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wicking assays for the rapid detection of west nile and st. louis encephalitis viral antigens in mosquitoes (diptera: culicidae).the recent outbreaks of west nile (wn) encephalitis and st. louis encephalitis (sle) in the united states have highlighted the need for rapid and specific methods of detecting arboviral antigens in mosquitoes. we evaluated rapid, field-usable assays for detecting and differentiating wn and sle viruses in mosquito pools, based on a patent-pending, immunochromatographic technology (vectest) formatted on a dipstick. the device provides results in less than 20 min and can be used in laboratories wit ...200312597660
epidemiology of hepatitis c virus infection in australia.since the discovery in 1989 of hepatitis c virus (hcv) as the infectious agent responsible for the vast majority of post-transfusion non-a non-b hepatitis the patterns of transmission and clinical consequences of this highly prevalent flavivirus have been widely studied.200312600649
tick-borne encephalitis.tick-borne encephalitis (tbe) is one of the most dangerous human infections occurring in europe and many parts of asia. the etiological agent tick-borne encephalitis virus (tbev), is a member of the virus genus flavivirus, of the family flaviviridae. tbev is believed to cause at least 11,000 human cases of encephalitis in russia and about 3000 cases in the rest of europe annually. related viruses within the same group, louping ill virus (liv), langat virus (lgtv) and powassan virus (powv), also ...200312615309
enhancing biosynthesis and secretion of premembrane and envelope proteins by the chimeric plasmid of dengue virus type 2 and japanese encephalitis virus.we have constructed a series of plasmids encoding premembrane (prm) and envelope (e) protein genes of dengue virus type 2 (den-2). these plasmids included an authentic den-2 prm-e construct (pcbd2-14-6), and two chimeric constructs, 90% den-2 e-10% japanese encephalitis (je) virus e (pcb9d2-1j-4-3) and 80% den-2 e-20% je e (pcb8d2-2j-2-9-1). monoclonal antibody (mab) reactivity indicated that all three plasmids expressed authentic den-2 virus e protein epitopes representative of flavivirus domai ...200312620809
epitope-blocking enzyme-linked immunosorbent assays for the detection of serum antibodies to west nile virus in multiple avian species.we report the development of epitope-blocking enzyme-linked immunosorbent assays (elisas) for the rapid detection of serum antibodies to west nile virus (wnv) in taxonomically diverse north american avian species. a panel of flavivirus-specific monoclonal antibodies (mabs) was tested in blocking assays with serum samples from wnv-infected chickens and crows. selected mabs were further tested against serum samples from birds that represented 16 species and 10 families. serum samples were collecte ...200312624027
steady-state cleavage kinetics for dengue virus type 2 ns2b-ns3(pro) serine protease with synthetic peptides.the n-terminal part of the ns3 protein from dengue virus contains a trypsin-like serine protease responsible for processing the nonstructural region of the viral polyprotein. enzymatic activity of the ns2b-ns3(pro) precursor incorporating a full-length ns2b cofactor of dengue virus type 2 was examined by using synthetic dodecamer peptide substrates encompassing native cleavage sequences of the ns2a/ns2b, ns2b/ns3, ns3/ns4a and ns4b/ns5 polyprotein junctions. cleavage of the dansylated substrates ...200312625822
role of type i and type ii interferon responses in recovery from infection with an encephalitic flavivirus.we have investigated the contribution of the interferon (ifn)-alpha/beta system, ifn-gamma and nitric oxide to recovery from infection with murray valley encephalitis virus, using a mouse model for flaviviral encephalitis where a small dose of virus was administered to 6-week-old wild-type and gene knockout animals by the intravenous route. we show that a defect in the ifn-alpha/beta responses results in uncontrolled extraneural virus growth, rapid virus entry into the brain and 100 % mortality. ...200312604807
role of the transmembrane domains of prm and e proteins in the formation of yellow fever virus envelope.flavivirus envelope proteins have been shown to play a major role in virus assembly. these proteins are anchored into cellular and viral membranes by their c-terminal domain. these domains are composed of two hydrophobic stretches separated by a short hydrophilic segment containing at least one charged residue. we investigated the role of the transmembrane domains of prm and e in the envelope formation of the flavivirus yellow fever virus (yfv). alanine scanning insertion mutagenesis has been us ...200312502797
antibody and interleukin-12 treatment in murine models of encephalitogenic flavivirus (st. louis encephalitis, tick-borne encephalitis) and alphavirus (venezuelan equine encephalitis) infection.early and sustained treatment with interleukin-12 (il-12) ameliorated disease in a mouse model of infection with the encephalitogenic flavivirus, st. louis encephalitis virus (slev, japanese encephalitis serogroup). however, this effect was not reproduced in murine infections with either the flavivirus tick-bore encephalitis virus (tbev) or the alphavirus venezuelan equine encephalitis virus (veev). il-12 exacerbated tbev disease when used in conjunction with monoclonal antibody (mab), suggestin ...200312639298
the importance of being outer: consequences of the distinction between the outer and inner surfaces of flavivirus glycoprotein e.glycoprotein e of west nile, dengue and other flaviviruses is the principal stimulus for the development of neutralizing antibodies and contains a fusion peptide responsible for inserting the virus into the host cell membrane. this glycoprotein lies flat on the surface of the virion and therefore only epitopes on the outer or lateral surface are important immunogens. changes in antigen recognition after exposure of the virus to low ph have yielded clues to the fusion process.200312648938
spinal cord disease in west nile virus infection. 200312575663
mhc class i up-regulation by flaviviruses: immune interaction with unknown advantage to host or pathogen.in contrast to many other viruses that escape from cytotoxic t cell recognition by down-regulating major histocompatibility complex class i-restricted antigen presentation, flavivirus infection of mammalian cells up-regulates cell surface expression of major histocompatibility complex class i molecules. two putative mechanisms for flavivirus-induced major histocompatibility complex class i up-regulation, one via activation of the transcription factor nf-kappab, the second by augmentation of pept ...200312752686
impact of direct virus-induced neuronal dysfunction and immunological damage on the progression of flavivirus (modoc) encephalitis in a murine model.flavivirus encephalitis is believed to be the result of two main mechanisms: (i) direct damage to and dysfunction of neurons as a result of viral replication and (ii) destruction of the brain tissue by an inflammatory response. the differential impact of both mechanisms on the progression of flavivirus encephalitis has not been clearly determined. we have now studied the encephalitis caused by modoc virus (modv) infection in (i) severe combined immunodeficiency (scid) mice, (ii) immunocompetent ...200312587070
