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immune interference after sequential alphavirus vaccine vaccinations.we compared the effect of order of administration of investigational alphavirus vaccines on neutralizing antibody response. volunteers who received the inactivated eastern and western equine encephalitis (eee and wee) vaccines before live attenuated venezuelan (vee) vaccine had significantly lower rates of antibody response than those receiving vee vaccine before eee and wee vaccines (66.7% vs. 80.6%; p=0.026). the odds of having a vee antibody non-response among those initially receiving eee an ...200919576665
performance of the realstar chikungunya virus real-time reverse transcription-pcr kit.a novel commercial chikungunya virus real-time reverse transcription-pcr (rt-pcr) kit was evaluated on a comprehensive panel of original patient samples. the assay was 100% sensitive and specific in comparison to a published real-time rt-pcr. viral loads from both assays were highly correlated. the kit proved to be suitable for routine use in patient care.200919625474
environmental and biological factors influencing culex pipiens quinquefasciatus say (diptera: culicidae) vector competence for saint louis encephalitis virus.complex interactions between environmental and biological factors influence the susceptibility of culex pipiens quinquefasciatus to st. louis encephalitis virus and could affect the epidemiology of virus transmission. similar interactions could have epidemiologic implications for other vector-virus systems. we conducted an experiment to examine four such factors in combination: mosquito age, extrinsic incubation temperature (eit), virus dose, and colony. the proportion of mosquitoes with body in ...200919635881
similarities and differences in antagonism of neuron alpha/beta interferon responses by venezuelan equine encephalitis and sindbis alphaviruses.venezuelan equine encephalitis virus (veev) is highly virulent in adult laboratory mice, while sindbis virus (sinv) is avirulent regardless of dose or inoculation route, dependent upon functioning alpha/beta interferon (ifn-alpha/beta) responses. we have examined each virus' resistance to and/or antagonism of ifn-alpha/beta responses in neurons, a cell type targeted by both viruses in mice, by infecting ifn-alpha/beta-treated or untreated primary cultures with viruses or virus-derived replicons ...200919641001
genomic sequencing of highlands j virus: a comparison to western and eastern equine encephalitis viruses.highlands j virus (hjv) is a member of the genus alphavirus, family togaviridae. hjv is the sole representative of the western equine encephalitis (wee) serocomplex found in the eastern united states, and circulates in nature in an apparently identical transmission cycle as eastern equine encephalitis virus (eeev). north american representatives of the wee serocomplex [hjv, wee virus (weev), and fort morgan virus (fmv)] are believed to be derived from a recombination event involving eeev and a s ...200919651164
emerging viral infections of the central nervous system: part 1.in this 2-part review, i will focus on emerging virus infections of the central nervous system (cns). part 1 will introduce the basic features of emerging infections, including their definition, epidemiology, and the frequency of cns involvement. important mechanisms of emergence will be reviewed, including viruses spreading into new host ranges as exemplified by west nile virus (wnv), japanese encephalitis (je) virus, toscana virus, and enterovirus 71 (ev71). emerging infections also result fro ...200919667214
exclusion of west nile virus superinfection through rna replication.superinfection exclusion is the ability of an established viral infection to interfere with a second viral infection. using west nile virus (wnv) as a model, we show that replicating replicons in bhk-21 cells suppress subsequent wnv infection. the wnv replicon also suppresses superinfections of other flaviviruses but not nonflaviviruses. mode-of-action analysis indicates that the exclusion of wnv superinfection occurs at the step of rna synthesis. the continuous culturing of wnv in the replicon- ...200919726510
replication cycle of chikungunya: a re-emerging arbovirus.arboviruses (or arthropod-borne viruses), represent a threat for the new century. the 2005-2006 year unprecedented epidemics of chikungunya virus (chikv) in the french reunion island in the indian ocean, followed by several outbreaks in other parts of the world such as india, have attracted the attention of clinicians, scientists, and state authorities about the risks linked to this re-emerging mosquito-borne virus. chikv, which belongs to the alphaviruses genus, was not previously regarded as a ...200919732931
aedes triseriatus females transovarially infected with la crosse virus mate more efficiently than uninfected mosquitoes.the mating efficiencies (the percentage of females inseminated by males) of field-collected and laboratory-colonized aedes triseriatus (say) (diptera: culicidae) female mosquitoes transovarially infected or uninfected with la crosse virus (lacv) were compared. the females were placed in cages with age-matched males, and the insemination rates (number of inseminated females of the total number of females examined) were determined daily by detection of sperm in the spermathecae. lacv-infected mosq ...200919769048
stone lakes virus (family togaviridae, genus alphavirus), a variant of fort morgan virus isolated from swallow bugs (hemiptera: cimicidae) west of the continental divide.multiple isolates of an alphaviruses within the western equine encephalomyelitis-serocomplex that were related closely to ft. morgan and its variant buggy creek virus were made from swallow bugs, oeciacus vicarius horvath (hemiptera: cimicidae), collected from cliff swallow (petrochelidon pyrrhonota) nests at the stone lakes national wildlife refuge, sacramento county, ca, during the summers of 2005 and 2006. this virus (hereafter stone lakes virus, family togaviridae, genus alphavirus, stlv) wa ...200919769055
encephalitic alphaviruses.this review will cover zoonotic, encephalitic alphaviruses in the family togaviridae. encephalitic alphaviruses, i.e. western- (weev), eastern- (eeev), venezuelan equine encephalitis virus (veev) and, more rarely, ross river virus, chikungunya virus and highlands j virus (hjv), are neuroinvasive and may cause neurological symptoms ranging from mild (e.g., febrile illness) to severe (e.g., encephalitis) in humans and equines. among the naturally occurring alphaviruses, weev, eeev and veev have wi ...201019775836
a multisystem approach for development and evaluation of inactivated vaccines for venezuelan equine encephalitis virus (veev).a multisystem approach was used to assess the efficiency of several methods for inactivation of venezuelan equine encephalitis virus (veev) vaccine candidates. a combination of diverse assays (plaque, in vitro cytopathology and mouse neurovirulence) was used to verify virus inactivation, along with the use of a specific elisa to measure retention of veev envelope glycoprotein epitopes in the development of several inactivated veev candidate vaccines derived from an attenuated strain of veev (v35 ...201019903494
