PMID(sorted descending)
a survey of mosquito-borne and insect-specific viruses in hospitals and livestock markets in western kenya.aedes aegypti and culex pipiens complex mosquitoes are prolific vectors of arboviruses that are a global threat to human and animal health. increased globalization and ease of travel have facilitated the worldwide dissemination of these mosquitoes and the viruses they transmit. to assess disease risk, we determined the frequency of arboviruses in western kenyan counties bordering an area of high arboviral activity. in addition to pathogenic viruses, insect-specific flaviviruses (isfs), some of w ...202134048473
targeting lentiviral vectors to primordial germ cells (pgcs): an efficient strategy for generating transgenic chickens.recent advances in avian transgenic studies highlight the possibility of utilizing lentiviral vectors as tools to generate transgenic chickens. however, low rates of gonadal chimerism and germ line transmission efficiency still limit the broad usage of this method in creating transgenic chickens. in this study, we implemented a simple strategy using modified lentiviral vectors targeted to chicken primordial germ cells (pgcs) to generate transgenic chickens. the lentiviral vectors were pseudotype ...202032274905
arboviruses and blood meal sources in zoophilic mosquitoes at human-wildlife interfaces in kenya.background: zoophilic mosquitoes play an important role in the transmission of arboviruses of medical importance at human-wildlife interfaces, yet arbovirus surveillance efforts have been focused mostly on anthropophilic mosquitoes. understanding the diversity of zoophilic mosquitoes and their associated feeding patterns and arboviruses can inform better vector control strategies. materials and methods: we morphologically identified mosquitoes collected from two game reserves in kenya, the maasa ...202032155389
sindbis virus- a wild bird associated zoonotic arbovirus circulates in germany.sindbis virus (sinv) is an arbovirus causing clinical symptoms such as arthritis, rash and fever following human infections in fennoscandia. its transmission cycle involves mosquito species as vectors as well as wild birds that act as natural reservoir hosts. in germany, sinv was first time observed in 2009 in different mosquito species in the upper rhine valley and one year later in a hooded crow in berlin. recently, sinv was also detected repeatedly at various locations in germany in the conte ...201931767092
activation of rnase l in egyptian rousette bat-derived roni/7 cells is dependent primarily on oas3 and independent of mavs signaling.bats are reservoirs for many rna viruses that are highly pathogenic in humans yet relatively apathogenic in the natural host. it has been suggested that differences in innate immunity are responsible. the antiviral oas-rnase l pathway is well characterized in humans, but there is little known about its activation and antiviral activity in bats. during infection, oass, upon sensing double-stranded rna (dsrna), produce 2'-5' oligoadenylates (2-5a), leading to activation of rnase l which degrades v ...201931719180
infection of western gray kangaroos (macropus fuliginosus) with australian arboviruses associated with human infection.more than 75 arboviruses (arthropod-borne viruses) have been identified in australia. while alfuy virus (alfv), barmah forest virus (bfv), edge hill virus (ehv), kokobera virus (kokv), murray valley encephalitis virus (mvev), sindbis virus (sinv), ross river virus (rrv), stratford virus (strv), and west nile virus strain kunjin (kunv) have been associated with human infection, there remains a paucity of data regarding their respective transmission cycles and any potential nonhuman vertebrate hos ...202031556842
common vole (microtus arvalis) and bank vole (myodes glareolus) derived permanent cell lines differ in their susceptibility and replication kinetics of animal and zoonotic viruses.pathogenesis and reservoir host adaptation of animal and zoonotic viruses are poorly understood due to missing adequate cell culture and animal models. the bank vole (myodes glareolus) and common vole (microtus arvalis) serve as hosts for a variety of zoonotic pathogens. for a better understanding of virus association to a putative animal host, we generated two novel cell lines from bank voles of different evolutionary lineages and two common vole cell lines and assayed their susceptibility, rep ...201931513859
host-feeding patterns of culex mosquitoes in iran.different mosquito-borne pathogens are circulating in iran including sindbis virus, west nile virus, filarioid worms and malaria parasites. however, the local transmission cycles of these pathogenic agents are poorly understood, especially because ecological data on vector species are scarce and there is limited knowledge about the host range; this understanding could help to direct species-specific vector control measurements or to prioritize research.201830587194
arboviruses isolated from the barkedji mosquito-based surveillance system, 2012-2013.a mosquito-based arbovirus surveillance system was set up at barkedji, senegal after the first outbreak of rift valley fever in west africa in 1988. this system was recently updated using more sampling methods and collecting in greater number of ponds and villages sites.201830541472
linkages between chiropteran diversity and ecosystem services for sustainable fragmented forest conservation.this data article informs about chiropteran diversity, new records, ecosystem services and possible pathogen carriers in fragmented forests (sub-divided by utility corridors, man-made structures, untouched and secondary plantations) within districts setiu (setiu research station), hulu terengganu (saok and lasir waterfalls) and besut (gunung tebu forest reserve) of state terengganu, peninsular malaysia. these bats were captured using harp traps and mist nets that were set 10 m apart across flywa ...201830533456
h-ras exerts opposing effects on type i interferon responses depending on its activation status.using shrna high-throughput screening, we identified h-ras as a regulator of antiviral activity, whose depletion could enhance sindbis virus replication. further analyses indicated that depletion of h-ras results in a robust increase in vesicular stomatitis virus infection and a decrease in sendai virus (sev)-induced retinoic acid-inducible gene-i-like receptor (rlr) signaling. interestingly, however, ectopic expression of wild-type h-ras results in a biphasic mode of rlr signaling regulation: w ...201728848563
dorsal and ventral parts of thalamic nucleus submedius project to different areas of rat orbitofrontal cortex: a single neuron-tracing study using virus vectors.the rodent orbitofrontal cortex is involved in a variety of cognitive and behavioral functions that require thalamic input to be successfully expressed. although the thalamic nucleus submedius (sm) is a major source of afferents to the orbitofrontal cortex, thalamocortical projection from the sm has not been fully elucidated. in the present study, we first divided the rat sm into dorsal and ventral parts according to the distribution of vesicular glutamate transporter 2-immunoreactive varicositi ...201728833113
