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rna structures required for production of subgenomic flavivirus rna.flaviviruses are a group of single-stranded, positive-sense rna viruses causing ∼100 million infections per year. we have recently shown that flaviviruses produce a unique, small, noncoding rna (∼0.5 kb) derived from the 3' untranslated region (utr) of the genomic rna (grna), which is required for flavivirus-induced cytopathicity and pathogenicity (g. p. pijlman et al., cell host microbe, 4: 579-591, 2008). this rna (subgenomic flavivirus rna [sfrna]) is a product of incomplete degradation of gr ...201020719943
an rna pseudoknot is required for production of yellow fever virus subgenomic rna by the host nuclease xrn1.cells and mice infected with arthropod-borne flaviviruses produce a small subgenomic rna that is colinear with the distal part of the viral 3'-untranslated region (utr). this small subgenomic flavivirus rna (sfrna) results from the incomplete degradation of the viral genome by the host 5'-3' exonuclease xrn1. production of the sfrna is important for the pathogenicity of the virus. this study not only presents a detailed description of the yellow fever virus (yfv) sfrna but, more importantly, des ...201020739539
identification and characterization of a virus-specific continuous b-cell epitope on the prm/m protein of japanese encephalitis virus: potential application in the detection of antibodies to distinguish japanese encephalitis virus infection from west nile virus and dengue virus infections.differential diagnose of japanese encephalitis virus (jev) infection from other flavivirus especially west nile virus (wnv) and dengue virus (dv) infection was greatly hindered for the serological cross-reactive. virus specific epitopes could benefit for developing jev specific antibodies detection methods. to identify the jev specific epitopes, we fully mapped and characterized the continuous b-cell epitope of the prm/m protein of jev.201020858291
direct interaction between two viral proteins, the nonstructural protein 2c and the capsid protein vp3, is required for enterovirus morphogenesis.in spite of decades-long studies, the mechanism of morphogenesis of plus-stranded rna viruses belonging to the genus enterovirus of picornaviridae, including poliovirus (pv), is not understood. numerous attempts to identify an rna encapsidation signal have failed. genetic studies, however, have implicated a role of the non-structural protein 2c(atpase) in the formation of poliovirus particles. here we report a novel mechanism in which protein-protein interaction is sufficient to explain the spec ...201020865167
dendritic cells transfected with her2 antigen-encoding rna replicons cross-prime cd8 t cells and protect mice against tumor challenge.antigen-specific t cells can be induced by direct priming and cross-priming. to investigate cross-priming as a vaccination approach dendritic cells were transfected with cytopathogenic viral rna-replicons that expressed domains of the tumor-associated her2-antigen and injected into mhc-discordant mice that did not allow direct priming. upon tumor challenge 75% of the vaccinated, but none of the mock-vaccinated mice remained tumor-free. the anti-tumor effect required t cells and correlated with t ...201020887827
evaluation of the influenza a replicon for transient expression of recombinant proteins in mammalian cells.recombinant protein expression in mammalian cells has become a very important technique over the last twenty years. it is mainly used for production of complex proteins for biopharmaceutical applications. transient recombinant protein expression is a possible strategy to produce high quality material for preclinical trials within days. viral replicon based expression systems have been established over the years and are ideal for transient protein expression. in this study we describe the evaluat ...201020949004
structural and functional parameters of the flaviviral protease: a promising antiviral drug target.flaviviruses have a single-strand, positive-polarity rna genome that encodes a single polyprotein. the polyprotein is comprised of seven nonstructural (ns) and three structural proteins. the n- and c-terminal parts of ns3 represent the serine protease and the rna helicase, respectively. the cleavage of the polyprotein by the protease is required to produce the individual viral proteins, which assemble a new viral progeny. conversely, inactivation of the protease blocks viral infection. both the ...201021076642
arboviral diseases and malaria in australia, 2008-09: annual report of the national arbovirus and malaria advisory committee.the national notifiable diseases surveillance system received 8,677 notifications of diseases transmitted by mosquitoes in australia from 1 july 2008 to 30 june 2009. the alphaviruses, barmah forest and ross river, accounted for 6,574 (78%) of these notifications during 2008-09. there were 1,009 notifications of dengue virus infection locally-acquired in north queensland and 484 notified cases resulted from overseas travel. notification rates of dengue virus infection for 2008-09, regardless of ...201021090179
phylogeography of west nile virus: from the cradle of evolution in africa to eurasia, australia, and the americas.west nile virus (wnv) is the most widely distributed of the encephalitic flaviviruses and is a major cause of encephalitis, with isolates obtained from all continents, apart from antarctica. subsequent to its divergence from the other members of the japanese encephalitis virus complex, presumably in africa, wnv has diverged into individual lineages that mostly correspond with geographic distribution. here we elucidate the phylogeography and evolutionary history of isolates from lineage 1 of wnv. ...201021159871
west nile virus differentially modulates the unfolded protein response to facilitate replication and immune evasion.for intracellular survival it is imperative that viruses have the capacity to manipulate various cellular responses, including metabolic and biosynthetic pathways. the unfolded protein response (upr) is induced by various external and internal stimuli, including the accumulation of misfolded proteins in the endoplasmic reticulum (er). our previous studies have indicated that the replication and assembly of the flavivirus west nile virus strain kunjin virus (wnv(kun)) is intimately associated wit ...201021191014
successful propagation of flavivirus infectious cdnas by a novel method to reduce the cryptic bacterial promoter activity of virus genomes.reverse genetics is a powerful tool to study single-stranded rna viruses. despite tremendous efforts having been made to improve the methodology for constructing flavivirus cdnas, the cause of toxicity of flavivirus cdnas in bacteria remains unknown. here we performed mutational analysis studies to identify escherichia coli promoter (ecp) sequences within nucleotides (nt) 1 to 3000 of the dengue virus type 2 (denv2) and japanese encephalitis virus (jev) genomes. eight and four active ecps were d ...201121228244