emergence of zoonotic arboviruses by animal trade and migration.abstract: arboviruses are transmitted in nature exclusively or to a major extend by arthropods. they belong to the most important viruses invading new areas in the world and their occurrence is strongly influenced by climatic changes due to the life cycle of the transmitting vectors. several arboviruses have emerged in new regions of the world during the last years, like west nile virus (wnv) in the americas, usutu virus (usuv) in central europe, or rift valley fever virus (rvfv) in the arabian ...201020377873
prevalence and pathology of west nile virus in naturally infected house sparrows, western nebraska, 2008.nestling birds are rarely sampled in the field for most arboviruses, yet they may be important in arbovirus amplification cycles. we sampled both nestling and adult house sparrows (passer domesticus) in western nebraska for west nile virus (wnv) or wnv-specific antibodies throughout the summer of 2008 and describe pathology in naturally infected nestlings. across the summer, 4% of nestling house sparrows were wnv-positive; for the month of august alone, 12.3% were positive. two wnv-positive nest ...201020439979
persistence of buggy creek virus (togaviridae, alphavirus) for two years in unfed swallow bugs (hemiptera: cimicidae: oeciacus vicarius).alphaviruses (togaviridae) have rarely been found to persist for long in the adult insects that serve as their vectors. the ectoparasitic swallow bug (hemiptera: cimicidae: oeciacus vicarius horvath), the vector for buggy creek virus (bcrv; togaviridae, alphavirus), lives year-round in the mud nests of its host, the cliff swallow (petrochelidon pyrrhonota vieillot). we measured the prevalence of bcrv in swallow bugs at sites with cliff swallows present and at the same sites after cliff swallows ...201020496591
detection of all species of the genus alphavirus by reverse transcription-pcr with diagnostic sensitivity.clinical arbovirus screening requires exclusion of a broad range of viruses with as few assays as possible. we present a reverse transcription-pcr (rt-pcr) for the detection of all species of the genus alphavirus qualified for exclusion screening (limit of detection [lod], 5 to 100 rna copies per reaction across all alphavirus species; detection of viremia down to ca. 10,000 copies per ml).201020504990
environmental and biological factors influencing culex pipiens quinquefasciatus (diptera: culicidae) vector competence for west nile virus.interactions between environmental and biological factors affect the vector competence of culex pipiens quinquefasciatus for west nile virus. three age cohorts from two cx. p. quinquefasciatus colonies were fed blood containing a low- or high-virus dose, and each group was held at two different extrinsic incubation temperatures (eit) for 13 days. the colonies differed in the way that they responded to the effects of the environment on vector competence. the effects of mosquito age on aspects of ...201020595491
characterization of dengue virus resistance to brequinar in cell culture.brequinar is an inhibitor of dihydroorotate dehydrogenase, an enzyme that is required for de novo pyrimidine biosynthesis. here we report that brequinar has activity against a broad spectrum of viruses. the compound not only inhibits flaviviruses (dengue virus, west nile virus, yellow fever virus, and powassan virus) but also suppresses a plus-strand rna alphavirus (western equine encephalitis virus) and a negative-strand rna rhabdovirus (vesicular stomatitis virus). using dengue virus serotype ...201020606073
evaluation of antiviral activity of aqueous extracts from achyrocline satureioides against western equine encephalitis virus.achyrocline satureioides (asteraceae) is a medicinal plant traditionally used in argentina for the treatment of intestinal infections and various digestive disorders. its infusion is widely utilised for respiratory problems and viral infections. the objective of this study was to investigate cytotoxicity, virucidal and antiviral properties of the cold aqueous extract (cae) and hot aqueous extract (hae) of this plant against western equine encephalitis virus (weev). cytotoxicity in vero cells was ...201020623427
structure of the recombinant alphavirus western equine encephalitis virus revealed by cryoelectron microscopy.western equine encephalitis virus (weev; togaviridae, alphavirus) is an enveloped rna virus that is typically transmitted to vertebrate hosts by infected mosquitoes. weev is an important cause of viral encephalitis in humans and horses in the americas, and infection results in a range of disease, from mild flu-like illnesses to encephalitis, coma, and death. in addition to spreading via mosquito vectors, human weev infections can potentially occur directly via aerosol transmission. due to its ae ...201020631130
arboviral encephalitides: transmission, emergence, and pathogenesis.arthropod-borne viruses (arboviruses) are of paramount concern as a group of pathogens at the forefront of emerging and re-emerging diseases. although some arboviral infections are asymptomatic or present with a mild influenza-like illness, many are important human and veterinary pathogens causing serious illness ranging from rash and arthritis to encephalitis and hemorrhagic fever. here, we discuss arboviruses from diverse families (flaviviruses, alphaviruses, and the bunyaviridae) that are cau ...201020652430
structure of a venezuelan equine encephalitis virus assembly intermediate isolated from infected cells.venezuelan equine encephalitis virus (veev) is a prototypical enveloped ssrna virus of the family togaviridae. to better understand alphavirus assembly, we analyzed newly formed nucleocapsid particles (termed pre-viral nucleocapsids) isolated from infected cells. these particles were intermediates along the virus assembly pathway, and ultimately bind membrane-associated viral glycoproteins to bud as mature infectious virus. purified pre-viral nucleocapsids were spherical with a unimodal diameter ...201020701942
development of human-murine chimeric immunoglobulin g for use in the serological detection of human flavivirus and alphavirus antibodies.diagnosis of human arboviral infections relies heavily on serological techniques such as the immunoglobulin m (igm) antibody capture enzyme-linked immunosorbent assay (mac-elisa) and the indirect igg elisa. broad application of these assays is hindered by the lack of standardized positive human control sera that react with a wide variety of flaviviruses (e.g., dengue, west nile, yellow fever, japanese encephalitis, saint louis encephalitis, and powassan viruses), or alphaviruses (e.g., eastern e ...201020739503
fast duplex one-step reverse transcriptase pcr for rapid differential detection of west nile and japanese encephalitis viruses.the aim of this study was to develop a highly sensitive and specific one-step duplex reverse transcriptase pcr (rt-pcr) assay for the simultaneous and differential detection of west nile (wnv) and japanese encephalitis (jev) viruses. the bioinformatic analysis of published sequences of wnv and jev revealed conserved regions not targeted by previously reported primers. a total of 13 primers were designed based on these regions to detect all of the wnv and jev lineages and to discriminate between ...201020844215
antibody to the e3 glycoprotein protects mice against lethal venezuelan equine encephalitis virus infection.six monoclonal antibodies were isolated that exhibited specificity for a furin cleavage site deletion mutant (v3526) of venezuelan equine encephalitis virus (veev). these antibodies comprise a single competition group and bound the e3 glycoprotein of veev subtype i viruses but failed to bind the e3 glycoprotein of other alphaviruses. these antibodies neutralized v3526 virus infectivity but did not neutralize the parental strain of trinidad donkey (trd) veev. however, the e3-specific antibodies d ...201020926570
