an application of pap (peroxidase-anti-peroxidase) staining technique for the rapid titration of dengue virus type 4 infectivity. 1977328006
development of dengue virus type 4 in bhk21 cells.the development of dengue type 4 virus in bhk21 and vero cells was observed by electron microscopy. in bhk21 cells, completed virions appeared in vacuoles after marked vacuolization of the cytoplasm. budding of dengue virions from internal membranous structures was observed in some of these vacuoles. a new type of vesicles with a marginal, rosary-like arrangement of particles was observed associated with masses of electron-dense particles. these vesicles were suggested to be related to virus pro ...1976825104
construction and characterization of chimeric tick-borne encephalitis/dengue type 4 viruses.dengue type 4 virus (den4) cdna was used as a vector to express genes of the distantly related tick-borne encephalitis virus (tbev). full-length chimeric tbev/den4 cdnas were constructed by substituting tbev genes coding for proteins such as capsid (c); pre-membrane, which is the precursor of membrane (m); envelope (e); or nonstructural protein ns1 for the corresponding den4 sequences. rna transcripts prepared from cdnas were used to transfect permissive simian cells. two viable chimeric viruses ...19921438242
correlation of e protein binding with cell susceptibility to dengue 4 virus infection.supernatant culture fluids from dengue virus type 4 (den-4)-infected cultures of monkey kidney vero cells and aedes albopictus c6/36 cells contained the virion structural proteins; secreted ns1 was found only in supernatants from infected vero cells. using supernatant culture fluids from [35s]methionine-labelled, virus-infected vero and c6/36 cells, binding of radiolabelled viral proteins was examined with various cell lines varying in susceptibility to den-4 infection. binding of viral e protei ...19921645154
both nonstructural proteins ns2b and ns3 are required for the proteolytic processing of dengue virus nonstructural proteins.the cleavages at the junctions of the flavivirus nonstructural (ns) proteins ns2a/ns2b, ns2b/ns3, ns3/ns4a, and ns4b/ns5 share an amino acid sequence motif and are presumably catalyzed by a virus-encoded protease. we constructed recombinant vaccinia viruses expressing various portions of the ns region of the dengue virus type 4 polyprotein. by analyzing immune precipitates of 35s-labeled lysates of recombinant virus-infected cells, we could monitor the ns2a/ns2b, ns2b/ns3, and ns3/ns4a cleavages ...19912016768
immunization of mice with recombinant vaccinia virus expressing authentic dengue virus nonstructural protein ns1 protects against lethal dengue virus encephalitis.the protective immunity conferred by a set of recombinant vaccinia viruses containing the entire coding sequence of dengue virus type 4 nonstructural glycoprotein ns1 plus various flanking sequences was evaluated by using a mouse encephalitis model. mice immunized with recombinant vns1-ns2a, which expresses authentic ns1, were solidly protected against intracerebral dengue virus challenge. however, mice immunized with recombinants vns1-15%ns2a and vrsvg/ns1-15%ns2a, which express aberrant forms ...19902143542
in vitro processing of dengue virus structural proteins: cleavage of the pre-membrane protein.processing of dengue virus structural proteins was assessed in vitro. rna transcripts for cell-free translation were prepared from cloned dna (dengue virus type 4, strain 814669 genome) encoding capsid, pre-membrane (prm), and the first 23 amino acids of envelope (e). processing of a 33-kilodalton precursor polypeptide encoded by wild-type rna transcripts occurred only in the presence of added microsomal membranes. under these conditions, cleavage at the capsid-prm and prm-e sites and glycosylat ...19892501515
dengue virus-specific cross-reactive cd8+ human cytotoxic t lymphocytes.stimulation with live dengue virus of peripheral blood mononuclear cells from a dengue virus type 4-immune donor generated virus-specific, serotype-cross-reactive, cd8+, class i-restricted cytotoxic t lymphocytes (ctl) capable of lysing dengue virus-infected cells and cells pulsed with dengue virus antigens of all four serotypes. these ctl lysed autologous fibroblasts infected with vaccinia virus-dengue virus recombinant viruses containing the e gene or several nonstructural dengue virus type 4 ...19892511337
cloning full-length dengue type 4 viral dna sequences: analysis of genes coding for structural proteins.dna sequences (approximately 11,000 nucleotides) representing the full-length genome of the dengue virus type 4 were cloned. the sequence of the first 2,429 nucleotides at the 5' terminus which includes the coding region for the structural proteins is presented. the virion structural proteins are encoded in one long open reading frame specifying a polyprotein precursor which is apparently proteolytically cleaved by a mechanism resembling that proposed for expression of structural proteins of oth ...19863022479
rapid titration of dengue virus type 4 infectivity by counting fluorescent foci. 19744218091
[monoclonal antibodies specific to dengue virus type-4]. 19836228313
delayed-type hypersensitivity (dth) response to dengue virus in mice: effect of route of sensitization and splenectomy.this study was designed to determine the role of the sensitization route and the spleen in the development of delayed-type hypersensitivity (dth) to dengue virus in mice. dth was measured by footpad swelling response. strong but transient dth was produced in cyclophosphamide (cy) pretreated mice sensitized subcutaneously (s.c.) or intravenously (i.v.) with dengue virus type 4. subcutaneous inoculation of virus in incomplete freund's adjuvant (ifa) further enhanced the dth elicited. the time cour ...19836669077
induction and characterization of delayed-type hypersensitivity to dengue virus in mice.delayed-type hypersensitivity (dth) to dengue virus type 4 in mice is described. the dth reaction (measured by footpad swelling) was maximal at 24 hr after challenge and was enhanced significantly when mice were pretreated with cyclophosphamide. the response was maximal six days after infection with an immunizing dose of approximately 10(7.3) 50% lethal doses per mouse. histologic examination of the test footpad showed a predominantly mononuclear cell infiltrate at 24 hr after challenge. fibrin ...19827108274
[dengue outbreak by virus type 4 in french polynesia. i. general epidemiology - clinical specific aspects (author's transl)].authors describe an extensive outbreak of dengue 4 virus which is observed for the first time out of its asian original focus. the whole archipelagos of french polynesia have been contaminated in a space of four months. usual form of the disease is typical with lightness of the symptoms. this outbreak has been implicated in the death of a child by shock; and two cases of reversible encephalitis have been certainly caused by this virus. the neurotropism is the main characteristic of dengue virus ...19817278574
