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essential role of cyclization sequences in flavivirus rna replication.a possible role in rna replication for interactions between conserved complementary (cyclization) sequences in the 5'- and 3'-terminal regions of flavivirus rna was previously suggested but never tested in vivo. using the m-fold program for rna secondary-structure predictions, we examined for the first time the base-pairing interactions between the covalently linked 5' genomic region (first ~160 nucleotides) and the 3' untranslated region (last ~115 nucleotides) for a range of mosquito-borne fla ...200111413342
infectious cdna clone of attenuated langat tick-borne flavivirus (strain e5) and a 3' deletion mutant constructed from it exhibit decreased neuroinvasiveness in immunodeficient mice.forty-five years ago a naturally attenuated tick-borne flavivirus, langat (lgt) strain tp21, was recovered from ticks in malaysia. subsequently, it was tested as a live attenuated vaccine for virulent tick-borne encephalitis viruses. in a large clinical trial its attenuation was confirmed but there was evidence of a low level of residual virulence. thirty-five years ago further attenuation of lgt tp21 was achieved by multiple passages in eggs to yield mutant e5. to study the genetic determinants ...200111289811
self-replicative rna vaccines elicit protection against influenza a virus, respiratory syncytial virus, and a tickborne encephalitis virus.in genetic vaccination, recipients are immunized with antigen-encoding nucleic acid, usually dna. this study addressed the possibility of using the recombinant alpha virus rna molecule, which replicates in the cytoplasm of transfected cells, as a novel approach for genetic vaccination. mice were immunized with recombinant semliki forest virus rna-encoding envelope proteins from one of 3 viruses: influenza a virus, a tickborne flavivirus (louping ill virus), or respiratory syncytial virus (rsv). ...200111294672
the degree of attenuation of tick-borne encephalitis virus depends on the cumulative effects of point mutations.an infectious clone (pggvs) of the tick-borne encephalitis complex virus vasilchenko (vs) was constructed previously. virus recovered from pggvs produced slightly smaller plaques than the vs parental virus. sequence analysis demonstrated five nucleotide differences between the original vs virus and pggvs; four of these mutations resulted in amino acid substitutions, while the fifth mutation was located in the 3' untranslated region (3'utr). two mutations were located in conserved regions and thr ...200111413378
virus evolution: how does an enveloped virus make a regular structure? 200111300997
the fusion glycoprotein shell of semliki forest virus: an icosahedral assembly primed for fusogenic activation at endosomal ph.semliki forest virus (sfv) has been extensively studied as a model for analyzing entry of enveloped viruses into target cells. here we describe the trace of the polypeptide chain of the sfv fusion glycoprotein, e1, derived from an electron density map at 3.5 a resolution and describe its interactions at the surface of the virus. e1 is unexpectedly similar to the flavivirus envelope protein, with three structural domains disposed in the same primary sequence arrangement. these results introduce a ...200111301009
phylogenetic analysis of brazilian flavivirus using nucleotide sequences of parts of ns5 gene and 3' non-coding regions.viruses of the genus flavivirus, which are arboviruses, of the flaviviridae family, are amongst the most important agents of infectious disease in brazil, causing human infections with a high morbility and mortality. in this work, the phylogeny of 14 virus amplicon sequences that were obtained by rt-pcr with universal primers for mosquito-borne flavivirus were studied. the amplicons included a region of the flavivirus genome of 129 nucleotides at the 3' terminus of the ns5 gene and the 145 initi ...200111311426
phylogenetic and evolutionary relationships among yellow fever virus isolates in africa.previous studies with a limited number of strains have indicated that there are two genotypes of yellow fever (yf) virus in africa, one in west africa and the other in east and central africa. we have examined the prm/m and a portion of the e protein for a panel of 38 wild strains of yf virus from africa representing different countries and times of isolation. examination of the strains revealed a more complex genetic relationship than previously reported. overall, nucleotide substitutions varie ...200111435580
safety and immunogenicity of attenuated dengue virus vaccines (aventis pasteur) in human volunteers.a randomized, controlled, double-blinded study was conducted to determine safety and immunogenicity of five live attenuated dengue vaccines produced by aventis pasteur (avp). the study was completed with 40 flavivirus non-immune volunteers: five recipients of each monovalent (dengue-1, dengue-2, dengue-3, or dengue-4) vaccine, ten recipients of tetravalent (dengue-1, dengue-2, dengue-3, and dengue-4) vaccine, and ten recipients of vaccine vehicle alone. all vaccines were administered in a single ...200111312014
a small region of the dengue virus-encoded rna-dependent rna polymerase, ns5, confers interaction with both the nuclear transport receptor importin-beta and the viral helicase, ns3.the dengue virus rna-dependent rna polymerase, ns5, and the protease/helicase, ns3, are multidomain proteins that have been shown to interact both in vivo and in vitro. a hyperphosphorylated form of ns5 that does not interact with ns3 has been detected in the nuclei of virus-infected cells, presumably as the result of the action of a functional nuclear localization sequence within the interdomain region of ns5 (residues 369-405). in this study, it is shown by using the yeast two-hybrid system th ...200111257177
langat virus m protein is structurally homologous to prm.langat (lgt) virus m protein has been generated in a recombinant system. antiserum raised against the lgt virus m protein neutralizes tick-borne encephalitis serocomplex flaviviruses but not mosquito-borne flaviviruses, indicating that the m protein is exposed on the surface of virions. the antiserum recognizes intracellular lgt virus prm/m and binds to prm and m in western blots of whole-cell lysates and purified virus, respectively. these data suggest that the prm and m proteins are structural ...200111264391