a duplex real-time reverse transcriptase polymerase chain reaction assay for detecting western equine and eastern equine encephalitis viruses.in order to establish an accurate, ready-to-use assay for simultaneous detection of eastern equine encephalitis virus (eeev) and western equine encephalitis virus (weev), we developed one duplex taqman real-time reverse transcriptase polymerase chain reaction (rt-pcr) assay, which can be used in human and vector surveillance. first, we selected the primers and fam-labeled taqman-probe specific for weev from the consensus sequence of nsp3 and the primers and hex-labeled taqman-probe specific for ...201020977706
recovery from viral encephalomyelitis: immune-mediated noncytolytic virus clearance from neurons.viral encephalomyelitis is caused by virus infections of neurons in the brain and spinal cord. recovery is dependent on immune-mediated control and clearance of virus from these terminally differentiated essential cells. preservation of neuronal function is essential for prevention of neurologic sequelae such as paralysis, seizures and cognitive deficits. using the model system of sindbis virus-induced encephalomyelitis in mice, we have shown that immune-mediated clearance of infectious virus fr ...201020087684
pathology and virus detection in tissues of nestling house sparrows naturally infected with buggy creek virus (togaviridae).alphaviruses (togaviridae) infect wild birds, but clinical illness and death attributable to virus in naturally infected birds is rarely reported, particularly for small passerine species or nestlings. buggy creek virus is a unique alphavirus in the western equine encephalomyelitis virus (weev) complex that is vectored by the cimicid swallow bug (oeciacus vicarius), an ectoparasite of the colonially nesting cliff swallow (petrochelidon pyrrhonota) and the introduced house sparrow (passer domesti ...201020090015
transmission potential of two chimeric western equine encephalitis vaccine candidates in culex tarsalis.western equine encephalitis virus (weev) is a zoonotic alphavirus that circulates in western north america between passerine birds and mosquitoes, primarily culex tarsalis. since it was isolated in 1930, weev has caused tens of thousands of equine deaths in addition to thousands of human cases. in addition because weev is a virus of agricultural importance in addition to a public health threat, we developed two live-attenuated chimeric vaccine candidates that have been shown to be immunogenic an ...201020134017
mechanisms of viral emergence.a number of virologic and environmental factors are involved in the emergence and re-emergence of viral disease. viruses do not conservatively occupy a single and permanent ecological niche. rather, due to their intrinsic capacity for genetic change, and to the evolvability of fitness levels, viruses display a potential to parasitize alternative host species. mutation, recombination and genome segment reassortment, and combination of these molecular events, produce complex and phenotypically div ...201020167200
identification of western equine encephalitis virus structural proteins that confer protection after dna vaccination.dna vaccines encoding different portions of the structural proteins of western equine encephalitis virus were tested for the efficacy of their protection in a 100% lethal mouse model of the virus. the 6k-e1 structural protein encoded by the dna vaccine conferred complete protection against challenge with the homologous strain and limited protection against challenge with a heterologous strain.201019923571
a trans-complementing recombination trap demonstrates a low propensity of flaviviruses for intermolecular recombination.intermolecular recombination between the genomes of closely related rna viruses can result in the emergence of novel strains with altered pathogenic potential and antigenicity. although recombination between flavivirus genomes has never been demonstrated experimentally, the potential risk of generating undesirable recombinants has nevertheless been a matter of concern and controversy with respect to the development of live flavivirus vaccines. as an experimental system for investigating the abil ...201019864381
insights into arbovirus evolution and adaptation from experimental studies.arthropod-borne viruses (arboviruses) are maintained in nature by cycling between vertebrate hosts and haematophagous invertebrate vectors. these viruses are responsible for causing a significant public health burden throughout the world, with over 100 species having the capacity to cause human disease. arbovirus outbreaks in previously naïve environments demonstrate the potential of these pathogens for expansion and emergence, possibly exacerbated more recently by changing climates. these recen ...201021994633
midgut barrier imparts selective resistance to filarial worm infection in culex pipiens pipiens.mosquitoes in the culex pipiens complex thrive in temperate and tropical regions worldwide, and serve as efficient vectors of bancroftian lymphatic filariasis (lf) caused by wuchereria bancrofti in asia, africa, the west indies, south america, and micronesia. however, members of this mosquito complex do not act as natural vectors for brugian lf caused by brugia malayi, or for the cat parasite b. pahangi, despite their presence in south asia where these parasites are endemic. previous work with t ...201021072236
single-dose intranasal administration with mdef201 (adenovirus vectored mouse interferon-alpha) confers protection from mortality in a lethal sars-cov balb/c mouse model.interferons (ifns) are a first line of defense against viral infection. herein we describe the use of an adenovirus vectored mouse ifn alpha gene (mdef201) as a prophylactic and treatment countermeasure in a sars-cov-infected balb/c mouse model. complete survival protection was observed in mice given a single dose of mdef201 administered intranasally 1, 3, 5, 7, or 14 days prior to lethal sars-cov challenge (p<0.001), and body weights of these treated mice were unaffected by the challenge. in ad ...201021093489
dissemination of western equine encephalomyelitis virus in the potential vector, culex pipiens pallens.two western equine encephalomyelitis virus (weev) strains have been isolated in china. our previous studies have verified that the mosquito culex pipiens pallens coquillett (diptera: culicidae) infected with weev was capable of transmitting this arbovirus, but it was not clear how the sequential multiplication and spread of virus occurred within the mosquito. in this study, we observed the distribution of weev antigen in orally-infected cx. p. pallens by immunohistochemistry in order to better u ...201021175937
alphavirus transducing system: tools for visualizing infection in mosquito vectors.alphavirus transducing systems (atss) are important tools for expressing genes of interest (goi) during infection. atss are derived from cdna clones of mosquito-borne rna viruses (genus alphavirus; family togaviridae). the alphavirus genus contains about 30 different mosquito-borne virus species. alphaviruses are enveloped viruses and contain single-stranded rna genomes (~11.7 kb). alphaviruses transcribe a subgenomic mrna that encodes the structural proteins of the virus required for encapsidat ...201021178952