a single nine-amino acid peptide induces virus-specific, cd8+ human cytotoxic t lymphocyte clones of heterogeneous serotype is generally accepted that virus-specific cd8+ cytotoxic t lymphocytes (ctls) recognize nine-amino acid peptides in conjunction with hla class i molecules. we recently reported that dengue virus-specific cd8+ ctls of two different serotype specificities, which were established by stimulation with dengue virus, recognize a single nine-amino acid peptide of the nonstructural protein ns3 of dengue virus type 4 (d4v) in an hla-b35-restricted fashion. to further analyze the relationships between t ...19957544398
growth-restricted dengue virus mutants containing deletions in the 5' noncoding region of the rna genome.the dengue type 4 virus (den4) rna genome contains a 101-nt 5' noncoding (nc) sequence which is predicted to form a stable secondary structure. den4 cdna from which infectious rna can be transcribed was used to engineer deletions in the 5' nc region for functional analysis of rna structure and for isolation of den4 mutants that could be evaluated as candidates for use in a live attenuated vaccine. eleven distinct deletions in the region of the den4 genome between nts 18 and 98 were constructed; ...19957871753
recognition of envelope protein by dengue virus serotype-specific human cd4+ cd8- cytotoxic t-cell clones.we analyzed dengue virus-specific cd4+ cd8- cytotoxic t lymphocytes (ctl) at the clonal level to further understand their role in dengue virus infections. stimulation of peripheral blood mononuclear cells from two dengue virus type 4 (d4v)-immune donors with live d4v or noninfectious d4v antigen generated 17 hla class ii-restricted cd4+ ctl capable of specific lysis of dengue virus antigen-treated autologous lymphoblastoid cell lines. thirteen clones were d4v specific, three clones were cross-re ...19947908702
dengue virus. detection and pathogeny.sequences front a cdna of dengue virus type 4 were cloned into transcription vectors. these sequences included the e, ns1, ns2a, ns2b, ns3 genes. rna transcripts produced in vitro from these plasmids were used in hybridization assays to detect dengue viral sequences. with these rna-probes we have been able to detect molecules of serotype-specific dengue 4 viral rna. moreover, the riboprobes detected viral sequences of other serotypes in the following order of sensitivity 4 > 2 > 3 > 1, and might ...19947919815
preferential usage of t-cell receptor v beta 17 by dengue virus-specific human t lymphocytes in a donor with immunity to dengue virus type order to better understand human t-cell responses to dengue viruses (dv), we analyzed t-cell receptor (tcr) v beta gene usage in dv-specific t lymphocytes. peripheral blood t lymphocytes from a dv type 4 immune donor were stimulated in vitro with dv type 4 antigen, and tcr usage was examined by reverse transcriptase pcr. tcr v beta 17 was preferentially used (p = 0.020) among t cells stimulated by dv type 4 in bulk culture. furthermore, 8 of 19 dv type 4-specific cd4+ t-cell clones establishe ...19947933152
processing of flavivirus structural glycoproteins: stable membrane insertion of premembrane requires the envelope signal peptide.the flavivirus structural proteins capsid (c), premembrane (prm), and envelope (e) are cleaved in that order from the n-terminus of the polyprotein by the er intralumenal enzyme signal peptidase. the prm-e and e-ns1 junctions contain hydrophobic domains with both transmembrane and signal function. these domains reside at the c-termini of prm and e, respectively, after cleavage. we studied the functions of the 37-amino-acid c-terminus of the dengue virus type 4 (den4) prm (amino acids 243-279 of ...19947941319
[riboprobes: an alternative in the detection of viral sequences].the use of riboprobes for the detection of rna viral sequences is analyzed. subgenomic fragments of cdna from poliovirus type 2, dengue virus type 4 and human immunodeficiency virus type 1, were inserted downstream sp6 and/or t7 promoters in the transcription vectors pgem-4z or psp64. rnas obtained by in vitro transcription in the presence of utp infinity (32p) were used as probes for the detection of rna viral sequences from infected cell lines in slot and northern blot assays. the poliovirus r ...19948073329
chimeric tick-borne encephalitis and dengue type 4 viruses: effects of mutations on neurovirulence in mice.two new chimeric flaviviruses were constructed from full-length cdnas that contained tick-borne encephalitis virus (tbev) cme or me structural protein genes and the remaining genes derived from dengue type 4 virus (den4). studies involving mice inoculated intracerebrally with the me chimeric virus indicated that it retained the neurovirulence of its tbev parent from which its pre-m and e genes were derived. however, unlike parental tbev, the chimeric virus did not produce encephalitis when mice ...19938331735
dengue virus-specific human cd4+ t-lymphocyte responses in a recipient of an experimental live-attenuated dengue virus type 1 vaccine: bulk culture proliferation, clonal analysis, and precursor frequency determination.we analyzed the cd4+ t-lymphocyte responses to dengue, west nile, and yellow fever viruses 4 months after immunization of a volunteer with an experimental live-attenuated dengue virus type 1 vaccine (den-1 45az5). we examined bulk culture proliferation to noninfectious antigens, determined the precursor frequency of specific cd4+ t cells by limiting dilution, and established and analyzed cd4+ t-cell clones. bulk culture proliferation was predominantly dengue virus type 1 specific with a lesser d ...19938371350
deletion analysis of dengue virus type 4 nonstructural protein ns2b: identification of a domain required for ns2b-ns3 protease activity.most proteolytic cleavages in the nonstructural protein (ns) region of the flavivirus polyprotein are effected by a virus-encoded protease composed of two viral proteins, ns2b and ns3. the n-terminal 180-amino-acid-region of ns3 includes sequences with homology to the active sites of serine proteases, and there is evidence that this portion of ns3 can mediate proteolytic cleavages. in contrast, nothing is known about required sequences in ns2b. we constructed a series of deletion mutations in th ...19938383225
genetic determinants of dengue type 4 virus neurovirulence for mice.mouse-adapted dengue type 4 virus (den4) strain h241 is highly neurovirulent for mice, whereas its non-mouse-adapted parent is rarely neurovirulent. the genetic basis for the neurovirulence of the mouse-adapted mutant was studied by comparing intratypic chimeric viruses that contained the three structural protein genes from the parental virus or the neurovirulent mutant in the background sequence of nonneurovirulent den4 strain 814669. the chimera that contained the three structural protein gene ...19938411360
