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vaccinia virus-free rescue of fluorescent replication-defective vesicular stomatitis virus and pseudotyping with puumala virus glycoproteins for use in neutralization tests.puumala virus (puuv) grows slowly in cell culture. to study antigenic properties of puuv, an amenable method for their expression would be beneficial. to achieve this, a replication-defective recombinant vesicular stomatitis virus, rvsvδg*egfp, was rescued using bsrt7/5 and encephalomyocarditis virus (emcv) internal ribosomal entry site (ires)-enabled rescue plasmids. using these particles, pseudotypes bearing puuv sotkamo strain glycoproteins were produced, with titres in the range 105-108, and ...201626916544
poxvirus uracil-dna glycosylase-an unusual member of the family i uracil-dna glycosylases.uracil-dna glycosylases are ubiquitous enzymes, which play a key role repairing damages in dna and in maintaining genomic integrity by catalyzing the first step in the base excision repair pathway. within the superfamily of uracil-dna glycosylases family i enzymes or ungs are specific for recognizing and removing uracil from dna. these enzymes feature conserved structural folds, active site residues and use common motifs for dna binding, uracil recognition and catalysis. within this family the e ...201627684934
structural analysis of point mutations at the vaccinia virus a20/d4 interface.the vaccinia virus polymerase holoenzyme is composed of three subunits: e9, the catalytic dna polymerase subunit; d4, a uracil-dna glycosylase; and a20, a protein with no known enzymatic activity. the d4/a20 heterodimer is the dna polymerase cofactor, the function of which is essential for processive dna synthesis. the recent crystal structure of d4 bound to the first 50 amino acids of a20 (d4/a201-50) revealed the importance of three residues, forming a cation-π interaction at the dimerization ...201627599859
position on reproductive donors and smallpox vaccine: a committee opinion.although there is presently no definitive evidence linking vaccinia virus transmission through reproductive cells, the society for assisted reproductive technology (sart) and the american society for reproductive medicine (asrm) accordingly recommend that assisted reproductive technology (art) practitioners consider deferring individuals who are planning on donating gametes for reproductive use (reproductive donors) who have recently received smallpox vaccine or contracted symptomatic vaccinia v ...201626829211
occurrence of pseudocowpox virus associated to bovine viral diarrhea virus-1, brazilian amazon.in 2011, an outbreak of severe vesicular disease occurred in the state of pará, amazon region. besides proliferative or verrucous lesions, cattle showed atypical clinical signs such as diarrhea and leading to death. the animals were submitted to clinical, pathological and molecular diagnosis, and laboratory tests have confirmed the presence of pseudocowpox virus (pcpv), a parapoxvirus genus member, and have also found bovine viral diarrhea virus-1 (bvdv-1), probably causing persistent infection. ...201627865267
an orthopoxvirus-based vaccine reduces virus excretion after mers-cov infection in dromedary camels.middle east respiratory syndrome coronavirus (mers-cov) infections have led to an ongoing outbreak in humans, which was fueled by multiple zoonotic mers-cov introductions from dromedary camels. in addition to the implementation of hygiene measures to limit further camel-to-human and human-to-human transmissions, vaccine-mediated reduction of mers-cov spread from the animal reservoir may be envisaged. here we show that a modified vaccinia virus ankara (mva) vaccine expressing the mers-cov spike p ...201626678878
development and virucidal activity of a novel alcohol-based hand disinfectant supplemented with urea and citric acid.hand disinfectants are important for the prevention of virus transmission in the health care system and environment. the development of broad antiviral spectrum hand disinfectants with activity against enveloped and non-enveloped viruses is limited due to a small number of permissible active ingredients able to inactivate viruses.201626864562
evaluation of quantum dot immunofluorescence and a digital cmos imaging system as an alternative to conventional organic fluorescence dyes and laser scanning for quantifying protein microarrays.organic fluorescent dyes are widely used for the visualization of bound antibody in a variety of immunofluorescence assays. however, the detection equipment is often expensive, fragile, and hard to deploy widely. quantum dots (qdot) are nanocrystals made of semiconductor materials that emit light at different wavelengths according to the size of the crystal, with increased brightness and stability. here, we have evaluated a small benchtop "personal" optical imager (arraycam) developed for quanti ...201626842269
live-vaccinia virus encapsulation in ph-sensitive polymer increases safety of a reservoir-targeted lyme disease vaccine by targeting gastrointestinal release.the incidence of lyme disease has continued to rise despite attempts to control its spread. vaccination of zoonotic reservoirs of human pathogens has been successfully used to decrease the incidence of rabies in raccoons and foxes. we have previously reported on the efficacy of a vaccinia virus vectored vaccine to reduce carriage of borrelia burgdorferi in reservoir mice and ticks. one potential drawback to vaccinia virus vectored vaccines is the risk of accidental infection of humans. to reduce ...201627502570
the ep152r orf of african swine fever virus strain georgia encodes for an essential gene that interacts with host protein bag6.african swine fever virus (asfv) is the etiological agent of a contagious and often lethal disease of domestic pigs that has significant economic consequences for the swine industry. the viral genome encodes for more than 150 genes, and only a select few of these genes have been studied in some detail. here we report the characterization of open reading frame ep152r that has a predicted complement control module/scr domain. this domain is found in vaccinia virus proteins that are involved in blo ...201627497620
response of three different viruses to interferon priming and dithiothreitol treatment of avian cells.we have previously shown that the replication of avian reovirus (arv) in chicken cells is much more resistant to interferon (ifn) than the replication of vesicular stomatitis virus (vsv) or vaccinia virus (vv). in this study, we have investigated the role that the double-stranded rna (dsrna)-activated protein kinase (pkr) plays in the sensitivity of these three viruses toward the antiviral action of chicken interferon. our data suggest that while interferon priming of avian cells blocks vaccinia ...201627440902
interferon induction by avian reovirus.we have previously shown that the replication of avian reovirus (arv) in chicken embryo fibroblasts (cef) is more resistant to the antiviral action of interferon (ifn) than the replication of vesicular stomatitis virus (vsv) or vaccinia virus (vv). in this study we examined the capacity of these three viruses to induce the expression of ifn when infecting avian cells. efficient expression of both type-α and type-β ifns, as well as of the double-stranded rna (dsrna)-activated protein kinase (pkr) ...201626517397