review paper: pathology of animal models of alphavirus encephalitis.the encephalitides caused by venezuelan (veev), eastern (eeev), and western (weev) equine encephalitis viruses are important natural diseases of horses and humans and potential agents of biowarfare or bioterrorism. no licensed vaccines or specific therapies exist to prevent or treat human infections with veev, eeev, or weev. well-characterized animal models are needed to support the development of such medical countermeasures under the united states food and drug administration's "animal rule." ...201020551475
temporal connections between culex tarsalis abundance and transmission of western equine encephalomyelitis virus in california.definition of targets for vector control requires an understanding of the relationship between vector abundance and the intensity of arbovirus transmission. using an extensive surveillance dataset with observations from sentinel chicken flocks and mosquito traps paired in time and space, hierarchical autoregressive logistic regression models were developed to predict the probability of seroconversion in chickens for western equine encephalomyelitis virus (weev) based on the relative abundance of ...201020519621
structural basis for substrate specificity of alphavirus nsp2 proteases.the alphavirus nsp2 protease is essential for correct processing of the alphavirus nonstructural polyprotein (nsp1234) and replication of the viral genome. we have combined molecular dynamics simulations with our structural studies to reveal features of the nsp2 protease catalytic site and s1'-s4 subsites that regulate the specificity of the protease. the catalytic mechanism of the nsp2 protease appears similar to the papain-like cysteine proteases, with the conserved catalytic dyad forming a th ...201020483643
treatment with cationic liposome-dna complexes (cldcs) protects mice from lethal western equine encephalitis virus (weev) challenge.having recently characterized a cd-1 outbred mouse model of pathogenesis for western equine encephalitis virus, we examined the possible protective effects of cationic liposome-dna complexes (cldcs) against encephalitic arboviral infection. in this investigation, mice were pre-treated, co-treated, or post-treated with cldc then challenged with a subcutaneous or aerosol dose of the highly virulent weev-mcmillan strain, lethal in mice 4-5 days after inoculation. pre-treatment with cldcs provided a ...201020452378
antiviral compounds in the pipeline to tackle h1n1 influenza infection.the recent pandemic of h1n1 has demonstrated the potential vulnerability of the human population to novel influenza viruses. while there is recent increased interest and effort in developing effective anti-influenza agents, few new products have entered clinical studies. this review will highlight the limited armamentarium of licensed influenza agents, and discuss novel compounds and strategies that have entered clinical studies and may therefore be imminently available to the treating clinician ...201021738286
envelope protein e1 as vaccine target for western equine encephalitis virus.western equine encephalitis virus (weev) is a mosquito-borne rna virus which causes lethal infection in humans and equines. there are no commercial vaccines or anti-weev drugs available for humans. we used replication-defective, human adenovirus serotype-5 (had5) as a delivery vector for developing weev vaccine. our previous study found delivery of both e1 and e2 envelope proteins of weev by had5 vector offers complete protection against lethal challenge of weev. in this paper, we constructed a ...201021084062
serologic evidence of the recent circulation of saint louis encephalitis virus and high prevalence of equine encephalitis viruses in horses in the nhecolândia sub-region in south pantanal, central-west brazil.as in humans, sub-clinical infection by arboviruses in domestic animals is common; however, its detection only occurs during epizootics and the silent circulation of some arboviruses may remain undetected. the objective of the present paper was to assess the current circulation of arboviruses in the nhecolândia sub-region of south pantanal, brazil. sera from a total of 135 horses, of which 75 were immunized with bivalent vaccine composed of inactive eastern equine encephalitis virus (eeev) and w ...201020945001
migratory birds and the dispersal of arboviruses in california.each spring large numbers of neotropical migrants traversing the pacific flyway pass through the coachella valley enroute to northern destinations, providing an opportunity to test the hypothesis that mosquito-borne encephalitis viruses are introduced annually into california by migratory birds. a total of 5,632 sera were collected from 43 species of migrants during spring (april-june), of which 34 (0.61%) comprised of 14 species tested positive by enzyme immunoassay; only 10 were confirmed by p ...201020889869
principles of antidote pharmacology: an update on prophylaxis, post-exposure treatment recommendations and research initiatives for biological agents.the use of biological agents has generally been confined to military-led conflicts. however, there has been an increase in non-state-based terrorism, including the use of asymmetric warfare, such as biological agents in the past few decades. thus, it is becoming increasingly important to consider strategies for preventing and preparing for attacks by insurgents, such as the development of pre- and post-exposure medical countermeasures. there are a wide range of prophylactics and treatments being ...201020860656
why do rna viruses recombine?recombination occurs in many rna viruses and can be of major evolutionary significance. however, rates of recombination vary dramatically among rna viruses, which can range from clonal to highly recombinogenic. here, we review the factors that might explain this variation in recombination frequency and show that there is little evidence that recombination is favoured by natural selection to create advantageous genotypes or purge deleterious mutations, as predicted if recombination functions as a ...201121725337
crystallization, high-resolution data collection and preliminary crystallographic analysis of aura virus capsid protease and its complex with dioxane.the c-terminal protease domain of capsid protein from aura virus expressed in a bacterial expression system has been purified to homogeneity and crystallized. crystals suitable for x-ray diffraction analysis were obtained by the vapour-diffusion method using 0.1 m bis-tris and polyethylene glycol monomethyl ether 2000. crystals of the c-terminal protease domain of capsid protein in complex with dioxane were also produced and crystal data were obtained. both crystals belonged to space group c2, w ...201122102240
a translation inhibitor that suppresses dengue virus in vitro and in vivo.we describe a novel translation inhibitor that has anti-dengue virus (denv) activity in vitro and in vivo. the inhibitor was identified through a high-throughput screening using a denv infection assay. the compound contains a benzomorphan core structure. mode-of-action analysis indicated that the compound inhibits protein translation in a viral rna sequence-independent manner. analysis of the stereochemistry demonstrated that only one enantiomer of the racemic compound inhibits viral rna transla ...201121730119