dengue type 4 virus mutants containing deletions in the 3' noncoding region of the rna genome: analysis of growth restriction in cell culture and altered viremia pattern and immunogenicity in rhesus monkeys.the dengue type 4 virus (den4) genome contains a 384-nucleotide (nt) 3' noncoding sequence in which the last 81 nt, predicted to form a secondary structure, are thought to be essential for virus replication. immediately upstream of the secondary structure, short rna sequences that are conserved among mosquito-borne flaviviruses have been identified. a series of deletions that range from 30 to 262 nt were introduced into this upstream region of full-length den4 cdna to create viable deletion muta ...19968648730
monkeys immunized with intertypic chimeric dengue viruses are protected against wild-type virus challenge.dengue epidemics caused by the four dengue virus serotypes continue to pose a major public health problem in most tropical and subtropical regions. a safe and effective vaccine against dengue is still not available. the current strategy for dengue immunization favors the use of a vaccine containing each of the four serotypes. we previously employed full-length dengue type 4 virus (den4) cdna to construct a viable intertypic dengue virus of type 1 or type 2 antigenic specificity that contained th ...19968648761
recognition of synthetic oligopeptides from nonstructural proteins ns1 and ns3 of dengue-4 virus by sera from dengue virus-infected children.the nonstructural proteins ns1 and ns3 from dengue virus are involved in the immune response during natural infection in humans. to analyze the immunogeneity of some epitopes present in ns1 and ns3 proteins from dengue virus type-4, six oligopeptides were synthesized; five from ns1 (ns1.1, ns1.2, ns1.3, ns1.4, and ns1.5) and one from ns3 (ns3.1). peptides ns1.1, ns1.2, ns1.3, ns1.5, and ns3.1 were recognized by sera from dengue virus-infected children, suggesting that they represent exposed epit ...19979158059
attenuation of the langat tick-borne flavivirus by chimerization with mosquito-borne flavivirus dengue type 4.langat virus (lgt) strain tp21 is the most attenuated of the tick-borne flaviviruses for humans. even though lgt has low-level neurovirulence for humans, it, and its more attenuated egg-passage derivative, strain e5, exhibit significant neurovirulence and neuroinvasiveness in normal mice, albeit less than that associated with tick-borne encephalitis virus (tbev), the most virulent of the tick-borne flaviviruses. we sought to reduce or ablate these viral phenotypes of tp21 and e5 by using a strat ...19989465088
evaluation of molecular strategies to develop a live dengue vaccine.millions of individuals are estimated to become infected with dengue virus each year, particularly in tropical and subtropical regions. mortality is low but infection can lead to a severe form of dengue, characterised by haemorrhage and shock. a safe and effective vaccine against dengue is still not available.19989741643
identification of brazilian flaviviruses by a simplified reverse transcription-polymerase chain reaction method using flavivirus universal primers.we report a simplified reverse transcription-polymerase chain reaction (rt-pcr) method for identification of brazilian flaviviruses based on the patterns of electrophoretic separation of the amplicons. the rt-pcr was done on the culture fluids of aedes albopictus c6/36 cells infected with brazilian flaviviruses, without previous extraction of viral rna, using flavivirus universal primers that anneal to highly conserved sequences within the nonstructural protein 5 and 3'- non translated region of ...19989749625
vector competence of aedes notoscriptus (diptera: culicidae) for barmah forest virus and of this species and aedes aegypti (diptera: culicidae) for dengue 1-4 viruses in queensland, australia.aedes notoscriptus (skuse) mosquitoes colonized from brisbane, queensland, australia, were fed on blood suspensions containing either barmah forest (bf) virus, dengue virus type 1 (den-1), dengue virus type 2 (den-2), dengue virus type 3 (den-3), or dengue virus type 4 (den-4). ae. notoscriptus was shown to be moderately susceptible to oral infection with bf virus, id50 = 10(3.3) ccid50 per mosquito, with titers in infected mosquitoes peaking 10 d after infection. bf virus transmission occurred ...199910467781
immunization of rhesus monkeys with a recombinant of modified vaccinia virus ankara expressing a truncated envelope glycoprotein of dengue type 2 virus induced resistance to dengue type 2 virus challenge.dengue epidemics increasingly pose a public health problem in most countries of the tropical and subtropical areas. despite decades of research, development of a safe and effective live dengue virus vaccine is still at the experimental stage. to explore an alternative vaccine strategy, we employed the highly attenuated, replication-deficient modified vaccinia ankara (mva) as a vector to construct recombinants for expression of the major envelope glycoprotein of one or more dengue virus serotypes ...200010856791
comparison of two colorimetric assays to determine viral infectivity in micro culture virus titration.efficacy of two colorimetric assays, viz. mtt (3-4,5-dimethylthiazol-2-(yl-2,5-diphenyl tetrazolium bromide) and neutral red (nr) assays, performed by integrating them to micro culture virus titration (mcvt), was compared with the conventional mcvt method in terms of percentages of infectivity and 50% infectivity end points by employing polio virus type-3 and dengue virus type 4 as the candidate viruses. the results suggested that mtt assay has an edge over nr assay as well as conventional mcvt ...199910865890
isolation and partial characterization of dengue virus type 2 and 4 strains from dengue fever and dengue haemorrhagic fever patients from mindanao, republic of the philippines.isolation of dengue virus from dengue fever and dengue haemorrhagic fever cases from mindanao, republic of the philippines.200010886794
invited commentary: dengue lessons from 18-year interval between a dengue virus type 1 outbreak in 1977-1979 and a dengue virus type 2 outbreak in 1997 in santiago de cuba, cuba, provided a unique opportunity to evaluate risk factors for dengue disease. all patients with symptomatic dengue, including 205 cases of dengue hemorrhagic fever and 12 deaths, were adults born before the dengue virus type 1 epidemic, and nearly all (98%) experienced secondary dengue virus infections. in contrast, almost all of those who seroconverted witho ...200011085390
[monoclonal antibodies to dengue virus 4: the spectrum of the reactivity to 4 serotypes of dengue].these types of monoclonal antibodies 8e8, 3f7 and 1e9 to dengue 4 virus h-241 strain. these monoclonal antibodies show various patterns of reactivity to the four dengue serotypes and different antigen preparations of serotype 4 when they were tested in various serological methods. the monoclonal antibody 8e8 exhibited a specificity of serotype (d-2; by hemagglutination inhibition); subcomplex (d-2 and d-4 by immunofluorescence) and complex (by immunoperoxidase technique). it was able to neutrali ...200011107906