polystyrene derivatives substituted with arginine interact with babanki (togaviridae) and kedougou (flaviviridae) viruses.outbreaks of new or old diseases appear primarily in tropical zones such as africa, south and central america, or asia. among these diseases, those induced by arboviruses (the best known of which are being yellow fever, dengue, ebola, and sindbis) are under intensive observation by the world health organization. rapid isolation and identification of the viral species is the first step in the diagnosis, study, and control of epidemics. one major problem with the isolation of viruses is capturing ...200312601758
isolation of a new flavivirus related to cell fusing agent virus (cfav) from field-collected flood-water aedes mosquitoes sampled from a dambo in central kenya.cell fusing agent virus (cfav) is an rna insect virus that was isolated from a line of aedes aegypti mosquito cells and has been assigned to the family flaviviridae, genus flavivirus. we report here the first isolation of a cfa-like virus from field-collected mosquitoes. mosquito larvae and pupae were sampled from flooded dambos in central province, kenya during the short rain season of 1999. specimens were reared to adults, identified and pooled by species and were tested for the presence of vi ...200312756616
genetic and phenotypic characterization of the newly described insect flavivirus, kamiti river virus.we have described in the accompanying paper by sang, et al., ([57], arch virol 2003, in press) the isolation and identification of a new flavivirus, kamiti river virus (krv), from ae. macintoshi mosquitoes that were collected as larvae and pupae from flooded dambos in central province, kenya. among known flaviviruses, krv was shown to be most similar to, but genetically and phenotypically distinct from, cell fusing agent virus (cfav). krv was provisionally identified as an insect-only flavivirus ...200312756617
enzyme-linked immunosorbent assay using recombinant antigens expressed in mammalian cells for serodiagnosis of tick-borne encephalitis.a recombinant plasmid that expresses the tick-borne encephalitis (tbe) virus premembrane (prm) and envelope (e) proteins in mammalian cells was constructed. recombinant proteins retained antigenic and conformational structures similar to those of native virus proteins, and transfected cells released virus-like particles (vlps), which were 1.13-1.14 g/ml in density and 20-30 nm in diameter, into the culture medium. recombinant e proteins were used for the development of an enzyme-linked immunosor ...200312609684
diagnosis of tick-borne encephalitis.the actual diagnosis of a tick-borne encephalitis (tbe) must be established in the laboratory because of the non-specific clinical features it presents. the method of choice is the demonstration of specific igm- and igg-serum antibodies by enzyme-linked immuno-sorbent assay (elisa), since these antibodies are detectable in practically every case at the time of hospitalization. early after onset of disease in the cerebrospinal fluid specific antibodies can only be found in 50% of the patients, bu ...200312628812
flavivirus capsid is a dimeric alpha-helical protein.the capsid proteins of two flaviviruses, yellow fever virus and dengue virus, were expressed in escherichia coli and purified to near homogeneity suitable for biochemical characterization and structure determination by nuclear magnetic resonance. the oligomeric properties of the capsid protein in solution were investigated. in the absence of nucleic acid, both proteins were predominantly dimeric in solution. further analysis of both proteins with far-uv circular dichroism spectroscopy indicated ...200312768036
structures of immature flavivirus particles.structures of prm-containing dengue and yellow fever virus particles were determined to 16 and 25 a resolution, respectively, by cryoelectron microscopy and image reconstruction techniques. the closely similar structures show 60 icosahedrally organized trimeric spikes on the particle surface. each spike consists of three prm:e heterodimers, where e is an envelope glycoprotein and prm is the precursor to the membrane protein m. the pre-peptide components of the prm proteins in each spike cover th ...200312773377
history of tbe vaccines.tick borne encephalitis was a frequent viral cns disease in europe in the last century, with several hundred hospitalisation cases per year. the causative agent is tick-borne encephalitis virus (tbev), a flavivirus that is transmitted by ticks. the first tissue culture derived vaccine was developed in the early 1970s and this was commercialised as a collaboration between the institute of virology, vienna, austria and immuno ag vienna (now baxter healthcare). this vaccine was highly successful du ...200312628813
inoculation of plasmids encoding japanese encephalitis virus prm-e proteins with colloidal gold elicits a protective immune response in balb/c mice.we established a simple and effective method for dna immunization against japanese encephalitis virus (jev) infection with plasmids encoding the viral prm and e proteins and colloidal gold. inoculation of plasmids mixed with colloidal gold induced the production of specific anti-jev antibodies and a protective response against jev challenge in balb/c mice. when we compared the efficacy of different inoculation routes, the intravenous and intradermal inoculation routes were found to elicit strong ...200312634382
traditional and novel approaches to flavivirus vaccines.yellow fever, dengue, japanese encephalitis and tick-borne encephalitis viruses are the medically most important members of the flavivirus genus composed primarily of arboviruses. in this paper, we review the commercially available traditional flavivirus vaccines against yellow fever, japanese encephalitis, and tick-borne encephalitis, as well as modern approaches to flavivirus vaccines. formalin inactivation technology has been employed to produce killed vaccines. flaviviruses have been attenua ...200312782056
dendritic-cell-specific icam3-grabbing non-integrin is essential for the productive infection of human dendritic cells by mosquito-cell-derived dengue viruses.dengue virus (dv) is a mosquito-borne flavivirus that causes haemorrhagic fever in humans. dv primarily targets immature dendritic cells (dcs) after a bite by an infected mosquito vector. here, we analysed the interactions between dv and human-monocyte-derived dcs at the level of virus entry. we show that the dc-specific icam3-grabbing non-integrin (dc-sign) molecule, a cell-surface, mannose-specific, c-type lectin, binds mosquito-cell-derived dvs and allows viral replication. conclusive evidenc ...200312783086
intracellular assembly and secretion of recombinant subviral particles from tick-borne encephalitis virus.it is believed that flavivirus assembly occurs by intracellular budding of the nucleocapsid into the lumen of the endoplasmic reticulum (er). recombinant expression of tick-borne encephalitis (tbe) virus envelope proteins prm and e in mammalian cells leads to their incorporation into enveloped recombinant subviral particles (rsps), which have been used as a model system for studying assembly and entry processes and are also promising vaccine candidates. in this study, we analyzed the formation a ...200312634393