partially uncleaved alphavirus replicase forms spherule structures in the presence and absence of rna template.alphaviruses are positive-strand rna viruses expressing their replicase as a polyprotein, p1234, which is cleaved to four final products, nonstructural proteins nsp1 to nsp4. the replicase proteins together with viral rna and host factors form membrane invaginations termed spherules, which act as the replication complexes producing progeny rnas. we have previously shown that the wild-type alphavirus replicase requires a functional rna template and active polymerase to generate spherule structure ...201728701392
identification of interactions between sindbis virus capsid protein and cytoplasmic vrna as novel virulence determinants.alphaviruses are arthropod-borne viruses that represent a significant threat to public health at a global level. while the formation of alphaviral nucleocapsid cores, consisting of cargo nucleic acid and the viral capsid protein, is an essential molecular process of infection, the precise interactions between the two partners are ill-defined. a clip-seq approach was used to screen for candidate sites of interaction between the viral capsid protein and genomic rna of sindbis virus (sinv), a model ...201728662211
application of alphaviral vectors for immunomodulation in cancer therapy.the lack of specific and efficient cancer therapies has influenced the development of novel approaches, such as immunotherapy, which from its original application of immunogenic protein delivery has developed into the use of more sophisticated recombinant gene delivery methods to achieve better safety and efficacy profiles. this approach involves viral and non-viral delivery systems.201728641531
wolbachia elevates host methyltransferase expression to block an rna virus early during infection.wolbachia pipientis is an intracellular endosymbiont known to confer host resistance against rna viruses in insects. however, the causal mechanism underlying this antiviral defense remains poorly understood. to this end, we have established a robust arthropod model system to study the tripartite interaction involving sindbis virus and wolbachia strain wmel within its native host, drosophila melanogaster. by leveraging the power of drosophila genetics and a parallel, highly tractable d. melanogas ...201728617844
development and characterization of sindbis virus with encoded fluorescent rna aptamer spinach2 for imaging of replication and immune-mediated changes in intracellular viral rna.viral rna studies often rely on in situ hybridization and reverse transcriptase-pcr to provide snapshots of rna dynamics in infected cells. to facilitate analysis of cellular rnas, aptamers spinach and spinach2 that bind and activate the conditional fluorophore 3, 5-difluoro-4-hydroxybenzylidene imidazolinon have been developed. to determine the feasibility of applying this technology to viral rna, we have used cdna clones of the te strain of sindbis virus (sinv) to construct multiple viruses co ...201728555544
glutamine antagonist-mediated immune suppression decreases pathology but delays virus clearance in mice during nonfatal alphavirus encephalomyelitis.infection of weanling c57bl/6 mice with the te strain of sindbis virus (sinv) causes nonfatal encephalomyelitis associated with hippocampal-based memory impairment that is partially prevented by treatment with 6-diazo-5-oxo-l-norleucine (don), a glutamine antagonist (potter et al., j neurovirol 21:159, 2015). to determine the mechanism(s) of protection, lymph node and central nervous system (cns) tissues from sinv-infected mice treated daily for 1 week with low (0.3mg/kg) or high (0.6mg/kg) dose ...201728531865
operational mosquito and vector-borne diseases surveillance at incirlik air base, turkey.arboviruses on incirlik air base, turkey, pose a threat to military personnel and civilians, but might also be relevant for understanding the threats in neighboring conflict zones such as syria. we reviewed 6 years of mosquito and arbovirus surveillance at incirlik air base. over 6,000 mosquitoes were identified as aedes caspius, anopheles claviger, culex mimeticus, cx. perexiguus, cx. pipiens, cx. sinaiticus, and culiseta longiareolata. almost all of the mosquitoes (more than 90%) were cx. pere ...201728511277
biological safety of a highly purified 10% liquid intravenous immunoglobulin preparation from human plasma.a highly purified 10% liquid intravenous immunoglobulin, iqymune(®), has been developed using an innovative manufacturing process including an affinity chromatography step for the removal of anti-a and anti-b hemagglutinins.201728508264
imaging of viral neuroinvasion in the zebrafish reveals that sindbis and chikungunya viruses favour different entry routes.alphaviruses, such as chikungunya virus (chikv) and sindbis virus (sinv), are vector-borne pathogens that cause acute illnesses in humans and are sometimes associated with neuropathies, especially in infants and elderly patients. little is known about their mechanism of entry into the central nervous system (cns), even for sinv, which has been used extensively as a model for viral encephalopathies. we previously established a chikv infection model in the optically transparent zebrafish larva; he ...201728483796
identification of a newly isolated getah virus in the china-laos border, this study, we isolated a virus strain (yn12031) from specimens of armigeres subalbatus collected in the china-laos border. bhk-21 cells infected with yn12031 exhibited an evident cytopathic effect (cpe) 32 h post-infection. the virus particles were spherical, 70 nm in diameter, and enveloped; they also featured surface fibers. molecular genetic analysis revealed that yn12031 was closely related to alpha viruses such as chikungunya virus and sindbis virus, and located in the same clade as mm2 ...201728427491
oncolytic alphaviruses in cancer immunotherapy.oncolytic viruses show specific targeting and killing of tumor cells and therefore provide attractive assets for cancer immunotherapy. in parallel to oncolytic viral vectors based on adenoviruses and herpes simplex viruses, oncolytic rna viruses and particularly alphaviruses have been evaluated as delivery vehicles. immunization studies in experimental rodent models for various cancers including glioblastoma, hematologic, hepatocellular, colon, cervix, and lung cancer as well as melanoma have be ...201728417936
alphavirus-based vaccines.alphavirus-based vectors have been engineered from semliki forest virus, sindbis virus, and venezuelan equine encephalitis virus and applied for vaccine development. immunization in preclinical animal models has been conducted with naked rna replicons, recombinant viral particles and layered dna-rna vectors. most commonly, the targets for the immunization have been viral surface proteins and tumor antigens, which have elicited strong immune responses and even provided protection against challeng ...201728374252
an icosahedral virus as a fluorescent calibration standard: a method for counting protein molecules in cells by fluorescence microscopy.the ability to replace genes coding for cellular proteins with dna that codes for fluorescent protein-tagged versions opens the way to counting the number of molecules of each protein component of macromolecular assemblies in vivo by measuring fluorescence microscopically. converting fluorescence to absolute numbers of molecules requires a fluorescent standard whose molecular composition is known precisely. in this report, the construction, properties and mode of using a set of fluorescence cali ...201728328099