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
development of a monoclonal antibody specific to a recombinant envelope protein from dengue virus type 4 expressed in pichia pastoris.a mouse monoclonal antibody (mab, 4b6) was able to recognize dengue virus type 4 envelope (e) protein both as a recombinant protein in pichia pastoris and when it was present in infected brains of suckling mice. 4b6 was characterized by enzyme-linked immunoadsorbent assay (elisa), hemaglutination inhibition, neutralization, and immunoblot. the mab was isotyped as igg2a. it was serotype 4 specific and it inhibited hemaglutination and neutralized homologous virus. it did not enhance infection of p ...200111289225
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
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
attenuation and immunogenicity in humans of a live dengue virus type-4 vaccine candidate with a 30 nucleotide deletion in its 3'-untranslated region.the recombinant dengue virus type-4 vaccine candidate 2aa30 was attenuated in rhesus monkeys due to an engineered 30-nucleotide deletion in the 3'-untranslated region of the viral genome. a clinical trial to evaluate the safety and immunogenicity of a single dose of 2adelta30 was conducted with 20 adult human volunteers. the vaccine candidate was well tolerated and did not cause systemic illness in any of the 20 volunteers. viremia was detectable in 14 volunteers at a mean level of 1.6 log10 pla ...200111716091
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
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
genetic basis of attenuation of dengue virus type 4 small plaque mutants with restricted replication in suckling mice and in scid mice transplanted with human liver cells.mutations that restrict replication of dengue virus have been sought for the generation of recombinant live-attenuated dengue virus vaccines. dengue virus type 4 (den4) was previously grown in vero cells in the presence of 5-fluorouracil, and the characterization of 1248 mutagenized, vero cell passaged clones identified 20 temperature-sensitive (ts) mutant viruses that were attenuated (att) in suckling mouse brain (j. e. blaney, jr., d. h. johnson, c. y. firestone, c. t. hanson, b. r. murphy, an ...200212202213
emergence of dengue virus type 4 genotype iia in malaysia.phylogenetic analyses of the envelope (e) gene sequence of five recently isolated dengue virus type 4 (denv-4) suggested the emergence of a distinct geographical and temporal denv-4 subgenotype iia in malaysia. four of the isolates had direct ancestral lineage with denv-4 indonesia 1973 and showed evidence of intra-serotypic recombination with the other recently isolated denv-4, my01-22713. the e gene of isolate my01-22713 had strong evidence of an earlier recombination involving denv-4 genotype ...200212237425
a live, attenuated dengue virus type 1 vaccine candidate with a 30-nucleotide deletion in the 3' untranslated region is highly attenuated and immunogenic in monkeys.the delta30 deletion mutation, which was originally created in dengue virus type 4 (den4) by the removal of nucleotides 172 to 143 from the 3' untranslated region (3' utr), was introduced into a homologous region of wild-type (wt) dengue virus type 1 (den1). the resulting virus, rden1delta30, was attenuated in rhesus monkeys to a level similar to that of the rden4delta30 vaccine candidate. rden1delta30 was more attenuated in rhesus monkeys than the previously described vaccine candidate, rden1mu ...200312502885
temperature sensitive mutations in the genes encoding the ns1, ns2a, ns3, and ns5 nonstructural proteins of dengue virus type 4 restrict replication in the brains of mice.using reverse genetics, it is possible to readily add well-defined attenuating mutations to the genome of wild type or incompletely attenuated dengue (den) viruses to generate vaccine candidates that exhibit the desired balance between attenuation and immunogenicity. here, we describe the identification of eight temperature sensitive missense mutations distributed in four non-structural protein genes that specify a 60- to 10,000-fold range of restricted replication in the suckling mouse brain co ...200312721805
tick-borne encephalitis with hemorrhagic syndrome, novosibirsk region, russia, 1999.eight fatal cases of tick-borne encephalitis with an unusual hemorrhagic syndrome were identified in 1999 in the novosibirsk region, russia. to study these strains, we sequenced cdna fragments of protein e gene from six archival formalin-fixed brain samples. phylogenetic analysis showed tick-borne encephalitis variants clustered with a far eastern subtype (homology 94.7%) but not with the siberian subtype (82%).200312781020
a trade-off in replication in mosquito versus mammalian systems conferred by a point mutation in the ns4b protein of dengue virus type acceptable live-attenuated dengue virus vaccine candidate should have low potential for transmission by mosquitoes. we have identified and characterized a mutation in dengue virus type 4 (den4) that decreases the ability of the virus to infect mosquitoes. a panel of 1248 mutagenized virus clones generated previously by chemical mutagenesis was screened for decreased replication in mosquito c6/36 cells but efficient replication in simian vero cells. one virus met these criteria and contained a ...200312890635
induction of neutralizing antibodies and partial protection from viral challenge in macaca fascicularis immunized with recombinant dengue 4 virus envelope glycoprotein expressed in pichia pastoris.a recombinant vaccine that expresses the envelope (e) gene of dengue virus type 4 was tested for immunogenicity and protection in macaca fascicularis. one hundred micrograms of semipurified recombinant e protein (e4rec) expressed in pichia pastoris was used to immunize three animals. neutralizing antibodies to dengue 4 virus with a titer of 1:30 were detected in all immunized monkeys prior to challenge. animals were challenged with 10(5) plaque-forming units of dengue 4 virus. one vaccine-immuni ...200313677367
substitution of the structural genes of dengue virus type 4 with those of type 2 results in chimeric vaccine candidates which are attenuated for mosquitoes, mice, and rhesus monkeys.antigenic chimeric viruses in which the structural genes of dengue virus type 4 (den4) have been replaced with those derived from dengue virus type 2 (den2) have been created and evaluated as a first step in generating a live attenuated tetravalent dengue virus vaccine. specifically, the capsid, membrane precursor, and envelope (cme) or the membrane precursor and envelope (me) gene regions of den2 were substituted for the corresponding genes of wild-type rden4 or vaccine candidate rden4delta30 w ...200314505913