inhibition of dengue virus through suppression of host pyrimidine biosynthesis.viral replication relies on host to supply nucleosides. host enzymes involved in nucleoside biosynthesis are potential targets for antiviral development. ribavirin (a known antiviral drug) is such an inhibitor that suppresses guanine biosynthesis; depletion of intracellular gtp pool was shown to be the major mechanism to inhibit flavivirus. along a similar concept, inhibitors of pyrimidine biosynthesis pathway could be targeted for potential antiviral development. here we report a novel antivira ...201121507975
validation of assays to monitor immune responses in the syrian golden hamster (mesocricetus auratus).the syrian golden hamster (mesocricetus auratus) is a valuable but under-utilized animal model for studies of human viral pathogens such as bunyaviruses, arenaviruses, flaviviruses, henipaviruses, and sars-coronavirus. a lack of suitable reagents and specific assays for monitoring host responses has limited the use of this animal model to clinical observations, pathology and humoral immune responses. the objective of this study was to establish and validate assays to monitor host immune response ...201121334343
simultaneous detection of rift valley fever, bluetongue, rinderpest, and peste des petits ruminants viruses by a single-tube multiplex reverse transcriptase-pcr assay using a dual-priming oligonucleotide system.the aim of this study was to develop a highly sensitive and specific one-step multiplex reverse transcriptase pcr assay for the simultaneous and differential detection of rift valley fever virus (rvfv), bluetongue virus (btv), rinderpest virus (rpv), and peste des petits ruminants virus (pprv). these viruses cause mucosal lesions in cattle, sheep, and goats, and they are difficult to differentiate from one another based solely on their clinical presentation in suspected disease cases. in this st ...201121307219
Inhibition of dengue virus by targeting viral NS4B protein.We report a novel inhibitor that selectively suppresses dengue virus (DENV) by targeting viral NS4B protein. The inhibitor was identified by screening a 1.8-million-compound library using a luciferase replicon of DENV serotype 2 (DENV-2). The compound specifically inhibits all four serotypes of DENV (50% effective concentration [EC(50)], 1 to 4 µM; and 50% cytotoxic concentration [CC(50)], >40 µM), but it does not inhibit closely related flaviviruses (West Nile virus and yellow fever virus) or n ...201121865382
factors affecting recombinant western equine encephalitis virus glycoprotein production in the baculovirus system.in an effort to produce processed, soluble western equine encephalitis virus (weev) glycoproteins for subunit therapeutic vaccine studies, we isolated twelve recombinant baculoviruses designed to express four different weev glycoprotein constructs under the transcriptional control of three temporally distinct baculovirus promoters. the weev glycoprotein constructs encoded full-length e1, the e1 ectodomain, an e26ke1 polyprotein precursor, and an artificial, secretable e2e1 chimera. the three dif ...201121864686
chimeric chikungunya viruses are nonpathogenic in highly sensitive mouse models but efficiently induce a protective immune response.chikungunya virus (chikv) is an important pathogen causing outbreaks of highly debilitating and often chronic, arthralgic human disease. we have designed chimeric alphaviruses encoding chikv-specific structural proteins but no structural or nonstructural proteins capable of interfering with development of cellular antiviral response. these chimeras demonstrate a highly attenuated phenotype in both immunocompetent and immunocompromised (a129) mice. however, after a single vaccination, they induce ...201121697494
replication of alphaviruses: a review on the entry process of alphaviruses into cells.alphaviruses are small, enveloped viruses, ~70 nm in diameter, containing a single-stranded, positive-sense, rna genome. viruses belonging to this genus are predominantly arthropod-borne viruses, known to cause disease in humans. their potential threat to human health was most recently exemplified by the 2005 chikungunya virus outbreak in la reunion, highlighting the necessity to understand events in the life-cycle of these medically important human pathogens. the replication and propagation of ...201122312336
temporal and spatial alterations in mutant swarm size of st. louis encephalitis virus in mosquito hosts.st. louis encephalitis virus (slev; flaviviridae; flavivirus) is a member of the japanese encephalitis serocomplex and a close relative of west nile virus (wnv). although slev remains endemic to the us, both levels of activity and geographical dispersal are relatively constrained when compared to the widespread distribution of wnv. in recent years, wnv appears to have displaced slev in california, yet both viruses currently coexist in texas and several other states. it has become clear that vira ...201121215334
variation in western equine encephalomyelitis viral strain growth in mammalian, avian, and mosquito cells fails to explain temporal changes in enzootic and epidemic activity in california.the decrease in western equine encephalomyelitis virus (weev; togaviridae, alphavirus) activity in north america over the past 20-30 years has prompted research to determine if there have been concurrent declines in virulence. six (weev) strains isolated from culex tarsalis mosquitoes from california during each of the six preceding decades failed to show a consistent declining temporal trend in virus titer using mosquito (c6/36), avian (duck embryo fibroblast), or mammalian (vero) cells, result ...201121395409
fatal human case of western equine encephalitis, uruguay.to the editor: the genus alphavirus (family togaviridae) comprises 29 viral species (1), grouped in at least 7 antigenic complexes by their serologic cross-reactivity (2). they are maintained in nature through enzootic cycles involving arthropods as vectors with subsequent amplification in small mammals or birds, and epizootic cycles between mosquitoes and large mammals such as horses or humans.201121529429
transmission potential of two chimeric chikungunya vaccine candidates in the urban mosquito vectors, aedes aegypti and ae. albopictus.abstract. chikungunya virus (chikv) is an emerging, mosquito-borne alphavirus that has caused major epidemics in africa and asia. we developed chimeric vaccine candidates using the non-structural protein genes of either venezuelan equine encephalitis virus (veev) attenuated vaccine strain tc-83 or a naturally attenuated strain of eastern equine encephalitis virus (eeev) and the structural genes of chikv. because the transmission of genetically modified live vaccine strains is undesirable because ...201121633043
viruses in reptiles.abstract: the etiology of reptilian viral diseases can be attributed to a wide range of viruses occurring across different genera and families. thirty to forty years ago, studies of viruses in reptiles focused mainly on the zoonotic potential of arboviruses in reptiles and much effort went into surveys and challenge trials of a range of reptiles with eastern and western equine encephalitis as well as japanese encephalitis viruses. in the past decade, outbreaks of infection with west nile virus i ...201121933449