translation of sindbis subgenomic mrna is independent of eif2, eif2a and eif2d.translation of sindbis virus subgenomic mrna (sgmrna) can occur after inactivation of eif2 by phosphorylation in mammalian cells. several studies have suggested that eif2 can be replaced by eif2a or eif2d. hap1 human cell lines knocked-out for eif2a, eif2d or both by crispr/cas9 genome engineering were compared with wild-type (wt) cells to test the potential role of eif2a and eif2d in translation. sindbis virus infection was comparable between the four cell lines. moreover, synthesis of viral pr ...201728240315
discovery of a novel antiviral agent targeting the nonstructural protein 4 (nsp4) of chikungunya virus.chikungunya fever (chikf) is caused by chikungunya virus (chikv) infection which is a re-emerging mosquito-borne zoonosis. at present, there are no approved therapeutics for chikf. herein, we have investigated candidate compounds which can inhibit chikv infection. screening of chemical compound libraries were performed and one candidate, a benzimidazole-related compound designated compound-a was found to inhibit infection by several chikv strains and a sindbis virus strain at nanomolar concentra ...201728236746
microbiota activates imd pathway and limits sindbis infection in aedes aegypti.aedes aegypti is the main vector of important arboviruses such as dengue, zika and chikungunya. during infections mosquitoes can activate the immune pathways toll, imd and jak/stat to limit pathogen replication.201728231846
inactivation of viruses by pasteurization at 60 °c for 10 h with and without 40% glucose as stabilizer during a new manufacturing process of α2-macroglobulin from cohn fraction iv.pasteurization is regularly used to inactivate viruses for the safety of plasma derivatives. influence of pasteurization at 60 °c for 10 h on α2-macroglobulin activity and virus inactivation were studied. with 40% sugar as stabilizers more than 70% α2-macroglobulin activity was reserved after pasteurization compared with 20% in control. glucose presented a better activity protection effect than sucrose and maltose. by pasteurization without stabilizer the virus titers of pseudorabies virus, sind ...201728215695
mitochondrial damage elicits a tcdd-inducible poly(adp-ribose) polymerase-mediated antiviral response.the innate immune system senses rna viruses by pattern recognition receptors (prrs) and protects the host from virus infection. prrs mediate the production of immune modulatory factors and direct the elimination of rna viruses. here, we show a unique prr that mediates antiviral response. tetrachlorodibenzo-p-dioxin (tcdd)-inducible poly(adp ribose) polymerase (tiparp), a cysteine3 histidine (ccch)-type zinc finger-containing protein, binds to sindbis virus (sinv) rna via its zinc finger domain a ...201728213497
trim25 is required for the antiviral activity of zinc-finger antiviral protein (zap).zinc-finger antiviral protein (zap) is a host factor that specifically inhibits the replication of certain viruses by binding to viral mrnas, and repressing the translation and/or promoting the degradation of target mrna. in addition, zap regulates the expression of certain cellular genes. here, we report that tripartite motif-containing protein 25 (trim25), a ubiquitin e3 ligase, is required for the antiviral activity of zap. downregulation of endogenous trim25 abolished zap's antiviral activit ...201728202764
spatial and temporal analysis of alphavirus replication and assembly in mammalian and mosquito cells.sindbis virus (sinv [genus alphavirus, family togaviridae]) is an enveloped, mosquito-borne virus. alphaviruses cause cytolytic infections in mammalian cells while establishing noncytopathic, persistent infections in mosquito cells. mosquito vector adaptation of alphaviruses is a major factor in the transmission of epidemic strains of alphaviruses. though extensive studies have been performed on infected mammalian cells, the morphological and structural elements of alphavirus replication and ass ...201728196962
selective transduction of astrocytic and neuronal cns subpopulations by lentiviral vectors pseudotyped with chikungunya virus envelope.lentiviral vectors are gene delivery vehicles that integrate into the host genome of dividing and non-dividing mammalian cells facilitating long-term transgene expression. lentiviral vector versatility is greatly increased by incorporating heterologous viral envelope proteins onto the vector particles instead of the native envelope, conferring on these pseudotyped vectors a modified tropism and host range specificity. we investigated the pseudotyping efficiency of hiv-1 based lentiviral vectors ...201728152379
neurotropic alphaviruses can propagate without capsid. 201728129638
dynamics of molecular evolution in rna virus populations depend on sudden versus gradual environmental change.understanding the dynamics of molecular adaptation is a fundamental goal of evolutionary biology. while adaptation to constant environments has been well characterized, the effects of environmental complexity remain seldom studied. one simple but understudied factor is the rate of environmental change. here we used experimental evolution with rna viruses to investigate whether evolutionary dynamics varied based on the rate of environmental turnover. we used whole-genome next-generation sequencin ...201728121018
trim25 enhances the antiviral action of zinc-finger antiviral protein (zap).the host factor and interferon (ifn)-stimulated gene (isg) product, zinc-finger antiviral protein (zap), inhibits a number of diverse viruses by usurping and intersecting with multiple cellular pathways. to elucidate its antiviral mechanism, we perform a loss-of-function genome-wide rnai screen to identify cellular cofactors required for zap antiviral activity against the prototype alphavirus, sindbis virus (sinv). in order to exclude off-target effects, we carry out stringent confirmatory assay ...201728060952
a quantitative real-time rt-pcr assay for the detection of venezuelan equine encephalitis virus utilizing a universal alphavirus control rna.venezuelan equine encephalitis virus (veev) is an alphavirus from the family togaviridae that causes epizootic outbreaks in equids and humans in central and south america. so far, most studies use conventional reverse transcriptase pcr assays for the detection of the different veev subtypes. here we describe the development of a taqman quantitative real-time reverse transcriptase pcr assay for the specific detection and quantitation of all veev subtypes which uses in parallel a universal equine ...201628042576
high insecticides resistance in culex pipiens (diptera: culicidae) from tehran, capital of iran.during recent years transmission of dirofilaria immitis (dog heart worm) by culex pipiens and west nile virus have been reported from iran. the present study was preformed for evaluating the susceptibility status of cx. pipiens collected from capital city of tehran, iran.201628032100
use of aptamers as diagnostics tools and antiviral agents for human viruses.appropriate diagnosis is the key factor for treatment of viral diseases. time is the most important factor in rapidly developing and epidemiologically dangerous diseases, such as influenza, ebola and sars. chronic viral diseases such as hiv-1 or hcv are asymptomatic or oligosymptomatic and the therapeutic success mainly depends on early detection of the infective agent. over the last years, aptamer technology has been used in a wide range of diagnostic and therapeutic applications and, concretel ...201627999271