engineered nanoconstructs for the multiplexed and sensitive detection of high-risk pathogens.many countries categorize the causative agents of severe infectious diseases as high-risk pathogens. given their extreme infectivity and potential to be used as biological weapons, a rapid and sensitive method for detection of high-risk pathogens (e.g., bacillus anthracis, francisella tularensis, yersinia pestis, and vaccinia virus) is highly desirable. here, we report the construction of a novel detection platform comprising two units: (1) magnetic beads separately conjugated with multiple capt ...201626462853
the vaccinia virus h3 envelope protein, a major target of neutralizing antibodies, exhibits a glycosyltransferase fold and binds udp-glucose.the highly conserved h3 poxvirus protein is a major target of the human antibody response against poxviruses and is likely a key contributor to protection against infection. here, we present the crystal structure of h3 from vaccinia virus at a 1.9-å resolution. h3 looks like a glycosyltransferase, a family of enzymes that transfer carbohydrate molecules to a variety of acceptor substrates. like glycosyltransferases, h3 binds udp-glucose, as shown by saturation transfer difference (std) nuclear m ...201626937025
pkr transduces mda5-dependent signals for type i ifn induction.sensing invading pathogens early in infection is critical for establishing host defense. two cytosolic rig-like rna helicases, rig-i and mda5, are key to type i interferon (ifn) induction in response to viral infection. mounting evidence suggests that another viral rna sensor, protein kinase r (pkr), may also be critical for ifn induction during infection, although its exact contribution and mechanism of action are not completely understood. using pkr-deficient cells, we found that pkr was requi ...201626939124
detection of vaccinia virus in dairy cattle serum samples from 2009, uruguay.we detected orthopoxvirus in 28 of 125 serum samples collected during 2009 from cattle in uruguay. two samples were pcr-positive for vaccinia virus and had sequences similar to those for vaccinia virus associated with outbreaks in brazil. autochthonous circulation of vaccinia virus in uruguay and other south american countries cannot be ruled out.201627869601
rapid and sensitive point-of-care detection of orthopoxviruses by abicap immunofiltration.the rapid and reliable detection of infectious agents is one of the most challenging tasks in scenarios lacking well-equipped laboratory infrastructure, like diagnostics in rural areas of developing countries. commercially available point-of-care diagnostic tests for emerging and rare diseases are particularly scarce.201627938377
domain organization of vaccinia virus helicase-primase d5.poxviridae are viruses with a large linear double-stranded dna genome coding for up to 250 open reading frames and a fully cytoplasmic replication. the double-stranded dna genome is covalently circularized at both ends. similar structures of covalently linked extremities of the linear dna genome are found in the african swine fever virus (asfarvirus) and in the phycodnaviridae we are studying the machinery which replicates this peculiar genome structure. from our work with vaccinia virus, we giv ...201626912611
destabilization of α-helical structure in solution improves bactericidal activity of antimicrobial peptides: opposite effects on bacterial and viral targets.we have previously examined the mechanism of antimicrobial peptides on the outer membrane of vaccinia virus. we show here that the formulation of peptides ll37 and magainin-2b amide in polysorbate 20 (tween 20) results in greater reductions in virus titer than formulation without detergent, and the effect is replicated by substitution of polysorbate 20 with high-ionic-strength buffer. in contrast, formulation with polysorbate 20 or high-ionic-strength buffer has the opposite effect on bactericid ...201626824944
suppression of kaposi's sarcoma-associated herpesvirus infection and replication by 5'-amp-activated protein kinase.the host intracellular antiviral restriction factors inhibit viral infection and replication. the 5'-amp-activated protein kinase (ampk) is a cellular energy sensor regulating metabolic homeostasis. activated ampk inhibits the replication of numerous rna viruses but enhances the entry of vaccinia virus. however, the role of ampk in herpesvirus infection is unclear. in this study, we showed that the constitutive ampk activity restricted kaposi's sarcoma-associated herpesvirus (kshv) lytic replica ...201627147746
high-throughput screening to enhance oncolytic virus immunotherapy.high-throughput screens can rapidly scan and capture large amounts of information across multiple biological parameters. although many screens have been designed to uncover potential new therapeutic targets capable of crippling viruses that cause disease, there have been relatively few directed at improving the efficacy of viruses that are used to treat disease. oncolytic viruses (ovs) are biotherapeutic agents with an inherent specificity for treating malignant disease. certain ov platforms - i ...201627579293
activation of rnase l is dependent on oas3 expression during infection with diverse human viruses.the 2',5'-oligoadenylate (2-5a) synthetase (oas)-rnase l system is an ifn-induced antiviral pathway. rnase l activity depends on 2-5a, synthesized by oas. although all three enzymatically active oas proteins in humans--oas1, oas2, and oas3--synthesize 2-5a upon binding dsrna, it is unclear which are responsible for rnase l activation during viral infection. we used clustered regularly interspaced short palindromic repeats (crispr)-crispr-associated protein-9 nuclease (cas9) technology to enginee ...201626858407
use of reporter genes in the generation of vaccinia virus-derived vectors.vaccinia virus (vacv) is one of the most extensively-studied viruses of the poxviridae family. it is easy to genetically modify, so it has become a key tool for many applications. in this context, reporter genes facilitate the study of the role of foreign genes introduced into the genome of vacv. in this review, we describe the type of reporter genes that have been used to generate reporter-expressing vacv and the applications of the recombinant viruses obtained. reporter-expressing vacv are cur ...201627213433
myxoma virus m156 is a specific inhibitor of rabbit pkr but contains a loss-of-function mutation in australian virus isolates.myxoma virus (myxv) is a rabbit-specific poxvirus, which is highly virulent in european rabbits. the attenuation of myxv and the increased resistance of rabbits following the release of myxv in australia is one of the best-documented examples of host-pathogen coevolution. to elucidate the molecular mechanisms that contribute to the restriction of myxv infection to rabbits and myxv attenuation in the field, we have studied the interaction of the myxv protein m156 with the host antiviral protein k ...201626903626
antitumor effect of the livp-gfp recombinant vaccinia virus. 201623975469