mutations which enhance the replication of dengue virus type 4 and an antigenic chimeric dengue virus type 2/4 vaccine candidate in vero cells.mutations which increase the replication of dengue viruses in cell culture would greatly facilitate the manufacture of both a live attenuated or inactivated dengue virus vaccine. we have identified eight missense mutations in dengue virus type 4 (den4) that increase the plaque size and kinetics of replication of recombinant den4 virus in vero cells. den4 viruses bearing these vero cell adaptation mutations were also evaluated for the level of replication in the brains of mice. two of these eight ...200314505914
isolation of putative dengue virus receptor molecules by affinity chromatography using a recombinant e protein ligand.nucleotide sequences coding for the full-length envelope (e) glycoprotein gene of dengue virus type 4 was amplified using an rt-pcr method from infected c6/36 cells and cloned into pproex-hta expression vector. the expression of the recombinant e protein in escherichia coli was confirmed by western blot using a polyclonal anti-dengue polyclonal antibody. the his-tagged fusion protein was obtained from the bacterial cellular extracts in almost pure form by immobilized metal affinity chromatograph ...200414715312
uncertainties regarding dengue modeling in rio de janeiro, brazil.dengue fever is currently the most important arthropod-borne viral disease in brazil. mathematical modeling of disease dynamics is a very useful tool for the evaluation of control measures. to be used in decision-making, however, a mathematical model must be carefully parameterized and validated with epidemiological and entomological data. in this work, we developed a simple dengue model to answer three questions: (i) which parameters are worth pursuing in the field in order to develop a dengue ...200314765541
enhanced vector competence of aedes aegypti (diptera: culicidae) from the torres strait compared with mainland australia for dengue 2 and 4 viruses.australian aedes aegypti (l.) mosquitoes colonized from the torres strait and three mainland localities (charters towers, townsville, and cairns) were fed on blood suspensions containing dengue virus type 2 (den-2) or dengue virus type 4 (den-4). variation was found in oral susceptibility to den-2 (59 -99% infection) and den-4 (28-79% infection) among ae. aegypti assayed for virus at 8, 12, 16, or 20 d after ingestion of infected blood. torres strait ae. aegypti were the most susceptible to den- ...200314765675
alterations of pr-m cleavage and virus export in pr-m junction chimeric dengue viruses.during the export of flavivirus particles through the secretory pathway, a viral envelope glycoprotein, prm, is cleaved by the proprotein convertase furin; this cleavage is required for the subsequent rearrangement of receptor-binding e glycoprotein and for virus infectivity. similar to many furin substrates, prm in vector-borne flaviviruses contains basic residues at positions p1, p2, and p4 proximal to the cleavage site; in addition, a number of charged residues are found at position p3 and be ...200414963133
introduction of mutations into the non-structural genes or 3' untranslated region of an attenuated dengue virus type 4 vaccine candidate further decreases replication in rhesus monkeys while retaining protective immunity.a dengue virus vaccine candidate, rden4delta30, has been previously reported to be safe and immunogenic in humans, but a subset of vaccinees developed asymptomatic rash, elevation of liver enzymes and/or mild neutropenia. in the current study, mutations that had previously been shown to reduce replication of den4 virus in suckling mice and/or in scid mice engrafted with human liver cells (scid-huh-7 mice) were introduced into rden4delta30 in an attempt to further attenuate this virus. three of t ...200415308370
[the cloning and sequence analysis of cdna of the partial e genomic region of dengue type 4 virus from a chinese patient].the partial envelope (e) protein gene of dengue virus type 4 (dv4), derived from the serum of a patient with dengue fever in an epidemic during october 1993 in guangdong province of china, was cloned and sequenced. dv rna was converted to cdna by reverse transcription with random primers before the polymerase chain reaction using dv4 specific primers was performed. the amplified product of 421bp was subsequently filled in 3' recessed ends, isolated, purified and inserted into puc18 and puc19 pla ...199715619909
rden4delta30, a live attenuated dengue virus type 4 vaccine candidate, is safe, immunogenic, and highly infectious in healthy adult volunteers.the live attenuated dengue virus type 4 (den-4) vaccine candidate virus rden4 delta 30 was previously found to be safe and immunogenic at a dose of 10(5) plaque-forming units (pfu).200515688284
secondary dengue virus type 4 infections in vietnam.this study was designated to describe clinical and biological features of patients with a suspected diagnosis of dengue fever/dengue hemorrhagic fever during an outbreak in central vietnam. one hundred and twenty-five consecutive patients hospitalized at khanh hoa and binh thuan provincial hospitals between november 2001 and january 2002 with a diagnosis of suspected dengue infection were included in the present study. viruses were isolated in c6/36 and vero e6 cell cultures or detected by rt-pc ...200515906664
comparison of live and inactivated tick-borne encephalitis virus vaccines for safety, immunogenicity and efficacy in rhesus monkeys.three antigenic chimeric live attenuated tick-borne encephalitis virus (tbev) vaccine candidates were compared for level of replication in murine and human neuroblastoma cells, for neurovirulence and neuroinvasiveness in mice, and for safety, immunogenicity and efficacy in rhesus monkeys. two chimeric viruses were generated by replacing the membrane precursor and envelope protein genes of dengue type 4 virus (den4) with the corresponding genes of a far eastern tbev, sofjin strain, in the presenc ...200616115704
individual donor nucleic acid amplification testing for detection of west nile virus.we have developed an economical, high-throughput nucleic acid amplification test (nat) for blood-borne viruses, suitable for use in the screening of plasma samples from individual blood donors. this assay system includes a semiautomated procedure, using 96-well glass fiber plates for the extraction of viral nucleic acids from plasma and "universal beacon" technology which permits the detection of all genotypes of highly variable viruses (e.g., human immunodeficiency virus and hepatitis c virus). ...200516207971
invasion and maintenance of dengue virus type 2 and type 4 in the americas.dengue virus type 4 (denv-4) was first reported in the americas in 1981, where it caused epidemics of dengue fever throughout the region. in the same year, the region's first epidemic of dengue hemorrhagic fever was reported, caused by an asian strain of dengue virus type 2 (denv-2) that was distinct from the american subtype circulating previously. despite the importance of these epidemics, little is known about the rates or determinants of viral spread among island and mainland populations or ...200516282468