identification and characterization of the host protein dnajc14 as a broadly active flavivirus replication modulator.viruses in the flavivirus genus of the flaviviridae family are arthropod-transmitted and contribute to staggering numbers of human infections and significant deaths annually across the globe. to identify cellular factors with antiviral activity against flaviviruses, we screened a cdna library using an iterative approach. we identified a mammalian hsp40 chaperone protein (dnajc14) that when overexpressed was able to mediate protection from yellow fever virus (yfv)-induced cell death. further stud ...201121249176
seroprevalence of west nile virus in wild birds in far eastern russia using a focus reduction neutralization test.west nile (wn) virus has been spreading geographically to non-endemic areas in various parts of the world. however, little is known about the extent of wn virus infection in russia. japanese encephalitis (je) virus, which is closely related to wn virus, is prevalent throughout east asia. we evaluated the effectiveness of a focus reduction neutralization test in young chicks inoculated with je and wn viruses, and conducted a survey of wn infection among wild birds in far eastern russia. following ...201121363987
fuse binding protein 1 interacts with untranslated regions of japanese encephalitis virus rna and negatively regulates viral replication.the untranslated regions (utrs) located at the 5' and 3' ends of the japanese encephalitis virus (jev) genome, a positive-sense rna, are involved in viral translation, the initiation of rna synthesis, and the packaging of nascent virions. the cellular and viral proteins that participate in these processes are expected to interact with the utrs. in this study, we used biotinylated rna-protein pulldown and liquid chromatography-mass spectrometry/mass spectrometry (lc-ms/ms) analyses to identify th ...201121367899
identification of a conserved jev serocomplex b-cell epitope by screening a phage-display peptide library with a mab generated against west nile virus capsid protein.the west nile virus (wnv) capsid (c) protein is one of the three viral structural proteins, encapsidates the viral rna to form the nucleocapsid, and is necessary for nuclear and nucleolar localization. the antigenic sites on c protein that are targeted by humoral immune responses have not been studied thoroughly, and well-defined b-cell epitopes on the wnv c protein have not been reported.201121375771
stat2 mediates innate immunity to dengue virus in the absence of stat1 via the type i interferon receptor.dengue virus (denv) is a mosquito-borne flavivirus, and symptoms of infection range from asymptomatic to the severe dengue hemorrhagic fever/dengue shock syndrome (dhf/dss). high viral loads correlate with disease severity, and both type i & ii interferons (ifns) are crucial for controlling viral replication. we have previously reported that signal transducer and activator of transcription (stat) 1-deficient mice are resistant to denv-induced disease, but little is known about this stat1-indepen ...201121379341
assembly of alphavirus replication complexes from rna and protein components in a novel trans-replication system in mammalian cells.for positive-strand rna viruses, the viral genomic rna also acts as an mrna directing the translation of the replicase proteins of the virus. replication takes place in association with cytoplasmic membranes, which are heavily modified to create specific replication compartments. here we have expressed by plasmid dna transfection the large replicase polyprotein of semliki forest virus (sfv) in mammalian cells from a nonreplicating mrna and provided a separate rna containing the replication signa ...201121389137
the capsid-binding nucleolar helicase ddx56 is important for infectivity of west nile virus.recent findings suggest that in addition to its role in packaging genomic rna, the west nile virus (wnv) capsid protein is an important pathogenic determinant, a scenario that requires interaction of this viral protein with host cell proteins. we performed an extensive multitissue yeast two-hybrid screen to identify capsid-binding proteins in human cells. here we describe the interaction between wnv capsid and the nucleolar rna helicase ddx56/noh61. coimmunoprecipitation confirmed that capsid pr ...201121411523
virulence determinants between new york 99 and kunjin strains of west nile virus.an attenuated australian strain of west nile virus (wnv), kunjin (kun), shares ~98% amino acid homology with the pathogenic new york 99 ny99 strain (ny99). to investigate the viral factors involved in ny99 virulence we generated an infectious cdna clone of the wnv ny99 4132 isolate from which virus was recovered and was shown to be indistinguishable from the parental isolate. we then introduced the regions of the ny99 non-structural (ns) proteins and/or untranslated regions (utrs) into the kun b ...201121477835
identification of a cryptic prokaryotic promoter within the cdna encoding the 5' end of dengue virus rna genome.infectious cdna clones of rna viruses are important research tools, but flavivirus cdna clones have proven difficult to assemble and propagate in bacteria. this has been attributed to genetic instability and/or host cell toxicity, however the mechanism leading to these difficulties has not been fully elucidated. here we identify and characterize an efficient cryptic bacterial promoter in the cdna encoding the dengue virus (denv) 5' utr. following cryptic transcription in e. coli, protein express ...201121483867
delayed cytosolic exposure of japanese encephalitis virus double-stranded rna impedes interferon activation and enhances viral dissemination in porcine cells.interferon is a principal component of the host antiviral defense system. in this study, abortive focus formation by japanese encephalitis virus (jev) in primate cells was accompanied with early interferon induction, while productive focus formation in porcine cells was associated with a late interferon response. neutralization antibodies against interferon relieved the restricted infection in primate cells, and increasingly larger foci were generated as treatment with exogenous interferon was d ...201121525349
genetic characterization of west nile virus lineage 2, greece, 2010.we conducted a complete genome analysis of a west nile virus detected in culex pipiens mosquitoes during a severe outbreak of human west nile disease in greece 2010. the virus showed closest genetic relationship to the lineage 2 strain that emerged in hungary in 2004; increased virulence may be associated with amino acid substitution h249p.201121529413
defective interfering viral particles in acute dengue infections.while much of the genetic variation in rna viruses arises because of the error-prone nature of their rna-dependent rna polymerases, much larger changes may occur as a result of recombination. an extreme example of genetic change is found in defective interfering (di) viral particles, where large sections of the genome of a parental virus have been deleted and the residual sub-genome fragment is replicated by complementation by co-infecting functional viruses. while most reports of di particles h ...201121559384