oncolytic virotherapy for pediatric malignancies: future prospects.pediatric solid tumors remain a major health concern, with nearly 16,000 children diagnosed each year. of those, ~2,000 succumb to their disease, and survivors often suffer from lifelong disability secondary to toxic effects of current treatments. countless multimodality treatment regimens are being explored to make advances against this deadly disease. one targeted treatment approach is oncolytic virotherapy. conditionally replicating viruses can infect tumor cells while leaving normal cells un ...201627579298
silk-elastin-like protein polymer matrix for intraoperative delivery of an oncolytic vaccinia virus.oncolytic viral efficacy may be limited by the penetration of the virus into tumors. this may be enhanced by intraoperative application of virus immediately after surgical resection.201625244076
thymic stromal lymphopoietin and il-33 promote skin inflammation and vaccinia virus replication in a mouse model of atopic dermatitis. 201626830114
engineering of double recombinant vaccinia virus with enhanced oncolytic potential for solid tumor virotherapy.vaccinia virus (vacv) oncolytic therapy has been successful in a number of tumor models. in this study our goal was to generate a double recombinant vaccinia virus (vv-gmcsf-lact) with enhanced antitumor activity that expresses exogenous proteins: the antitumor protein lactaptin and human granulocyte-macrophage colony-stimulating factor (gm-csf). lactaptin has previously been demonstrated to act as a tumor suppressor in mouse hepatoma as well as mda-mb-231 human adenocarcinoma cells grafted into ...201627708236
npf motifs in the vaccinia virus protein a36 recruit intersectin-1 to promote cdc42:n-wasp-mediated viral release from infected cells.during its egress, vaccinia virus transiently recruits ap-2 and clathrin after fusion with the plasma membrane. this recruitment polarizes the viral protein a36 beneath the virus, enhancing actin polymerization and the spread of infection. we now demonstrate that three npf motifs in the c-terminus of a36 recruit ap-2 and clathrin by interacting directly with the epsin15 homology domains of eps15 and intersectin-1. a36 is the first identified viral npf motif containing protein shown to interact w ...201627670116
dok-1 and dok-2 regulate the formation of memory cd8+ t cells.diverse signals received by cd8(+) t cells are integrated to achieve the required magnitude of cell expansion and the appropriate balance of effector/memory cd8(+) t cell generation. notably, the strength and nature of tcr signaling influence the differentiation and functional capacity of effector and memory cd8(+) t cells. dok-1 and dok-2, the two members of the dok family expressed in t cells, negatively regulate tcr signaling in vitro. however, the role of dok proteins in modulating t cell fu ...201627664281
opposing roles of double-stranded rna effector pathways and viral defense proteins revealed with crispr-cas9 knockout cell lines and vaccinia virus mutants.vaccinia virus (vacv) decapping enzymes and cellular exoribonuclease xrn1 catalyze successive steps in mrna degradation and prevent double-stranded rna (dsrna) accumulation, whereas the viral e3 protein can bind dsrna. we showed that dsrna and e3 colocalized within cytoplasmic viral factories in cells infected with a decapping enzyme mutant as well as with wild-type vacv and that they coprecipitated with antibody. an e3 deletion mutant induced protein kinase r (pkr) and eukaryotic translation in ...201627334583
multiple bcl-2 family immunomodulators from vaccinia virus regulate mapk/ap-1 activation.vaccinia virus (vacv) is a poxvirus and encodes many proteins that modify the host cell metabolism or inhibit the host response to infection. for instance, it is known that vacv infection can activate the mitogen-activated protein kinase (mapk)/activator protein 1 (ap-1) pathway and inhibit activation of the pro-inflammatory transcription factor nf-κb. since nf-κb and mapk/ap-1 share common upstream activators we investigated whether six different vacv bcl-2-like nf-κb inhibitors can also influe ...201627312213
single-particle characterization of oncolytic vaccinia virus by flow virometry.vaccinia virus (vv) is an oncolytic virus that is currently being evaluated as a promising cancer vaccine in several phase i, ii and iii clinical trials. although several quality control tests are performed on each new batch of virus, these do not routinely include a systematic characterization of virus particle homogeneity, or relate the infectious titer to the total number of submicron sized particles (ssps) present in the sample. ssps are comprised of infectious virus and non-infectious viral ...201627614781
vaccinia virus uses retromer-independent cellular retrograde transport pathways to facilitate the wrapping of intracellular mature virions during viral morphogenesis.poxviruses such as vaccinia virus (vacv) undertake a complex cytoplasmic replication cycle which involves morphogenesis through four distinct virion forms, and includes a crucial "wrapping" step whereby intracellular mature virions (imvs) are wrapped in two additional membranes to form intracellular enveloped virions (ievs). to determine if cellular retrograde transport pathways were required for this wrapping step we examined vacv morphogenesis in cells with reduced expression of the tetrameric ...201627581988
expression of dai by an oncolytic vaccinia virus boosts the immunogenicity of the virus and enhances antitumor immunity.in oncolytic virotherapy, the ability of the virus to activate the immune system is a key attribute with regard to long-term antitumor effects. vaccinia viruses bear one of the strongest oncolytic activities among all oncolytic viruses. however, its capacity for stimulation of antitumor immunity is not optimal, mainly due to its immunosuppressive nature. to overcome this problem, we developed an oncolytic vv that expresses intracellular pattern recognition receptor dna-dependent activator of ifn ...201627626058
vanilloid receptor 1 agonists, capsaicin and resiniferatoxin, enhance mhc class i-restricted viral antigen presentation in virus-infected dendritic cells.dcs, like the sensory neurons, express vanilloid receptor 1 (vr1). here we demonstrate that the vr1 agonists, capsaicin (cp) and resiniferatoxin (rtx), enhance antiviral ctl responses by increasing mhc class i-restricted viral antigen presentation in dendritic cells (dcs). bone marrow-derived dcs (bm-dcs) were infected with a recombinant vaccinia virus (vv) expressing ova (vv-ova), and then treated with cp or rtx. both cp and rtx increased mhc class i-restricted presentation of virus-encoded end ...201627574502
protein primary structure of the vaccinia virion at increased resolutionhere we examine the protein covalent structure of the vaccinia virus virion. within two virion preparations, >88% of the theoretical vaccinia virus-encoded proteome was detected with high confidence, including the first detection of products from 27 open reading frames (orfs) previously designated "predicted," "uncharacterized," "inferred," or "hypothetical" polypeptides containing as few as 39 amino acids (aa) and six proteins whose detection required nontryptic proteolysis. we also detected th ...201627558425