chimeric west nile/dengue virus vaccine candidate: preclinical evaluation in mice, geese and monkeys for safety and immunogenicity.a live attenuated virus vaccine is being developed to protect against west nile virus (wn) disease in humans. previously, it was found that chimeric west nile/dengue viruses (wn/den4 and wn/den4delta30) bearing the membrane precursor and envelope protein genes of wn on a backbone of dengue type 4 virus (den4) with or without a deletion of 30 nucleotides (delta30) in the 3' noncoding region of the den4 part of the chimeric genome were attenuated and efficacious in mice and monkeys against wn chal ...200616831498
bacterially expressed and refolded envelope protein (domain iii) of dengue virus type-4 binds heparan arboviral infection like dengue fever/dengue hemorrhagic fever (dhf) with high morbidity and mortality rate are extensively prevalent in several parts of the world. global efforts have been directed towards development of vaccine for prevention of dengue. however, lack of thorough understanding about biology and pathogenesis of dengue virus restricts us from development of an effective vaccine. here we report molecular interaction of domain iii of envelope protein of dengue virus type-4 with ...200717011249
the four serotypes of dengue recognize the same putative receptors in aedes aegypti midgut and ae. albopictus cells.dengue viruses (denv) attach to the host cell surface and subsequently enter the cell by receptor-mediated endocytosis. several primary and low affinity co-receptors for this flavivirus have been identified. however, the presence of these binding molecules on the cell surface does not necessarily render the cell susceptible to infection. determination of which of them serve as bona fide receptors for this virus in the vector may be relevant to treating denv infection and in designing control str ...200617014723
secondary structure of dengue virus type 4 3' untranslated region: impact of deletion and substitution mutations.several studies have generated computer-based predictions of secondary structure of the 3' untranslated region (utr) of dengue virus (den); however, experimental verification of the formation of these structures in vitro is lacking. this study assessed the congruence of mfold predictions of secondary structure of the core region of the den type 4 3' utr with nuclease maps of this region. maps and predictions were largely consistent. maps supported the existence of previously predicted pseudoknot ...200617030863
reemergence of dengue virus type 4, french antilles and french guiana, 2004-2005. 200617283628
vaccine candidates for dengue virus type 1 (den1) generated by replacement of the structural genes of rden4 and rden4delta30 with those of den1.antigenic chimeric viruses have previously been generated in which the structural genes of recombinant dengue virus type 4 (rden4) have been replaced with those derived from den2 or den3. two vaccine candidates were identified, rden2/4delta30(me) and rden3/4delta30(me), which contain the membrane (m) precursor and envelope (e) genes of den2 and den3, respectively, and a 30 nucleotide deletion (delta30) in the 3' untranslated region of the den4 backbone. based on the promising preclinical phenoty ...200717328799
panmicrobial oligonucleotide array for diagnosis of infectious facilitate rapid, unbiased, differential diagnosis of infectious diseases, we designed greenechippm, a panmicrobial microarray comprising 29,455 sixty-mer oligonucleotide probes for vertebrate viruses, bacteria, fungi, and parasites. methods for nucleic acid preparation, random primed pcr amplification, and labeling were optimized to allow the sensitivity required for application with nucleic acid extracted from clinical materials and cultured isolates. analysis of nasopharyngeal aspirates, b ...200717370518
efficacy of a live attenuated tetravalent candidate dengue vaccine in naïve and previously infected cynomolgus macaques.the development of a safe and effective vaccine against dengue is a public health priority. attempts to evaluate candidate vaccine formulations in human volunteers were largely unsuccessful, at least in part due to too high reactogenicity of some of the candidate vaccines tested. we evaluated a live attenuated tetravalent dengue vaccine candidate in flavivirus naïve and dengue virus type 3 immune non-human primates. immune responses were measured both at the humoral and the cellular level and th ...200717560694
incorporation of dengue virus replicon into virus-like particles by a cell line stably expressing precursor membrane and envelope proteins of dengue virus type 2.while virus-like particles (vlps) containing subgenomic replicons, which can transduce replicons into target cells efficiently for studying viral replication and vectors of gene therapy and vaccine, have been established for several flaviviruses, none has been reported for the four serotypes of dengue virus, the causal agent of the most important arboviral diseases in this century. in this study, we successfully established a cell line stably expressing the precursor membrane/envelope (prm/e) pr ...200817768670
rapid determination of viral rna sequences in mosquitoes collected in the field.a method for rapid determination of viral rna sequences (rdv) was applied to homogenates of aedes aegypti collected in thailand in an area in which dengue fever (dengue hemorrhagic fever) is endemic, using the mosquito cell line c6/36. nucleic acid sequences of dengue virus type 4 and cell fusing agent virus were detected. this rdv method has the potential to become a standard method for detection of both known and newly emerging, unknown mosquito-borne viruses.200717869351
epitope determinants of a chimpanzee dengue virus type 4 (denv-4)-neutralizing antibody and protection against denv-4 challenge in mice and rhesus monkeys by passively transferred humanized antibody.the chimpanzee monoclonal antibody (mab) 5h2 is specific for dengue virus type 4 (denv-4) and neutralizes the virus at a high titer in vitro. the epitope detected by the antibody was mapped by sequencing neutralization escape variants of the virus. one variant contained a lys174-glu substitution and another contained a pro176-leu substitution in domain i of the denv-4 envelope protein (e). these mutations reduced binding affinity for the antibody 18- to >100-fold. humanized immunoglobulin g (igg ...200717881450
human cd4+ t cell epitopes from vaccinia virus induced by vaccination or infection.despite the importance of vaccinia virus in basic and applied immunology, our knowledge of the human immune response directed against this virus is very limited. cd4(+) t cell responses are an important component of immunity induced by current vaccinia-based vaccines, and likely will be required for new subunit vaccine approaches, but to date vaccinia-specific cd4(+) t cell responses have been poorly characterized, and cd4(+) t cell epitopes have been reported only recently. classical approaches ...200717937498