[kunjin virus replicon--a novel viral vector].viral replicon is a kind of self-replicating viral rna sourced from viral genome, which contains viral non-structural genes that are critical for viral genome replication with structural proteins deleted or replaced by foreign genes. kunjin virus is a member of the flavivirida family, flavivirus genus, and kunjin virus replicon is the first and the clearly defined flavivirus replicon. kunjun virus replicon has been regarded as an excellent viral vector on account of its high expression, lower cy ...201121650037
pdna-lipoplexes engrafted with flagellin-related peptide induce potent immunity and anti-tumour effects.complexes of cationic lipids and dna (lipoplexes) are widely used for non-viral gene delivery and dna vaccine development, but cationic lipids are toxic and promote non-specific interactions with cells, leading to poor efficacy. near-neutral lipoplexes, on the other hand, can obviate toxicity, but a convenient means to target them to specific cells such as dendritic cells (dcs) has been lacking. here, we show that a his-tagged flagellin-derived peptide (denoted 9flg), previously reported to prom ...201121798298
genotype v japanese encephalitis virus is emerging.japanese encephalitis (je) is a global public health issue that has spread widely to more than 20 countries in asia and has extended its geographic range to the south pacific region including australia. je has become the most important cause of viral encephalitis in the world. japanese encephalitis viruses (jev) are divided into five genotypes, based on the nucleotide sequence of the envelope (e) gene. the muar strain, isolated from patient in malaya in 1952, is the sole example of genotype v je ...201121750744
identification of two linear b-cell epitopes from west nile virus ns1 by screening a phage-displayed random peptide library.abstract:201121729328
a conserved peptide in west nile virus ns4a protein contributes to proteolytic processing and is essential for replication.the west nile virus strain kunjin virus (wnv(kun)) ns4a protein is a multifunctional protein involved in membrane proliferation, stimulation of cellular pathways, and evasion of host defense and is a major component of the wnv(kun) rna replication complex. we identified a highly conserved region ((120)p-e-p-e(123)) upstream of the viral protease dibasic cleavage site and investigated whether this motif was required for wnv(kun) replication. single point mutations to alanine and a pepe deletion m ...201121880777
the cell biology of the unfolded protein response.the unfolded protein response (upr) is an ensemble of signal transduction pathways that respond to perturbations in the oxidative, pro-folding environment of the endoplasmic reticulum. during the past decade, ongoing research implicated these pathways in maintaining homeostasis of cells and organisms exposed to various stresses. herein, we highlight recent findings regarding the functional role of the upr in both normal and pathophysiologic processes.201121620842
reverse genetic analysis of ourmiaviruses reveals the nucleolar localization of the coat protein in nicotiana benthamiana and unusual requirements for virion formation.ourmia melon virus (oumv) is the type member of the genus ourmiavirus. these viruses have a trisegmented genome, each part of which encodes a single protein. ourmiaviruses share a distant similarity with other plant viruses only in their movement proteins (mp), whereas their rna-dependent rna polymerase (rdrp) shares features only with fungal viruses of the family narnaviridae. thus, ourmiaviruses are in a unique phylogenetic position among existing plant viruses. here, we developed an agroinocu ...201121411534
west nile virus replication requires fatty acid synthesis but is independent on phosphatidylinositol-4-phosphate lipids.west nile virus (wnv) is a neurovirulent mosquito-borne flavivirus, which main natural hosts are birds but it also infects equines and humans, among other mammals. as in the case of other plus-stranded rna viruses, wnv replication is associated to intracellular membrane rearrangements. based on results obtained with a variety of viruses, different cellular processes have been shown to play important roles on these membrane rearrangements for efficient viral replication. as these processes are re ...201121949814
identification of a functional, crm-1-dependent nuclear export signal in hepatitis c virus core protein.hepatitis c virus (hcv) infection is a major cause of chronic liver disease worldwide. hcv core protein is involved in nucleocapsid formation, but it also interacts with multiple cytoplasmic and nuclear molecules and plays a crucial role in the development of liver disease and hepatocarcinogenesis. the core protein is found mostly in the cytoplasm during hcv infection, but also in the nucleus in patients with hepatocarcinoma and in core-transgenic mice. hcv core contains nuclear localization sig ...201122039426
west nile virus (kunjin subtype) disease in the northern territory of australia--a case of encephalitis and review of all reported cases.west nile virus kunjin subtype (wnv/kunv) is enzootic across the tropical north of australia, with epizootic spread into other jurisdictions. the clinical spectrum of illness in humans is poorly described. we report a clinical case of wnv/kunv encephalitis and performed a retrospective chart audit of all cases of wnv/kunv notified in the northern territory from 1992 to 2010. thirteen cases of wnv/kunv disease were identified; case notes were available for 10 of these presentations. six of these ...201122049056
small noncoding rna modulates japanese encephalitis virus replication and translation in trans.sequence and structural elements in the 3'-untranslated region (utr) of japanese encephalitis virus (jev) are known to regulate translation and replication. we previously reported an abundant accumulation of small subgenomic flaviviral rna (sfrna) which is collinear with the highly conserved regions of the 3'-utr in jev-infected cells. however, function of the sfrna in jev life cycle remains unknown.201122040380
the role of interferon antagonist, non-structural proteins in the pathogenesis and emergence of arboviruses.a myriad of factors favor the emergence and re-emergence of arthropod-borne viruses (arboviruses), including migration, climate change, intensified livestock production, an increasing volume of international trade and transportation, and changes to ecosystems (e.g., deforestation and loss of biodiversity). consequently, arboviruses are distributed worldwide and represent over 30% of all emerging infectious diseases identified in the past decade. although some arboviral infections go undetected o ...201121994750
Tembusu virus in ducks, china.In China in 2010, a disease outbreak in egg-laying ducks was associated with a flavivirus. The virus was isolated and partially sequenced. The isolate exhibited 87%-91% identity with strains of Tembusu virus, a mosquito-borne flavivirus of the Ntaya virus group. These findings demonstrate emergence of Tembusu virus in ducks.201122000358
diagnostic strategy used to establish etiologies of encephalitis in a prospective cohort of patients in england.the laboratory diagnostic strategy used to determine the etiology of encephalitis in 203 patients is reported. an etiological diagnosis was made by first-line laboratory testing for 111 (55%) patients. subsequent testing, based on individual case reviews, resulted in 17 (8%) further diagnoses, of which 12 (71%) were immune-mediated and 5 (29%) were due to infection. seventy-five cases were of unknown etiology. sixteen (8%) of 203 samples were found to be associated with either n-methyl-d-asparta ...201121865429