obatoclax inhibits alphavirus membrane fusion by neutralizing the acidic environment of endocytic new pathogenic viruses continue to emerge, it is paramount to have intervention strategies that target a common denominator in these pathogens. the fusion of viral and cellular membranes during viral entry is one such process that is used by many pathogenic viruses, including chikungunya virus, west nile virus, and influenza virus. obatoclax, a small-molecule antagonist of the bcl-2 family of proteins, was previously determined to have activity against influenza a virus and also sindbis virus ...201727993855
intercellular extensions are induced by the alphavirus structural proteins and mediate virus transmission.alphaviruses are highly organized enveloped rna viruses with an internal nucleocapsid surrounded by a membrane containing the e2 and e1 transmembrane proteins. alphavirus budding takes place at the plasma membrane and requires the interaction of the cytoplasmic domain of e2 with the capsid protein. here we used wt alphaviruses and sindbis virus in which e2 was fused to a fluorescent protein to characterize virus exit from host cells. our results show that alphavirus infection induced striking mo ...201627977778
the conserved coronavirus macrodomain promotes virulence and suppresses the innate immune response during severe acute respiratory syndrome coronavirus infection.adp-ribosylation is a common posttranslational modification that may have antiviral properties and impact innate immunity. to regulate this activity, macrodomain proteins enzymatically remove covalently attached adp-ribose from protein targets. all members of the coronavirinae, a subfamily of positive-sense rna viruses, contain a highly conserved macrodomain within nonstructural protein 3 (nsp3). however, its function or targets during infection remain unknown. we identified several macrodomain ...201627965448
first report on human-biting culex pipiens in sweden.culex mosquitoes are vectors of several bird-hosted arboviruses that cause outbreaks in europe, such as sindbis virus and west nile virus. recently, the human-biting form of culex pipiens, cx. pipiens biotype molestus, was found causing big nuisance in a housing cooperative in gothenburg in southern sweden, confirmed by molecular identification. this is the first report of human-biting culex in scandinavia, signalling increased risk of arbovirus infection in northern europe.201627927233
visualization of neutrophil extracellular traps and fibrin meshwork in human fibrinopurulent inflammatory lesions: iii. correlative light and electron microscopic study.neutrophil extracellular traps (nets) released from dead neutrophils at the site of inflammation represent webs of neutrophilic dna stretches dotted with granule-derived antimicrobial proteins, including lactoferrin, and play important roles in innate immunity against microbial infection. we have shown the coexistence of nets and fibrin meshwork in varied fibrinopurulent inflammatory lesions at both light and electron microscopic levels. in the present study, correlative light and electron micro ...201627917008
an mrna-specific trnai carrier eif2a plays a pivotal role in cell proliferation under stress conditions: stress-resistant translation of c-src mrna is mediated by eif2a.c-src, a non-receptor protein tyrosine kinase, activates nf-κb and stat3, which in turn triggers the transcription of anti-apoptosis- and cell cycle-related genes. c-src protein regulates cell proliferation, cell motility and programmed cell death. and the elevated level of activated c-src protein is related with solid tumor generation. translation of c-src mrna is directed by an ires element which mediates persistent translation under stress conditions when translation of most mrnas is inhibite ...201627899592
interferon regulatory factor 2 protects mice from lethal viral neuroinvasion.the host responds to virus infection by activating type i interferon (ifn) signaling leading to expression of ifn-stimulated genes (isgs). dysregulation of the ifn response results in inflammatory diseases and chronic infections. in this study, we demonstrate that ifn regulatory factor 2 (irf2), an isg and a negative regulator of ifn signaling, influences alphavirus neuroinvasion and pathogenesis. a sindbis virus strain that in wild-type (wt) mice only causes disease when injected into the brain ...201627899441
acyclic nucleoside phosphonates containing the amide study the influence of a linker rigidity and donor-acceptor properties, the p-ch2-o-chr- fragment in acyclic nucleoside phosphonates (e.g., acyclovir, tenofovir) was replaced by the p-ch2-hn-c(o)- residue. the respective phosphonates were synthesized in good yields by coupling the straight chain of ω-aminophosphonates and nucleobase-derived acetic acids with edc. based on the (1)h and (13)c nmr data, the unrestricted rotation within the methylene and 1,2-ethylidene linkers in phosphonates fro ...201627881885
(in)validating experimentally derived knowledge about influenza a defective interfering particles.a defective interfering particle (dip) in the context of influenza a virus is a virion with a significantly shortened rna segment substituting one of eight full-length parent rna segments, such that it is preferentially amplified. hence, a cell co-infected with dips will produce mainly dips, suppressing infectious virus yields and affecting infection kinetics. unfortunately, the quantification of dips contained in a sample is difficult because they are indistinguishable from standard virus (stv) ...027881801
actin is an evolutionarily-conserved damage-associated molecular pattern that signals tissue injury in drosophila melanogaster.damage-associated molecular patterns (damps) are molecules released by dead cells that trigger sterile inflammation and, in vertebrates, adaptive immunity. actin is a damp detected in mammals by the receptor, dngr-1, expressed by dendritic cells (dcs). dngr-1 is phosphorylated by src-family kinases and recruits the tyrosine kinase syk to promote dc cross-presentation of dead cell-associated antigens. here we report that actin is also a damp in invertebrates that lack dcs and adaptive immunity. a ...201627871362
molecular evolution of type 2 porcine reproductive and respiratory syndrome viruses circulating in vietnam from 2007 to 2015.porcine respiratory and reproductive syndrome (prrs) virus is one of the most economically significant pathogens in the vietnamese swine industry. orf5, which participates in many functional processes, including virion assembly, entry of the virus into the host cell, and viral adaptation to the host immune response, has been widely used in molecular evolution and phylogeny studies. knowing of molecular evolution of prrsv fields strains might contribute to prrs control in vietnam.201627855667