Therapy and long-term prophylaxis of vaccinia virus respiratory infections in mice with an adenovirus-vectored interferon alpha (mDEF201).An adenovirus 5 vector encoding for mouse interferon alpha, subtype 5 (mDEF201) was evaluated for efficacy against lethal vaccinia virus (WR strain) respiratory infections in mice. mDEF201 was administered as a single intranasal treatment either prophylactically or therapeutically at doses of 10(6) to 10(8) plaque forming units/mouse. When the prophylactic treatment was given at 56 days prior to infection, it protected 90% of animals from death (100% protection for treatments given between 1-49 ...201122022603
tick cell lines for study of crimean-congo hemorrhagic fever virus and other arboviruses.abstract continuous cell lines derived from many of the vectors of tick-borne arboviruses of medical and veterinary importance are now available. their role as tools in arbovirus research to date is reviewed and their potential application in studies of tick cell responses to virus infection is explored, by comparison with recent progress in understanding mosquito immunity to arbovirus infection. a preliminary study of propagation of the human pathogen crimean-congo hemorrhagic fever virus (cc ...201121955214
The costs of infection and resistance as determinants of West Nile virus susceptibility in Culex mosquitoes.Understanding the phenotypic consequences of interactions between arthropod-borne viruses (arboviruses) and their mosquito hosts has direct implications for predicting the evolution of these relationships and the potential for changes in epidemiological patterns. Although arboviruses are generally not highly pathogenic to mosquitoes, pathology has at times been noted. Here, in order to evaluate the potential costs of West Nile virus (WNV) infection and resistance in a primary WNV vector, and to ...201121975028
development of a human-murine chimeric immunoglobulin m for use in the serological detection of human alphavirus antibodies.diagnosis of human alphaviral infections relies on serological techniques, such as the immunoglobulin m antibody capture-enzyme-linked immunosorbent assay (mac-elisa). we have humanized the alphavirus broadly cross-reactive murine monoclonal antibody 1a4b-6 to create a reagent capable of replacing human positive sera in the mac-elisa for diagnosis of human alphaviral infections.201121976225
Metagenomic analysis of fever, thrombocytopenia and leukopenia syndrome (FTLS) in Henan Province, China: discovery of a new bunyavirus.Since 2007, many cases of fever, thrombocytopenia and leukopenia syndrome (FTLS) have emerged in Henan Province, China. Patient reports of tick bites suggested that infection could contribute to FTLS. Many tick-transmitted microbial pathogens were tested for by PCR/RT-PCR and/or indirect immunofluorescence assay (IFA). However, only 8% (24/285) of samples collected from 2007 to 2010 tested positive for human granulocytic anaplasmosis (HGA), suggesting that other pathogens could be involved. Here ...201122114553
SH3 domain-mediated recruitment of host cell amphiphysins by alphavirus nsP3 promotes viral RNA replication.Among the four non-structural proteins of alphaviruses the function of nsP3 is the least well understood. NsP3 is a component of the viral replication complex, and composed of a conserved aminoterminal macro domain implicated in viral RNA synthesis, and a poorly conserved carboxyterminal region. Despite the lack of overall homology we noted a carboxyterminal proline-rich sequence motif shared by many alphaviral nsP3 proteins, and found it to serve as a preferred target site for the Src-homology ...201122114558
rapid molecular detection methods for arboviruses of livestock of importance to northern europe.arthropod-borne viruses (arboviruses) have been responsible for some of the most explosive epidemics of emerging infectious diseases over the past decade. their impact on both human and livestock populations has been dramatic. the early detection either through surveillance or diagnosis of virus will be a critical feature in responding and resolving the emergence of such epidemics in the future. although some of the most important emerging arboviruses are human pathogens, this paper aims to high ...201222219660
mayaro virus infection in traveler returning from amazon basin, northern peru. 201222469145
effects of virus dose and extrinsic incubation temperature on vector competence of culex nigripalpus (diptera: culicidae) for st. louis encephalitis virus.culex nigripalpus theobald is a primary vector of st. louis encephalitis virus in the southeastern united states. cx. nigripalpus females were fed blood containing a low (4.0 +/- 0.01 log10 plaque-forming unit equivalents (pfueq) /ml) or high (4.7 +/- 0.1 log10 pfueq/ml) st. louis encephalitis virus dose and maintained at extrinsic incubation temperatures (eit) of 25 or 28 degrees c for 12 d. vector competence was measured via quantitative real-time reverse transcriptase polymerase chain reactio ...201223270182
health assessment and seroepidemiologic survey of potential pathogens in wild antillean manatees (trichechus manatus manatus).the antillean manatee (trichechus manatus manatus), a subspecies of the west indian manatee, inhabits fresh, brackish, and warm coastal waters distributed along the eastern border of central america, the northern coast of south america, and throughout the wider caribbean region. threatened primarily by human encroachment, poaching, and habitat degradation, antillean manatees are listed as endangered by the international union for the conservation of nature. the impact of disease on population vi ...201222984521
attenuation of chikungunya virus vaccine strain 181/clone 25 is determined by two amino acid substitutions in the e2 envelope glycoprotein.chikungunya virus (chikv) is the mosquito-borne alphavirus that is the etiologic agent of massive outbreaks of arthralgic febrile illness that recently affected millions of people in africa and asia. the only chikv vaccine that has been tested in humans, strain 181/clone 25, is a live-attenuated derivative of southeast asian human isolate strain af15561. the vaccine was immunogenic in phase i and ii clinical trials; however, it induced transient arthralgia in 8% of the vaccinees. there are five ...201222457519
interplay between viruses and host mrna degradation.messenger rna degradation is a fundamental cellular process that plays a critical role in regulating gene expression by controlling both the quality and the abundance of mrnas in cells. naturally, viruses must successfully interface with the robust cellular rna degradation machinery to achieve an optimal balance between viral and cellular gene expression and establish a productive infection in the host. in the past several years, studies have discovered many elegant strategies that viruses have ...201223274304
effects of larval rearing temperature on immature development and west nile virus vector competence of culex tarsalis.temperature is known to induce changes in mosquito physiology, development, ecology, and in some species, vector competence for arboviruses. since colonized mosquitoes are reared under laboratory conditions that can be significantly different from their field counterparts, laboratory vector competence experiments may not accurately reflect natural vector-virus interactions.201222967798
novel inhibitors of neurotropic alphavirus replication that improve host survival in a mouse model of acute viral encephalitis.arboviral encephalitis is a potentially devastating human disease with no approved therapies that target virus replication. we previously discovered a novel class of thieno[3,2-b]pyrrole-based inhibitors active against neurotropic alphaviruses such as western equine encephalitis virus (weev) in cultured cells. in this report, we describe initial development of these novel antiviral compounds, including bioisosteric replacement of the 4h-thieno[3,2-b]pyrrole core with indole to improve metabolic ...201222428985