biophysical characterization and vector-specific antagonist activity of domain iii of the tick-borne flavivirus envelope protein.the molecular determinants responsible for flavivirus host cell binding and tissue tropism are largely unknown, although domain iii of the envelope protein has been implicated in these functions. we examined the solution properties and antagonist activity of langat virus domain iii. our results suggest that domain iii adopts a stably folded structure that can mediate binding of tick-borne flaviviruses but not mosquito-borne flaviviruses to their target cells. three clusters of phylogenetically c ...200111264392
effect of naturally occurring active site mutations on hepatitis c virus ns3 protease specificity.a comparison of the dna sequences from all available genotypes of hcv indicate that the active site residues of the ns3 protease are strictly conserved with the exception of positions 123 and 168, which border the s(4) subsite. in genotype 3, the canonic arginine and aspartic acid have been replaced with threonine and glutamine, respectively. to determine if these differences contribute to an altered specificity, we characterized single-chain ns3 proteases from strains 1a, 1b, and 3a with peptid ...200111276078
a new strategy in design of +rna virus infectious clones enabling their stable propagation in e. coli.infectious clone methodology is a valuable tool of modern experimental virology. however, its use is often constrained by the instability of infectious clone constructs during propagation in e. coli. to circumvent this problem, we have devised a strategy that could be suitable for design of +rna virus molecular clones in general. an infectious clone is assembled as "infectious dna," and expression of problem regions present in the viral cdna is prevented during propagation in e. coli by insertio ...200111277699
diagnostic immunoassays for tick-borne encephalitis virus based on recombinant baculovirus protein expression.the baculovirus expression system that utilizes autographa californica nuclear polyhedrosis virus was used to express the highly antigenic envelope protein e of a tick-borne encephalitis (tbe) complex virus, as well as a c-terminally truncated form of protein e (etr). the recombinant proteins were produced with a histidine-tag at their carboxy-terminus. protein purification by nickel agarose chromatography resulted in high concentrations of pure etr protein, but only poor yields of e protein. th ...200111164488
comparison of six different commercial igg-elisa kits for the detection of tbev-antibodies.tick-borne encephalitis virus (tbev) is a pathogenic human flavivirus endemic in some parts of europe and asia. commercial enzyme immunoassays (eia) for the detection of igg antibodies are often used in tbev-seroprevalence studies, as well as for the confirmation of a successful vaccination against tbev. however, the detection of tbev-specific antibodies can be biased by the cross-reactivity between different flavivirus genera.200111166668
antibody to the nonstructural protein ns1 of japanese encephalitis virus: potential application of mab-based indirect elisa to differentiate infection from vaccination.an indirect enzyme-linked immunosorbent assay (elisa) was developed to detect and differentiate the antibody responses to japanese encephalitis (je) virus nonstructural protein ns1 between infected and vaccinated individuals. the results showed that all convalescent sera from je patients contained ns1-specific igg antibodies, while 65 and 40% of these sera showed detectable ns1-specific igm and iga antibodies, respectively. specificity analysis showed that ns1-specific igm and iga antibodies fro ...200111166901
innate resistance to flavivirus infection in mice controlled by flv is nitric oxide-independent.innate resistance to flaviviruses in mice is active in the brain where it restricts virus replication. this resistance is controlled by a single genetic locus, flv, located on mouse chromosome 5 near the locus encoding the neuronal form of nitric oxide synthase (nos1). since nitric oxide (no) has been implicated in antiviral activity, its involvement in natural resistance to flaviviruses has been hypothesized. here we present data on no production before and during flavivirus infection in both b ...200111172102
[analysis of genetic variability of strains of tick-borne encephalitis virus by primary structure of a fragment of the membrane protein e gene].primary structures of gene fragments of e protein (160 n.b.) have been determined for 29 tick-borne encephalitis (tbe) strains isolated from different parts of a territory. analysis of homology of nucleotide sequences of these strains and data on 6 tbe strains published by other authors showed that they can be divided into 6 groups (genotypes) by the following gene typing criteria: strain structure within the genotypes differing by no more than 9%, differences between strains of different genoty ...200111233280
[genetic typing of tick-borne encephalitis virus based on an analysis of the levels of homology of a membrane protein gene fragment].all heretofore known genomic structures of tick-borne encephalitis (tbe) virus are analyzed. the authors prove the adequacy of using short fragment of e protein gene for characterization of philogenetic relationships between tbe strains. three main genotypes of the virus are distinguished, one corresponding to far eastern variant, one to west, and the third includes strains belonging to ural siberian and central siberian and transbaikal variants. results of genetic typing by nucleotide sequences ...200111233281
[protective activity of a bacterial plasmid, bearing the gene for the tick-borne encephalitis virus ns1 nonstructural protein].three intramuscular injections (50 micrograms each) with bacterial plasmid pmv45 carrying nonstructural gene of ns1 protein of tick-borne encephalitis (tbe) virus protected 88% balb/c mice from lethal challenge with the virus. antibodies to ns1 nonstructural protein were detected in the sera of vaccinated mice after the challenge. absence of antibodies to e structural protein indicated absence of manifest infectious process in mice vaccinated with plasmid and challenged with a lethal dose of tbe ...200111233282
antiviral cytotoxic t cells cross-reactively recognize disparate peptide determinants from related viruses but ignore more similar self- and foreign determinants.we have investigated the reactivities of cytotoxic t (tc) cells against the two immunodominant, h-2k(k)-restricted determinants from the flavivirus: murray valley encephalitis virus (mve), mve(1785) (rehsgnei) and mve(1971) (degegrvi). the respective tc cell populations cross-reactively lysed target cells pulsed with determinants from the mve(1785)- and mve(1971)-corresponding positions of six other flaviviruses, despite low sequence homology in some cases. notably, anti-mve(1785) tc cells recog ...200111238625
