has been implicated in dephosphorylating phospho-ser20 and phospho-ser37 in the p53 transactivation domain. in this paper, we report the 1.68 å crystal structure of a catalytically inactive mutant (cys152ser) of dusp26 lacking the first 60 n-terminal residues (δn60-c/s-dusp26). this structure r ...201323298255
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
kinetics of antigen expression and epitope presentation during virus infection.current knowledge about the dynamics of antigen presentation to t cells during viral infection is very poor despite being of fundamental importance to our understanding of anti-viral immunity. here we use an advanced mass spectrometry method to simultaneously quantify the presentation of eight vaccinia virus peptide-mhc complexes (epitopes) on infected cells and the amounts of their source antigens at multiple times after infection. the results show a startling 1000-fold range in abundance as we ...201323382674
multifunctional electrochemical aptasensor for aptamer clones screening, virus quantitation in blood and viability assessment.a novel attempt was made to develop a disposable multifunctional sensor for analysis of vaccinia virus (vacv), a promising oncolytic agent that can replicate in and kill tumor cells. briefly, we developed aptamers specific to vacv that were negatively selected against human serum as well as human and mouse blood to be further utilized for viral analysis directly in serum and blood. in addition, the aptamers were negatively selected against heat-inactivated vacv to enable them to distinguish betw ...201323381386
the tanapoxvirus 142r protein is a serine-threonine kinase that phosphorylates the tumor suppressor p53.to effectively respond to viral infections, mammals rely on the innate and adaptive immune systems. additionally, host cellular responses, such as apoptosis also play a vital role in the host defense mechanisms. to accomplish a successful replicative strategy in vivo, animal viruses have evolved a variety of molecular mechanisms that interfere with host responses. poxviruses in particular, represents a prime example of where animal viruses encode a wide variety of proteins necessary for replicat ...201323359106
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
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
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
small cell ovarian carcinoma: genomic stability and responsiveness to therapeutics.the biology of small cell ovarian carcinoma of the hypercalcemic type (sccoht), which is a rare and aggressive form of ovarian cancer, is poorly understood. tumourigenicity, in vitro growth characteristics, genetic and genomic anomalies, and sensitivity to standard and novel chemotherapeutic treatments were investigated in the unique sccoht cell line, bin-67, to provide further insight in the biology of this rare type of ovarian cancer.201323433318
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
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
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
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
l1r, a27l, a33r and b5r vaccinia virus genes expressed by fowlpox recombinants as putative novel orthopoxvirus vaccines.the traditional smallpox vaccine, administered by scarification, was discontinued in the general population from 1980, because of the absence of new smallpox cases. however, the development of an effective prophylactic vaccine against smallpox is still necessary, to protect from the threat of deliberate release of the variola virus for bioterrorism and from new zoonotic infections, and to improve the safety of the traditional vaccine. preventive vaccination still remains the most effective contr ...201323578094
dicer-2 processes diverse viral rna species.rna silencing pathways play critical roles in gene regulation, virus infection, and transposon control. rna interference (rnai) is mediated by small interfering rnas (sirnas), which are liberated from double-stranded (ds)rna precursors by dicer and guide the rna-induced silencing complex (risc) to targets. although principles governing small rna sorting into risc have been uncovered, the spectrum of rna species that can be targeted by dicer proteins, particularly the viral rnas present during an ...201323424633
reduced interleukin-4 receptor α expression on cd8+ t cells correlates with higher quality anti-viral immunity.with the hope of understanding how interleukin (il)-4 and il-13 modulated quality of anti-viral cd8(+) t cells, we evaluated the expression of receptors for these cytokines following a range of viral infections (e.g. pox viruses and influenza virus). results clearly indicated that unlike other il-4/il-13 receptor subunits, il-4 receptor α (il-4rα) was significantly down-regulated on anti-viral cd8(+) t cells in a cognate antigen dependent manner. the infection of gene knockout mice and wild-type ...201323383283