[study of the genetical stability of the system of expression of protein e of dengue virus 4 in methyltrophic yeast pichia pastoris].the plasmidic stability of methyltrophic yeast pichia pastoris expressing the recombinant protein e of dengue virus 4 was analyzed. to this end, the number of generations from the growth process in petri plaque to the propagation in zaranda was estimated, as well as the fermentation process. besides, in the selected colonies the integration pattern was determined by dot-blot and neuclotide sequence of the gene e of dengue virus 4. this study allowed to prove the conservation and integrity of the ...200517969269
evaluation of the langat/dengue 4 chimeric virus as a live attenuated tick-borne encephalitis vaccine for safety and immunogenicity in healthy adult volunteers.with the steady rise in tick-borne encephalitis virus (tbev) infections in europe, development of a live attenuated vaccine that will generate long-lasting immunity would be of considerable benefit. a chimeric flavivirus, designated lgt/den4, was previously constructed to have a genome containing the prm and e protein genes of langat virus (lgt), a naturally attenuated member of the tbev complex, and the remaining genetic sequences derived from dengue 4 virus (den4). lgt/den4 was highly attenuat ...200818207289
universal primers that amplify rna from all three flavivirus subgroups.species within the flavivirus genus pose public health problems around the world. increasing cases of dengue and japanese encephalitis virus in asia, frequent outbreaks of yellow fever virus in africa and south america, and the ongoing spread of west nile virus throughout the americas, show the geographical burden of flavivirus diseases. flavivirus infections are often indistinct from and confused with other febrile illnesses. here we review the specificity of published primers, and describe a n ...200818218114
production and characterization of recombinant dengue virus type 4 envelope domain iii protein.dengue fever, a mosquito-borne viral disease has become a major worldwide public health problem with a dramatic expansion in recent years. cultivation process for production of recombinant dengue virus type 4 envelope domain iii (rden 4 ediii) protein in escherichia coli was developed for its diagnostic use as well as for further studies in immunoprophylaxis. the dissolved oxygen level was maintained at 20-30% of air saturation. the culture was induced with 1mm of isopropyl beta-d-thiogalactosid ...200818342971
high genetic stability of dengue virus propagated in mrc-5 cells as compared to the virus propagated in vero cells.this work investigated the replication kinetics of the four dengue virus serotypes (den-1 to den-4), including dengue virus type 4 (den-4) recovered from an infectious cdna clone, in vero cells and in mrc-5 cells grown on cytodex 1 microcarriers. den-1 strain hawaii, den-2 strain ngc, den-3 strain h-87, and den-4 strain h-241 , and den-4 strain 814669 derived from cloned dna, were used to infect vero cells and mrc-5 cells grown in serum-free or serum-containing microcarrier cultures. serum-free ...200818350148
comparative neuropathogenesis and neurovirulence of attenuated flaviviruses in nonhuman primates.based on previous preclinical evaluation in mice and monkeys, the chimeric tbev/den4delta30 virus, carrying the prm and e protein genes from a highly virulent far eastern strain of tick-borne encephalitis virus (tbev) on the backbone of a nonneuroinvasive dengue type 4 virus (den4), has been identified as a promising live attenuated virus vaccine candidate against disease caused by tbev. however, prior to use of this vaccine candidate in humans, its neurovirulence in nonhuman primates needed to ...200818353947
dengue virus type 4, manaus, brazil.we report dengue virus type 4 (denv-4) in amazonas, brazil. this virus was isolated from serum samples of 3 patients treated at a tropical medicine reference center in manaus. all 3 cases were confirmed by serologic and molecular tests; 1 patient was co-infected with denv-3 and denv-4.200818394292
evaluation of st. louis encephalitis virus/dengue virus type 4 antigenic chimeric viruses in mice and rhesus develop a live attenuated virus vaccine against st. louis encephalitis (sle) virus, two antigenic chimeric viruses were generated by replacing the membrane precursor and envelope protein genes of dengue virus type 4 (den4) with those from sle with or without a 30 nucleotide deletion in the den4 3' untranslated region of the chimeric genome. chimeric viruses were compared with parental wild-type sle for level of neurovirulence and neuroinvasiveness in mice and for safety, immunogenicity, and p ...200818586359
immunogenicity of a recombinant envelope domain iii protein of dengue virus type-4 with various adjuvants in mice.dengue fever, a mosquito borne viral disease, has become a major public health problem with dramatic expansion in recent decades. several dengue vaccines are at developing stage, none are yet available for humans. there is no vaccine or antiviral therapy available for dengue fever till date. domain iii of envelope protein is involved in binding to host receptors and it contains type and subtype-specific epitopes that elicit virus neutralizing antibodies. hence domain iii is an attractive vaccine ...200818640168
introduction of dengue virus 4 (denv-4) genotype i into brazil from asia? 200919399169
passage of dengue virus type 4 vaccine candidates in fetal rhesus lung cells selects heparin-sensitive variants that result in loss of infectivity and immunogenicity in rhesus macaques.three dengue virus type 4 (denv-4) vaccine candidates containing deletions in the 3' noncoding region were prepared by passage in dbs-frhl-2 (frhl) cells. unexpectedly, these vaccine candidates and parental denv-4 similarly passaged in the same cells failed to elicit either viremia or a virus-neutralizing antibody response. consensus sequence analysis revealed that each of the three viruses, as well as the parental denv-4 when passaged in frhl cells, rapidly acquired a single glu327-gly substitu ...200919656873
seasonal dynamics of aedes aegypti (diptera: culicidae) in the northernmost state of brazil: a likely port-of-entry for dengue virus 4.roraima is the northernmost state of brazil, bordering both venezuela and guyana. appropriate climate and vector conditions for dengue transmission together with its proximity to countries where all four dengue serotypes circulate make this state, particularly the capital boa vista, strategically important for dengue surveillance in brazil. nonetheless, few studies have addressed the population dynamics of aedes aegypti in boa vista. in this study, we report temporal and spatial variations in ae ...200919722086
increment of interleukin 6, tumour necrosis factor alpha, nitric oxide, c-reactive protein and apoptosis in dengue.this study evaluated the levels of tnfalpha, il-6, il-1beta, nitric oxide (no), crp, c3 and apoptosis in 36 patients with dengue fever (df), 34 patients with dengue haemorrhagic fever (dhf) and in virus-infected monocyte cultures. il-6, tnfalpha, no (nitrites) and crp levels were increased and c3 diminished in patients with df and dhf. il-6, tnfalpha, cpr and c3 values were associated with disease severity (dhf). nitrite content was incremented in df patients. tnfalpha, no and crp levels were as ...201019733377