Reduced avian virulence and viremia of West Nile virus isolates from Mexico and Texas.A West Nile virus (WNV) isolate from Mexico (TM171-03) and BIRD1153, a unique genotype from Texas, have exhibited reduced murine neuroinvasive phenotypes. To determine if murine neuroinvasive capacity equates to avian virulence potential, American crow (Corvus brachyrhynchos) and house sparrows (Passer domesticus) were experimentally inoculated with representative murine neuroinvasive/non-neuroinvasive strains. In both avian species, a plaque variant from Mexico that was E-glycosylation competen ...201121976584
flavivirus ns3 and ns5 proteins interaction network: a high-throughput yeast two-hybrid screen.the genus flavivirus encompasses more than 50 distinct species of arthropod-borne viruses, including several major human pathogens, such as west nile virus, yellow fever virus, japanese encephalitis virus and the four serotypes of dengue viruses (denv type 1-4). each year, flaviviruses cause more than 100 million infections worldwide, some of which lead to life-threatening conditions such as encephalitis or haemorrhagic fever. among the viral proteins, ns3 and ns5 proteins constitute the major e ...201122014111
novel flaviviruses detected in different species of mosquitoes in spain.abstract we report the characterization of three novel flaviviruses isolated in spain. marisma mosquito virus, a novel mosquito borne virus, was isolated from ochlerotatus caspius mosquitoes; spanish ochlerotatus flavivirus and spanish culex flavivirus, two novel insect flaviviruses, were isolated from oc. caspius and culex pipiens, respectively. during this investigation, we designed a sensitive rt-nested polymerase chain reaction method that amplifies a 1019bp fragment of the flavivirus ns5 ...201122022811
Globalization, land use, and the invasion of West Nile virus.Many invasive species that have been spread through the globalization of trade and travel are pathogens. A paradigmatic case is the introduction of West Nile virus (WNV) into North America in 1999. A decade of research on the ecology and evolution of WNV includes three findings that provide insight into the outcome of future pathogen introductions. First, WNV transmission in North America is highest in urbanized and agricultural habitats, in part because the hosts and vectors of WNV are abundant ...201122021850
heterogeneous nuclear ribonucleoprotein a2 participates in the replication of japanese encephalitis virus through an interaction with viral proteins and rna.japanese encephalitis virus (jev) is a mosquito-borne flavivirus that is kept in a zoonotic transmission cycle between pigs and mosquitoes. jev causes infection of the central nervous system with a high mortality rate in dead-end hosts, including humans. many studies have suggested that the flavivirus core protein is not only a component of nucleocapsids but also an important pathogenic determinant. in this study, we identified heterogeneous nuclear ribonucleoprotein a2 (hnrnp a2) as a binding p ...201121865391
kunjin virus replicon-based vaccines expressing ebola virus glycoprotein gp protect the guinea pig against lethal ebola virus infection.pre- or postexposure treatments against the filoviral hemorrhagic fevers are currently not available for human use. we evaluated, in a guinea pig model, the immunogenic potential of kunjin virus (kun)-derived replicons as a vaccine candidate against ebola virus (ebov). virus like particles (vlps) containing kun replicons expressing ebov wild-type glycoprotein gp, membrane anchor-truncated gp (gp/ctr), and mutated gp (d637l) with enhanced shedding capacity were generated and assayed for their pro ...201121987742
west nile virus population genetics and evolution.west nile virus (wnv) (flaviviridae: flavivirus) is transmitted from mosquitoes to birds, but can cause fatal encephalitis in infected humans. since its introduction into north america in new york in 1999, it has spread throughout the western hemisphere. multiple outbreaks have also occurred in europe over the last 20 years. this review highlights recent efforts to understand how host pressures impact viral population genetics, genotypic and phenotypic changes which have occurred in the wnv geno ...201122226703
descriptive overview of the 2011 epidemic of arboviral disease in horses in australia.to provide an overview and descriptive analysis of the 2011 arboviral disease epidemic in horses that involved three important australian mosquito-borne viruses: murray valley encephalitis virus, west nile virus (kunjin strain) and ross river virus.201223356366
novel vaccine against venezuelan equine encephalitis combines advantages of dna immunization and a live attenuated vaccine.dna vaccines combine remarkable genetic and chemical stability with proven safety and efficacy in animal models, while remaining less immunogenic in humans. in contrast, live-attenuated vaccines have the advantage of inducing rapid, robust, long-term immunity after a single-dose vaccination. here we describe novel idna vaccine technology that is based on an infectious dna platform and combines advantages of dna and live attenuated vaccines. we applied this technology for vaccination against infe ...201223287629
west nile virus neurologic disease in humans, south africa, september 2008-may 2009.we investigated west nile virus (wnv) as a possible disease etiology in persons hospitalized in south africa. of 206 specimens tested, 36 had wnv neutralizing antibodies, significantly more than in similar earlier serosurveys. seven probable acute wnv cases were identified, suggesting wnv may be overlooked as an etiology of severe disease in south africa.201223171668
highly similar structural frames link the template tunnel and ntp entry tunnel to the exterior surface in rna-dependent rna polymerases.rna-dependent rna polymerase (rdrp) is essential to viral replication and is therefore one of the primary targets of countermeasures against these dangerous infectious agents. development of broad-spectrum therapeutics targeting polymerases has been hampered by the extreme sequence variability of these sequences. rdrps range in length from 400-800 residues, yet contain only ∼20 residues that are conserved in most species. in this study, we made structure-based comparisons that are independent of ...201223275546
etiology of acute, non-malaria, febrile illnesses in jayapura, northeastern papua, indonesia.abstract. we conducted a prospective, inpatient fever study in malaria-endemic papua, indonesia to determine non-malaria fever etiologies. investigations included malaria blood films, blood culture, paired serologic samples analysis for dengue, japanese encephalitis, leptospirosis, scrub typhus, murine typhus, and spotted fever group rickettsia. during 1997-2000, 226 patients (127 males and 99 females) 1-80 years of age (median age = 25 years) were enrolled. positive blood cultures (n = 34, 15%) ...201222232450