purification and characterization of west nile virus nucleoside triphosphatase (ntpase)/helicase: evidence for dissociation of the ntpase and helicase activities of the enzyme.the nucleoside triphosphatase (ntpase)/helicase associated with nonstructural protein 3 of west nile (wn) virus was purified from cell culture medium harvested from virus-infected vero cells. the purification procedure included sequential chromatography on superdex-200 and reactive red 120 columns, followed by a concentration step on an ultrogel hydroxyapatite column. the nature of the purified protein was confirmed by immunoblot analysis using a wn virus-positive antiserum, determination of its ...200111238848
a personal account of the role of peptide research in drug discovery: the case of hepatitis c.although peptides themselves are not usually the end products of a drug discovery effort, peptide research often plays a key role in many aspects of this process. this will be illustrated by reviewing the experience of peptide research carried out at irbm in the course of our study of hepatitis c virus (hcv). the target of our work is the ns3/4a protease, which is essential for maturation of the viral polyprotein. after a thorough examination of its substrate specificity we fine-tuned several su ...200111245202
a novel model for the study of the therapy of flavivirus infections using the modoc virus.the murine flavivirus modoc replicates well in vero cells and appears to be as equally sensitive as both yellow fever and dengue fever virus to a selection of antiviral agents. infection of scid mice, by either the intracerebral, intraperitoneal, or intranasal route, results in 100% mortality. immunocompetent mice and hamsters proved to be susceptible to the virus only when inoculated via the intranasal or intracerebral route. animals ultimately die of (histologically proven) encephalitis with f ...200111145886
stable expression of noncytopathic kunjin replicons simulates both ultrastructural and biochemical characteristics observed during replication of kunjin virus.this report focuses mainly on the characterization of a vero cell line stably expressing the flavivirus kunjin (kun) replicon c20sdrep (c20sdrepvero). we showed by immunofluorescence and cryoimmunoelectron microscopy that unique flavivirus-induced membrane structures, termed convoluted membranes/paracrystalline structures, were induced in the c20sdrepvero cells. these induced cytoplasmic foci were immunolabeled with kun virus anti-ns3 antibodies and with antibodies to the cellular markers ergic5 ...200111145899
coupling between replication and packaging of flavivirus rna: evidence derived from the use of dna-based full-length cdna clones of kunjin virus.in order to study whether flavivirus rna packaging is dependent on rna replication, we generated two dna-based kunjin virus constructs, pkun1 and pkun1dgdd, allowing continuous production of replicating (wild-type) and nonreplicating (with a deletion of the ns5 gene rna-polymerase motif gdd) full-length kunjin virus rnas, respectively, via nuclear transcription by cellular rna polymerase ii. as expected, transfection of pkun1 plasmid dna into bhk cells resulted in the recovery of secreted infect ...200111312333
experimental yellow fever virus infection in the golden hamster (mesocricetus auratus). i. virologic, biochemical, and immunologic studies.this report describes the clinical laboratory findings in golden hamsters experimentally infected with yellow fever (yf) virus. an accompanying paper describes the pathologic findings. following intraperitoneal inoculation of a virulent strain of yf virus, hamsters developed a high-titered viremia (up to 109/ml) lasting 5--6 days and abnormal liver function tests. yf hemagglutination-inhibiting antibodies appeared 4 or 5 days after infection, often while viremia was still present. the mortality ...200111319679
comparison of flavivirus universal primer pairs and development of a rapid, highly sensitive heminested reverse transcription-pcr assay for detection of flaviviruses targeted to a conserved region of the ns5 gene sequences.arthropod-transmitted flaviviruses are responsible for considerable morbidity and mortality, causing severe encephalitic, hemorrhagic, and febrile illnesses in humans. because there are no specific clinical symptoms for infection by a determined virus and because different arboviruses could be present in the same area, a genus diagnosis by pcr would be a useful first-line diagnostic method. the six published flavivirus genus primer pairs localized in the ns1, ns3, ns5, and 3' nc regions were eva ...200111326014
detection and isolation of a flavivirus-like agent from a leopard tortoise (geochelone paradalis) in the united kingdom. 200111338718
evolution of base composition and codon usage bias in the genus flavivirus.the extent to which base composition and codon usage vary among rna viruses, and the possible causes of this bias, is undetermined in most cases. a maximum-likelihood statistical method was used to test whether base composition and codon usage bias covary with arthropod association in the genus flavivirus, a major source of disease in humans and animals. flaviviruses are transmitted by mosquitoes, by ticks, or directly between vertebrate hosts. those viruses associated with ticks were found to h ...200111343134
pathologic and immunohistochemical findings in naturally occuring west nile virus infection in horses.the pathologic and peroxidase immunohistochemical features of west nile flavivirus (wnv) infection were compared in four horses from the northeastern united states and six horses from central italy. in all 10 animals, there were mild to severe polioencephalomyelitis with small t lymphocyte and lesser macrophage perivascular infiltrate, multifocal glial nodules, neutrophils, and occasional neuronophagia. perivascular hemorrhages, also noted macroscopically in two animals, were observed in 50% of ...200111467475
the influence of downstream protein-coding sequence on internal ribosome entry on hepatitis c virus and other flavivirus rnas.some studies suggest that the hepatitis c virus (hcv) internal ribosome entry site (ires) requires downstream 5' viral polyprotein-coding sequence for efficient initiation of translation, but the role of this rna sequence in internal ribosome entry remains unresolved. we confirmed that the inclusion of viral sequence downstream of the aug initiator codon increased ires-dependent translation of a reporter rna encoding secretory alkaline phosphatase, but found that efficient translation of chloram ...200111345437