polymeric cups for cavitation-mediated delivery of oncolytic vaccinia virus.oncolytic viruses (ov) could become the most powerful and selective cancer therapies. however, the limited transport of ov into and throughout tumors following intravenous injection means their clinical administration is often restricted to direct intratumoral dosing. application of physical stimuli, such as focused ultrasound, offers a means of achieving enhanced mass transport. in particular, shockwaves and microstreaming resulting from the instigation of an ultrasound-induced event known as i ...201627375160
artificial ribonucleases inactivate a wide range of viruses using their ribonuclease, membranolytic, and chaotropic-like activities.artificial ribonucleases (arnases) are small compounds catalysing rna cleavage. recently we demonstrated that arnases readily inactivate various viruses in vitro. here, for three series of arnases (1,4-diazabicyclo [2.2.2]octane-based and peptide-like compounds) we show that apart from ribonuclease activity the arnases display chaotropic-like and membranolytic activities. the levels of membranolytic and chaotropic-like activities correlate well with the efficiency of various viruses inactivation ...201627476043
vaccinia virus dissemination requires p21-activated kinase 1.the orthopoxvirus vaccinia virus (vacv) interacts with both actin and microtubule cytoskeletons in order to generate and spread progeny virions. here, we present evidence demonstrating the involvement of pak1 (p21-activated kinase 1) in the dissemination of vacv. although pak1 activation has previously been associated with optimal vacv entry via macropinocytosis, its absence does not affect the production of intracellular mature virions (imvs) and extracellular enveloped virions (eevs). our data ...201627465567
oncolytic vaccinia virus inhibits human hepatocellular carcinoma mhcc97-h cell proliferation via endoplasmic reticulum stress, autophagy and wnt pathways.hepatocellular carcinoma (hcc) is a highly lethal malignancy. vaccinia virus (vv) possessed many inherent advantages with respect to being engineered as a vector for cancer gene therapy, although the mechanism of action remains to be explored further.201627441866
virusmapper: open-source nanoscale mapping of viral architecture through super-resolution microscopy.the nanoscale molecular assembly of mammalian viruses during their infectious life cycle remains poorly understood. their small dimensions, generally bellow the 300nm diffraction limit of light microscopes, has limited most imaging studies to electron microscopy. the recent development of super-resolution (sr) light microscopy now allows the visualisation of viral structures at resolutions of tens of nanometers. in addition, these techniques provide the added benefit of molecular specific labell ...201627374400
a general method for rapid and cost-efficient large-scale production of 5' capped rna.the eukaryotic mrna 5' cap structure is indispensible for pre-mrna processing, mrna export, translation initiation, and mrna stability. despite this importance, structural and biophysical studies that involve capped rna are challenging and rare due to the lack of a general method to prepare mrna in sufficient quantities. here, we show that the vaccinia capping enzyme can be used to produce capped rna in the amounts that are required for large-scale structural studies. we have therefore designed ...201627368341
a simple and efficient approach to construct mutant vaccinia virus vectors.the crispr-associated endonuclease cas9 can edit particular genomic loci directed by a single guide rna (grna). the crispr/cas9 system has been successfully employed for editing genomes of various organisms. here we describe a protocol for editing the vaccinia virus (vv) genome in the cytoplasm of vv-infected cv-1 cells using the rna-guided cas9. rna-guided cas9 induces double-stranded dna breaks facilitating homologous recombination efficiently and specifically in the targeted site of vv and a ...201627842362
a conserved tripeptide sequence at the c terminus of the poxvirus dna processivity factor d4 is essential for protein integrity and function.vaccinia virus (vacv) is a poxvirus, and the vacv d4 protein serves both as a uracil-dna glycosylase and as an essential component required for processive dna synthesis. the vacv a20 protein has no known catalytic function itself but associates with d4 to form the d4-a20 heterodimer that functions as the poxvirus dna processivity factor. the heterodimer enables the dna polymerase to efficiently synthesize extended strands of dna. upon characterizing the interaction between d4 and a20, we observe ...201627836975
genome sequence of vaccinia virus strain lister-butantan, a lister vaccine variant used during a smallpox eradication campaign in brazil.here, we report the 187.8-kb genome sequence of vaccinia virus lister-butantan, which was used in brazil during the who smallpox eradication campaign. its genome showed an average similarity of 98.18% with the original lister isolate, highlighting the low divergence among related vaccinia virus vaccine strains, even after several passages in animals and cell culture.201627340056
oncolytic vaccinia virus combined with radiotherapy induces apoptotic cell death in sarcoma cells by down-regulating the inhibitors of apoptosis.advanced extremity melanoma and sarcoma present a significant therapeutic challenge, requiring multimodality therapy to treat or even palliate disease. these aggressive tumours are relatively chemo-resistant, therefore new treatment approaches are urgently required. we have previously reported on the efficacy of oncolytic virotherapy (ov) delivered by isolated limb perfusion. in this report, we have improved therapeutic outcomes by combining ov with radiotherapy. in vitro, the combination of onc ...201627783991
oncolytic vaccinia virus as a vector for therapeutic sodium iodide symporter gene therapy in prostate cancer.oncolytic strains of vaccinia virus are currently in clinical development with clear evidence of safety and promising signs of efficacy. addition of therapeutic genes to the viral genome may increase the therapeutic efficacy of vaccinia. we evaluated the therapeutic potential of vaccinia virus expressing the sodium iodide symporter (nis) in prostate cancer models, combining oncolysis, external beam radiotherapy and nis-mediated radioiodide therapy. the nis-expressing vaccinia virus (vv-nis), glv ...201626814609
dogs and opossums positive for vaccinia virus during outbreak affecting cattle and humans, são paulo state, brazil.during a vaccinia virus (vacv) outbreak in são paulo state, brazil, blood samples were collected from cows, humans, other domestic animals, and wild mammals. samples from 3 dogs and 3 opossums were positive for vacv by pcr. results of gene sequencing yielded major questions regarding other mammalian species acting as reservoirs of vacv.201626812352
oncolytic vaccine virus harbouring the il-24 gene suppresses the growth of lung cancer by inducing apoptosis.lung cancer has an especially high incidence rate worldwide, and its resistance to cell death and chemotherapeutic drugs increases its intractability. the vaccinia virus has been shown to destroy neoplasm within a short time and disseminate rapidly and extensively as an enveloped virion throughout the circulatory system, and this virus has also demonstrated a strong ability to overexpress exogenous genes. interleukin-24 (il-24/mda-7) is an important cytokine that belongs to the activating caspas ...201627208781