crystal structure of a novel conformational state of the flavivirus ns3 protein: implications for polyprotein processing and viral replication.the flavivirus genome comprises a single strand of positive-sense rna, which is translated into a polyprotein and cleaved by a combination of viral and host proteases to yield functional proteins. one of these, nonstructural protein 3 (ns3), is an enzyme with both serine protease and ntpase/helicase activities. ns3 plays a central role in the flavivirus life cycle: the ns3 n-terminal serine protease together with its essential cofactor ns2b is involved in the processing of the polyprotein, where ...200919793813
targeted mutagenesis as a rational approach to dengue virus vaccine development.the recombinant dengue virus type 4 (rden4) vaccine candidate, rden4delta30, was found to be highly infectious, immunogenic and safe in human volunteers. at the highest dose (10(5) pfu) evaluated in volunteers, 25% of the vaccinees had mild elevations in liver enzymes that were rarely seen at lower doses. here, we describe the generation and selection of additional mutations that were introduced into rden4delta30 to further attenuate the virus in animal models and ultimately human vaccinees. bas ...201019802584
phase 1 trial of the dengue virus type 4 vaccine candidate rden4{delta}30-4995 in healthy adult volunteers.rden4delta30-4995 is a live attenuated dengue virus type 4 (denv4) vaccine candidate specifically designed as a further attenuated derivative of the rden4delta30 parent virus. in a previous study, 5 of 20 vaccinees who received 10(5) plaque-forming units (pfu) of rden4delta30 developed a transient elevation of the serum alanine aminotransferase (alt) level and an asymptomatic maculopapular rash developed in 10 of 20. in the current study, 28 healthy adult volunteers were randomized to receive 10 ...200919861619
high-throughput procedure for tick surveys of tick-borne encephalitis virus and its application in a national surveillance study in switzerland.tick-borne encephalitis (tbe), a viral infection of the central nervous system, is endemic in many eurasian countries. in switzerland, tbe risk areas have been characterized by geographic mapping of clinical cases. since mass vaccination should significantly decrease the number of tbe cases, alternative methods for exposure risk assessment are required. we established a new pcr-based test for the detection of tbe virus (tbev) in ticks. the protocol involves an automated, high-throughput nucleic ...201020453126
construction of a dengue virus type 4 reporter replicon and analysis of temperature-sensitive mutations in non-structural proteins 3 and 5.replicon systems have been useful to study mechanisms of translation and replication of flavivirus rnas. in this study, we constructed a dengue virus 4 replicon encoding a renilla luciferase (r(luc)) reporter, and six single-residue substitution mutants were generated: l128f and s158p in the non-structural protein (ns) 3 protease domain gene, and n96i, n390a, k437r and m805i in the ns5 gene. the effects of these substitutions on viral rna translation and/or replication were examined by measuring ...201020631089
emergence of dengue virus type 4 (genotype i) in india.summarydengue is an emerging arboviral disease and currently poses the greatest arboviral threat to human health. in recent decades, there has been a substantial increase in dengue outbreaks in many parts of the world including india. we performed an in-depth investigation of a major dengue outbreak in andhra pradesh, southern india in 2007 by serology, virus isolation, rt-pcr and genotyping. the results revealed an unusual emergence of dengue virus type 4 (denv-4) along with the prevailing denv ...201120670467
insertion of microrna targets into the flavivirus genome alters its highly neurovirulent phenotype.flaviviruses such as west nile, japanese encephalitis, and tick-borne encephalitis (tbev) viruses are important neurotropic human pathogens, causing a devastating and often fatal neuroinfection. here, we demonstrate that incorporation into the viral genome of a target sequence for cellular micrornas expressed in the central nervous system (cns) enables alteration of the neurovirulence of the virus and control of the neuropathogenesis of flavivirus infection. as a model virus for this type of mod ...201021123372
dengue virus rna purification from human plasma: a comparison of two techniques.dengue virus rna purification from human plasma is useful for research and clinical purposes. dengue is endemic in the espirito santo state, brazil, and it is progressively becoming a hard-to-control public health problem [1]. dengue virus types 1, 2 and 3 are currently found in brazilian territory, and recently dengue virus type 4 has been reported to enter brazilian borders. this virus spreads rapidly during epidemic outbreaks, and thousands of patients are infected annually, with an underesti ...201021161397
evolutionary history of dengue virus type 4: insights into genotype phylodynamics.dengue virus type 4 (denv-4) circulates in tropical and subtropical countries from asia and the americas. despite the importance of dengue virus distribution, little is known about the worldwide viral spread. following a bayesian phylogenetic approach we inferred the evolutionary history of 310 isolates sampled from 37 countries during the time period 1956-2008 and the spreading dynamics for genotypes i and ii. the region (tropical rainforest biome) comprised by malaysia-thailand was the most li ...201121335103
microarray hybridization for assessment of the genetic stability of chimeric west nile/dengue 4 virus.genetic stability is an important characteristic of live viral vaccines because an accumulation of mutants can cause reversion to a virulent phenotype as well as a loss of immunogenic properties. this study was aimed at evaluating the genetic stability of a live attenuated west nile (wn) virus vaccine candidate that was generated by replacing the pre-membrane and envelope protein genes of dengue 4 virus with those from wn. chimeric virus was serially propagated in vero, sh-sy5y human neuroblasto ...201121360544
evaluation of two molecular methods for the detection of yellow fever virus genome.yellow fever virus (yfv), a member of the family flaviviridae, genus flavivirus is endemic to tropical areas of africa and south america and is among the arboviruses that pose a threat to public health. recent outbreaks in brazil, bolivia, and paraguay and the observation that vectors capable of transmitting yfv are presenting in urban areas underscore the urgency of improving surveillance and diagnostic methods. two novel methods (rt-hemi-nested-pcr and sybr(®) green qrt-pcr) for efficient dete ...201121419803
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