tick-borne langat/mosquito-borne dengue flavivirus chimera, a candidate live attenuated vaccine for protection against disease caused by members of the tick-borne encephalitis virus complex: evaluation in rhesus monkeys and in mosquitoes.langat virus (lgt), strain tp21, a naturally avirulent tick-borne flavivirus, was used to construct a chimeric candidate virus vaccine which contained lgt genes for premembrane (prem) and envelope (e) glycoprotein and all other sequences derived from dengue type 4 virus (den4). the live virus vaccine was developed to provide resistance to the highly virulent, closely related tick-borne flaviviruses that share protective e epitopes among themselves and with lgt. toward that end the chimera, initi ...200111483771
assessment of substrate specificity of hepatitis g virus ns3 protease by a genetic method.the rna genome of hepatitis g virus (hgv) encodes a large polyprotein that is processed to mature proteins by viral-encoded proteases. the hgv ns3 protease is responsible for the cleavage of the hgv polyprotein at four different locations. no conserved sequence motif has been identified for the cleavage sites of the ns3 protease. to determine the substrate specificity of the ns3 protease, amino acid sequences cleaved by the ns3 protease were obtained from randomized sequence libraries by using a ...200111485324
characterisation of inter- and intra-molecular interactions of the dengue virus rna dependent rna polymerase as potential drug targets.our research is directed towards enhancing the understanding of the molecular biology of dengue virus replication with the ultimate goal being to develop novel antiviral strategies based on preventing critical inter- or intra-molecular interactions required for the normal virus life cycle. the viral rna-dependent rna polymerase (ns5) and the viral helicase (ns3) interaction offers a possible target for inhibitors to bind and prevent replication. in this study the yeast-two hybrid system was used ...200111347963
rapid elimination of gb virus c (hepatitis g virus) in the mosquito aedes aegypti.the transmissibility of the gb virus c (hepatitis g virus; hgv), a member of the flaviviridae, by a typical flavivirus vector was investigated. female mosquitoes of the species aedes aegypti were fed with hgv-infected human blood and assayed 1, 24, 48, 72 and 96 h after the blood meal for viral rna, human glyceraldehyde-3-phosphate dehydrogenase mrna, human beta-actin dna and a. aegypti actin mrna by total nucleic acid extraction, reverse transcription and pcr. viral rna had already disappeared ...200111489416
adaptation of tick-borne encephalitis virus to bhk-21 cells results in the formation of multiple heparan sulfate binding sites in the envelope protein and attenuation in vivo.propagation of the flavivirus tick-borne encephalitis virus in bhk-21 cells selected for mutations within the large surface glycoprotein e that increased the net positive charge of the protein. in the course of 16 independent experiments, 12 different protein e mutation patterns were identified. these were located in all three of the structural domains and distributed over almost the entire upper and lateral surface of protein e. the mutations resulted in the formation of local patches of predom ...200111356970
[pathogenesis of hemorrhagic fever]. 200111496775
modulation of transporter associated with antigen processing (tap)-mediated peptide import into the endoplasmic reticulum by flavivirus infection.in contrast to many other viruses that escape the cellular immune response by downregulating major histocompatibility complex (mhc) class i molecules, flavivirus infection can upregulate their cell surface expression. previously we have presented evidence that during flavivirus infection, peptide supply to the endoplasmic reticulum is increased (a. müllbacher and m. lobigs, immunity 3:207-214, 1995). here we show that during the early phase of infection with different flaviviruses, the transport ...200111356974
dengue and other emerging flaviviruses.flaviviruses are among the most important emerging viruses known to man. most are arboviruses (arthropod-borne) being transmitted by mosquitoes or ticks. they derived from a common ancestor 10-20000 years ago and are evolving rapidly to fill new ecological niches. many are spreading to new geographical areas and causing increased numbers of infections. traditionally, three clinical syndromes are recognized: fever-arthralgia-rash, viral haemorrhagic fever, and neurological disease, though for som ...200111531316
west nile virus recombinant dna vaccine protects mouse and horse from virus challenge and expresses in vitro a noninfectious recombinant antigen that can be used in enzyme-linked immunosorbent assays.introduction of west nile (wn) virus into the united states in 1999 created major human and animal health concerns. currently, no human or veterinary vaccine is available to prevent wn viral infection, and mosquito control is the only practical strategy to combat the spread of disease. starting with a previously designed eukaryotic expression vector, we constructed a recombinant plasmid (pcbwn) that expressed the wn virus prm and e proteins. a single intramuscular injection of pcbwn dna induced ...200111287553
solution structure and dynamics of the single-chain hepatitis c virus ns3 protease ns4a cofactor complex.the backbone assignments, secondary structure, topology, and dynamics of the single-chain hepatitis c virus ns3 protease ns4a cofactor complex have been determined by nmr spectroscopy. residues i34 to s181 of ns3 and the central three residues of the ns4a cofactor were assigned and the secondary structure was verified for these residues. in several x-ray structures of ns4a-bound ns3 protease, residues 1 to 28 are stabilized by crystal packing, which allows for the formation of the a0 strand and ...200111162117
infection with gb virus c and reduced mortality among hiv-infected patients.the flavivirus gb virus c (gbv-c, also designated hepatitis g virus) was identified in a search for hepatitis viruses, but no disease is currently known to be associated with it. we investigated the relation between coinfection with gbv-c and the long-term outcome in patients infected with the human immunodeficiency virus (hiv).200111547740