phase 1 study of intravenous oncolytic poxvirus (vvdd) in patients with advanced solid cancers.we have conducted a phase 1 study of intravenous vvdd, a western reserve strain oncolytic vaccinia virus, on 11 patients with standard treatment-refractory advanced colorectal or other solid cancers. the primary endpoints were maximum tolerated dose and associated toxicity while secondary endpoints were pharmacokinetics, pharmacodynamics, immune responses, and antitumor activity. no dose-limiting toxicities and treatment related severe adverse events were observed. the most common adverse events ...201627203445
nucleoside triphosphate phosphohydrolase i (nph i) functions as a 5' to 3' translocase in transcription termination of vaccinia early genes.vaccinia virus early genes are transcribed immediately upon infection. nucleoside triphosphate phosphohydrolase i (nph i) is an essential component of the early gene transcription complex. nph i hydrolyzes atp to release transcripts during transcription termination. the atpase activity of nph i requires single-stranded (ss) dna as a cofactor; however, the source of this cofactor within the transcription complex is not known. based on available structures of transcription complexes it has been hy ...201627189950
impaired innate, humoral, and cellular immunity despite a take in smallpox vaccine recipients.smallpox vaccine is highly effective, inducing protective immunity to smallpox and diseases caused by related orthopoxviruses. smallpox vaccine efficacy was historically defined by the appearance of a lesion or "take" at the vaccine site, which leaves behind a characteristic scar. both the take and scar are readily recognizable and were used during the eradication effort to indicate successful vaccination and to categorize individuals as "protected." however, the development of a typical vaccine ...201627177944
complete genome sequence of vaccinia virus strain l-ivp.most of the live vaccine doses of vaccinia virus donated to the intensified smallpox eradication programme after 1971 were prepared using the l-ivp strain. a mixture of three clones of the l-ivp strain was sequenced using myseq. consensus sequence similarity with the vaccinia virus lister strain is 99.5%.201627174282
both cd8+ and cd4+ t cells contribute to corneal clouding and viral clearance following vaccinia virus infection in c57bl/6 mice.vaccinia virus (vacv) keratitis is a serious complication following smallpox vaccination and can lead to blindness. the pathological mechanisms involved in ocular vacv infection are poorly understood. previous studies have used rabbits, but the lack of immune reagents and transgenic or knockout animals makes them less suitable for mechanistic studies. we report that infection of c57bl/6 mice with 1 × 10(7) pfu of vaccinia virus strain wr results in blepharitis, corneal neovascularization, and st ...201627170749
an improved high pressure freezing and freeze substitution method to preserve the labile vaccinia virus nucleocapsid.in recent years, high pressure freezing and freeze substitution have been widely used for electron microscopy to reveal viral and cellular structures that are difficult to preserve. vaccinia virus, a member of the poxviridae family, presents one of the most complex viral structures. the classical view of vaccinia virus structure consists of an envelope surrounding a biconcave core, with a lateral body in each concavity of the core. this classical view was challenged by peters and muller (1963), ...201627155322
oncolytic and immunologic cancer therapy with gm-csf-armed vaccinia virus of tian tan strain guang9.targeted oncolytic vaccinia viruses are being developed as a novel strategy in cancer therapy. arming vaccinia viruses with immunostimulatory cytokines can enhance antitumor efficacy. such engineered oncolytic viruses, like jx-594, a wyeth strain vaccinia virus modified with human granulocyte-macrophage colony-stimulating factor (gm-csf), have shown promising results and have proceeded rapidly in clinical trials. however, the oncolytic potential of the chinese vaccine strain tian tan (vtt) has n ...201626803055
features of the antitumor effect of vaccinia virus lister strain.oncolytic abilities of vaccinia virus (vacv) served as a basis for the development of various recombinants for treating cancer; however, "natural" oncolytic properties of the virus are not examined in detail. our study was conducted to know how the genetically unmodified l-ivp strain of vacv produces its antitumor effect. human a431 carcinoma xenografts in nude mice and murine ehrlich carcinoma in c57bl mice were used as targets for vacv, which was injected intratumorally. a set of virological m ...201626771631
protection against lethal vaccinia virus infection in mice using an sirna targeting the a5r gene.although the world health organization has declared the eradication of smallpox in 1980, the fear of its potential use in bioterrorism has become a reality. since the effectiveness of current vaccines and antiviral drugs is limited, development of new therapeutic strategies is needed. in this study, we investigated small interfering rna (sirna) as a therapeutic approach for preventing and treating smallpox infection.201626741698
crispr-cas9 as a powerful tool for efficient creation of oncolytic viruses.the development of oncolytic viruses has led to an emerging new class of cancer therapeutics. although the safety profile has been encouraging, the transition of oncolytic viruses to the clinical setting has been a slow process due to modifications. therefore, a new generation of more potent oncolytic viruses needs to be exploited, following our better understanding of the complex interactions between the tumor, its microenvironment, the virus, and the host immune response. the conventional meth ...201626959050
a cancer-favoring oncolytic vaccinia virus shows enhanced suppression of stem-cell like colon cancer.stem cell-like colon cancer cells (sccs) pose a major challenge in colon cancer treatment because of their resistance to chemotherapy and radiotherapy. oncolytic virus-based therapy has shown promising results in uncured cancer patients; however, its effects on sccs are not well studied yet. here, we engineered a cancer-favoring oncolytic vaccinia virus (cvv) as a potent biotherapeutic and investigated its therapeutic efficacy in terms of killing sccs. cvv is an evolved wyeth strain vaccinia vir ...201626918725
cd40 ligand-expressing recombinant vaccinia virus promotes the generation of cd8(+) central memory t cells.central memory cd8(+) t cells (tcm ) play key roles in the protective immunity against infectious agents, cancer immunotherapy, and adoptive treatments of malignant and viral diseases. cd8(+) tcm cells are characterized by specific phenotypes, homing, and proliferative capacities. however, cd8(+) tcm -cell generation is challenging, and usually requires cd4(+) cd40l(+) t-cell "help" during the priming of naïve cd8(+) t cells. we have generated a replication incompetent cd40 ligand-expressing rec ...201626561341
