determination of the intramolecular disulfide bond arrangement and biochemical identification of the glycosylation sites of the nonstructural protein ns1 of murray valley encephalitis virus.the 12 cysteine residues in the flavivirus ns1 protein are strictly conserved, suggesting that they form disulfide bonds that are critical for folding the protein into a functional structure. in this study, we examined the intramolecular disulfide bond arrangement of ns1 of murray valley encephalitis virus and elucidated three of the six cysteine-pairing arrangements. disulfide linkages were identified by separating tryptic-digested ns1 by reverse-phase high pressure liquid chromatography and an ...200111514736
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
[pathogenesis of hemorrhagic fever]. 200111496775
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
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
recent advances in prevention and treatment of hepatitis c virus infections.hepatitis c virus (hcv) is the leading cause of chronic hepatitis in humans. as members of the flavivirus family, hcvs are a group of small single-stranded, positive-sense rna viruses. upon translation of the genome, a polyprotein precursor is synthesized and further processed by both cellular and viral proteases to generate functional viral proteins. treatment options are currently limited to the administration of alpha-interferon alone or in combination with ribavirin. unfortunately, these app ...200111548211
effector cytolotic function but not ifn-gamma production in cytotoxic t cells triggered by virus-infected target cells in vitro.cytolysis and interferon(ifn)-gamma production are two independent effector functions of activated cytotoxic t (tc) cells. we have used the tc-cell response against the flavivirus, murray valley encephalitis virus (mve), to investigate the requirements for inducing these two functions with regard to antigen-concentration and cd8 coreceptor involvement. cognate peptide-pulsed target cells triggered cytolysis by primary ex vivo mve-immune as well as in vitro peptide-restimulated splenocytes at low ...200111555403
nucleotide sequencing and serological evidence that the recently recognized deer tick virus is a genotype of powassan virus.deer tick virus (dtv) is a recently recognized north american virus isolated from ixodes dammini ticks. nucleotide sequencing of fragments of structural and non-structural protein genes suggested that this virus was most closely related to the tick-borne flavivirus powassan (pow), which causes potentially fatal encephalitis in humans. to determine whether dtv represents a new and distinct member of the flavivirus genus of the family flaviviridae, we sequenced the structural protein genes and 5' ...200111551648
construction, characterization and immunogenicity of recombinant yellow fever 17d-dengue type 2 viruses.chimeric yellow fever (yf)-dengue type 2 (den 2) viruses were constructed by replacing the premembrane (prm) and envelope (e) genes of yf 17d virus with those from den 2 virus strains of south-east asian genotype. whereas viable chimeric viruses were successfully recovered when the yf 17d c gene and the den 2 prm gene were fused at the signalase cleavage site, no virus could be rescued from the constructions fused at the viral protease cleavage site. unlike yf virus that replicated in all the ce ...200111551641
chemical mutagenesis of dengue virus type 4 yields mutant viruses which are temperature sensitive in vero cells or human liver cells and attenuated in mice.a recombinant live attenuated dengue virus type 4 (den4) vaccine candidate, 2adelta30, was found previously to be generally well tolerated in humans, but a rash and an elevation of liver enzymes in the serum occurred in some vaccinees. 2adelta30, a non-temperature-sensitive (non-ts) virus, contains a 30-nucleotide deletion (delta30) in the 3' untranslated region (utr) of the viral genome. in the present study, chemical mutagenesis of den4 was utilized to generate attenuating mutations which may ...200111559806
mutagenesis of the dengue virus type 2 ns3 protein within and outside helicase motifs: effects on enzyme activity and virus replication.the protein ns3 of dengue virus type 2 (den-2) is the second largest nonstructural protein specified by the virus and is known to possess multiple enzymatic activities, including a serine proteinase located in the n-terminal region and an ntpase-helicase in the remaining 70% of the protein. the latter region has seven conserved helicase motifs found in all members of the family flaviviridae. den-2 ns3 lacking the proteinase region was synthesized as a fusion protein with glutathione s-transferas ...200111559795
the relationships between west nile and kunjin viruses.until recently, west nile (wn) and kunjin (kun) viruses were classified as distinct types in the flavivirus genus. however, genetic and antigenic studies on isolates of these two viruses indicate that the relationship between them is more complex. to better define this relationship, we performed sequence analyses on 32 isolates of kun virus and 28 isolates of wn virus from different geographic areas, including a wn isolate from the recent outbreak in new york. sequence comparisons showed that th ...200111585535