palmitoylation of sindbis virus tf protein regulates its plasma membrane localization and subsequent incorporation into virions.palmitoylation is a reversible, posttranslational modification that helps target proteins to cellular membranes. the alphavirus small membrane proteins 6k and tf have been reported to be palmitoylated and to positively regulate budding. 6k and tf are isoforms that are identical in their n termini but unique in their c termini due to a -1 ribosomal frameshift during translation. in this study, we used cysteine (cys) mutants to test differential palmitoylation of the sindbis virus 6k and tf protei ...201727852864
ablation of programmed -1 ribosomal frameshifting in venezuelan equine encephalitis virus results in attenuated neuropathogenicity.the alphaviruses venezuelan equine encephalitis virus (veev), eastern equine encephalitis virus (eeev), and western equine encephalitis virus (weev) are arthropod-borne positive-strand rna viruses that are capable of causing acute and fatal encephalitis in many mammals, including humans. veev was weaponized during the cold war and is recognized as a select agent. currently, there are no fda-approved vaccines or therapeutics for these viruses. the spread of veev and other members of this family d ...201727852852
yellow fever virus capsid protein is a potent suppressor of rna silencing that binds double-stranded rna.mosquito-borne flaviviruses, including yellow fever virus (yfv), zika virus (zikv), and west nile virus (wnv), profoundly affect human health. the successful transmission of these viruses to a human host depends on the pathogen's ability to overcome a potentially sterilizing immune response in the vector mosquito. similar to other invertebrate animals and plants, the mosquito's rna silencing pathway comprises its primary antiviral defense. although a diverse range of plant and insect viruses has ...201627849599
chikungunya virus infectivity, rna replication and non-structural polyprotein processing depend on the nsp2 protease's active site cysteine residue.chikungunya virus (chikv), genus alphavirus, family togaviridae, has a positive-stand rna genome approximately 12 kb in length. in infected cells, the genome is translated into non-structural polyprotein p1234, an inactive precursor of the viral replicase, which is activated by cleavages carried out by the non-structural protease, nsp2. we have characterized chikv nsp2 using both cell-free and cell-based assays. first, we show that cys478 residue in the active site of chikv nsp2 is indispensable ...201627845418
replicon rna viral vectors as vaccines.single-stranded rna viruses of both positive and negative polarity have been used as vectors for vaccine development. in this context, alphaviruses, flaviviruses, measles virus and rhabdoviruses have been engineered for expression of surface protein genes and antigens. administration of replicon rna vectors has resulted in strong immune responses and generation of neutralizing antibodies in various animal models. immunization of mice, chicken, pigs and primates with virus-like particles, naked r ...201627827980
peroxisomal protein pex13 functions in selective autophagy.pex13 is an integral membrane protein on the peroxisome that regulates peroxisomal matrix protein import during peroxisome biogenesis. mutations in pex13 and other peroxin proteins are associated with zellweger syndrome spectrum (zss) disorders, a subtype of peroxisome biogenesis disorder characterized by prominent neurological, hepatic, and renal abnormalities leading to neonatal death. the lack of functional peroxisomes in zss patients is widely accepted as the underlying cause of disease; how ...201627827795
a universal next-generation sequencing protocol to generate noninfectious barcoded cdna libraries from high-containment rna viruses.several biosafety level 3 and/or 4 (bsl-3/4) pathogens are high-consequence, single-stranded rna viruses, and their genomes, when introduced into permissive cells, are infectious. moreover, many of these viruses are select agents (sas), and their genomes are also considered sas. for this reason, cdnas and/or their derivatives must be tested to ensure the absence of infectious virus and/or viral rna before transfer out of the bsl-3/4 and/or sa laboratory. this tremendously limits the capacity to ...201627822536
mosquito-transmitted viruses - the great brazilian challenge.arboviruses pose a serious threat to public health worldwide, overloading the healthcare system and causing economic losses. these viruses form a very diverse group, and in brazil, arboviruses belonging to the families flaviviridae and togaviridae are predominant. unfortunately, the number of arboviruses increases in proportion with factors such as deforestation, poor sanitation, climate changes, and introduction of new viruses like chikungunya virus and zika virus. in brazil, dengue is endemic, ...201627818091
inactivation of the type i interferon pathway reveals long double-stranded rna-mediated rna interference in mammalian cells.rna interference (rnai) elicited by long double-stranded (ds) or base-paired viral rna constitutes the major mechanism of antiviral defence in plants and invertebrates. in contrast, it is controversial whether it acts in chordates. rather, in vertebrates, viral rnas induce a distinct defence system known as the interferon (ifn) response. here, we tested the possibility that the ifn response masks or inhibits antiviral rnai in mammalian cells. consistent with that notion, we find that sequence-sp ...201627815315
deep sequencing analysis reveals the mycoviral diversity of the virome of an avirulent isolate of rhizoctonia solani ag-2-2 iv.rhizoctonia solani represents an important plant pathogenic basidiomycota species complex and the host of many different mycoviruses, as indicated by frequent detection of dsrna elements in natural populations of the fungus. to date, eight different mycoviruses have been characterized in rhizoctonia and some of them have been reported to modulate its virulence. dsrna extracts of the avirulent r. solani isolate dc17 (ag2-2-iv) displayed a diverse pattern, indicating multiple infections with mycov ...201627814394
perspectives for therapeutic hpv vaccine development.human papillomavirus (hpv) infections and associated diseases remain a serious burden worldwide. it is now clear that hpv serves as the etiological factor and biologic carcinogen for hpv-associated lesions and cancers. although preventative hpv vaccines are available, these vaccines do not induce strong therapeutic effects against established hpv infections and lesions. these concerns create a critical need for the development of therapeutic strategies, such as vaccines, to treat these existing ...201627809842
genome investigations of vector competence in aedes aegypti to inform novel arbovirus disease control approaches.dengue (denv), yellow fever, chikungunya, and zika virus transmission to humans by a mosquito host is confounded by both intrinsic and extrinsic variables. besides virulence factors of the individual arboviruses, likelihood of virus transmission is subject to variability in the genome of the primary mosquito vector, aedes aegypti. the "vectorial capacity" of a. aegypti varies depending upon its density, biting rate, and survival rate, as well as its intrinsic ability to acquire, host and transmi ...201627809220
experimental piscine alphavirus rna recombination in vivo yields both viable virus and defective viral rna.rna recombination in non-segmented rna viruses is important for viral evolution and documented for several virus species through in vitro studies. here we confirm viral rna recombination in vivo using an alphavirus, the sav3 subtype of salmon pancreas disease virus. the virus causes pancreas disease in atlantic salmon and heavy losses in european salmonid aquaculture. atlantic salmon were injected with a sav3 6k-gene deleted cdna plasmid, encoding a non-viable variant of sav3, together with a he ...201627805034