Comprehensive mapping of West Nile virus (WNV)- and Japanese encephalitis virus serocomplex-specific linear B-cell epitopes from WNV non-structural protein 1.West Nile virus (WNV) non-structural protein 1 (NS1) elicits protective immune responses during infection of animals. WNV NS1-specific antibody responses can provide the basis for serological diagnostic reagents, so the antigenic sites in NS1 that are targeted by host immune responses need to be identified and the conservation of these sites among the Japanese encephalitis virus (JEV) serocomplex members also needs to be defined. The present study describes the mapping of linear B-cell epitopes ...201221940411
Structural gene (prME) chimeras of St Louis encephalitis virus and West Nile virus exhibit altered in vitro cytopathic and growth phenotypes.Despite utilizing the same avian hosts and mosquito vectors, St Louis encephalitis virus (SLEV) and West Nile virus (WNV) display dissimilar vector-infectivity and vertebrate-pathogenic phenotypes. SLEV exhibits a low oral infection threshold for Culex mosquito vectors and is avirulent in avian hosts, producing low-magnitude viraemias. In contrast, WNV is less orally infective to mosquitoes and elicits high-magnitude viraemias in a wide range of avian species. In order to identify the genetic de ...201221940408
characterization of west nile viruses isolated from captive american flamingoes (phoenicopterus ruber) in medellin, colombia.serum samples from a total of 71 healthy captive birds belonging to 18 species were collected in july of 2008 in medellin (colombia) and tested for flaviviruses. eighteen of 29 samples from american flamingoes (phoenicopterus ruber) were positive for west nile virus (wnv) by reverse transcription-polymerase chain reaction. selected positive samples were serially passaged and wnv was confirmed by immunofluorescence. two isolates (524/08, 9835/08) were characterized in vitro and in vivo. sequence ...201222802436
characterization of rabensburg virus, a flavivirus closely related to west nile virus of the japanese encephalitis antigenic group.rabensburg virus (rabv), a flavivirus with ∼76% nucleotide and 90% amino acid identity with representative members of lineage one and two west nile virus (wnv), previously was isolated from culex pipiens and aedes rossicus mosquitoes in the czech republic, and phylogenetic and serologic analyses demonstrated that it was likely a new lineage of wnv. however, no direct link between rabv and human disease has been definitively established and the extent to which rabv utilizes the typical wnv transm ...201222724010
a noncoding rna produced by arthropod-borne flaviviruses inhibits the cellular exoribonuclease xrn1 and alters host mrna stability.all arthropod-borne flaviviruses generate a short noncoding rna (sfrna) from the viral 3' untranslated region during infection due to stalling of the cellular 5'-to-3' exonuclease xrn1. we show here that formation of sfrna also inhibits xrn1 activity. cells infected with dengue or kunjin viruses accumulate uncapped mrnas, decay intermediates normally targeted by xrn1. xrn1 repression also resulted in the increased overall stability of cellular mrnas in flavivirus-infected cells. importantly, a m ...201223006624
biogenesis and dynamics of the coronavirus replicative structures.coronaviruses are positive-strand rna viruses that are important infectious agents of both animals and humans. a common feature among positive-strand rna viruses is their assembly of replication-transcription complexes in association with cytoplasmic membranes. upon infection, coronaviruses extensively rearrange cellular membranes into organelle-like replicative structures that consist of double-membrane vesicles and convoluted membranes to which the nonstructural proteins involved in rna synthe ...201223202524
a three-dimensional comparison of tick-borne flavivirus infection in mammalian and tick cell lines.tick-borne flaviviruses (tbfv) are sustained in nature through cycling between mammalian and tick hosts. in this study, we used african green monkey kidney cells (vero) and ixodes scapularis tick cells (ise6) to compare virus-induced changes in mammalian and arthropod cells. using confocal microscopy, transmission electron microscopy (tem), and electron tomography (et), we examined viral protein distribution and the ultrastructural changes that occur during tbfv infection. within host cells, fla ...201223112871
new insights into control of arbovirus replication and spread by insect rna interference pathways.arthropod-borne (arbo) viruses are transmitted by vectors, such as mosquitoes, to susceptible vertebrates. recent research has shown that arbovirus replication and spread in mosquitoes is not passively tolerated but induces host responses to control these pathogens. small rna-mediated host responses are key players among these antiviral immune strategies. studies into one such small rna-mediated antiviral response, the exogenous rna interference (rnai) pathway, have generated a wealth of informa ...201226466541
west nile virus noncoding subgenomic rna contributes to viral evasion of the type i interferon-mediated antiviral response.we previously showed that a noncoding subgenomic flavivirus rna (sfrna) is required for viral pathogenicity, as a mutant west nile virus (wnv) deficient in sfrna production replicated poorly in wild-type mice. to investigate the possible immunomodulatory or immune evasive functions of sfrna, we utilized mice and cells deficient in elements of the type i interferon (ifn) response. replication of the sfrna mutant wnv was rescued in mice and cells lacking interferon regulatory factor 3 (irf-3) and ...201222379089
amp-activated kinase restricts rift valley fever virus infection by inhibiting fatty acid synthesis.the cell intrinsic innate immune responses provide a first line of defense against viral infection, and often function by targeting cellular pathways usurped by the virus during infection. in particular, many viruses manipulate cellular lipids to form complex structures required for viral replication, many of which are dependent on de novo fatty acid synthesis. we found that the energy regulator ampk, which potently inhibits fatty acid synthesis, restricts infection of the bunyavirus, rift valle ...201222532801
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
a novel coding-region rna element modulates infectious dengue virus particle production in both mammalian and mosquito cells and regulates viral replication in aedes aegypti mosquitoes.dengue virus (denv) is an enveloped flavivirus with a positive-sense rna genome transmitted by aedes mosquitoes, causing the most important arthropod-borne viral disease affecting humans. relatively few cis-acting rna regulatory elements have been described in the denv coding-region. here, by introducing silent mutations into a denv-2 infectious clone, we identify the conserved capsid-coding region 1 (ccr1), an rna sequence element that regulates viral replication in mammalian cells and to a gre ...201222840606