mutations in the ns3 gene and 3'-ncr of japanese encephalitis virus isolated from an unconventional ecosystem and implications for natural attenuation of the virus.the t1p1 strain of japanese encephalitis (je) virus was recently isolated from paddy-free liu-chiu islet in which natural je antibody has been prevalent. in mouse neuroblastoma-derived neuro-2a cells, t1p1 appeared significantly lower in virus productivity than another local isolate, ch1392. it implied that this new isolate possesses a characteristic viral replication pattern other than that of ch1392. t1p1 has also shown lower neurovirulence, which was reflected by a significantly higher ld(50) ...200111601924
transcriptional regulation of major histocompatibility complex class i by flavivirus west nile is dependent on nf-kappab activation.infection by the flavivirus west nile (wnv) is associated with a virus-specific increase of major histocompatibility complex class i (mhc-i) molecules on the cell surface of diploid vertebrate cells. the increased mhc-i cell surface expression is functional and is associated with increased susceptibility to secondary wnv-immune and alloimmune cytotoxic t cells. wnv-induced up-regulation of cell surface mhc-i expression is associated with nf-kappab activation and increased transcription of mhc-i ...200111574908
activity of recombinant dengue 2 virus ns3 protease in the presence of a truncated ns2b co-factor, small peptide substrates, and inhibitors.recombinant forms of the dengue 2 virus ns3 protease linked to a 40-residue co-factor, corresponding to part of ns2b, have been expressed in escherichia coli and shown to be active against para-nitroanilide substrates comprising the p6-p1 residues of four substrate cleavage sequences. the enzyme is inactive alone or after the addition of a putative 13-residue co-factor peptide but is active when fused to the 40-residue co-factor, by either a cleavable or a noncleavable glycine linker. the ns4b/n ...200111581268
a phylogenetic approach to following west nile virus in connecticut.the 1999 outbreak of west nile (wn) virus in the northeastern united states was the first known natural occurrence of this flavivirus in the western hemisphere. in 1999 and 2000, 82 independent connecticut wn virus isolates were cultured from nine species of birds, five species of mosquitoes, and one striped skunk. nucleotide sequences obtained from these isolates identified 30 genetic changes, compared with wn-ny99, in a 921-nt region of the viral genome beginning at nucleotide position 205 and ...200111606791
complete coding sequence of the alkhurma virus, a tick-borne flavivirus causing severe hemorrhagic fever in humans in saudi date, tick-borne flaviviruses responsible for hemorrhagic fever in humans have been isolated in siberia (omsk hemorrhagic fever virus), india (kyasanur forest disease virus, kfdv), and in saudi arabia (alkhurma virus, alkv). prior to this study, only partial coding sequences of these severe pathogens had been determined. we report here the complete coding sequence of alk virus, which was determined to be 10,248 nucleotides (nt) long, and to encode a single 3,416 amino acid polyprotein. indepe ...200111554750
isolation and characterization of noncytopathic pestivirus mutants reveals a role for nonstructural protein ns4b in viral cytopathogenicity.isolates of bovine viral diarrhea virus (bvdv), the prototype pestivirus, are divided into cytopathic (cp) and noncytopathic (ncp) biotypes according to their effect on cultured cells. the cp viruses also differ from ncp viruses by the production of viral nonstructural protein ns3. however, the mechanism by which cp viruses induce cytopathic effect in cell culture remains unknown. here we used a genetic approach to isolate ncp variants that arose from a cp virus at low frequency. a bicistronic b ...200111602707
assembly and maturation of the flavivirus kunjin virus appear to occur in the rough endoplasmic reticulum and along the secretory pathway, respectively.the intracellular assembly site for flaviviruses in currently not known but is presumed to be located within the lumen of the rough endoplasmic reticulum (rer). building on previous studies involving immunofluorescence (if) and cryoimmunoelectron microscopy of kunjin virus (kun)-infected cells, we sought to identify the steps involved in the assembly and maturation of kun. thus, using antibodies directed against envelope protein e in if analysis, we found the accumulation of e within regions coi ...200111602720
evolution, epidemiology, and dispersal of flaviviruses revealed by molecular phylogenies. 200111680389
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
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
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