rabies control and treatment: from prophylaxis to strategies with curative potential.rabies is an acute, fatal, neurological disease that affects almost all kinds of mammals. vaccination (using an inactivated rabies vaccine), combined with administration of rabies immune globulin, is the only approved, effective method for post-exposure prophylaxis against rabies in humans. in the search for novel rabies control and treatment strategies, live-attenuated viruses have recently emerged as a practical and promising approach for immunizing and controlling rabies. unlike the conventio ...201627801824
crystal structure of glycoprotein c from a hantavirus in the post-fusion conformation.hantaviruses are important emerging human pathogens and are the causative agents of serious diseases in humans with high mortality rates. like other members in the bunyaviridae family their m segment encodes two glycoproteins, gn and gc, which are responsible for the early events of infection. hantaviruses deliver their tripartite genome into the cytoplasm by fusion of the viral and endosomal membranes in response to the reduced ph of the endosome. unlike phleboviruses (e.g. rift valley fever vi ...201627783673
interferon gamma: influence on neural stem cell function in neurodegenerative and neuroinflammatory disease.interferon-gamma (ifnγ), a pleiotropic cytokine, is expressed in diverse neurodegenerative and neuroinflammatory conditions. its protective mechanisms are well documented during viral infections in the brain, where ifnγ mediates non-cytolytic viral control in infected neurons. however, ifnγ also plays both protective and pathological roles in other central nervous system (cns) diseases. of the many neural cells that respond to ifnγ, neural stem/progenitor cells (nspcs), the only pluripotent cell ...201627774000
mutagenic effects of ribavirin on hepatitis e virus-viral extinction versus selection of fitness-enhancing mutations.hepatitis e virus (hev), an important agent of viral hepatitis worldwide, can cause severe courses of infection in pregnant women and immunosuppressed patients. to date, hev infections can only be treated with ribavirin (rbv). major drawbacks of this therapy are that rbv is not approved for administration to pregnant women and that the virus can acquire mutations, which render the intra-host population less sensitive or even resistant to rbv. one of the proposed modes of action of rbv is a direc ...201627754363
antiviral activities of niclosamide and nitazoxanide against chikungunya virus entry and transmission.chikungunya disease results from an infection with the arbovirus, chikungunya virus (chikv). symptoms of chikv include fever and persistent, severe arthritis. in recent years, several antiviral drugs have been evaluated in clinical trials; however, no registered antivirals have been approved for clinical therapy. in this study, we established a high-throughput screening (hts) system based on chikv 26s mediated insect cell fusion inhibition assay. our screening system was able to search potential ...201627742486
ancient recombination events and the origins of hepatitis e virus.hepatitis e virus (hev) is an enteric, single-stranded, positive sense rna virus and a significant etiological agent of hepatitis, causing sporadic infections and outbreaks globally. tracing the evolutionary ancestry of hev has proved difficult since its identification in 1992, it has been reclassified several times, and confusion remains surrounding its origins and ancestry.201627733122
serosurveillance of viral pathogens circulating in west africa.sub-saharan africa is home to a variety of pathogens, but disease surveillance and the healthcare infrastructure necessary for proper management and control are severely limited. lassa virus, the cause of lassa fever, a severe hemorrhagic fever in humans is endemic in west africa. in sierra leone at the kenema government hospital lassa diagnostic laboratory, up to 70 % of acute patient samples suspected of lassa fever test negative for lassa virus infection. this large amount of acute undiagnose ...201627716429
quantification of virus particles using nanopore-based resistive-pulse sensing techniques.viruses have drawn much attention in recent years due to increased recognition of their important roles in virology, immunology, clinical diagnosis, and therapy. because the biological and physical properties of viruses significantly impact their applications, quantitative detection of individual virus particles has become a critical issue. however, due to various inherent limitations of conventional enumeration techniques such as infectious titer assays, immunological assays, and electron micro ...201627713738
cobalt complexes as antiviral and antibacterial agents.metal ion complexes are playing an increasing role in the development of antimicrobials. we review here the antimicrobial properties of cobalt coordination complexes in oxidation state 3+. in addition to reviewing the cobalt complexes containing polydentate donor ligands, we also focus on the antimicrobial activity of the homoleptic [co(nh₃)₆](3+) ion.201027713325
cell-penetrating peptides for antiviral drug development.viral diseases affect hundreds of millions of people worldwide, and the few available drugs to treat these diseases often come with limitations. the key obstacle to the development of new antiviral agents is their delivery into infected cells in vivo. cell-penetrating peptides (cpps) are short peptides that can cross the cellular lipid bilayer with the remarkable capability to shuttle conjugated cargoes into cells. cpps have been successfully utilized to enhance the cellular uptake and intracell ...201027713263
amyloid-β effects on synapses and memory require ampa receptor subunit glua3.amyloid-β (aβ) is a prime suspect for causing cognitive deficits during the early phases of alzheimer's disease (ad). experiments in ad mouse models have shown that soluble oligomeric clusters of aβ degrade synapses and impair memory formation. we show that all aβ-driven effects measured in these mice depend on ampa receptor (ampar) subunit glua3. hippocampal neurons that lack glua3 were resistant against aβ-mediated synaptic depression and spine loss. in addition, aβ oligomers blocked long-term ...201627708157
molecular detection of flaviviruses and alphaviruses in mosquitoes (diptera: culicidae) from coastal ecosystems in the colombian caribbean.arboviruses belonging to the genera flavivirus and alphavirus were detected in mosquitoes in a rural area of san bernardo del viento (córdoba, colombia). a total of 22,180 mosquitoes were collected, sorted into 2,102 pools, and tested by generic/nested reverse transcription-polymerase chain reaction. venezuelan equine encephalitis virus, dengue virus, west nile virus, st. louis encephalitis virus, yellow fever virus, and culex flavivirus were detected and identified by sequencing. the detection ...201627706377