the contribution of rodent models to the pathological assessment of flaviviral infections of the central nervous system.members of the genus flavivirus are responsible for a spectrum of important neurological syndromes in humans and animals. rodent models have been used extensively to model flavivirus neurological disease, to discover host-pathogen interactions that influence disease outcome, and as surrogates to determine the efficacy and safety of vaccines and therapeutics. in this review, we discuss the current understanding of flavivirus neuroinvasive disease and outline the host, viral and experimental facto ...201222592957
a physical interaction between viral replicase and capsid protein is required for genome-packaging specificity in an rna virus.genome packaging is functionally coupled to replication in rna viruses pathogenic to humans (poliovirus), insects (flock house virus [fhv]), and plants (brome mosaic virus [bmv]). however, the underlying mechanism is not fully understood. we have observed previously that in fhv and bmv, unlike ectopically expressed capsid protein (cp), packaging specificity results from rna encapsidation by cp that has been translated from mrna produced from replicating genomic rna. consequently, we hypothesize ...201222438552
current ebola vaccines.ebolaviruses cause severe viral hemorrhagic fever in humans and non-human primates (nhps), with case fatality rates of up to 90%. currently, neither a specific treatment nor a vaccine licensed for use in humans is available. however, a number of vaccine candidates have been developed in the last decade that are highly protective in nhps, the gold standard animal model for ebola hemorrhagic fever.201222559078
evolution of mosquito-based arbovirus surveillance systems in australia.control of arboviral disease is dependent on the sensitive and timely detection of elevated virus activity or the identification of emergent or exotic viruses. the emergence of japanese encephalitis virus (jev) in northern australia revealed numerous problems with performing arbovirus surveillance in remote locations. a sentinel pig programme detected jev activity, although there were a number of financial, logistical, diagnostic and ethical limitations. a system was developed which detected vir ...201222505808
arboviral diseases and malaria in australia, 2009-10: annual report of the national arbovirus and malaria advisory committee.the national notifiable diseases surveillance system received 7,609 notified cases of disease transmitted by mosquitoes for the season 1 july 2009 to 30 june 2010. the alphaviruses barmah forest virus and ross river virus, accounted for 6,546 (79%) of these notifications during the 2009-10 season. there were 37 notifications of dengue virus infection locally-acquired from north queensland and 581 notified cases in australia that resulted from overseas travel. this number of overseas acquired cas ...201223153083
molecular evolution of lineage 2 west nile virus.since the 1990s west nile virus (wnv) has become an increasingly important public health problem and the cause of outbreaks of neurological disease. genetic analyses have identified multiple lineages with many studies focusing on lineage 1 due to its emergence in new york in 1999 and its neuroinvasive phenotype. until recently, viruses in lineage 2 were not thought to be of public health importance due to few outbreaks of disease being associated with viruses in this lineage. however, recent epi ...201223136360
sequential biogenesis of host cell membrane rearrangements induced by hepatitis c virus infection.like most positive-strand rna viruses, hepatitis c virus (hcv) forms a membrane-associated replication complex consisting of replicating rna, viral and host proteins anchored to altered cell membranes. we used a combination of qualitative and quantitative electron microscopy (em), immuno-em, and the 3d reconstruction of serial em sections to analyze the host cell membrane alterations induced by hcv. three different types of membrane alteration were observed: vesicles in clusters (vics), contiguo ...201223184194
is-98-st1 west nile virus derived from an infectious cdna clone retains neuroinvasiveness and neurovirulence properties of the original virus.infectious clones of west nile virus (wnv) have previously been generated and used to decipher the role of viral proteins in wnv virulence. the majority of molecular clones obtained to date have been derived from north american, australian, or african isolates. here, we describe the construction of an infectious cdna clone of a mediterranean wnv strain, is-98-st1. we characterized the biological properties of the recovered recombinant virus in cell culture and in mice. the growth kinetics of rec ...201223110088
chemoproteomic profiling identifies changes in dna-pk as markers of early dengue virus infection.many cellular factors are regulated via mechanisms affecting protein conformation, localization, and function that may be undetected by most commonly used rna- and protein-based profiling methods that monitor steady-state gene expression. mass-spectrometry-based chemoproteomic profiling provides alternatives for interrogating changes in the functional properties of proteins that occur in response to biological stimuli, such as viral infection. taking dengue virus 2 (dv2) infection as a model sys ...201222999307
cell culture-adaptive mutations promote viral protein-protein interactions and morphogenesis of infectious hepatitis c virus.recent genetic studies suggested that viral nonstructural (ns) proteins play important roles in morphogenesis of flaviviruses, particularly hepatitis c virus (hcv). adaptive and compensatory mutations occurring in different ns proteins were demonstrated to promote hcv production in cell culture. however, the underlying molecular mechanism of ns proteins in hcv morphogenesis is poorly understood. we have isolated a cell culture-adapted hcv of genotype 2a (jfh1) which grew to an infectious titer 3 ...201222674987
genetic characterization of an insect-specific flavivirus isolated from culex theileri mosquitoes collected in southern portugal.we describe the full genetic characterization of an insect-specific flavivirus (isf) from culex theileri (theobald) mosquitoes collected in portugal. this represents the first isolation and full characterization of an isf from portuguese mosquitoes. the virus, designated ctfv, for culex theileri flavivirus, was isolated in the c6/36 stegomyia albopicta (=aedes albopictus) cell line, and failed to replicate in vertebrate (vero) cells in common with other isfs. the ctfv genome encodes a single pol ...201222579596
proteomic profiling of the traf3 interactome network reveals a new role for the er-to-golgi transport compartments in innate immunity.tumor necrosis factor receptor-associated factor-3 (traf3) is a central mediator important for inducing type i interferon (ifn) production in response to intracellular double-stranded rna (dsrna). here, we report the identification of sec16a and p115, two proteins of the er-to-golgi vesicular transport system, as novel components of the traf3 interactome network. notably, in non-infected cells, traf3 was found associated with markers of the er-exit-sites (eres), er-to-golgi intermediate compartm ...201222792062