cross-protective and infection-enhancing immunity in mice vaccinated against flaviviruses belonging to the japanese encephalitis virus serocomplex.the japanese encephalitis virus serocomplex is a group of mosquito-borne flaviviruses that cause severe encephalitic disease in humans. the recent emergence of several members of this serocomplex in geographic regions where other closely related flaviviruses are endemic has raised urgent human health issues. thus, the impact of vaccination against one of these neurotropic virus on the outcome of infection with a second, serologically related virus is unknown. we show here that immunity against m ...200312639478
interferon alfa-2a in japanese encephalitis: a randomised double-blind placebo-controlled trial.japanese encephalitis virus (jev), although confined to asia, causes about 35000-50000 cases and 10000 deaths every year, and is the most important cause of encephalitis worldwide. there is no known antiviral treatment for any flavivirus. results from in-vitro studies and work in animals have shown inteferon alfa has antiviral activity on japanese encephalitis and other flaviviruses; therefore, we aimed to assess the efficacy of inteferon alfa-2a in japanese encephalitis.200312642049
characterization of rna synthesis, replication mechanism, and in vitro rna-dependent rna polymerase activity of japanese encephalitis virus.in vitro rna-dependent rna polymerase assays revealed that the jev replication complex (rc) synthesized viral rna utilizing a semiconservative and asymmetric mechanism. peak viral replicase activity and levels of viral rna observed 15-18 h postinfection (h p.i.) preceded maximum viral titers in the culture medium seen 21 h p.i. among divalent cations, mg(2+) was essential and exhibited cooperative binding for its two replicase-binding sites. mn(2+), despite sixfold higher affinity for the replic ...200312667804
interferons, interferon inducers, and interferon-ribavirin in treatment of flavivirus-induced encephalitis in mice.we evaluated the prophylactic and therapeutic efficacy of interferon alpha-2b, pegylated interferon alpha-2b, poly(i. c), and ampligen against modoc virus encephalitis in an animal model for flavivirus infections. all compounds significantly delayed virus-induced morbidity (paralysis) and mortality (due to progressive encephalitis). viral load (as measured on day 7 postinfection) was significantly reduced by 80 to 100% in the serum, brain, and spleen in mice that had been treated with either int ...200312543691
development of a quantitative real-time rt-pcr assay with internal control for the laboratory detection of tick borne encephalitis virus (tbev) rna.tick borne encephalitis virus (tbev), is a human flavivirus causing tick borne encephalitis (tbe), a viral infection of the central nervous system endemic in europe and asia.200312829035
kunjin virus replicon vectors for human immunodeficiency virus vaccine development.we have previously demonstrated the ability of the vaccine vectors based on replicon rna of the australian flavivirus kunjin (kun) to induce protective antiviral and anticancer cd8+ t-cell responses using murine polyepitope as a model immunogen (i. anraku, t. j. harvey, r. linedale, j. gardner, d. harrich, a. suhrbier, and a. a. khromykh, j. virol. 76:3791-3799, 2002). here we showed that immunization of balb/c mice with kun replicons encoding hiv-1 gag antigen resulted in induction of both gag- ...200312829819
a stable full-length yellow fever virus cdna clone and the role of conserved rna elements in flavivirus replication.yellow fever virus (yf) is the prototype member of the flavivirus genus. here, we report the successful construction of a full-length infectious cdna clone of the vaccine strain yf-17d. yf cdna was cloned into a low-copy-number plasmid backbone and stably maintained in several e. coli strains. transcribed rnas had a specific infectivity of 10(5)-10(6) p.f.u. ( micro g rna)(-1), and the resulting virus exhibited growth kinetics, plaque morphology and proteolytic processing similar to the parental ...200312692292
dengue virus type 2 ns3 protease and ns2b-ns3 protease precursor induce apoptosis.apoptosis was detected in vero cell cultures expressing transfected dengue virus type 2 (denv-2) genes. approximately 17.5 and 51.5 % of cells expressing ns3 serine protease and ns2b-ns3(185) serine protease precursor protein [ns2b-ns3(185)(pro)] genes, respectively, were apoptotic. the percentage of apoptotic cells was significantly higher in cell cultures expressing ns2b-ns3(185)(pro). ns2b-ns3(185)(pro) was detected as ns2b-ns3(185)(pro)-egfp fusion protein in cytoplasmic vesicular structures ...200312867651
novel, potent phenethylamide inhibitors of the hepatitis c virus (hcv) ns3 protease: probing the role of p2 aryloxyprolines with hybrid structures.synthesis of hybrid hcv ns3 protease/ns4a inhibitors having the 4,4-difluoroaminobutyric acid (difluoroabu) phenethylamides as p1-p1' and quinolyloxyprolines as p2 fragments led to 7 (ic(50) 54 nm). molecular modelling suggests that this potent tripeptide inhibitor utilizes interactions in the s1', s1, s2, s3 and s4 sites of the protease.200312873506
tick-borne encephalitis: a retrospective study of clinical cases in bornholm, denmark.tick-borne encephalitis (tbe) is caused by a flavivirus, tick-borne encephalitis virus (tbev). for decades tbe has been known to cause disease in bornholm, a danish island. a retrospective study was performed to identify undiagnosed cases of tbe among patients hospitalized with signs and symptoms of meningitis or encephalitis in the 5 y period 1994-1999. this investigation revealed 5 new, and initially undiagnosed cases of tbe. in total, 14 cases of tbe were found in the 7 y period 1994-2000; 2 ...200312875531
incorporation of tick-borne encephalitis virus replicons into virus-like particles by a packaging cell line.rna replicons derived from flavivirus genomes show considerable potential as gene transfer and immunization vectors. a convenient and efficient encapsidation system is an important prerequisite for the practical application of such vectors. in this work, tick-borne encephalitis (tbe) virus replicons and an appropriate packaging cell line were constructed and characterized. a stable cho cell line constitutively expressing the two surface proteins prm/m and e (named cho-me cells) was generated and ...200312885909
architecture of the flaviviral replication complex. protease, nuclease, and detergents reveal encasement within double-layered membrane compartments.flavivirus infection causes extensive proliferation and reorganization of host cell membranes to form specialized structures called convoluted membranes/paracrystalline arrays and vesicle packets (vp), the latter of which is believed to harbor flaviviral replication complexes. using detergents and trypsin and micrococcal nuclease, we provide for the first time biochemical evidence for a double membrane compartment that encloses the replicative form (rf) rna of the three pathogenic flaviviruses w ...200312700232