isolates of liao ning virus from wild-caught mosquitoes in the xinjiang province of china in 2005.liao ning virus (lnv) is related to banna virus, a known human-pathogen present in south-east asia. both viruses belong to the genus seadornavirus, family reoviridae. lnv causes lethal haemorrhage in experimentally infected mice. twenty seven isolates of lnv were made from mosquitoes collected in different locations within the xinjiang province of north-western china during 2005. these mosquitoes were caught in the accommodation of human patients with febrile manifestations, or in animal barns w ...201222649554
the alphavirus e3 glycoprotein functions in a clade-specific manner.the 80 trimeric, glycoprotein spikes that cover the surface of alphavirus particles are required for mediating viral entry into a host cell. spike assembly is a regulated process that requires interactions between five structural proteins, e3, e2, 6k and its translational frameshift product tf, and e1. e3 is a small, ∼65-amino-acid glycoprotein that has two known functions: e3 serves as the signal sequence for translocation of the e3-e2-6k-e1 polyprotein into the endoplasmic reticulum (er), and ...201223035234
serological evidence of flaviviruses and alphaviruses in livestock and wildlife in trinidad.seroprevalence rates of selected arboviruses in animal populations in trinidad were determined using serum samples collected between 2006 and 2009 from horses (n=506), cattle (n=163), sheep (n=198), goats (n=82), pigs (n=184), birds (n=140), rodents (n=116), and other vertebrates (n=23). the sera were screened for antibodies to west nile virus (wnv), st. louis encephalitis virus (slev), ilheus virus (ilhv), bussuquara virus (bsqv), venezuelan equine encephalitis virus (veev), eastern equine ence ...201222989182
dephosphorylation of hur protein during alphavirus infection is associated with hur relocalization to the cytoplasm.we have demonstrated previously that the cellular hur protein binds u-rich elements in the 3' untranslated region (utr) of sindbis virus rna and relocalizes from the nucleus to the cytoplasm upon sindbis virus infection in 293t cells. in this study, we show that two alphaviruses, ross river virus and chikungunya virus, lack the conserved high-affinity u-rich hur binding element in their 3' utrs but still maintain the ability to interact with hur with nanomolar affinities through alternative bind ...201222915590
global climate change and its potential impact on disease transmission by salinity-tolerant mosquito vectors in coastal zones.global climate change can potentially increase the transmission of mosquito vector-borne diseases such as malaria, lymphatic filariasis, and dengue in many parts of the world. these predictions are based on the effects of changing temperature, rainfall, and humidity on mosquito breeding and survival, the more rapid development of ingested pathogens in mosquitoes and the more frequent blood feeds at moderately higher ambient temperatures. an expansion of saline and brackish water bodies (water wi ...201222723781
a review of the invasive mosquitoes in europe: ecology, public health risks, and control options.there has been growing interest in europe in recent years in the establishment and spread of invasive mosquitoes, notably the incursion of aedes albopictus through the international trade in used tires and lucky bamboo, with onward spread within europe through ground transport. more recently, five other non-european aedine mosquito species have been found in europe, and in some cases populations have established locally and are spreading. concerns have been raised about the involvement of these ...201222448724
isolation and characterisation of a human-like antibody fragment (scfv) that inactivates veev in vitro and in vivo.venezuelan equine encephalitis virus (veev) belongs to the alphavirus genus and several species of this family are pathogenic to humans. the viruses are classified as potential agents of biological warfare and terrorism and sensitive detection as well as effective prophylaxis and antiviral therapies are required.in this work, we describe the isolation of the anti-veev single chain fragment variable (scfv), tor67-3b4, from a non-human primate (nhp) antibody gene library. we report its recloning i ...201222666347
rapid, specific detection of alphaviruses from tissue cultures using a replicon-defective reporter gene assay.we established a rapid, specific technique for detecting alphaviruses using a replicon-defective reporter gene assay derived from the sindbis virus xj-160. the pvaxj expression vector containing the xj-160 genome was engineered to form the expression vectors pvaxj-egfp expressing enhanced green fluorescence protein (egfp) or pvaxj-gluc expressing gaussia luciferase (gluc). the replicon-defective reporter plasmids pvaxj-egfpδnsp4 and pvaxj-glucδnsp4 were constructed by deleting 1139 bp in the non ...201222427930
development of an elisa-array for simultaneous detection of five encephalitis viruses.japanese encephalitis virus(jev), tick-borne encephalitis virus(tbev), and eastern equine encephalitis virus (eeev) can cause symptoms of encephalitis. establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. currently, there are still no multiple antigen detection methods available clinically. an elisa-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potent ...201222369052
impact of chikungunya virus on aedes albopictus females and possibility of vertical transmission using the actors of the 2007 outbreak in italy.we investigated the impact of chikv strains on some aedes albopictus (skuse) reproductive parameters and the possibility of vertical transmission. two strains were collected in the area where the epidemic occurred in 2007, one isolated from mosquitoes, the other one isolated from a viraemic patient. different types of blood meals, either infected or non-infected, were offered to ae. albopictus females, that were then analyzed at increasing time post infection. the virus titre, measured by two rt ...201222383945
specific and nonspecific host adaptation during arboviral experimental evolution.during the past decade or so, there has been a substantial body of work to dissect arboviral evolution and to develop models of adaptation during host switching. regardless of what species serve as host or vectors, and of the geographic distribution and the mechanisms of replication, arboviruses tend to have slow evolutionary rates in nature. the hypothesis that this is the result of replication in the disparate environments provided by host and vector did not receive solid experimental support ...201222248544
adaptive changes in alphavirus mrna translation allowed colonization of vertebrate hosts.members of the alphavirus genus are arboviruses that alternate replication in mosquitoes and vertebrate hosts. in vertebrate cells, the alphavirus resists the activation of antiviral rna-activated protein kinase (pkr) by the presence of a prominent rna structure (downstream loop [dlp]) located in viral 26s transcripts, which allows an eif2-independent translation initiation of these mrnas. this article shows that dlp structure is essential for replication of sindbis virus (sinv) in vertebrate ce ...201222761388