isg15 is counteracted by vaccinia virus e3 protein and controls the proinflammatory response against viral infection.conjugation of isg15 inhibits replication of several viruses. here, using an expression system for assaying human and mouse isg15 conjugations (isgylations), we have demonstrated that vaccinia virus e3 protein binds and antagonizes human and mouse isg15 modification. to study isgylation importance in poxvirus infection, we used a mouse model that expresses deconjugating proteases. our results indicate that isgylation restricts in vitro replication of the vaccinia virus vvδe3l mutant but unconjug ...201324257616
isg15 regulates peritoneal macrophages functionality against viral infection.upon viral infection, the production of type i interferon (ifn) and the subsequent upregulation of ifn stimulated genes (isgs) generate an antiviral state with an important role in the activation of innate and adaptive host immune responses. the ubiquitin-like protein (ubl) isg15 is a critical ifn-induced antiviral molecule that protects against several viral infections, but the mechanism by which isg15 exerts its antiviral function is not completely understood. here, we report that isg15 plays ...201324137104
il-10 suppresses il-17-mediated dermal inflammation and reduces the systemic burden of vaccinia virus in a mouse model of eczema vaccinatum.individuals with atopic dermatitis (ad) are susceptible to a severe, potentially fatal, systemic infection and inflammatory response following exposure to vaccinia virus (vv). il-10 acts both as an inducer of th2 responses and as a regulator of t cell activation. it has been shown to limit skin inflammation elicited by contact sensitizers. ad exacerbations have been associated with decreased il-10 function. we used il-10(-/-) mice to test the role of the cytokine in vv immunity. they exhibited l ...201324412909
cd4 t-cell memory responses to viral infections of humans show pronounced immunodominance independent of duration or viral persistence.little is known concerning immunodominance within the cd4 t-cell response to viral infections and its persistence into long-term memory. we tested cd4 t-cell reactivity against each viral protein in persons immunized with vaccinia virus (vv), either recently or more than 40 years ago, as a model self-limited viral infection. similar tests were done with persons with herpes simplex virus 1 (hsv-1) infection as a model chronic infection. we used an indirect method capable of counting the cd4 t cel ...201323255792
transcutaneous yellow fever vaccination of subjects with or without atopic dermatitis.atopic dermatitis (ad) is a common inflammatory skin disease with a global prevalence ranging from 3% to 20%. patients with ad have an increased risk for complications after viral infection (eg, herpes simplex virus), and vaccination of patients with ad with live vaccinia virus is contraindicated because of a heightened risk of eczema vaccinatum, a rare but potentially lethal complication associated with smallpox vaccination.201324331381
primary human leukocyte subsets differentially express vaccinia virus receptors enriched in lipid rafts.poxviruses, including vaccinia virus (vv) and canarypox virus (alvac), do not indiscriminately infect all cell types of the primary human leukocytes (phls) that they encounter but instead demonstrate an extremely strong bias toward infection of monocytes and monocyte lineage cells. we studied the specific molecular events that determine the vv tropism for major phl subsets including monocytes, b cells, neutrophils, nk cells, and t cells. we found that vv exhibited an extremely strong bias of cel ...201323785200
human endogenous retroviruses and cancer prevention: evidence and prospects.cancer is a significant and growing problem worldwide. while this increase may, in part, be attributed to increasing longevity, improved case notifications and risk-enhancing lifestyle (such as smoking, diet and obesity), hygiene-related factors resulting in immuno-regulatory failure may also play a major role and call for a revision of vaccination strategies to protect against a range of cancers in addition to infections.201323282240
susceptibility of the wild-derived inbred cast/ei mouse to infection by orthopoxviruses analyzed by live bioluminescence imaging.classical inbred mice are extensively used for virus research. however, we recently found that some wild-derived inbred mouse strains are more susceptible than classical strains to monkeypox virus. experiments described here indicated that the 50% lethal dose of vaccinia virus (vacv) and cowpox virus (cpxv) were two logs lower in wild-derived inbred cast/ei mice than classical inbred balb/c mice, whereas there was little difference in the susceptibility of the mouse strains to herpes simplex vir ...201324418545