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
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
nucleic acid sequence-based amplification assays for rapid detection of west nile and st. louis encephalitis viruses.the development and application of nucleic acid sequence-based amplification (nasba) assays for the detection of west nile (wn) and st. louis encephalitis (sle) viruses are reported. two unique detection formats were developed for the nasba assays: a postamplification detection step with a virus-specific internal capture probe and electrochemiluminescence (nasba-ecl assay) and a real-time assay with 6-carboxyfluorescein-labeled virus-specific molecular beacon probes (nasba-beacon assay). the sen ...200111724870
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
[patient with west nile fever in the netherlands].a 45-year-old patient was admitted with high fever and inadequate behaviour. she had recently stayed in israel. extensive investigation suggested a viral infection, but the cause was not established. her partner suggested the possibility of west nile virus infection, based upon information he had found on the internet. serology was performed and specific igm and igg antibodies were demonstrated, but a paired serum sample was not available. the patient recovered spontaneously. the west nile virus ...200111715596
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
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
kunjin virus: an australian variant of west nile?kunjin (kun) is a flavivirus in the japanese encephalitis antigenic complex that was first isolated from culex annulirostris mosquitoes captured in northern australia in 1960. it is the etiological agent of a human disease characterized by febrile illness with rash or mild encephalitis and, occasionally, of a neurological disease in horses. kun virus shares a similar epidemiology and ecology with the closely related murray valley encephalitis (mve) virus, the major causative agent of arboviral e ...200111797773
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
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
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
paired charge-to-alanine mutagenesis of dengue virus type 4 ns5 generates mutants with temperature-sensitive, host range, and mouse attenuation phenotypes.charge-to-alanine mutagenesis of dengue virus type 4 (den4) ns5 gene generated a collection of attenuating mutations for potential use in a recombinant live attenuated den vaccine. codons for 80 contiguous pairs of charged amino acids in ns5 were individually mutagenized to create uncharged pairs of alanine residues, and 32 recombinant mutant viruses were recovered from the 80 full-length mutant den4 cdna constructs. these mutant viruses were tested for temperature-sensitive (ts) replication in ...200211752143
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
dengue fever in pregnancy: a case report.background: dengue, a mosquito-borne flavivirus infection, is endemic in southeast asia. currently, the incidence has been increasing among adults. case presentation: a 26-year-old thai woman, g1p0 31 weeks pregnancy, presented with epigastric pain for 1 day. she also had a high-grade fever for 4 days. the physical examination, complete blood counts as well as serology confirmed dengue fever. the patient was under conservative treatment despite severe thrombocytopenia. she was well at the 3rd da ...200111747474
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
[a study on the pathogenicity of acute hepatitis g infection].to study the infection due to hepatitis g virus (hgv) in liver tissues of patients with acute hepatitis and to investigate its pathogenicity.199911798655
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
transport and budding at two distinct sites of visible nucleocapsids of west nile (sarafend) has been difficult to detect and visualize the physical nucleocapsid particles during the replication process of the flaviviruses. the use of cryo-immunoelectron microscopy has clearly revealed the capsid proteins and nucleocapsid particles of west nile (sarafend) virus (a flavivirus) for the first time. physical nucleocapsid particles accumulated in large numbers from 8 hr postinfection. double immunolabeling of the envelope and capsid proteins showed a close association of these structural ...200111745942
virologic and serologic surveillance for dengue fever in jeddah, saudi arabia, 1994-1999.dengue fever infection was first documented in jeddah, saudi arabia, by virus isolation of dengue type 2 virus in 1994 at the virology laboratory of dr. soliman fakeeh hospital. dengue virus surveillance was established after that time. blood samples were collected from 985 patients (710 male patients and 275 female patients) with suspected cases of dengue from february 1994 to december 1999. dengue virus isolates were obtained in 207 patients (21%; 162 male patients and 45 female patients). den ...200111791972