recent advances in japanese encephalitis.japanese encephalitis (je), the most important cause of epidemic encephalitis worldwide, is confined to asia, but its geographical area is spreading. west nile virus, and other closely related flaviviruses, cause similar disease elsewhere. recent cryoelectron microscopic studies have characterized the flavivirus envelope protein as a new class of viral fusion protein (class ii), and examined its arrangement on the virion surface. changes in the envelope protein's hinge region, or its putative re ...200312707858
interferon, ribavirin, 6-azauridine and glycyrrhizin: antiviral compounds active against pathogenic flaviviruses.ribavirin, interferon-alpha (ifn-alpha), 6-azauridine and glycyrrhizin were tested in vitro for their antiviral activities against 11 pathogenic flaviviruses belonging to principal antigenic complexes or individual serogroups of medical importance: dengue, japanese encephalitis, mammalian tick-borne and yellow fever virus (yfv) groups. antiviral activity was estimated by the reduction of the cytopathic effect of each flavivirus in vero cells and by the reduction in virus titer. cytotoxicity was ...200312719009
temperature sensitive mutations in the genes encoding the ns1, ns2a, ns3, and ns5 nonstructural proteins of dengue virus type 4 restrict replication in the brains of mice.using reverse genetics, it is possible to readily add well-defined attenuating mutations to the genome of wild type or incompletely attenuated dengue (den) viruses to generate vaccine candidates that exhibit the desired balance between attenuation and immunogenicity. here, we describe the identification of eight temperature sensitive missense mutations distributed in four non-structural protein genes that specify a 60- to 10,000-fold range of restricted replication in the suckling mouse brain co ...200312721805
[specific determination of flaviviruses by molecular hybridization with synthetic deoxyoligonucleotide probes].molecular probes were designed for the purpose of specific determination of flavioviruses transmitted by the ticks of tick-borne encephalitis (tbe) and omsk hemorrhagic fever (ohv) as well as by mosquitoes of japanese encephalitis (je), north nile (nn), murrey valley encephalitis (mve), saint-lois encephalitis (sle), dengue 1-4 and of yellow fever (yf). the probes are synthetic deoxyoligonucleotides with the 18-20 long basis and complementary for the rna fragments defined by computer analysis. t ...200312894476
antigen-expressed recombinant salmonella typhimurium driven by an in vivo-activated promoter is capable of inducing cellular immune response in transgenic mice.to explore the approaches and mechanisms for reversing the immune tolerance in transgenic mouse, and the pathogenicity of hepatitis g virus (hgv), the promoter of phop-activated gene (p(pagc)) of salmonella typhimurium was used as a transcriptionally regulating element to construct an attenuated s. typhimurium expressing hgv ns3. the recombinant s. typhimurium was orally administered to hgv transgenic mice. as the results, hgv antigen in serum and liver as well as hgv mrna in liver were decrease ...200312897963
viral internal ribosome entry site structures segregate into two distinct morphologies.an increasing number of viruses have been shown to initiate protein synthesis by a cap-independent mechanism involving internal ribosome entry sites (iress). predictions of the folding patterns of these rna motifs have been based primarily on sequence and biochemical analyses. biophysical confirmation of the models has been achieved only for the ires of hepatitis c virus (hcv), which adopts an open structure consisting of two major stems. we have conducted an extensive comparison of flavivirus a ...200312743317
evasion of innate and adaptive immunity by flaviviruses.after a virus infects an animal, antiviral responses are generated that attempt to prevent dissemination. interferons, antibody, complement, t and natural killer cells all contribute to the control and eradication of viral infections. most flaviviruses, with the exception of some of the encephalitic viruses, cause acute disease and do not establish persistent infection. the outcome of flavivirus infection in an animal is determined by a balance between the speed of viral replication and spread, ...200312752684
discrimination of west nile virus and japanese encephalitis virus strains using rt-pcr rflp analysis.west nile (wn) virus is a mosquito-borne flavivirus that induces lethal encephalitis in humans and horses. since an outbreak of wn encephalitis in humans and horses occurred in new york city in late august 1999, the possibility exists that wn virus will invade regions that have close links with the united states, such as japan. we developed a genetic diagnostic method that discriminates between strains of wn virus and japanese encephalitis (je) virus. the method involves rt-pcr restriction fragm ...200312906104
chronic hepatitis associated with gb virus b persistence in a tamarin after intrahepatic inoculation of synthetic viral rna.progress in understanding the pathogenesis of hepatitis c virus (hcv) has been slowed by the absence of tractable small animal models. whereas gb virus b (gbv-b, an unclassified flavivirus) shares a phylogenetic relationship and several biologic attributes with hcv, including hepatotropism, it is not known to cause persistent infection, a hallmark of hcv. here, we document persistent gbv-b infection in one of two healthy tamarins (saguinus oedipus) inoculated intrahepatically with infectious syn ...200312907703
interaction of flaviviruses with cells of the vertebrate host and decoy of the immune response.flaviviruses cause endemic and epidemic disease with significant morbidity and mortality throughout the world. in contrast to viruses that avoid the host immune response by down-regulating cell surface major histocompatibility complex expression, infection by members of the neurotropic japanese encephalitis serogroup induce virus-directed functional increases in expression of class i and ii major histocompatibility complex and various adhesion molecules, resulting in increased susceptibility to ...200312752685
is flavivirus resistance interferon type i-independent?interferon type i comprises a group of major virus-inducible host antiviral factors that control infection with a great number of human and animal viruses. they are ubiquitously expressed cytokines that interfere with virus replication within different cell types by activating a number of host genes and several parallel antiviral pathways. two major intracellular actors of ifn-i-induced antiviral states are ribonucleic acid-dependent protein kinase and 2'-5'-oligoadenylate synthetases/rnase l, b ...200312752687
evidence for antigen production in muscles by dengue and japanese encephalitis dna vaccines and a relation to their immunogenicity in mice.this study demonstrated viral antigen production in muscle tissues following inoculation with dna vaccines and examined its relation to antibody induction in mice using the flavivirus system. to achieve detectable levels of antigen production, we used a needle-free jet injector and examined 10% homogenate of quadriceps muscle for viral antigens in a sandwich enzyme-linked immunosorbent assay. we compared dna vaccines against dengue type 1 (designated pcd1me), dengue type 2 (pcd2me) and japanese ...200312922102