microbiota is an essential element for mice to initiate a protective immunity against vaccinia virus.the gastrointestinal tract of vertebrates harbors one of the most complex ecosystems known in microbial ecology and this indigenous microbiota almost always has a profound influence on host-parasite relationships, which can enhance or reduce the pathology of the infection. in this context, the impact of the microbiota during the infection of several viral groups remains poorly studied, including the family poxviridae. vaccinia virus (vacv) is a member of this family and is the causative agent of ...201626610433
integration of linear and dendritic actin nucleation in nck-induced actin comets.the nck adaptor protein recruits cytosolic effectors such as n-wasp that induce localized actin polymerization. experimental aggregation of nck sh3 domains at the membrane induces actin comet tails--dynamic, elongated filamentous actin structures similar to those that drive the movement of microbial pathogens such as vaccinia virus. here we show that experimental manipulation of the balance between unbranched/branched nucleation altered the morphology and dynamics of nck-induced actin comets. in ...201626609071
biochemical analysis of the multifunctional vaccinia mrna capping enzyme encoded by a temperature sensitive virus mutant.prior biochemical analysis of the heterodimeric vaccinia virus mrna capping enzyme suggests roles not only in mrna capping but also in early viral gene transcription termination and intermediate viral gene transcription initiation. prior phenotypic characterization of dts36, a temperature sensitive virus mutant affecting the large subunit of the capping enzyme was consistent with the multifunctional roles of the capping enzyme in vivo. we report a biochemical analysis of the capping enzyme encod ...201626496697
cd4(+) t-cell dependence of primary cd8(+) t-cell response against vaccinia virus depends upon route of infection and viral dose.cd4(+) t-cell help (cd4 help) plays a pivotal role in cd8(+) t-cell responses against viral infections. however, the role in primary cd8(+) t-cell responses remains controversial. we evaluated the effects of infection route and viral dose on primary cd8(+) t-cell responses to vaccinia virus (vacv) in mhc class ii(-/-) mice. cd4 help deficiency diminished the generation of vacv-specific cd8(+) t cells after intraperitoneal (i.p.) but not after intranasal (i.n.) infection. a large viral dose could ...201625544501
structure-function analysis of vaccinia virus h7 protein reveals a novel phosphoinositide binding fold essential for poxvirus replication.phosphoinositides and phosphoinositide binding proteins play a critical role in membrane and protein trafficking in eukaryotes. their critical role in replication of cytoplasmic viruses has just begun to be understood. poxviruses, a family of large cytoplasmic dna viruses, rely on the intracellular membranes to develop their envelope, and poxvirus morphogenesis requires enzymes from the cellular phosphoinositide metabolic pathway. however, the role of phosphoinositides in poxvirus replication re ...201525473060
phase 1 study of intratumoral pexa-vec (jx-594), an oncolytic and immunotherapeutic vaccinia virus, in pediatric cancer patients.pexa-vec (pexastimogene devacirepvec, jx-594) is an oncolytic and immunotherapeutic vaccinia virus designed to destroy cancer cells through viral lysis and induction of granulocyte-macrophage colony-stimulating factor (gm-csf)-driven tumor-specific immunity. pexa-vec has undergone phase 1 and 2 testing alone and in combination with other therapies in adult patients, via both intratumoral and intravenous administration routes. we sought to determine the safety of intratumoral administration in pe ...201525531693
mcl1 enhances the survival of cd8+ memory t cells after viral infection.viral infection results in the generation of massive numbers of activated effector cd8(+) t cells that recognize viral components. most of these are short-lived effector t cells (slecs) that die after clearance of the virus. however, a small proportion of this population survives and forms antigen-specific memory precursor effector cells (mpecs), which ultimately develop into memory cells. these can participate in a recall response upon reexposure to antigen even at protracted times postinfectio ...201525505074
a polymeric conjugate foreignizing tumor cells for targeted immunotherapy in vivo.antigen-specific cd8(+) cytotoxic t lymphocytes (ctls) are key elements of immunological rejection in transplantation as well as cancer immunotherapy. most tumors, however, are not immunologically rejected because they have self antigens, which are not recognized as the foreigner by ctls. in this study, we hypothesized that "foreignizing" tumor cells by delivering non-self foreign antigens into the tumors would result in rejection by foreign antigen-reactive ctls. as the model system to foreigni ...201525499555
programmed necrosis in the cross talk of cell death and inflammation.cell proliferation and cell death are integral elements in maintaining homeostatic balance in metazoans. disease pathologies ensue when these processes are disturbed. a plethora of evidence indicates that malfunction of cell death can lead to inflammation, autoimmunity, or immunodeficiency. programmed necrosis or necroptosis is a form of nonapoptotic cell death driven by the receptor interacting protein kinase 3 (ripk3) and its substrate, mixed lineage kinase domain-like (mlkl). ripk3 partners w ...201525493335
rab1a promotes vaccinia virus replication by facilitating the production of intracellular enveloped virions.vaccinia virus (vacv) is a large double-stranded dna virus with a complex cytoplasmic replication cycle that exploits numerous cellular proteins. this work characterises the role of a proviral cellular protein, the small gtpase rab1a, in vacv replication. using sirna, we identified rab1a as required for the production of extracellular enveloped virions (eevs), but not intracellular mature virions (imvs). immunofluorescence and electron microscopy further refined the role of rab1a as facilitating ...201525462347
neurotropin promotes ngf signaling through interaction of gm1 ganglioside with trk neurotrophin receptor in pc12 cells.activation of the high-affinity nerve growth factor (ngf) receptor trk occurs through multiple processes consisted of translocation and clustering within the plasma membrane lipid rafts, dimerization and autophosphorylation. here we found that a nonprotein extract of inflamed rabbit skin inoculated with vaccinia virus (neurotropin(®)) enhanced efficiency of ngf signaling. in rat pheochromocytoma pc12 cells overexpressing trk (pctrk cells), neurotropin augmented insufficient neurite outgrowth obs ...201525454796
a vaccinia virus armed with interleukin-10 is a promising therapeutic agent for treatment of murine pancreatic cancer.vaccinia virus has strong potential as a novel therapeutic agent for treatment of pancreatic cancer. we investigated whether arming vaccinia virus with interleukin-10 (il10) could enhance the antitumor efficacy with the view that il10 might dampen the host immunity to the virus, increasing viral persistence, thus maximizing the oncolytic effect and antitumor immunity associated with vaccinia virus.201525416195