climate change influences on the global potential distribution of the mosquito culex quinquefasciatus, vector of west nile virus and lymphatic filariasis.rapid emergence of most vector-borne diseases (vbds) may be associated with range expansion of vector populations. culex quinquefasciatus say 1823 is a potential vector of west nile virus, saint louis encephalitis virus, and lymphatic filariasis. we estimated the potential distribution of cx. quinquefasciatus under both current and future climate conditions. the present potential distribution of cx. quinquefasciatus showed high suitability across low-latitude parts of the world, reflecting the c ...201627695107
a 3'-end structure in rna2 of a crinivirus is essential for viral rna synthesis and contributes to replication-associated translation activity.the terminal ends in the genome of rna viruses contain features that regulate viral replication and/or translation. we have identified a y-shaped structure (yss) in the 3' terminal regions of the bipartite genome of lettuce chlorosis virus (lcv), a member in the genus crinivirus (family closteroviridae). the yss is the first in this family of viruses to be determined using selective 2'-hydroxyl acylation analyzed by primer extension (shape). using luciferase constructs/replicons, in vivo and in ...201627694962
a field-deployable reverse transcription recombinase polymerase amplification assay for rapid detection of the chikungunya virus.chikungunya virus (chikv) is a mosquito-borne virus currently transmitted in about 60 countries. chikv causes acute flu-like symptoms and in many cases prolonged musculoskeletal and joint pain. detection of the infection is mostly done using rt-rcr or elisa, which are not suitable for point-of-care diagnosis.201627685649
differential expression pattern of vago in bumblebee (bombus terrestris), induced by virulent and avirulent virus infections.viruses are one of the main drivers of the decline of domesticated and wild bees but the mechanisms of antiviral immunity in pollinators are poorly understood. recent work has suggested that next to the small interfering rna (sirna) pathway other immune-related pathways play a role in the defense of the bee hosts against viral infection. in addition, vago plays a role in the cross-talk between the innate immune pathways in culex mosquito cells. here we describe the vago orthologue in bumblebees ...201627680717
interferon gamma modulation of disease manifestation and the local antibody response to alphavirus encephalomyelitis.infection of mice with sindbis virus (sinv) produces encephalomyelitis and provides a model for examination of the central nervous system (cns) immune response to alphavirus infection. clearance of infectious virus is accomplished through a cooperative effort between sinv-specific antibody and ifn-γ, but the regulatory interactions are poorly understood. to determine the effects of ifn-γ on clinical disease and the antiviral immune response, c57bl/6 mice lacking ifn-γ (ifng-/-) or ifn-γ receptor ...201627667782
sindbis virus-pseudotyped lentiviral vectors carrying vegfr2-specific nanobody for potential transductional targeting of tumor vasculature.introduction of selectivity/specificity into viral-based gene delivery systems, such as lentiviral vectors (lvs), is crucial in their systemic administration for cancer gene therapy. the pivotal role of tumor-associated endothelial cells (taecs) in tumor angiogenesis and overexpression of vascular endothelial growth factor receptor-2 (vegfr2 or kdr) in taecs makes them a potent target in cancer treatment. herein, we report the development of vegfr2-targeted lvs pseudotyped with chimeric sindbis ...201627647452
morphological abnormalities and cell death in the asian citrus psyllid (diaphorina citri) midgut associated with candidatus liberibacter asiaticus.candidatus liberibacter asiaticus (clas) is a phloem-limited, gram-negative, fastidious bacterium that is associated with the development of citrus greening disease, also known as huanglongbing (hlb). clas is transmitted by the asian citrus psyllid (acp) diaphorina citri, in a circulative manner. two major barriers to transmission within the insect are the midgut and the salivary glands. we performed a thorough microscopic analysis within the insect midgut following exposure to clas-infected cit ...201627630042
lv305, a dendritic cell-targeting integration-deficient zvex(tm)-based lentiviral vector encoding ny-eso-1, induces potent anti-tumor immune response.we have engineered an integration-deficient lentiviral vector, lv305, to deliver the tumor antigen ny-eso-1 to human dendritic cells in vivo through pseudotyping with a modified sindbis virus envelop protein. mice immunized once with lv305 developed strong, dose-dependent, multifunctional, and cytotoxic ny-eso-1-specific cluster of differentiation 8 (cd8) t cells within 14 days post-immunization and could be boosted with lv305 at least twice to recall peak-level cd8 t-cell responses. immunizatio ...201627626061
inhibition of chikungunya virus by picolinate that targets viral capsid protein.the protein-protein interactions (ppis) of the transmembrane glycoprotein e2 with the hydrophobic pocket on the surface of capsid protein (cp) plays a critical role in alphavirus life cycle. dioxane based derivatives targeting ppis have been reported to possess antiviral activity against sindbis virus (sinv), the prototype alphavirus. in this study, the binding of picolinic acid (pca) to the conserved hydrophobic pocket of capsid protein was analyzed by molecular docking, isothermal titration ca ...201627614702
amphotericin b inhibits enterovirus 71 replication by impeding viral entry.enterovirus 71 (ev71) infection causes hand-foot-and-mouth disease that leads to cardiopulmonary complications and death in young children. there is thus an urgent need to find new treatments to control ev71 infection. in this study, we report potent inhibition of ev71 by a polyene antibiotic amphotericin b. amphotericin b profoundly diminished the expression of ev71 rna and viral proteins in the rd cells and the hek293 cells. as a result, ev71 production was inhibited by amphotericin b with an ...201627608771
m2bp inhibits hiv-1 virion production in a vimentin filaments-dependent manner.m2bp (also called 90k) is an interferon-stimulated gene product that is upregulated in hiv-1 infection. a recent study revealed that m2bp reduces the infectivity of hiv-1 by inhibiting the processing of the viral envelope protein. here we report that in addition to reducing viral infectivity, m2bp inhibits hiv-1 virion production. we provide evidence showing that m2bp inhibits hiv-1 gag trafficking to the plasma membrane in a vimentin-dependent manner. when vimentin filaments were collapsed by t ...201627604950
global profiling of the cellular alternative rna splicing landscape during virus-host interactions.alternative splicing (as) is a central mechanism of genetic regulation which modifies the sequence of rna transcripts in higher eukaryotes. as has been shown to increase both the variability and diversity of the cellular proteome by changing the composition of resulting proteins through differential choice of exons to be included in mature mrnas. in the present study, alterations to the global rna splicing landscape of cellular genes upon viral infection were investigated using mammalian reoviru ...201627598998