lipophilic derivatization of synthetic peptides belonging to ns3 and e2 proteins of gb virus-c (hepatitis g virus) and its effect on the interaction with model lipid membranes.the synthesis by solid-phase methodologies of peptides belonging to structural and non-structural proteins of gb virus c as well as its n-alpha-acylation with myristate and palmitate fatty acids is described. to explore the peptide-lipid interactions we have used liposomes composed of dipalmitoylphosphatidylcholine as model membranes and complementary spectroscopic and calorimetric techniques. our results show that structural and more clearly the structural lipophilic peptide sequences incorpora ...200312753379
[west nile fever may even be spread to sweden].west nile virus (wnv) is a flavivirus, which was first isolated in uganda 1937. this virus has attracted attention in the past years. wnv is one of the worlds most widespread flaviviruses and has caused recent outbreaks among humans and animals. west nile fever (wnf) is a mosquito borne zoonotic disease. the last decade the epidemiological pattern of the virus and the severity of the outbreaks have changed. this article reviews the epidemiological changes and current situation of wnf, presents a ...200312756695
growth characteristics of the veterinary vaccine candidate chimerivax-west nile (wn) virus in aedes and culex mosquitoes.in 1999 west nile (wn) virus was introduced to north america where this flavivirus has spread rapidly among wildlife (especially birds) transmitted by various species of mosquitoes (diptera: culicidae). increasing numbers of cases and deaths among humans, horses and other domestic animals require development of effective vaccines. 'chimerivax-west nile(vet)' is being developed for use as a veterinary vaccine to protect against wn infection. this chimeric virus contains the pre-membrane (prm) and ...200312941006
natural and nosocomial infection in a patient with west nile encephalitis and extrapyramidal movement disorders.since its first recognition in north america in 1999, west nile virus (wnv) has spread rapidly across the continent, but in many communities, rapid diagnostic tests for detection of wnv infection are not fully available. we describe a patient with extrapyramidal movement disorders and changes in the basal ganglia noted on magnetic resonance images that are characteristic of other flavivirus encephalitides and may help in the recognition of patients with west nile encephalitis. detailed molecular ...200312766856
[serological diagnosis of dengue and yellow fever infections in suspected cases from pará state, brazil, 1999].from june to december 1999, 785 serum samples were obtained from patients clinically suspected of having dengue or yellow fever. the patients were referred by public health centers distributed within the six mesoregions of par state, brazil. serum samples were tested for flavivirus antibodies by hemagglutination inhibition test and for dengue and yellow fever viruses by enzyme-linked immunosorbent assay for igm detection. of the sera collected, 563 (71.7%) were positive by hi test and out of th ...200312612738
molecular detection of west nile virus rna.west nile virus is a mosquito-borne flavivirus that is primarily maintained in nature in a mosquito-bird-mosquito transmission cycle. mammals, including humans and horses, are incidentally infected through biting by mosquitoes infected with west nile virus. since 1994, west nile virus outbreaks have occurred with a high incidence of severe disease in humans and horses. in the usa, west nile virus was first detected in 1999 in new york city and has since spread to 39 states in humans. the virus h ...200312779009
poliomyelitis and flaviviruses. 200312783413
[west nile fever].west nile fever is a mosquito-borne febrile illness seen in africa, asia, and europe, but reported in north america only once. west nile virus is of the family flaviviridae, genus flavivirus. several west nile fever outbreaks were reported from israel in the 1950s and in 1980. since the mid-1990s, frequency and apparent clinical severity of west nile virus outbreaks have increased. outbreaks in rumania (1996), russia (1999), and israel (2000) involved hundreds of persons with severe neurologic d ...200319753724
west nile virus infection in the united states: overview as a public health issue.in august 1999, west nile virus (wnv), a mosquito-transmitted flavivirus, appeared in new york city. this represented the first time a major outbreak of this old world virus caused an epidemic in the western hemisphere. by december 2002, the outbreak had spread, probably via avian migratory flyways, to involve 44 states and the district of columbia. the future epidemiology of wnv infection in the united states will be difficult to forecast. in the absence of an effective human vaccine, the only ...200321765762
a new cell line from the embryonic tissues of culex tritaeniorhynchus and its susceptibility to certain flaviviruses.a new cell line from the embryonic tissue of culex tritaeniorhynchus mosquito was established. morphological studies carried out at the 45th passage level (p-45) showed four different cell types viz. epithelial-like cells, fibroblast-like cells, giant cells and vacuolated cells. karyological studies indicated diploid (2n = 6) chromosomes in majority of cells irrespective of passage level. a twelve-fold increase of cell number was observed in 10 days at p-49. the cells could be preserved in liqui ...200212693860
in vitro determination of dengue virus type 2 ns2b-ns3 protease activity with fluorescent peptide substrates.the ns2b-ns3(pro) polyprotein segment from the dengue virus serotype 2 strain 16681 was purified from overexpressing e. coli by metal chelate affinity chromatography and gel filtration. enzymatic activity of the refolded ns2b-ns3(pro) protease complex was determined in vitro with dansyl-labeled peptide substrates, based upon native dengue virus type 2 cleavage sites. the 12mer substrate peptides and the cleavage products could be separated by reversed-phase hplc, and were identified by uv and fl ...200212297031
strategies for the identification of inhibitors of west nile virus and other flaviviruses.the recent outbreaks of west nile virus (wnv) and other emerging and re-emerging flaviviruses, have made prevention and treatment of flavivirus infections a global public health priority. no inhibitor is currently available for use as an antiflavivirus therapy. in this communication, i review (i) stages of flavivirus replication representing potential targets for antiviral drug discovery; (ii) strategies for identification of flavivirus inhibitors; and (iii) existing antiviral inhibitors reporte ...200212476954
effect of triethylamine on the recovery of selected south american alphaviruses, flaviviruses, and bunyaviruses from mosquito (diptera: culicidae) pools.we evaluated the effect of triethylamine (tea) on the recovery of infectious virus from pools of mosquitoes for two south american alphaviruses (eastern equine encephalomyelitis and venezuelan equine encephalomyelitis subtypes iiic and id), one flavivirus (ilheus) and two bunyaviruses (mirim [guama group] and itaqui [group c]). mosquitoes were inoculated intrathoracically with virus, held for 7-10 d at 26 degrees c, and handled under one of four regimens before testing for the presence of virus ...200212349865