human genome-wide rnai screen reveals a role for nuclear pore proteins in poxvirus morphogenesis.poxviruses are considered less dependent on host functions than other dna viruses because of their cytoplasmic site of replication and large genomes, which encode enzymes for dna and mrna synthesis. nevertheless, rnai screens with two independent human genome-scale libraries have identified more than 500 candidate genes that significantly inhibited and a similar number that enhanced replication and spread of infectious vaccinia virus (vacv). translational, ubiquitin-proteosome, and endoplasmic r ...201323401514
active vaccination with vaccinia virus a33 protects mice against lethal vaccinia and ectromelia viruses but not against cowpoxvirus; elucidation of the specific adaptive immune response.vaccinia virus protein a33 (a33vacv) plays an important role in protection against orthopoxviruses, and hence is included in experimental multi-subunit smallpox vaccines. in this study we show that single-dose vaccination with recombinant sindbis virus expressing a33vacv, is sufficient to protect mice against lethal challenge with vaccinia virus wr (vacv-wr) and ectromelia virus (ectv) but not against cowpox virus (cpxv), a closely related orthopoxvirus. moreover, a subunit vaccine based on the ...201323842430
effects of nasal or pulmonary delivered treatments with an adenovirus vectored interferon (mdef201) on respiratory and systemic infections in mice caused by cowpox and vaccinia viruses.an adenovirus 5 vector encoding for mouse interferon alpha, subtype 5 (mdef201) was evaluated for efficacy against lethal cowpox (brighton strain) and vaccinia (wr strain) virus respiratory and systemic infections in mice. two routes of mdef201 administration were used, nasal sinus (5-µl) and pulmonary (50-µl), to compare differences in efficacy, since the preferred treatment of humans would be in a relatively small volume delivered intranasally. lower respiratory infections (lri), upper respira ...201323874722
inhibition of enveloped viruses infectivity by curcumin.curcumin, a natural compound and ingredient in curry, has antiinflammatory, antioxidant, and anticarcinogenic properties. previously, we reported that curcumin abrogated influenza virus infectivity by inhibiting hemagglutination (ha) activity. this study demonstrates a novel mechanism by which curcumin inhibits the infectivity of enveloped viruses. in all analyzed enveloped viruses, including the influenza virus, curcumin inhibited plaque formation. in contrast, the nonenveloped enterovirus 71 r ...201323658730
growth inhibition of different human colorectal cancer xenografts after a single intravenous injection of oncolytic vaccinia virus glv-1h68.despite availability of efficient treatment regimens for early stage colorectal cancer, treatment regimens for late stage colorectal cancer are generally not effective and thus need improvement. oncolytic virotherapy using replication-competent vaccinia virus (vacv) strains is a promising new strategy for therapy of a variety of human cancers.201323531320
genome-wide comparison of cowpox viruses reveals a new clade related to variola virus.zoonotic infections caused by several orthopoxviruses (opv) like monkeypox virus or vaccinia virus have a significant impact on human health. in europe, the number of diagnosed infections with cowpox viruses (cpxv) is increasing in animals as well as in humans. cpxv used to be enzootic in cattle; however, such infections were not being diagnosed over the last decades. instead, individual cases of cowpox are being found in cats or exotic zoo animals that transmit the infection to humans. both ani ...201324312452
a vaccinia virus recombinant transcribing an alphavirus replicon and expressing alphavirus structural proteins leads to packaging of alphavirus infectious single cycle particles.poxviruses and alphaviruses constitute two promising viral vectors that have been used extensively as expression systems, or as vehicles for vaccine purposes. poxviruses, like vaccinia virus (vv) are well-established vaccine vectors having large insertion capacity, excellent stability, and ease of administration. in turn, replicons derived from alphaviruses like semliki forest virus (sfv) are potent protein expression and immunization vectors but stocks are difficult to produce and maintain. in ...201324130722
a systematic analysis of host factors reveals a med23-interferon-λ regulatory axis against herpes simplex virus type 1 replication.herpes simplex virus type 1 (hsv-1) is a neurotropic virus causing vesicular oral or genital skin lesions, meningitis and other diseases particularly harmful in immunocompromised individuals. to comprehensively investigate the complex interaction between hsv-1 and its host we combined two genome-scale screens for host factors (hfs) involved in virus replication. a yeast two-hybrid screen for protein interactions and a rna interference (rnai) screen with a druggable genome small interfering rna ( ...201323950709