experimental infections of pigs with japanese encephalitis virus and closely related australian flaviviruses.the flavivirus japanese encephalitis (je) virus has recently emerged in the australasian region. to investigate the involvement of infections with related enzootic flaviviruses, namely murray valley encephalitis (mve) virus and kunjin (kun) virus, on immunity of pigs to je virus and to provide a basis for interpretation of serologic data, experimental infections were conducted with combinations of these viruses. antibody responses to primary and secondary infections were evaluated using panels o ...200111693888
genomic sequencing of deer tick virus and phylogeny of powassan-related viruses of north america.powassan (pow) virus is responsible for central nervous system infection in humans in north america and the eastern parts of russia. recently, a new flavivirus, deer tick (dt) virus, related to pow virus was isolated in the united states, but neither its pathogenic potential in human nor the taxonomic relationship with pow virus has been elucidated. in this study, we obtained the near-full-length genomic sequence of the dt virus and complete sequences of 3 genomic regions of 15 strains of pow-re ...200111716135
characterization of tick-borne encephalitis virus from latvia: evidence for co-circulation of three distinct subtypes.viruses of the tick-borne encephalitis (tbe) antigenic complex within the family flaviviridae cause a variety of diseases, including uncomplicated febrile illness, meningoencephalitis, and hemorrhagic fever. different domesticated animals or wildlife species often act as reservoir hosts and ixodid ticks serve as vectors. although tbe is a serious problem in latvia, the knowledge concerning tbe virus (tbev) strains circulating in the country is most limited. only two strains (latvia-1-96 isolated ...200111745938
outbreak of powassan encephalitis--maine and vermont, 1999-2001.powassan (pow) virus, a north american tickborne flavivirus related to the eastern hemisphere's tickborne encephalitis viruses, was first isolated from a patient with encephalitis in 1958. during 1958-1998, 27 human pow encephalitis cases were reported from canada and the northeastern united states. during september 1999-july 2001, four maine and vermont residents with encephalitis were found to be infected with pow virus. these persons were tested for other arbovirus infections found in the nor ...200111787585
flavivirus dna vaccines: current status and potential.the use of dna-based vaccines is a novel and promising immunization approach for the development of flavivirus vaccines. this approach has been attempted in vaccine development for various virus species, including st. louis encephalitis, russian spring-summer encephalitis, central european encephalitis, dengue serotypes 1 and 2, murray valley encephalitis, japanese encephalitis, and west nile viruses. however, very little is known about the factors affecting its efficacy. recently, we demonstrat ...200111797784
sequence comparison and secondary structure analysis of the 3' noncoding region of flavivirus genomes reveals multiple pseudoknots.sequences of 191 flavivirus rnas belonging to four sero-groups were used to predict the secondary structure of the 3' noncoding region (3' ncr) directly upstream of the conserved terminal hairpin. in mosquito-borne flavivirus rnas (n = 164) a characteristic structure element was identified that includes a phylogenetically well-supported pseudoknot. this element is repeated in the dengue and japanese encephalitis rnas and centers around the conserved sequences cs2 and rcs2. in yellow fever virus ...200111680841
role of reactive oxygen intermediates in japanese encephalitis virus infection in murine neuroblastoma cells.infection with japanese encephalitis virus (jev), a mosquito-borne, neurotropic flavivirus, may cause acute encephalitis in humans and induce severe cytopathic effects in various types of cultured cells. this study attempted to determine whether jev infection induces free radical generation and whether oxidative stress contributes to virus-induced cell death in neuroblastoma cells. a rise in the intracellular level of free radicals indicated by the 2',7'-dichlorofluorescein fluorescence was obse ...200111711202
gb virus-c infection in patients infected with the human immunodeficiency virus.hepatitis virus infections are frequent in patients suffering from hiv infection due to similar transmission routes of these viruses. in addition, hepatitis virus infections lead to impaired survival in hiv positive patients. the recently discovered flavivirus gb virus c (alias hepatitis g virus) was initially believed to be another hepatitis virus. while there is still some minor discussion whether gb virus c (gbv-c) plays a role in fulminant hepatic failure, there is no evidence that this viru ...200111672817
phylogenetic relationships of flaviviruses correlate with their epidemiology, disease association and biogeography.phylogenetic analysis of the flavivirus genus, using either partial sequences of the non-structural 5 gene or the structural envelope gene, revealed an extensive series of clades defined by their epidemiology and disease associations. these phylogenies identified mosquito-borne, tick-borne and no-known-vector (nkv) virus clades, which could be further subdivided into clades defined by their principal vertebrate host. the mosquito-borne flaviviruses revealed two distinct epidemiological groups: ( ...200111457992
salicylates inhibit flavivirus replication independently of blocking nuclear factor kappa b activation.flaviviruses comprise a positive-sense rna genome that replicates exclusively in the cytoplasm of infected cells. whether flaviviruses require an activated nuclear factor(s) to complete their life cycle and trigger apoptosis in infected cells remains elusive. flavivirus infections quickly activate nuclear factor kappa b (nf-kappab), and salicylates have been shown to inhibit nf-kappab activation. in this study, we investigated whether salicylates suppress flavivirus replication and virus-induced ...200111483726
a 'minimal' approach in design of flavivirus infectious dna.the 'infectious dna' approach, which is based on in vivo transcription of (+)rna virus genome cdna cassettes from eukaryotic promoters in transfected cells, became a popular alternative to the classical scheme in the infectious clone methodology. its use, however, is often limited by the instability of plasmids due to a transcriptional activity of eukaryotic promoters in escherichia coli resulting in synthesis of products toxic for the bacterial host. using a highly unstable representative infec ...200111682130