west nile virus.west nile (wn) virus is a mosquito-borne flavivirus and human, equine, and avian neuropathogen. the virus is indigenous to africa, asia, europe, and australia, and has recently caused large epidemics in romania, russia, and israel. birds are the natural reservoir (amplifying) hosts, and wn virus is maintained in nature in a mosquito-bird-mosquito transmission cycle primarily involving culex sp mosquitoes. wn virus was recently introduced to north america, where it was first detected in 1999 duri ...200212206968
intrauterine west nile virus infection--new york, 2002.west nile virus (wnv), a mosquito-borne flavivirus and human neuropathogen, is epidemic in the united states. in 2002, newly recognized mechanisms of person-to-person wnv transmission were described, including possible transmission from mother to infant through breast milk. wnv has not been previously associated with intrauterine infection or adverse birth outcomes. this report describes a case of transplacental wnv transmission. pregnant women should take precautions to reduce their risk for wn ...200212537289
expression, purification, and characterization of the rna 5'-triphosphatase activity of dengue virus type 2 nonstructural protein 3.dengue virus type 2 (den2), a member of the flaviviridae family of positive-strand rna viruses, contains a single rna genome having a type i cap structure at the 5' end. the viral rna is translated to produce a single polyprotein precursor that is processed to yield three virion proteins and at least seven nonstructural proteins (ns) in the infected host. ns3 is a multifunctional protein having a serine protease catalytic triad within the n-terminal 180 amino acid residues which requires ns2b as ...200212167347
laboratory-acquired west nile virus infections--united states, 2002.west nile virus (wnv), a mosquito-borne flavivirus introduced recently to north america, is a human, equine, and avian neuropathogen. the majority of human infections with wnv are mosquito-borne; however, laboratory-acquired infections with wnv and other arboviruses also occur. this report summarizes two recent cases of wnv infection in laboratory workers without other known risk factors who acquired infection through percutaneous inoculation. laboratory workers handling fluids or tissues known ...200212537288
classical swine fever virus ns5b-gfp fusion protein possesses an rna-dependent rna polymerase activity.rna-dependent rna polymerase (rdrp) is the replicase of positive-strand rna viruses. expression and characterization of the replicase are the first steps in the elucidation of the virus replication mechanism. we expressed nonstructural protein 5b (ns5b) of classical swine fever virus (csfv) as a fusion protein with green fluorescent protein (gfp) in porcine kidney cells (pk-15 cells), natural host cells of csfv. the expressed csfv ns5b-gfp fusion protein possessed rdrp activity. by fluorescence ...200212209316
a nonsense mutation in the gene encoding 2'-5'-oligoadenylate synthetase/l1 isoform is associated with west nile virus susceptibility in laboratory mice.a mouse model has been established to investigate the genetic determinism of host susceptibility to west nile (wn) virus, a member of the genus flavivirus and family flaviviridae. whereas wn virus causes encephalitis and death in most laboratory inbred mouse strains after peripheral inoculation, most strains derived from recently trapped wild mice are completely resistant. the phenotype of resistance/susceptibility is determined by a major locus, wnv, mapping to chromosome 5 within the 0.4-cm-wi ...200212186974
the molecular biology of west nile virus: a new invader of the western hemisphere.west nile virus (wnv) is a mosquito-borne flavivirus that primarily infects birds but occasionally also infects humans and horses. in recent years, the frequency of wnv outbreaks in humans has increased, and these outbreaks have been associated with a higher incidence of severe disease. in 1999, the geographical distribution of wnv expanded to the western hemisphere. wnv has a positive strand rna genome of about 11 kb that encodes a single polyprotein. wnv replicates in the cytoplasm of infected ...200212142476
poly i:clc induction of the interferon system in mice: an initial study of four detection methods.induction of a large number of the components of the interferon (ifn) system (ifn genes, their mrnas, ifn proteins, ifn receptors, ifn signaling molecules, the ifn response genes, and their effector proteins) has been studied. less well studied is the comparative induction of these components in vivo. induction of ifn by double-stranded rna (dsrna) treatment mimics certain aspects of viral infection and induces the components of the ifn system. to determine the comparative sensitivity of detecti ...200212433283
sequence diversity of the capsid gene and the nonstructural gene ns2b of dengue-3 virus in vivo.previously, we studied the envelope (e) gene of dengue virus and reported that dengue-3 virus is present as a quasispecies. to investigate the extent of intrahost sequence variation of other dengue viral genes, we examined in this study the capsid (c) gene and the nonstructural gene, ns2b, derived directly from plasma dengue viruses from 18 confirmed dengue-3 patients. using reverse transcription-pcr, multiple clones of a 360-nucleotide region covering the c gene and of a 404-nucleotide region c ...200212482670
tick-borne encephalitis (tbe) in dogs.tick-borne encephalitis (tbe) is caused by a flavivirus and transmitted by ticks. it is known in dogs for nearly 30 years and the number of tbe cases is increasing. in addition to fever, cerebrocortical, thalamic, and brainstem symptoms occur simultaneously. not all tbe infections in dogs lead to clinical signs but peracute/lethal as well as subacute and chronic courses have been reported. tbe is a seasonal disease, depending on climate related tick activity. infected ticks are spreading the vir ...200212141763
hepatitis g virus (hgv): current perspectives.five viruses are usually associated with hepatitis in humans: a-e. in addition to these viruses as aetiological agents of hepatitis, there remain a number of patients with hepatitis in whom no virus could be identified. it was therefore postulated that there may be other agents which may be causing hepatitis. recently, two viruses have been associated with hepatitis: hepatitis g virus (hgv), and transfusion transmissible virus (ttv). hepatitis g virus (hgv) is a single stranded rna virus which r ...200212593580
infection of polarized epithelial cells with flavivirus west nile: polarized entry and egress of virus occur through the apical surface.both polarized epithelial vero (c1008) and non-polarized vero (control) cells were grown on permeable cell culture inserts and infected either apically or basolaterally with west nile (wn) or kunjin (kun) virus. kun virus (closely related to wn virus) was used as a comparison. using indirect immunofluorescence and plaque assays of productive virus titres, entry of wn and kun viruses was confined to the apical surface of polarized epithelial cells. for the first time, these results provided evide ...200212237424