excess cerebral tnf causing glutamate excitotoxicity rationalizes treatment of neurodegenerative diseases and neurogenic pain by anti-tnf agents.the basic mechanism of the major neurodegenerative diseases, including neurogenic pain, needs to be agreed upon before rational treatments can be determined, but this knowledge is still in a state of flux. most have agreed for decades that these disease states, both infectious and non-infectious, share arguments incriminating excitotoxicity induced by excessive extracellular cerebral glutamate. excess cerebral levels of tumor necrosis factor (tnf) are also documented in the same group of disease ...201627596607
sindbis virus can exploit a host antiviral protein to evade immune surveillance.viral infection induces production of type i interferons (ifns), which stimulate the expression of a variety of antiviral factors to inhibit viral replication. to establish effective infection, viruses need to develop strategies to evade the immune responses. a neurovirulent sindbis virus strain with neuroinvasive properties (svni) causes lethal encephalitis in mice, and its replication in cultured cells is inhibited by the zinc finger antiviral protein (zap), a host factor that specifically inh ...201627581990
temporal variation in sindbis virus antibody prevalence in bird hosts in an endemic area in sweden.sindbis virus (sinv) is a mosquito-borne bird virus that occasionally causes human disease in fennoscandia, suggested to have cyclic 7-year intervals between outbreaks. reliable data on human infections in sweden is however lacking. here we investigated the sinv antibody prevalence among birds in a swedish area endemic to sinv to scrutinize if a cyclic variation in antibody prevalence is present in the natural host of sinv. serum from birds were sampled in the summers of 2002-2004 and 2009 in th ...201627579607
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
cancer immunotherapy via combining oncolytic virotherapy with chemotherapy: recent advances.oncolytic viruses are multifunctional anticancer agents with huge clinical potential, and have recently passed the randomized phase iii clinical trial hurdle. both wild-type and engineered viruses have been selected for targeting of specific cancers, to elicit cytotoxicity, and also to generate antitumor immunity. single-agent oncolytic virotherapy treatments have resulted in modest effects in the clinic. there is increasing interest in their combination with cytotoxic agents, radiotherapy and i ...201627579292
trim14 inhibits hepatitis c virus infection by spry domain-dependent targeted degradation of the viral ns5a protein.tripartite motif 14 (trim14) was reported to function as a mitochondrial signaling adaptor in mediating innate immune responses. however, the involvement of trim14 in host defense against viral infection and molecular mechanisms remain unclear. here, we demonstrated that enforced expression of trim14 could potently inhibit the infection and replication of hcv in hepatocytes, whereas trim14 knockout cells became more susceptible to hcv infection. interestingly, further experiments revealed that s ...201627578425
development of 2, 7-diamino-1, 8-naphthyridine (danp) anchored hairpin primers for rt-pcr detection of chikungunya virus infection.a molecular diagnostic platform with danp-anchored hairpin primer was developed and evaluated for the rapid and cost-effective detection of chikungunya virus (chikv) with high sensitivity and specificity. the molecule 2, 7-diamino-1, 8-naphthyridine (danp) binds to a cytosine-bulge and emits fluorescence at 450 nm when it is excited by 400 nm light. thus, by measuring the decline in fluorescence emitted from danp-primer complexes after pcr reaction, we could monitor the pcr progress. by adapting ...201627571201
consecutive inhibition of isg15 expression and isgylation by cytomegalovirus regulators.interferon-stimulated gene 15 (isg15) encodes an ubiquitin-like protein that covalently conjugates protein. protein modification by isg15 (isgylation) is known to inhibit the replication of many viruses. however, studies on the viral targets and viral strategies to regulate isgylation-mediated antiviral responses are limited. in this study, we show that human cytomegalovirus (hcmv) replication is inhibited by isgylation, but the virus has evolved multiple countermeasures. hcmv-induced isg15 expr ...201627564865
type-1 angiotensin receptor signaling in central nervous system myeloid cells is pathogenic during fatal alphavirus encephalitis in mice.alphaviruses can cause fatal encephalitis in humans. natural infections occur via the bite of infected mosquitos, but aerosol transmissibility makes some of these viruses potential bioterrorism agents. central nervous system (cns) host responses contribute to alphavirus pathogenesis in experimental models and are logical therapeutic targets. we investigated whether reactive oxygen species (ros) generated by nicotinamide adenine dinucleotide phosphate (nadph) oxidase (nox) activity within the cns ...201627562117
the viral capping enzyme nsp1: a novel target for the inhibition of chikungunya virus infection.the chikungunya virus (chikv) has become a substantial global health threat due to its massive re-emergence, the considerable disease burden and the lack of vaccines or therapeutics. we discovered a novel class of small molecules ([1,2,3]triazolo[4,5-d]pyrimidin-7(6h)-ones) with potent in vitro activity against chikv isolates from different geographical regions. drug-resistant variants were selected and these carried a p34s substitution in non-structural protein 1 (nsp1), the main enzyme involve ...201627545976
outrunning the red queen: bystander activation as a means of outpacing innate immune subversion by intracellular pathogens.originally described by the late evolutionary biologist leigh van valen, the red queen hypothesis posits that the evolutionary arms race between hosts and their pathogens selects for discrete, genetically encoded events that lead to competitive advantages over the other species. examples of immune evasion strategies are seen throughout the co-evolution of the mammalian immune system and pathogens, such as the enzymatic inactivation of nuclear factor-κb signaling or host translation by pathogen-e ...201627545071
working with zika and usutu viruses in vitro.usutu (usuv) and zika (zikv) viruses are emerging arboviruses of significant medical and veterinary importance. these viruses have not been studied as well as other medically important arboviruses such as west nile (wnv), dengue (denv), or chikungunya (chikv) viruses. as such, information regarding the behavior of zikv and usuv viruses in the laboratory is dated. usutu virus re-emerged in austria in 2001 and has since spread throughout the european and asian continents causing significant mortal ...201627541001
deah-rha helicase•znf cofactor systems in kinetoplastid rna editing and evolutionarily distant rna processes.multi-zinc finger proteins are an emerging class of cofactors in deah-rha rna helicases across highly divergent eukaryotic lineages. deah-rha helicase•zinc finger cofactor partnerships predate the split of kinetoplastid protozoa, which include several human pathogens, from other eukaryotic lineages 100-400 ma. despite a long evolutionary history, the prototypical deah-rha domains remain highly conserved. this short review focuses on a recently identified deah-rha helicase•zinc finger cofactor sy ...201627540585
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