the west nile virus encephalitis outbreak in the united states (1999-2000): from flushing, new york, to beyond its borders.viruses cause most forms of encephalitis. the two main types responsible for epidemic encephalitis are enteroviruses and arboviruses. the city of new york reports about 10 cases of encephalitis yearly. establishing a diagnosis is often difficult. in august 1999, a cluster of five patients with fever, confusion, and weakness were admitted to a community hospital in flushing, new york. flaccid paralysis developed in four of the five patients, and they required ventilatory support. three, less seve ...200111797774
host factors involved in west nile virus replication.viruses use cell proteins during many stages of their replication cycles, including attachment, entry, translation, transcription/replication, and assembly. mutations in the cell proteins involved can cause disruptions of these critical host-virus interactions, which in turn can affect the efficiency of virus replication. these host-virus interactions also represent novel targets for the development of new antiviral agents. the different alleles of the murine flv gene confer resistance or suscep ...200111797778
use of live and inactivated vaccines in the control of west nile fever in domestic geese.the recent epizootic of west nile fever in israel affected predominantly young domestic geese between three and eight weeks old. clinically, the birds presented paralytic signs while morbidity and mortality were severe in affected flocks. the condition was encountered from early september through late november on goose farms located throughout the country. losses incurred by goose flocks were sufficiently great as to warrant investigation of ways to protect young geese against the neurological f ...200111797782
the machinery for flavivirus fusion with host cell membranes.a combination of structural, biochemical and functional studies with the flavivirus tick-borne encephalitis virus has revealed the characteristics of a new class of viral fusion protein, class ii, that is unrelated to the class i viral fusion proteins for which influenza virus hemagglutinin is the prototype. new structural data have shown that the alphaviruses, another group of icosahedral enveloped viruses, also have class ii fusion proteins, suggesting a common origin.200111495810
terminal nucleotidyl transferase activity of recombinant flaviviridae rna-dependent rna polymerases: implication for viral rna synthesis.recombinant hepatitis c virus (hcv) rna-dependent rna polymerase (rdrp) was reported to possess terminal transferase (tntase) activity, the ability to add nontemplated nucleotides to the 3' end of viral rnas. however, this tntase was later purported to be a cellular enzyme copurifying with the hcv rdrp. in this report, we present evidence that tntase activity is an inherent function of hcv and bovine viral diarrhea virus rdrps highly purified from both prokaryotic and eukaryotic cells. a change ...200111507207
current status of flavivirus vaccines.although there are approximately 68 flaviviruses recognized, vaccines have been developed to control very few human flavivirus diseases. licensed live attenuated vaccines have been developed for yellow fever (strain 17d) and japanese encephalitis (strain sa14-14-2) viruses, and inactivated vaccines have been developed for japanese encephalitis and tick-borne encephalitis viruses. the yellow fever live attenuated 17d vaccine is one of the most efficacious and safe vaccines developed to date and h ...200111797783
antibody prophylaxis and therapy for flavivirus encephalitis infections.the outbreak of west nile (wn) encephalitis in the united states has rekindled interest in developing direct methods for prevention and control of human flaviviral infections. although equine wn vaccines are currently being developed, a wn vaccine for humans is years away. there is also no specific therapeutic agent for flaviviral infections. the incidence of human wn virus infection is very low, which makes it difficult to target the human populations in need of vaccination and to assess the va ...200111797785
west nile virus transmission and ecology in birds.the ecology of the strain of west nile virus (wnv) introduced into the united states in 1999 has similarities to the native flavivirus, st. louis encephalitis (sle) virus, but has unique features not observed with sle virus or with wnv in the old world. the primary route of transmission for most of the arboviruses in north america is by mosquito, and infected native birds usually do not suffer morbidity or mortality. an exception to this pattern is eastern equine encephalitis virus, which has an ...200111797804
west nile virus infection in birds and mammals.west nile virus (wnv) was found throughout new york state in year 2000. the epicenter was located in new york city with a high level of activity in the immediately surrounding counties, including rockland, westchester, nassau, and suffolk. during 2000, wnv testing was performed by the wadsworth center on 3,687 dead birds, representing 153 species, 46 families, and 18 orders. there were 1,203 wnv-positive birds, representing 63 species, 30 families and 14 orders. the percentage of wnv-positive bi ...200111797807
identification of epitopes on the envelope (e) protein of dengue 2 and dengue 3 viruses using monoclonal antibodies.of a panel of forty-six anti-dengue 3 monoclonal antibodies (mabs) only three neutralised infection of bhk cells by dengue 3 virus. attempts to select neutralisation escape mutants (n.e.m.) with two of these antibodies failed. the n.e.m. population selected in the presence of the third neutralising antibody, 1h9, had a nucleotide change at position 1157 of the e protein gene resulting in a non-conservative amino acid change at e386 for a lys to an asn. a dengue 2 n.e.m. was selected with the fla ...200111811694
immunogenicity of hgv ns5 protein expressed from sf9 insect cells. 200111819741
immunization of mice against west nile virus with recombinant envelope protein.west nile (wn) virus is a mosquito-borne flavivirus that emerged in the united states in 1999 and can cause fatal encephalitis. envelope (e) protein cdna from a wn virus isolate recovered from culex pipiens in connecticut was expressed in escherichia coli. the recombinant e protein was purified and used as ag in immunoblot assays and immunization experiments. patients with wn virus infection had abs that recognized the recombinant e protein. c3h/hen mice immunized with e protein developed e prot ...200111673542