genome sequence analysis of tamana bat virus and its relationship with the genus flavivirus.tamana bat virus (tabv, isolated from the bat pteronotus parnellii) is currently classified as a tentative species in the genus flavivirus: we report here the determination and analysis of its complete coding sequence. low but significant similarity scores between tabv and member-viruses of the genus flavivirus were identified in the amino acid sequences of the structural, ns3 and ns5 genes. a series of cysteines located in the envelope protein and the most important enzymatic domains of the vir ...200212237426
positional cloning of the murine flavivirus resistance gene.inbred mouse strains exhibit significant differences in their susceptibility to viruses in the genus flavivirus, which includes human pathogens such as yellow fever, dengue, and west nile virus. a single gene, designated flv, confers this differential susceptibility and was mapped previously to a region of mouse chromosome 5. a positional cloning strategy was used to identify 22 genes from the flv gene interval including 10 members of the 2'-5'-oligoadenylate synthetase gene family. one 2'-5'-ol ...200212080145
complementation analysis of the flavivirus kunjin ns3 and ns5 proteins defines the minimal regions essential for formation of a replication complex and shows a requirement of ns3 in cis for virus assembly.we have previously reported successful trans-complementation of defective kunjin virus genomic rnas with a range of large lethal deletions in the nonstructural genes ns1, ns3, and ns5 (a. a. khromykh et al., j. virol. 74:3253-3263, 2000). in this study we have mapped further the minimal region in the ns5 gene essential for efficient trans-complementation of genome-length rnas in repbhk cells to the first 316 of the 905 codons. to allow amplification and easy detection of complemented defective r ...200212368319
modes of transmission of hemorrhagic fever. 200212150658
assessment of the factors associated with flavivirus seroprevalence in a population in southern vietnam.dengue and japanese encephalitis flaviviruses cause severe disease and are hyperendemic in southern vietnam. this study assesses associations between sociodemographic factors and flavivirus seroprevalence in this region. sera were collected from 308 community and hospital-based subjects between april 1996 and august 1997 and tested with an indirect elisa. the factors associated with seroprevalence were assessed using multivariate logistic regression. in this first report of adjusted prevalence o ...200212002539
mycophenolic acid inhibits dengue virus infection by preventing replication of viral rna.dengue fever is a mosquito-borne viral disease of global importance with no available antiviral therapy. we assessed the ability of mycophenolic acid (mpa), a drug currently used as an immunosuppressive agent, to inhibit dengue virus (dv) antigen expression, rna replication, and virus production. pharmacological concentrations of mpa effectively blocked dv infection, decreasing the percentage of infected cells by 99% and the levels of secreted virus by up to a millionfold. results were reproduce ...200212504563
cross-reactivity in flavivirus serology: new implications of an old finding?to investigate the influence of pre-existing antibodies against tick-borne encephalitis (tbe) or yellow fever (yf) viruses on dengue virus antibody test results, we examined sera from vaccinees and from individuals with previous tbe virus infection. distinct igg antibody cross-reactivity was found in about 15.1% in the yf-vaccinated group and in about 9.5% in the tbe-vaccinated group. altogether 15 out of a total of 80 samples tested (18.8%) had detectable dengue virus igg antibody titres. the s ...200212005333
reappearance of human cases due to murray valley encephalitis virus and kunjin virus in central australia after an absence of 26 years.murray valley encephalitis (mve) and kunjin virus disease are endemic in the tropical parts of the northern territory and western australia, but have been absent from central australia since 1974. in 2000, 5 laboratory-confirmed cases of encephalitis occurred over a short period in the normally dry inland region of central australia. the sudden occurrence of cases in march and april 2000 followed unusually high rainfall in the preceding months and evidence of flavivirus activity in the endemic a ...200211950201
single mutation in the flavivirus envelope protein hinge region increases neurovirulence for mice and monkeys but decreases viscerotropism for monkeys: relevance to development and safety testing of live, attenuated vaccines.a chimeric yellow fever (yf) virus/japanese encephalitis (je) virus vaccine (chimerivax-je) was constructed by insertion of the prm-e genes from the attenuated je virus sa14-14-2 vaccine strain into a full-length cdna clone of yf 17d virus. passage in fetal rhesus lung (frhl) cells led to the emergence of a small-plaque virus containing a single met-->lys amino acid mutation at e279, reverting this residue from the sa14-14-2 to the wild-type amino acid. a similar virus was also constructed by si ...200211799188
antibodies against prm protein distinguish between previous infection with dengue and japanese encephalitis viruses.in southeast asia, dengue viruses often co-circulate with other flaviviruses such as japanese encephalitis virus, and due to the presence of shared antigenic epitopes it is often difficult to use serological methods to distinguish between previous infections by these flaviviruses.200212019028
t cell responses to an hla-b*07-restricted epitope on the dengue ns3 protein correlate with disease severity.dengue hemorrhagic fever (dhf), the severe manifestation of dengue virus (dv) infection characterized by plasma leakage, is more common in secondary dv infections in previously infected individuals and is associated with high levels of immune activation. to determine the ag specificity of this immune response, we studied the response to an hla-b*07-restricted t cell epitope, residues 221-232 of the dv ns3 protein, in 10 hla-b*07(+) thai children who were studied during and after acute dv infecti ...200212023403
clinical proof of principle for chimerivax: recombinant live, attenuated vaccines against flavivirus infections.chimerivax is a live, attenuated recombinant virus constructed from yellow fever (yf) 17d in which the envelope protein genes of yf 17d are replaced with the corresponding genes of another flavivirus. a chimerivax vaccine was developed against japanese encephalitis (je). a randomized, double-blind, outpatient study was conducted to compare the safety and immunogenicity of chimerivax-je and yf 17d. six yf immune and six non-immune adults were randomized to receive a single sc inoculation of chime ...200211803060