an indirect immunofluorescence assay to detect antibodies against st. louis encephalitis in house indirect immmunofluorescence assay ( ifa ) in relation to neutralization (nt) reference test, was assessed as a fast and cheap method to carry out serological surveys for st. louis encephalitis virus (sle). sera obtained from 213 blood donors were analyzed by both tests. the prevalence of seropositivity obtained with ifa was lower than (30.98%) that observed on nt (41.78%). the relative specificity rate of ifa was 96.77% whereas its relative sensitivity rate was 69.66%. kappa index s ...200111781605
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
enzyme-linked immunosorbent assay specific to dengue virus type 1 nonstructural protein ns1 reveals circulation of the antigen in the blood during the acute phase of disease in patients experiencing primary or secondary infections.during flavivirus infection in vitro, nonstructural protein ns1 is released in a host-restricted fashion from infected mammalian cells but not vector-derived insect cells. in order to analyze the biological relevance of ns1 secretion in vivo, we developed a sensitive enzyme-linked immunosorbent assay (elisa) to detect the protein in the sera of dengue virus-infected patients. the assay was based on serotype 1 ns1-specific mouse and rabbit polyclonal antibody preparations for antigen immunocaptur ...200211825945
hepatitis c virus nonstructural proteins are localized in a modified endoplasmic reticulum of cells expressing viral subgenomic replicons.for many years our knowledge on hepatitis c virus (hcv) replication has been based on in vitro experiments or transfection studies. recently, the first reliable system for studying viral replication in tissue culture cells was developed. taking advantage of this system, we examined in detail the localization of viral nonstructural (ns) proteins in cells containing functional replication complexes. by fractionation experiments and immunomicroscopy, we observed that all ns proteins were associated ...200211853397
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
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
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
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
immunogenicity of hgv ns5 protein expressed from sf9 insect cells. 200111819741
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
[female patient with west-nile fever in the netherlands]. 200211820069
synergistic neutralizing antibody response to a dengue virus type 2 dna vaccine by incorporation of lysosome-associated membrane protein sequences and use of plasmid expressing gm-csf.we have previously shown that a dengue virus type 1 dna vaccine expressing premembrane (prm) and envelope (e) genes was immunogenic in mice and monkeys and that rhesus monkeys vaccinated with this construct were completely to partially protected from virus challenge. in order to improve the immunogenicity of dengue dna vaccines, we have evaluated the effect of lysosome targeting of antigens and coimmunization with a plasmid expressing gm-csf on antibody responses. a dengue virus type 2 candidate ...200111883007
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
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
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
immunization with heterologous flaviviruses protective against fatal west nile encephalitis.prior immunization of hamsters with three heterologous flaviviruses (japanese encephalitis virus [jev] sa14-2-8 vaccine, wild-type st. louis encephalitis virus [slev], and yellow fever virus [yfv] 17d vaccine) reduces the severity of subsequent west nile virus (wnv) infection. groups of adult hamsters were immunized with each of the heterologous flaviviruses; approximately 30 days later, the animals were injected intraperitoneally with a virulent new york strain of wnv. subsequent levels of vire ...200211927020
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
recently discovered blood-borne recent years, molecular biology advances have enabled many investigators to discover a number of viruses that have been difficult to characterise by cell culture techniques. two blood-borne viruses have been identified. these are gb virus c (gbv-c) and tt virus (ttv). gbv-c was discovered in 1995. it is a flavivirus-like enveloped particle measuring 50-100 nm in diameter with a density of 1.08-1.13 g/cm3. the genome of gbv-c is a single-stranded, positive strand ribonucleic acid of approximat ...200111948357
capsid protein c of tick-borne encephalitis virus tolerates large internal deletions and is a favorable target for attenuation of virulence.deletions ranging in size from 4 to 21 amino acid residues were introduced into the capsid protein of the flavivirus tick-borne encephalitis (tbe) virus. these deletions incrementally affected a hydrophobic domain which is present at the center of all flavivirus capsid protein sequences and part of which may form an amphipathic alpha-helix. in the context of the full-length tbe genome, the deletions did not measurably affect protein expression and up to a deletion length of 16 amino acid residue ...200211884577