a novel mechanism to explain protein abnormalities in schizophrenia based on the flavivirus resistance gene.the geographical distribution of schizophrenia was previously shown to correlate with the global distribution of tick-borne flaviviruses. the correlation suggests a natural resistance gene to flaviviruses could be involved in schizophrenia. the flavivirus resistance gene, flv, a gene found in wild mice and certain in-bred strains, confers resistance to flaviviruses through the interaction of cellular proteins with the flaviviral 3' untranslated regions (utrs). although the sequence and product o ...200111673799
de novo synthesis of rna by the dengue virus rna-dependent rna polymerase exhibits temperature dependence at the initiation but not elongation phase.replication of positive strand flaviviruses is mediated by the viral rna-dependent rna polymerases (rdrp). to study replication of dengue virus (den), a flavivirus family member, an in vitro rdrp assay was established using cytoplasmic extracts of den-infected mosquito cells and viral subgenomic rna templates containing 5'- and 3'-terminal regions (trs). evidence supported that an interaction between the trs containing conserved stem-loop, cyclization motifs, and pseudoknot structural elements i ...200111546770
immunopathogenesis of hepatitis c virus infection.hepatitis c virus, a recently identified member of the family flaviviridae, is an important cause of chronic viral hepatitis and cirrhosis. there are similarities in the nature of the immune response to this pathogen with immunity in other flavivirus and hepatotropic virus infections, such as hepatitis b. however, the high rate of viral persistence after primary hepatitis c infection, and the observation that neutralizing antibodies are not protective, would suggest that there are a number of im ...200111903612
lack of both fas ligand and perforin protects from flavivirus-mediated encephalitis in mice.the mechanism by which encephalitic flaviviruses enter the brain to inflict a life-threatening encephalomyelitis in a small percentage of infected individuals is obscure. we investigated this issue in a mouse model for flavivirus encephalitis in which the virus was administered to 6-week-old animals by the intravenous route, analogous to the portal of entry in natural infections, using a virus dose in the range experienced following the bite of an infectious mosquito. in this model, infection wi ...200211884544
derivation and characterization of a dengue type 1 host range-restricted mutant virus that is attenuated and highly immunogenic in monkeys.we recently described the derivation of a dengue serotype 2 virus (den2mutf) that exhibited a host range-restricted phenotype; it was severely impaired for replication in cultured mosquito cells (c6/36 cells). den2mutf virus had selected mutations in genomic sequences predicted to form a 3' stem-loop structure (3'-sl) that is conserved among all flavivirus species. the 3'-sl constitutes the downstream terminal similar95 nucleotides of the 3' noncoding region in flavivirus rna. here we report the ...200211884557
antiviral effects of an iminosugar derivative on flavivirus infections.endoplasmic reticulum (er) alpha-glucosidase inhibitors, which block the trimming step of n-linked glycosylation, have been shown to eliminate the production of several er-budding viruses. here we investigated the effects of one such inhibitor, n-nonyl-deoxynojirimycin (nn-dnj), a 9-carbon alkyl iminosugar derivative, on infection by japanese encephalitis virus (jev) and dengue virus serotype 2 (den-2). in the presence of nn-dnj, jev and den-2 infections were suppressed in a dose-dependent manne ...200211907199
membrane interactions of the tick-borne encephalitis virus fusion protein e at low ph.membrane fusion of the flavivirus tick-borne encephalitis virus is triggered by the mildly acidic ph of the endosome and is mediated by envelope protein e, a class ii viral fusion protein. the low-ph trigger induces an oligomeric rearrangement in which the subunits of the native e homodimers dissociate and the monomeric subunits then reassociate into homotrimers. here we provide evidence that membrane binding is mediated by the intermediate monomeric form of e, generated by low-ph-induced dissoc ...200211907218
kunjin virus replicon vaccine vectors induce protective cd8+ t-cell immunity.the ability of self-replicating rna (replicon) vaccine vectors derived from the australian flavivirus kunjin (kun) to induce protective alphabeta cd8+ t-cell responses was examined. kun replicons encoding a model immunogen were delivered by three different vaccine modalities: (i) as naked rna transcribed in vitro, (ii) as plasmid dna constructed to allow in vivo transcription of replicon rna by cellular rna polymerase ii (dna based), and (iii) as replicon rna encapsidated into virus-like particl ...200211907219
emergence of usutu virus, an african mosquito-borne flavivirus of the japanese encephalitis virus group, central europe.during late summer 2001 in austria, a series of deaths in several species of birds occurred, similar to the beginning of the west nile virus (wnv) epidemic in the united states. we necropsied the dead birds and examined them by various methods; pathologic and immunohistologic investigations suggested a wnv infection. subsequently, the virus was isolated, identified, partially sequenced, and subjected to phylogenetic analysis. the isolates exhibited 97% identity to usutu virus (usuv), a mosquito- ...200212095429
structural surprises from the flaviviruses and alphaviruses.recent structural studies demonstrate that the alphavirus and flavivirus fusion proteins, although very similar in overall fold, are arranged very differently in the two virions. these differences raise many interesting questions about virus assembly and fusion activity.200211931753
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
complete genome sequence of montana myotis leukoencephalitis virus, phylogenetic analysis and comparative study of the 3' untranslated region of flaviviruses with no known vector.montana myotis leukoencephalitis virus (mmlv), a virus isolated from bats, causes an encephalitis in small rodents reminiscent of flavivirus encephalitis in humans. the complete mmlv genome is 10690 nucleotides long and encodes a putative polyprotein of 3374 amino acids. the virus contains the same conserved motifs in genes that are believed to be interesting antiviral targets (ntpase/helicase, serine protease and rna-dependent rna polymerase) as flaviviruses of clinical importance. phylogenetic ...200212124451