genetic screen of a library of chimeric poxviruses identifies an ankyrin repeat protein involved in resistance to the avian type i interferon response.viruses must be able to resist host innate responses, especially the type i interferon (ifn) response. they do so by preventing the induction or activity of ifn and/or by resisting the antiviral effectors that it induces. poxviruses are no exception, with many mechanisms identified whereby mammalian poxviruses, notably, vaccinia virus (vacv), but also cowpox and myxoma viruses, are able to evade host ifn responses. similar mechanisms have not been described for avian poxviruses (avipoxviruses). ...201323427151
persistent suppression of type 1 diabetes by a multicomponent vaccine containing a cholera toxin b subunit-autoantigen fusion protein and complete freund's adjuvant.data presented here demonstrate multifunctional vaccination strategies that harness vaccinia virus mediated delivery of a gene encoding an immunoenhanced diabetes autoantigen in combination with complete freund's adjuvant (cfa) that can maintain safe and durable immunologic homeostasis in nod mice. systemic coinoculation of prediabetic mice with recombinant vaccinia virus rvv-ctb::gad and undiluted or 10-fold diluted cfa demonstrated a significant decrease in hyperglycemia and pancreatic islet i ...201324319466
coinfection by vaccinia virus and an orf virus-like parapoxvirus in an outbreak of vesicular disease in dairy cows in midwestern brazil.the current report describes an outbreak of vesicular disease affecting dairy cows in midwestern brazil in which a coinfection with 2 poxviruses-vaccinia virus (vacv) and a parapoxvirus-was demonstrated. milking cows presented vesicles, painful reddish or whitish papules, and scabby proliferative lesions in the teats and udder, in a clinical course of approximately 10-21 days. histologically, multifocal areas of moderate to severe acanthosis, spongiosis, hypergranulosis, and parakeratotic or ort ...201323404478
[research progress in the structure and fuction of orthopoxvirus host range genes].orthopoxvirus vector has a broad prospect in recombinant vaccine research, but the rarely severe side-effect impedes its development. vaccinia virus and cowpox virus of orthopoxvirus have broad host range, and they have typical host range genes as k1l, cp77 and c7l. these three genes affect host range of vaccinia virus, disturb the cell signaling pathways, suppress immune response and are related to virulence.201323895011
smallpox: can we still learn from the journey to eradication?one of the most celebrated achievements of immunology and modern medicine is the eradication of the dreaded plague smallpox. from the introduction of smallpox vaccination by edward jenner, to its popularization by louis pasteur, to the eradication effort led by donald henderson, this story has many lessons for us today, including the characteristics of the disease and vaccine that permitted its eradication, and the obviousness of the vaccine as a vector for other intractable infectious diseases. ...201323760373
comparison of host cell gene expression in cowpox, monkeypox or vaccinia virus-infected cells reveals virus-specific regulation of immune response genes.animal-borne orthopoxviruses, like monkeypox, vaccinia and the closely related cowpox virus, are all capable of causing zoonotic infections in humans, representing a potential threat to human health. the disease caused by each virus differs in terms of symptoms and severity, but little is yet know about the reasons for these varying phenotypes. they may be explained by the unique repertoire of immune and host cell modulating factors encoded by each virus. in this study, we analysed the specific ...201323425254
cowpox virus but not vaccinia virus induces secretion of cxcl1, il-8 and il-6 and chemotaxis of monocytes in vitro.orthopoxviruses are large dna viruses which can cause disease in numerous host species. today, after eradication of variola virus and the end of vaccination against smallpox, zoonotic orthopoxvirus infections are emerging as potential threat to human health. the most common causes of zoonotic orthopoxvirus infections are cowpox virus in europe, monkeypox virus in africa and vaccinia virus in south america. although all three viruses are genetically and antigenically closely related, the human di ...201323207068
prime-boost vaccination with plasmid dna followed by recombinant vaccinia virus expressing bggarp induced a partial protective immunity to inhibit babesia gibsoni proliferation in dogs.a heterologous prime-boost vaccination regime with dna and recombinant vaccinia virus (rvv) vectors expressing relevant antigens has been shown to induce effective immune responses against several infectious pathogens. in this study, we describe the effectiveness of the prime-boost strategy by immunizing dogs with a recombinant plasmid followed by vaccinia virus, both of which expressed the glutamic acid-rich protein (bggarp) of babesia gibsoni. the dogs immunized with the prime-boost regime dev ...201324338330