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
use of immunoglobulin m cross-reactions in differential diagnosis of human flaviviral encephalitis infections in the united 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
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
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
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
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
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
hemorrhagic fever viruses as biological weapons: medical and public health 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
antibodies against prm protein distinguish between previous infection with dengue and japanese encephalitis 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
iminosugars as possible broad spectrum anti hepatitis virus agents: the glucovirs and alkovirs.glucosidases in the endoplasmic reticulum (er) mediate the first step in processing n-linked oligosaccharides. recent evidence suggests that morphogenesis and secretion of members of the hepatitis b and flavivirus families are more dependent on these enzymes than are most host glycoproteins. thus, it is possible that glucosidase inhibitors can be designed that are safe and selective for the treatment of hepatitis b and possibly c (since hepatitis c virus is a member of the flavivirus family), ma ...200112018676
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
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
fatal yellow fever in a traveler returning from amazonas, brazil, 2002. 200212025823
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
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
emerging arboviral diseases. 200212054284
effect of downstream sequence on the cleavage of envelop protein 1 signal sequence in hepatitis c virus.the rna genome of hepatitis c virus encodes a polyprotein of 3 000 amino acids, which is processed into 10 viral proteins by proteases provided by host cells and virus itself. multiple precursors are produced due to inefficient processing. here, the study of e1 signal sequence (c/e1 site) processing in eukaryotic vaccinia virus/t7 system is reported. differently truncated hcv structural proteins were expressed in this system. it was found that the efficient cleavage of e1 signal sequence was aff ...200112035062
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
[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
regulation of immune recognition molecules by flavivirus, west nile.we have shown the flaviviruses can up-regulate the cell surface expression of the immune recognition molecules, major histocompatability complex class-i and class-ii (mhc-i, mhc-ii), icam-1, vcam, and e-selectin, in an interferon-independent and tumor necrosis factor-independent manner. this up-regulation is associated with an increased transcription of the relevant genes and is due to activation of the transcription factor, nuclear factor-kappa b. the level of up-regulation is determined in par ...200212081012
the japanese encephalitis serological group of flaviviruses: a brief introduction to the group. 200212082984
japanese encephalitis vaccines: current vaccines and future prospects.vaccination against je ideally should be practiced in all areas of asia where the virus is responsible for human disease. the who has placed a high priority on the development of a new vaccine for prevention of je. some countries in asia (japan, south korea, north korea, taiwan, vietnam, thailand, and the prc) manufacture je vaccines and practice childhood immunization, while other countries suffering endemic or epidemic disease (india, nepal, laos, cambodia, bangladesh, myanmar, malaysia, indon ...200212082985
host genetic resistance to japanese encephalitis group viruses. 200212082988
the emergence of west nile virus in north america: ecology, epidemiology, and 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
evolution of the japanese encephalitis serocomplex viruses. 200212082999
japanese encephalitis as an emerging virus: the emergence and spread of japanese encephalitis virus in australasia. 200212083000
immune responses to japanese encephalitis virus. 200212083002
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
clinical and immunological risk factors for severe disease in japanese encephalitis.eighty-five paediatric patients in thailand with acute japanese encephalitis (je) were studied in 1987-99 to determine risk factors present at hospital admission which were associated with severe disease. on univariate analysis, the following factors on admission were significantly associated with the combined end-point of death or a severe neurological deficit: depressed level of consciousness, elevated concentration of cerebrospinal fluid (csf) protein, low levels of serum and csf igg antibody ...200212055808
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