visualizing coronavirus rna synthesis in time by using click chemistry.coronaviruses induce in infected cells the formation of replicative structures, consisting of double-membrane vesicles (dmvs) and convoluted membranes, where viral rna synthesis supposedly takes place and to which the nonstructural proteins (nsp's) localize. double-stranded rna (dsrna), the presumed intermediate in rna synthesis, is localized to the dmv interior. however, as pores connecting the dmv interior with the cytoplasm have not been detected, it is unclear whether rna synthesis occurs at ...201222438542
small-molecule inhibitors of dengue-virus entry.flavivirus envelope protein (e) mediates membrane fusion and viral entry from endosomes. a low-ph induced, dimer-to-trimer rearrangement and reconfiguration of the membrane-proximal "stem" of the e ectodomain draw together the viral and cellular membranes. we found stem-derived peptides from dengue virus (dv) bind stem-less e trimer and mimic the stem-reconfiguration step in the fusion pathway. we adapted this experiment as a high-throughput screen for small molecules that block peptide binding ...201222496653
dengue virus entry as target for antiviral therapy.dengue virus (denv) infections are expanding worldwide and, because of the lack of a vaccine, the search for antiviral products is imperative. four serotypes of denv are described and they all cause a similar disease outcome. it would be interesting to develop an antiviral product that can interact with all four serotypes, prevent host cell infection and subsequent immune activation. denv entry is thus an interesting target for antiviral therapy. denv enters the host cell through receptor-mediat ...201222529868
kunjin flaviviral encephalomyelitis in an arabian gelding in new south wales, australia.flaviviruses, including kunjin virus, are arboviruses that cause encephalomyelitis in humans and horses. this case report describes an arabian gelding exhibiting neurological signs of flavivirus encephalomyelitis, the diagnostic investigation and confirmation of an unreported case of kunjin virus equine encephalomyelitis in australia.201222827627
recent progress in west nile virus diagnosis and vaccination.west nile virus (wnv) is a positive-stranded rna virus belonging to the flaviviridae family, a large family with 3 main genera (flavivirus, hepacivirus and pestivirus). among these viruses, there are several globally relevant human pathogens including the mosquito-borne dengue virus (denv), yellow fever virus (yfv), japanese encephalitis virus (jev) and west nile virus (wnv), as well as tick-borne viruses such as tick-borne encephalitis virus (tbev). since the mid-1990s, outbreaks of wn fever an ...201222380523
characterization of the 8-hydroxyquinoline scaffold for inhibitors of west nile virus serine protease.west nile virus (wnv) is a mosquito-borne member of flaviviruses that causes significant morbidity and mortality especially among children. there is currently no approved vaccine or antiviral therapeutic for human use. in a previous study, we described compounds containing the 8-hydroxyquinoline (8-hq) scaffold as inhibitors of wnv serine protease (ns2b/ns3pro) in a high throughput screen (hts) using the purified wnv ns2b/ns3pro as the target. in this study, we analyzed potencies of some commerc ...201222343093
comprehensive mapping of common immunodominant epitopes in the west nile virus nonstructural protein 1 recognized by avian antibody responses.west nile virus (wnv) is a mosquito-borne flavivirus that primarily infects birds but occasionally infects humans and horses. certain species of birds, including crows, house sparrows, geese, blue jays and ravens, are considered highly susceptible hosts to wnv. the nonstructural protein 1 (ns1) of wnv can elicit protective immune responses, including ns1-reactive antibodies, during infection of animals. the antigenicity of ns1 suggests that ns1-reactive antibodies could provide a basis for serol ...201222347477
novel atp-independent rna annealing activity of the dengue virus ns3 helicase.the flavivirus nonstructural protein 3 (ns3) bears multiple enzymatic activities and represents an attractive target for antiviral intervention. ns3 contains the viral serine protease at the n-terminus and atpase, rtpase, and helicase activities at the c-terminus. these activities are essential for viral replication; however, the biological role of rna remodeling by ns3 helicase during the viral life cycle is still unclear. secondary and tertiary rna structures present in the viral genome are cr ...201222558403
ivermectin is a potent inhibitor of flavivirus replication specifically targeting ns3 helicase activity: new prospects for an old drug.infection with yellow fever virus (yfv), the prototypic mosquito-borne flavivirus, causes severe febrile disease with haemorrhage, multi-organ failure and a high mortality. moreover, in recent years the flavivirus genus has gained further attention due to re-emergence and increasing incidence of west nile, dengue and japanese encephalitis viruses. potent and safe antivirals are urgently needed.201222535622
approaches for the development of rapid serological assays for surveillance and diagnosis of infections caused by zoonotic flaviviruses of the japanese encephalitis virus serocomplex.flaviviruses are responsible for a number of important mosquito-borne diseases of man and animals globally. the short vireamic period in infected hosts means that serological assays are often the diagnostic method of choice. this paper will focus on the traditional methods to diagnose flaviviral infections as well as describing the modern rapid platforms and approaches for diagnostic antigen preparation.201222570528
blocking double-stranded rna-activated protein kinase pkr by japanese encephalitis virus nonstructural protein 2a.japanese encephalitis virus (jev) is an enveloped flavivirus with a single-stranded, positive-sense rna genome encoding three structural and seven nonstructural proteins. to date, the role of jev nonstructural protein 2a (ns2a) in the viral life cycle is largely unknown. the interferon (ifn)-induced double-stranded rna (dsrna)-activated protein kinase (pkr) phosphorylates the eukaryotic translation initiation factor 2α subunit (eif2α) after sensing viral rna and results in global translation arr ...201222787234
japanese encephalitis virus infects neuronal cells through a clathrin-independent endocytic mechanism.japanese encephalitis virus (jev) is a mosquito-borne pathogenic flavivirus responsible for acute viral encephalitis in humans. the cellular entry of jev is poorly characterized in terms of molecular requirements and pathways. here we present a systematic study of the internalization mechanism of jev in fibroblasts and neuroblastoma cells. to verify the roles of distinct pathways of cell entry, we used fluorescently labeled virus particles, a combination of pharmacological inhibitors, rna interf ...201223055570
limited cross-reactivity of mouse monoclonal antibodies against dengue virus capsid protein among four serotypes.dengue illness is one of the important mosquito-borne viral diseases in tropical and subtropical regions. four serotypes of dengue virus (denv-1, denv-2, denv-3, and denv-4) are classified in the flavivirus genus of the family flaviviridae. we prepared monoclonal antibodies against denv capsid protein from mice immunized with denv-2 and determined the cross-reactivity with each serotype of denv and japanese encephalitis virus.201223209363