infection of scid mice with montana myotis leukoencephalitis virus as a model for flavivirus encephalitis.we have established a convenient animal model for flavivirus encephalitis using montana myotis leukoencephalitis virus (mmlv), a bat flavivirus. this virus has the same genomic organization, and contains the same conserved motifs in genes that encode potential antiviral targets, as flaviviruses that cause disease in man (n. charlier et al., accompanying paper), and has a similar particle size (approximately 40 nm). mmlv replicates well in vero cells and appears to be equally as sensitive as yell ...200212124452
west nile virus/dengue type 4 virus chimeras that are reduced in neurovirulence and peripheral virulence without loss of immunogenicity or protective efficacy.a candidate live attenuated vaccine strain was constructed for west nile virus (wn), a neurotropic flavivirus that has recently emerged in the u.s. considerable attenuation for mice was achieved by chimerization with dengue virus type 4 (den4). the genes for the structural premembrane and envelope proteins of den4 present in an infectious cdna clone were replaced by the corresponding genes of wn strain ny99. two of 18 cdna clones of a wn/den4 chimera yielded full-length rna transcripts that were ...200211880643
[localization of tick-borne encephalitis virus protein e antigenic determinant recognized by antihemagglutinating monoclonal antibodies using a phage-display peptide library].bacteriophages bearing peptides reacting with antihemagglutinating monoclonal antibodies (mab) 10h10 to tick-borne encephalitis (tbe) virus protein e were selected from a phage-display peptide library by affinity selection and enzyme immunoassay. the library contained randomized peptides that are 6 amino acids long, fused with protein piii and exposed on the surface of the bacteriophage. no significant homology between the sequences of selected peptides and tbe virus protein e was detected. comp ...200212046465
use of immunoglobulin m cross-reactions in differential diagnosis of human flaviviral encephalitis infections in the united states.to define the virus specificity of the immunoglobulin m (igm) antibody-capture enzyme-linked immunosorbent assay (mac-elisa) among the medically important members of the japanese encephalitis (je) virus serocomplex of flaviviruses, 103 igm-positive human serum samples from patients with confirmed west nile (wn) virus, st. louis encephalitis (sle) virus, or je virus infections were assembled and simultaneously tested against all three viral antigens in a standardized mac-elisa. of the serum sampl ...200211986257
hemorrhagic fever viruses as biological weapons: medical and public health management.to develop consensus-based recommendations for measures to be taken by medical and public health professionals if hemorrhagic fever viruses (hfvs) are used as biological weapons against a civilian population.200211988060
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
emerging arboviral diseases. 200212054284
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
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
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
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
the japanese encephalitis serological group of flaviviruses: a brief introduction to the group. 200212082984
a single amino acid is critical for the expression of b-cell epitopes on the helicase domain of the pestivirus ns3 protein.truncated ns3 proteins, expressed by recombinant baculoviruses, were used to investigate the location of conserved b-cell epitopes on this non-structural bovine viral diarrhoea virus (bvdv) protein. a goat anti-pestivirus antiserum, and a panel of anti-ns3 monoclonal antibodies, including the bvdv-1 specific antibody p1d8, were used to verify the presence or absence of the epitopes. interestingly, the monoclonal antibodies reacted only with the truncated protein encompassing the helicase domain ...200211900844
complete genome sequence, taxonomic assignment, and comparative analysis of the untranslated regions of the modoc virus, a flavivirus with no known vector.we recently developed a model for flavivirus infection in mice and hamsters using the modoc virus (modv), a flavivirus with no known vector (p. leyssen, a. van lommel, c. drosten, h. schmitz, e. de clercq, and j. neyts, 2001, virology 279, 27-37). we now present the coding and noncoding sequence of modv. the modoc virus genome was determined to be 10,600 nucleotides in length with a single open reading frame extending from nucleotides 110 to 10,234, encoding 3374 amino acids. the deduced gene or ...200211853406
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
human cytotoxic t lymphocyte responses to live attenuated 17d yellow fever vaccine: identification of hla-b35-restricted ctl epitopes on nonstructural proteins ns1, ns2b, ns3, and the structural protein e.yellow fever virus (yfv) is a re-emerging problem despite the existence of an effective live-attenuated vaccine. the induction of yfv-neutralizing antibodies undoubtedly contributes to vaccine efficacy, but t lymphocyte responses to yfv likely play a role in long-term efficacy. we studied the t lymphocyte responses to yfv in four vaccinees. proliferation and cytolytic responses to yfv were demonstrated in all subjects. we isolated 13 yfv-specific cd8(+) ctl lines that recognized epitopes on the ...200211853408
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
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
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
structure of dengue virus: implications for flavivirus organization, maturation, and fusion.the first structure of a flavivirus has been determined by using a combination of cryoelectron microscopy and fitting of the known structure of glycoprotein e into the electron density map. the virus core, within a lipid bilayer, has a less-ordered structure than the external, icosahedral scaffold of 90 glycoprotein e dimers. the three e monomers per icosahedral asymmetric unit do not have quasiequivalent symmetric environments. difference maps indicate the location of the small membrane protein ...200211893341
heparan sulfate-mediated binding of infectious dengue virus type 2 and yellow fever virus.dengue virus type 2 and yellow fever virus are arthropod-borne flaviviruses causing hemorrhagic fever in humans. identification of virus receptors is important in understanding flavivirus pathogenesis. the aim of this work was to study the role of cellular heparan sulfate in the adsorption of infectious yellow fever and dengue type 2 viruses. virus attachment was assessed by adsorbing virus to cells, washing unbound virus away, releasing cell-bound virus by freezing/thawing, and then titrating t ...200211878919
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