actin assessment in addition to specific immuno-fluorescence staining to demonstrate rickettsial growth in cell culture.rickettsiae are able to spread within infected cell mono-layers by modifying intra-cellular actin formations. the study analyzes whether a visualization of actin modifications in addition to specific immuno-fluorescence staining of rickettsiae might facilitate the proof of rickettsial growth in cell culture. cell mono-layers of vero e6 und bgm cells were infected with rickettsia honei. intra-cellular actin was fluorescence stained with tritc-(tetra-methyl-5,6-isothiocyanate)-labeled phalloidin i ...201324265939
use of a recombinant vaccinia virus expressing interferon gamma for post-exposure protection against vaccinia and ectromelia viruses.post-exposure vaccination with vaccinia virus (vacv) has been suggested to be effective in minimizing death if administered within four days of smallpox exposure. while there is anecdotal evidence for efficacy of post-exposure vaccination this has not been definitively studied in humans. in this study, we analyzed post-exposure prophylaxis using several attenuated recombinant vacv in a mouse model. a recombinant vacv expressing murine interferon gamma (ifn-γ) was most effective for post-exposure ...201324147092
genetic screen of a mutant poxvirus library identifies an ankyrin repeat protein involved in blocking induction of avian type i interferon.mammalian poxviruses, including vaccinia virus (vacv), have evolved multiple mechanisms to evade the host type i interferon (ifn) responses at different levels, with viral proteins targeting ifn induction, signaling, and antiviral effector functions. avian poxviruses (avipoxviruses), which have been developed as recombinant vaccine vectors for permissive (i.e., poultry) and nonpermissive (i.e., mammals, including humans) species, encode no obvious equivalents of any of these proteins. we show th ...201323427153
memory cd8⁺ t cells can outsource ifn-γ production but not cytolytic killing for antiviral protection.immunization with vaccinia virus (vacv), the virus comprising the smallpox vaccine, induces memory cd8(+) t cells that protect from subsequent infections with smallpox in humans or the related ectromelia virus (ectv) in mice. memory cd8(+) t cells largely mediate these effects by expanding into secondary effectors that secrete the antiviral cytokine interferon-γ (ifn-γ) and induce cytolysis via releasing factors such as perforin, which permeabilizes target cells. we show that protection from ect ...201323684306
the mature virion of ectromelia virus, a pathogenic poxvirus, is capable of intrahepatic spread and can serve as a target for delayed therapy.orthopoxviruses (opvs), which include the agent of smallpox (variola virus), the zoonotic monkeypox virus, the vaccine and zoonotic species vaccinia virus, and the mouse pathogen ectromelia virus (ectv), form two types of infectious viral particles: the mature virus (mv), which is cytosolic, and the enveloped virus (ev), which is extracellular. it is believed that mvs are required for viral entry into the host, while evs are responsible for spread within the host. following footpad infection of ...201323596297
nk cells and poxvirus infection.in recent years, our understanding of the role of natural killer (nk) cells in the response to viral infection has grown rapidly. not only do we realize viruses have many immune-evasion strategies to escape nk cell responses, but that stimulation of nk cell subsets during an antiviral response occurs through receptors seemingly geared directly at viral products and that nk cells can provide a memory response to viral pathogens. tremendous knowledge has been gained in this area through the study ...201323372568
development of specific dengue virus 2'-o- and n7-methyltransferase assays for antiviral drug screening.dengue virus (denv) protein ns5 carries two mrna cap methyltransferase (mtase) activities involved in the synthesis of a cap structure, (7me)gpppa(2'ome)-rna, at the 5'-end of the viral mrna. the methylation of the cap guanine at its n7-position (n7-mtase, (7me)gpppa-rna) is essential for viral replication. the development of high throughput methods to identify specific inhibitors of n7-mtase is hampered by technical limitations in the large scale synthesis of long capped rnas. in this work, we ...201323769894
reversible inactivation and desiccation tolerance of silicified viruses.long-distance host-independent virus dispersal is poorly understood, especially for viruses found in isolated ecosystems. to demonstrate a possible dispersal mechanism, we show that bacteriophage t4, archaeal virus sulfolobus spindle-shaped virus kamchatka, and vaccinia virus are reversibly inactivated by mineralization in silica under conditions similar to volcanic hot springs. in contrast, bacteriophage prd1 is not silicified. moreover, silicification provides viruses with remarkable desiccati ...201324109222
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