fine structure of the vaccinia virion determined by controlled degradation and immunolocalization.the vaccinia virion is a membraned, slightly flattened, barrel-shaped particle, with a complex internal structure featuring a biconcave core flanked by lateral bodies. although the architecture of the purified mature virion has been intensely characterized by electron microscopy, the distribution of the proteins within the virion has been examined primarily using biochemical procedures. thus, it has been shown that non-ionic and ionic detergents combined or not with a sulfhydryl reagent can be u ...201525486587
tailored immunity by skin antigen-presenting cells.skin vaccination aims at targeting epidermal and dermal antigen-presenting cells (apcs), indeed many subsets of different origin endowed with various functions populate the skin. the idea that the skin could represent a particularly potent site to induce adaptive and protective immune response emerged after the success of vaccinia virus vaccination by skin scarification. recent advances have shown that multiple subsets of apcs coexist in the skin and participate in immunity to infectious disease ...201525483512
vaccinia virus protein a49 is an unexpected member of the b-cell lymphoma (bcl)-2 protein family.vaccinia virus (vacv) encodes several proteins that inhibit activation of the proinflammatory transcription factor nuclear factor κb (nf-κb). vacv protein a49 prevents translocation of nf-κb to the nucleus by sequestering cellular β-trcp, a protein required for the degradation of the inhibitor of κb. a49 does not share overall sequence similarity with any protein of known structure or function. we solved the crystal structure of a49 from vacv western reserve to 1.8 å resolution and showed, surpr ...201525605733
novel therapeutic strategies in human malignancy: combining immunotherapy and oncolytic virotherapy.results from randomized clinical trials over the last several years have finally begun to demonstrate the potential of oncolytic viral therapies to treat a variety of cancers. one reason for these successes has been the realization that this platform is most effective when considered primarily as an immunotherapy. cancer immunotherapy has also made dramatic strides recently with antibodies capable of blocking immune checkpoint inhibitors and adoptive t-cell therapies, notably car t-cells, leadin ...201527512672
evolution of and evolutionary relationships between extant vaccinia virus strains.although vaccinia virus (vacv) was once used as a vaccine to eradicate smallpox on a worldwide scale, the biological origins of vacv are uncertain, as are the historical relationships between the different strains once used as smallpox vaccines. here, we sequenced additional vacv strains that either represent relatively pristine examples of old vaccines (e.g., dryvax, lister, and tashkent) or have been subjected to additional laboratory passage (e.g., ihd-w and wr). these genome sequences were c ...201525410873
a mouse model of atopic dermatitis.atopic dermatitis (ad) is a chronic or chronically relapsing, pruritic inflammatory skin disease. the incidence of ad has dramatically increased for the past three decades in industrialized countries. we established a highly efficient method to induce ad-like skin lesions using repeated epicutaneous treatments with house dust mite allergen and staphylococcal enterotoxin b (seb). the dermatitis-induced mice showed increased serum ige levels that were similar to human ad patients and also treatabl ...201525388270
enhanced efficacy of cidofovir combined with vaccinia immune globulin in treating progressive cutaneous vaccinia virus infections in immunosuppressed hairless mice.the treatment of progressive vaccinia in individuals has involved antiviral drugs, such as cidofovir (cdv), brincidofovir, and/or tecovirimat, combined with vaccinia immune globulin (vig). vig is costly, and its supply is limited, so sparing the use of vig during treatment is an important objective. vig sparing was modeled in immunosuppressed mice by maximizing the treatment benefits of cdv combined with vig to determine the effective treatments that delayed the time to death, reduced cutaneous ...201525385098
enhancement of cd8(+) t-cell memory by removal of a vaccinia virus nuclear factor-κb inhibitor.factors influencing t-cell responses are important for vaccine development but are incompletely understood. here, vaccinia virus (vacv) protein n1 is shown to impair the development of both effector and memory cd8(+) t cells and this correlates with its inhibition of nuclear factor-κb (nf-κb) activation. infection with vacvs that either have the n1l gene deleted (vδn1) or contain a i6e mutation (vn1.i6e) that abrogates its inhibition of nf-κb resulted in increased central and memory cd8(+) t-cel ...201525382035
viruses that ride on the coat-tails of actin nucleation.actin nucleation drives a diversity of critical cellular processes and the motility of a select group of viral pathogens. vaccinia virus and baculovirus, autographa californica multiple nucleopolyhedrovirus, recruit and activate the cellular actin nucleator, the arp2/3 complex, at the surface of virus particles thereby instigating highly localized actin nucleation. the extension of these filaments provides a mechanical force that bestows the ability to navigate the intracellular environment and ...201526459972
enhanced production of a single domain antibody with an engineered stabilizing extra disulfide bond.single domain antibodies derived from the variable region of the unique heavy chain antibodies found in camelids yield high affinity and regenerable recognition elements. adding an additional disulfide bond that bridges framework regions is a proven method to increase their melting temperature, however often at the expense of protein production. to fulfill their full potential it is essential to achieve robust protein production of these stable binding elements. in this work, we tested the hypot ...201526449768
decoding poxvirus genome. 201526429864
development of a high-content orthopoxvirus infectivity and neutralization assays.currently, a number of assays measure orthopoxvirus neutralization with serum from individuals, vaccinated against smallpox. in addition to the traditional plaque reduction neutralization test (prnt), newer higher throughput assays are based on neutralization of recombinant vaccinia virus, expressing reporter genes such as β-galactosidase or green fluorescent protein. these methods could not be used to evaluate neutralization of variola virus, since genetic manipulations of this virus are prohib ...201526426117
expression of anti-vegf antibody together with anti-egfr or anti-fap enhances tumor regression as a result of vaccinia virotherapy.the tumor microenvironment plays an important role in tumor growth and progression. here we demonstrate that vaccinia virus-mediated, constitutively expressed intratumoral antibodies against vascular endothelial growth factor (vegf), epidermal growth factor receptor (egfr), and fibroblast activation protein (fap) significantly improved tumor regression and oncolytic virotherapy through suppression of angiogenesis, cell proliferation, and stromagenesis in virus-colonized tumors. in contrast to th ...201527119102