a novel bacterium-free method for generation of flavivirus infectious dna by circular polymerase extension reaction allows accurate recapitulation of viral heterogeneity.a novel bacterium-free approach for rapid assembly of flavivirus infectious cdnas using circular polymerase extension reaction was applied to generate infectious cdna for the virulent new south wales isolate of the kunjin strain of west nile virus (kunv) that recently emerged in australia. recovered virus recapitulated the genetic heterogeneity present in the original isolate. the approach was utilized to generate viral mutants with designed phenotypic properties and to identify e protein glycos ...201223236063
Uncoupling cis-Acting RNA Elements from Coding Sequences Revealed a Requirement of the N-Terminal Region of Dengue Virus Capsid Protein in Virus Particle Formation.Little is known about the mechanism of flavivirus genome encapsidation. Here, functional elements of the dengue virus (DENV) capsid (C) protein were investigated. Study of the N-terminal region of DENV C has been limited by the presence of overlapping cis-acting RNA elements within the protein-coding region. To dissociate these two functions, we used a recombinant DENV RNA with a duplication of essential RNA structures outside the C coding sequence. By the use of this system, the highly conserve ...201222072762
west nile virus in the transfusion setting with a special focus on italian preventive measures adopted in 2008-2012 and their impact on blood safety. 201324120610
vaccines in development against west nile virus.west nile encephalitis emerged in 1999 in the united states, then rapidly spread through the north american continent causing severe disease in human and horses. since then, outbreaks appeared in europe, and in 2012, the united states experienced a new severe outbreak reporting a total of 5,387 cases of west nile virus (wnv) disease in humans, including 243 deaths. so far, no human vaccine is available to control new wnv outbreaks and to avoid worldwide spreading. in this review, we discuss the ...201324084235
natural exposure of horses to mosquito-borne flaviviruses in south-east queensland, australia.in 2011 an unprecedented epidemic of equine encephalitis occurred in south-eastern (se) australia following heavy rainfall and severe flooding in the preceding 2-4 months. less than 6% of the documented cases occurred in queensland, prompting the question of pre-existing immunity in queensland horses. a small-scale serological survey was conducted on horses residing in one of the severely flood-affected areas of se-queensland. using a flavivirus-specific blocking-elisa we found that 63% (39/62) ...201324048209
ecology of west nile virus in north america.the introduction, dispersal and establishment of west nile virus in north america were reviewed, focusing on factors that may have enhanced receptivity and enabled the invasion process. the overwintering persistence of this tropical virus within temperate latitudes was unexpected, but was key in the transition from invasion to endemic establishment. the cascade of temporal events allowing sporadic amplification to outbreak levels was discussed within a future perspective.201324008376
identification of conserved motifs in the west nile virus envelope essential for particle secretion.enveloped viruses utilize cellular membranes to bud from infected cells. the process of virion assembly and budding is often facilitated by the presence of certain conserved motifs within viral proteins in conjunction with cellular factors. we hence examined the west nile virus (wnv) envelope protein for the presence of any such motifs and their functional characterization.201324007503
west nile viral infection of equids.west nile virus (wnv) is a flavivirus transmitted between certain species of birds and mosquito vectors. tangential infections of equids and subsequent equine epizootics have occurred historically. although the attack rate has been estimated to be below 10%, mortality rates can approach 50% in horses that present clinical disease. symptoms are most commonly presenting in the form of encephalitis with ataxia as well as limb weakness, recumbency and muscle fasciculation. the most effective strateg ...201324035480
the effect of vaccination coverage and climate on japanese encephalitis in sarawak, malaysia.japanese encephalitis (je) is the leading cause of viral encephalitis across asia with approximately 70,000 cases a year and 10,000 to 15,000 deaths. because je incidence varies widely over time, partly due to inter-annual climate variability effects on mosquito vector abundance, it becomes more complex to assess the effects of a vaccination programme since more or less climatically favourable years could also contribute to a change in incidence post-vaccination. therefore, the objective of this ...201323951373
case definitions, diagnostic algorithms, and priorities in encephalitis: consensus statement of the international encephalitis consortium.encephalitis continues to result in substantial morbidity and mortality worldwide. advances in diagnosis and management have been limited, in part, by a lack of consensus on case definitions, standardized diagnostic approaches, and priorities for research.201323861361
altered protein networks and cellular pathways in severe west nile disease in mice.the recent west nile virus (wnv) outbreaks in developed countries, including europe and the united states, have been associated with significantly higher neuropathology incidence and mortality rate than previously documented. the changing epidemiology, the constant risk of (re-)emergence of more virulent wnv strains, and the lack of effective human antiviral therapy or vaccines makes understanding the pathogenesis of severe disease a priority. thus, to gain insight into the pathophysiological pr ...201323874584
west nile virus. 201324179475
increased pathogenicity of west nile virus (wnv) by glycosylation of envelope protein and seroprevalence of wnv in wild birds in far eastern russia.in this review, we discuss the possibility that the glycosylation of west nile (wn) virus e-protein may be associated with enhanced pathogenicity and higher replication of wn virus. the results indicate that e-protein glycosylation allows the virus to multiply in a heat-stable manner and therefore, has a critical role in enhanced viremic levels and virulence of wn virus in young-chick infection model. the effect of the glycosylation of the e protein on the pathogenicity of wn virus in young chic ...201324351738
west nile virus genome with glycosylated envelope protein and deletion of alpha helices 1, 2, and 4 in the capsid protein is noninfectious and efficiently secretes subviral particles.flavivirus genomes with deletions in the capsid (c) gene are attractive vaccine candidates, as they secrete highly immunogenic subviral particles (svps) without generating infectious virus. here, we report that cytomegalovirus promoter-driven cdna of west nile virus kunjin (kunv) containing a glycosylation motif in the envelope (e) gene and a combined deletion of alpha helices 1, 2, and 4 in c produces significantly more svps than kunv cdnas with nonglycosylated e and various other deletions in ...201324049184
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