etiology of encephalitis syndrome among hospitalized children and adults in takeo, cambodia, 1999-2000.whether or not japanese encephalitis virus (jev) is an important causative agent of acute encephalitis in cambodia remains unclear. this study was carried out to determine the cause of encephalitis syndrome among children and adults admitted to takeo provincial hospital from october 1999 to september 2000. ninety-nine cases were included in the study: 52 pediatric cases (12 were fatal) and 47 adult cases (10 were fatal). a causative agent such as human herpesvirus (hhv-3 or hhv-4), cryptococcus ...200212135294
the pathogenicity of louping ill virus for mice and lambs.mice and lambs were infected with the li/i, li/31 or ma54 strain of louping ill virus (liv) to provide information relevant to testing the efficacy and biosafety of a new generation of flavivirus vaccines based on a semliki forest virus (sfv) vector. whereas clinical signs and neuropathological lesions were consistently severe in mice, the majority of lambs showed lesions of moderate severity and only lambs with severe lesions were clinically affected. for both species, dispersal of viral antige ...200211945002
mutations in the yellow fever virus nonstructural protein ns2a selectively block production of infectious particles.little is known about the function of flavivirus nonstructural protein ns2a. two forms of ns2a are found in yellow fever virus-infected cells. full-length ns2a (224 amino acids) is the product of cleavage at the ns1/2a and ns2a/2b sites. ns2aalpha, a c-terminally truncated form of 190 amino acids, results from partial cleavage by the viral ns2b-3 serine protease at the sequence qk /t within ns2a. exchange of serine for lysine at this site (qkt-->qst) blocks the production of both ns2aalpha and i ...200211967294
catalytically active dengue virus ns3 protease forms aggregates that are separable by size exclusion chromatography.an active form of the dengue virus protease ns3 (cf40.gly.ns3pro) was expressed in escherichia coli. this construct consists of a critical 40 amino acid cofactor domain from ns2b fused to the n-terminal 184 amino acid protease domain of ns3 via a flexible, covalent linker (gly(4)sergly(4)). the recombinantly produced protein is soluble and has a hexa-histidine tag engineered at the n-terminus for ease of purification using metal affinity chromatography. however, the presence of lower molecular w ...200212135556
fatal yellow fever in a traveler returning from amazonas, brazil, 2002.yellow fever (yf) is a mosquitoborne viral disease that has caused deaths in u.s. and european travelers to sub-saharan africa and tropical south america. although no specific treatment exists for yf and the case-fatality rate for severe yf is approximately 20%, an effective vaccine is available. this report describes a case of fatal yf in an unvaccinated traveler who had returned from a 6-day fishing trip on the rio negro west of manaus in the state of amazonas, brazil. because information from ...200211990236
folding and dimerization of tick-borne encephalitis virus envelope proteins prm and e in the endoplasmic reticulum.flavivirus envelope proteins are synthesized as part of large polyproteins that are co- and posttranslationally cleaved into their individual chains. to investigate whether the interaction of neighboring proteins within the precursor protein is required to ensure proper maturation of the individual components, we have analyzed the folding of the flavivirus tick-borne encephalitis (tbe) virus envelope glycoproteins prm and e by using a recombinant plasmid expression system and virus-infected cell ...200211991976
langat flavivirus protease ns3 binds caspase-8 and induces apoptosis.the flavivirus ns3 protein plays an important role in the cleavage and processing of the viral polyprotein and in the synthesis of the viral rna. ns3 recruits ns2b and ns5 proteins to form complexes possessing protease and replicase activities through protease and nucleoside triphosphatase/helicase domains. we have found that ns3 also induces apoptosis. expression of the langat (lgt) virus ns3 protein resulted in a cleavage of cellular dna and reduced the viability of cells. coexpression of ns3 ...200211991998
fatal yellow fever in a traveler returning from amazonas, brazil, 2002. 200212025823
an rna cap (nucleoside-2'-o-)-methyltransferase in the flavivirus rna polymerase ns5: crystal structure and functional characterization.viruses represent an attractive system with which to study the molecular basis of mrna capping and its relation to the rna transcription machinery. the rna-dependent rna polymerase ns5 of flaviviruses presents a characteristic motif of s-adenosyl-l-methionine-dependent methyltransferases at its n-terminus, and polymerase motifs at its c-terminus. the crystal structure of an n-terminal fragment of dengue virus type 2 ns5 is reported at 2.4 a resolution. we show that this ns5 domain includes a typ ...200212032088
mouse neuroinvasive phenotype of west nile virus strains varies depending upon virus genotype.despite recent advances in the genetics of west nile (wn) virus, relatively little is known about the molecular basis of virulence of this virus. in particular, although the genotype of the wn virus strain that was recently introduced into north america has been determined, there have been few experimental studies on the virulence phenotype of the virus. we compared genetic and neurovirulence properties of 19 strains of wn virus, including 2 from north america, and observed significant differenc ...200212036314
flavivirus susceptibility in aedes aegypti.aedes aegypti is the primary vector of yellow fever (yf) and dengue fever (df) flaviviruses worldwide. in this review we focus on past and present research on genetic components and environmental factors in aedes aegypti that appear to control flavivirus transmission. we review genetic relationships among ae. aegypti populations throughout the world and discuss how variation in vector competence is correlated with overall genetic differences among populations. we describe current research into h ...200212234528
emerging arboviral diseases. 200212054284
ns3 protease of langat tick-borne flavivirus cleaves serine protease substrates.langat (lgt) virus, initially isolated in 1956 from ticks in malaysia, is a naturally occurring nonpathogenic virus with a very close antigenicity to the highly pathogenic tick-borne encephalitis (tbe) western subtype virus and tbe far eastern subtype virus. ns3, the second largest viral protein of lgt virus, is highly conserved among flaviviruses and contains a characteristic protease moiety (ns3 pro). ns3 pro represents an attractive target for anti-protease molecules against tbe virus. we rep ...200212054734
the emergence of west nile virus in north america: ecology, epidemiology, and surveillance.in late summer 1999, the first domestically acquired human cases of wn encephalitis were documented in the usa. aggressive vector-control and public education efforts by state and local public health officials limited the extent of human involvement. the discovery of virus-infected, overwintering mosquitoes during the winter of 1999-2000, predicted renewed virus activity for the following spring, and prompted early season vector-control activities and disease surveillance efforts in nyc and the ...200212082991
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