cell-based analysis of chikungunya virus e1 protein in membrane fusion.chikungunya fever is a pandemic disease caused by the mosquito-borne chikungunya virus (chikv). e1 glycoprotein mediation of viral membrane fusion during chikv infection is a crucial step in the release of viral genome into the host cytoplasm for replication. how the e1 structure determines membrane fusion and whether other chikv structural proteins participate in e1 fusion activity remain largely unexplored.201222520648
neurotropic arboviruses induce interferon regulatory factor 3-mediated neuronal responses that are cytoprotective, interferon independent, and inhibited by western equine encephalitis virus capsid.cell-intrinsic innate immune responses mediated by the transcription factor interferon regulatory factor 3 (irf-3) are often vital for early pathogen control, and effective responses in neurons may be crucial to prevent the irreversible loss of these critical central nervous system cells after infection with neurotropic pathogens. to investigate this hypothesis, we used targeted molecular and genetic approaches with cultured neurons to study cell-intrinsic host defense pathways primarily using t ...201223192868
in memoriam: dr h fred clark (1937-2012).dr h fred clark, a colleague and friend, passed away on april 28, 2012. drs frederick murphy, stanley plotkin, and paul offit, who knew fred at different stages of his career, recall his life.201222906941
b cell maintenance of subcapsular sinus macrophages protects against a fatal viral infection independent of adaptive immunity.neutralizing antibodies have been thought to be required for protection against acutely cytopathic viruses, such as the neurotropic vesicular stomatitis virus (vsv). utilizing mice that possess b cells but lack antibodies, we show here that survival upon subcutaneous (s.c.) vsv challenge was independent of neutralizing antibody production or cell-mediated adaptive immunity. however, b cells were absolutely required to provide lymphotoxin (lt) α1β2, which maintained a protective subcapsular sinus ...201222386268
phage display identifies an eastern equine encephalitis virus glycoprotein e2-specific b cell epitope.the present study identified a linear b-cell epitope in the eastern equine encephalitis virus (eeev) e2 glycoprotein by screening a phage-displayed random 12-mer peptide library using an eeev e2 specific monoclonal antibody (mab) 7c11 and defined l/f-e/r-y-t-w-g/r-n-h/w-p as the consensus binding motif. a sequence ((321)egleytwgnhpp(332)) encompassing this consensus motif was found in the eeev e2 glycoprotein and synthesized for further epitope confirmation. meanwhile, the corresponding epitope ...201222824180
vector competence of five common mosquito species in the people's republic of china for western equine encephalitis virus.two strains of the western equine encephalitis virus (weev) were first detected and isolated in china in 2001. the maintenance and transmission cycles of weev in china are currently not well understood, and the mosquito vectors involved in these cycles are unknown. to understand the ability of the local mosquitoes in china to transmit weev, the vector competence of five mosquito species, namely, culex pipiens pallens coquillett, cx. p. quinquefasciatus say, aedes (stegomyia) albopictus skuse, ae ...201222276651
assessment of plaque assay methods for alphaviruses.viruses from the alphavirus genus are responsible for numerous arboviral diseases impacting human health throughout the world. confirmation of acute alphavirus infection is based on viral isolation, identification of viral rna, or a fourfold or greater increase in antibody titers between acute and convalescent samples. in convalescence, the specificity of antibodies to an alphavirus may be confirmed by plaque reduction neutralization test. to identify the best method for alphavirus and neutraliz ...201323085307
validation of a cell-based elisa as a screening tool identifying anti-alphavirus small-molecule inhibitors.venezuelan (veev), eastern, and western equine encephalitis viruses, members of the genus alphavirus, are causative agents of debilitative and sometimes fatal encephalitis. although human cases are rare, these viruses pose a threat to military personnel, and to public health, due to their potential use as bioweapons. currently, there are no licensed therapeutics for treating alphavirus infections. to address this need, small-molecules with potential anti-alphavirus activity, provided by collabor ...201323764417
ten years of dengue drug discovery: progress and prospects.to combat neglected diseases, the novartis institute of tropical diseases (nitd) was founded in 2002 through private-public funding from novartis and the singapore economic development board. one of nitd's missions is to develop antivirals for dengue virus (denv), the most prevalent mosquito-borne viral pathogen. neither vaccine nor antiviral is currently available for denv. here we review the progress in dengue drug discovery made at nitd as well as the major discoveries made by academia and ot ...201324076358
activation of the type i interferon pathway is enhanced in response to human neuronal differentiation.despite the crucial role of innate immunity in preventing or controlling pathogen-induced damage in most, if not all, cell types, very little is known about the activity of this essential defense system in central nervous system neurons, especially in humans. in this report we use both an established neuronal cell line model and an embryonic stem cell-based system to examine human neuronal innate immunity and responses to neurotropic alphavirus infection in cultured cells. we demonstrate that ne ...201323505563
discovery of potent broad spectrum antivirals derived from marine actinobacteria.natural products provide a vast array of chemical structures to explore in the discovery of new medicines. although secondary metabolites produced by microbes have been developed to treat a variety of diseases, including bacterial and fungal infections, to date there has been limited investigation of natural products with antiviral activity. in this report, we used a phenotypic cell-based replicon assay coupled with an iterative biochemical fractionation process to identify, purify, and characte ...201324349254
effects of nasal or pulmonary delivered treatments with an adenovirus vectored interferon (mdef201) on respiratory and systemic infections in mice caused by cowpox and vaccinia viruses.an adenovirus 5 vector encoding for mouse interferon alpha, subtype 5 (mdef201) was evaluated for efficacy against lethal cowpox (brighton strain) and vaccinia (wr strain) virus respiratory and systemic infections in mice. two routes of mdef201 administration were used, nasal sinus (5-µl) and pulmonary (50-µl), to compare differences in efficacy, since the preferred treatment of humans would be in a relatively small volume delivered intranasally. lower respiratory infections (lri), upper respira ...201323874722
west nile virus drug discovery.the outbreak of west nile virus (wnv) in 1999 in the usa, and its continued spread throughout the americas, parts of europe, the middle east and africa, underscored the need for wnv antiviral development. here, we review the current status of wnv drug discovery. a number of approaches have been used to search for inhibitors of wnv, including viral infection-based screening, enzyme-based screening, structure-based virtual screening, structure-based rationale design, and antibody-based therapy. th ...201324300672
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