gene therapy using therapeutic and diagnostic recombinant oncolytic vaccinia virus glv-1h153 for management of colorectal peritoneal carcinomatosis.peritoneal carcinomatosis (pc) is a terminal progression of colorectal cancer (crc). poor response to cytoreductive operation and chemotherapy coupled with the inability to reliably track disease progression by the use of established diagnostic methods, make this a deadly disease. we examined the effectiveness of the oncolytic vaccinia virus glv-1h153 as a therapeutic and diagnostic vehicle. we believe that viral expression of the human sodium iodide transporter (hnis) provides both real-time mo ...201525616946
dermal-resident versus recruited γδ t cell response to cutaneous vaccinia virus infection.the study of t cell immunity at barrier surfaces has largely focused on t cells bearing the αβ tcr. however, t cells that express the γδ tcr are disproportionately represented in peripheral tissues of mice and humans, suggesting they too may play an important role responding to external stimuli. in this article, we report that, in a murine model of cutaneous infection with vaccinia virus, dermal γδ t cell numbers increased 10-fold in the infected ear and resulted in a novel γδ t cell population ...201525609844
overcoming tumor resistance by heterologous adeno-poxvirus combination therapy.successful cancer control relies on overcoming resistance to cell death and on activation of host antitumor immunity. oncolytic viruses are particularly attractive in this regard, as they lyse infected tumor cells and trigger robust immune responses during the infection. however, repeated injections of the same virus promote antiviral rather than antitumor immunity and tumors may mount innate antiviral defenses to restrict oncolytic virus replication. in this article, we have explored if alterna ...201527119097
vaccinia virus, a promising new therapeutic agent for pancreatic cancer.the poor prognosis of pancreatic cancer patients signifies a need for radically new therapeutic strategies. tumor-targeted oncolytic viruses have emerged as attractive therapeutic candidates for cancer treatment due to their inherent ability to specifically target and lyse tumor cells as well as induce antitumor effects by multiple action mechanisms. vaccinia virus has several inherent features that make it particularly suitable for use as an oncolytic agent. in this review, we will discuss the ...201526595180
redundancy complicates the definition of essential genes for vaccinia virus.vaccinia virus (vacv) genes are characterized as either essential or non-essential for growth in culture. it seems intuitively obvious that if a gene can be deleted without imparting a growth defect in vitro it does not have a function related to basic replication or spread. however, this interpretation relies on the untested assumption that there is no redundancy across the genes that have roles in growth in cell culture. first, we provide a comprehensive summary of the literature that describe ...201526290187
mapping vaccinia virus dna replication origins at nucleotide level by deep sequencing.poxviruses reproduce in the host cytoplasm and encode most or all of the enzymes and factors needed for expression and synthesis of their double-stranded dna genomes. nevertheless, the mode of poxvirus dna replication and the nature and location of the replication origins remain unknown. a current but unsubstantiated model posits only leading strand synthesis starting at a nick near one covalently closed end of the genome and continuing around the other end to generate a concatemer that is subse ...201526286988
the role of vaccinia termination factor and cis-acting elements in vaccinia virus early gene transcription termination.vaccinia virus early gene transcription termination requires the virion form of the viral rna polymerase (vrnap), nucleoside triphosphate phosphohydrolase i (nphi), atp, the vaccinia termination factor (vtf), and a u5nu termination signal in the nascent transcript. vtf, also the viral mrna capping enzyme, binds u5nu, and nphi hydrolyzes atp to release the transcript. nphi can release transcripts independent of vtf and u5nu if vrnap is not actively elongating. however, vtf and u5nu are required f ...201526280468
detection of vaccinia virus in milk: evidence of a systemic and persistent infection in experimentally infected cows.bovine vaccinia (bv) is a zoonosis caused by vaccinia virus (vacv), which affects lactating cows and milkers. vacv dna and infectious particles have been detected in milk of naturally infected cows. however, the period and pattern of vacv shedding in milk is unknown, as is whether the presence of vacv in milk is due to a localized or a systemic infection. to address those questions, eight lactating cows were inoculated with vacv in previously scarified teats. the experiment was divided in two ph ...201526545169
quantitative analysis of micrornas in vaccinia virus infection reveals diversity in their susceptibility to modification and suppression.vaccinia virus (vacv) is a large cytoplasmic dna virus that causes dramatic alterations to many cellular pathways including microrna biogenesis. the virus encodes a poly(a) polymerase which was previously shown to add poly(a) tails to the 3' end of cellular mirnas, resulting in their degradation by 24 hours post infection (hpi). here we used small rna sequencing to quantify the impact of vacv infection on cellular mirnas in human cells at both early (6 h) and late (24 h) times post infection. a ...201526161560
are bites the "missing link" in cancer therapy?conventional treatment for cancer routinely includes surgical resection and some combination of chemotherapy and radiation. these approaches are frequently accompanied by unintended and highly toxic collateral damage to healthy tissues, which are offset by only marginal prognostic improvements in patients with advanced cancers. this unfortunate balance has driven the development of novel therapies that aim to target tumors both safely and efficiently. over the past decade, mounting evidence has ...201526155413
evaluating anti-orthopoxvirus antibodies in individuals from brazilian rural areas prior to the bovine vaccinia era.vaccinia virus naturally circulates in brazil and is the causative agent of a zoonotic disease known as bovine vaccinia (bv). we retrospectively evaluated two populations from the amazon and southeast regions. bv outbreaks had not been reported in these regions before sample collection. neutralising antibodies were found in 13 individuals (n = 132) with titres ranging from 100 ≥ 6,400 neutralising units/ml. univariate analysis identified age and vaccination as statistically significant risk fact ...201526517662
vaccinia virus as a subhelper for aav replication and packaging.adeno-associated virus (aav) has been widely used as a gene therapy vector to treat a variety of disorders. while these vectors are increasingly popular and successful in the clinic, there is still much to learn about the viruses. understanding the biology of these viruses is essential in engineering better vectors and generating vectors more efficiently for large-scale use. aav requires a helper for production and replication making this aspect of the viral life cycle crucial. vaccinia virus (v ...201526636113
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