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age-associated differences in clinical manifestations and laboratory parameters during a dengue virus type 4 outbreak in argentina.infection by any of the four dengue virus (denv) serotypes produces a wide spectrum of clinical illness in humans. differences in clinical manifestation and severity have been associated with secondary heterologous infection, patient age, and virus serotype. in this context, this retrospective study sought to analyze the presentation of dengue in patients during the 2014 denv-4 outbreak affecting the city of orán, salta province, argentina. demographic data, clinical manifestations, and laborato ...201828941278
dengue type 4 in rio de janeiro, brazil: case characterization following its introduction in an endemic region.due to the populations' susceptibility, denv-4 introduction in 2010 led to the occurrence of explosive epidemics in the following years in brazil. in 2011, denv-4 was identified in rio de janeiro (rj) and it was prevalent in 2012 and 2013. here, we aimed to characterize clinical, epidemiological and laboratorial aspects of denv-4 cases after this serotype introduction in an endemic scenario.201728599640
"locked in a cage"-a case of dengue virus 4 encephalitis. 201728472166
computer-aided analysis of phytochemicals as potential dengue virus inhibitors based on molecular docking, admet and dft studies.dengue fever, caused by dengue virus (denv), has become a serious threat to human lives. phytochemicals are known to have great potential to eradicate viral, bacterial and fungal-borne diseases in human beings. this study was aimed at in silico drug development against nonstructural protein 4b (ns4b) of dengue virus 4 (denv4).201729097641
complete genomic sequence of dengue virus serotype 4 isolated from plasma collected from a haitian child in 2014.while data are limited, there is increasing evidence that infections by dengue viruses are endemic in haiti. in 2014, an outbreak caused by dengue virus 4 (denv-4) followed a chikungunya fever outbreak. we present here the complete genome sequence of one isolate grouped within the genotype ii south america and caribbean denv-4 clades.201728983011
detection of zika virus in april 2013 patient samples, rio de janeiro, brazil.we tested 210 dengue virus‒negative samples collected from febrile patients during a dengue virus type 4 outbreak in rio de janeiro in april 2013 and found 3 samples positive for zika virus. our findings support previously published entomological data suggesting zika virus was introduced into brazil during october 2012-may 2013.201728953451
recombinant dengue virus 4 envelope glycoprotein virus-like particles derived from pichia pastoris are capable of eliciting homotypic domain iii-directed neutralizing antibodies.dengue is a viral pandemic caused by four dengue virus serotypes (denv-1, 2, 3, and 4) transmitted by aedes mosquitoes. reportedly, there has been a 2-fold increase in dengue cases every decade. an efficacious tetravalent vaccine, which can provide long-term immunity against all four serotypes in all target populations, is still unavailable. despite the progress being made in the live virus-based dengue vaccines, the world health organization strongly recommends the development of alternative ap ...201727821688
letter to the editor "drosha, dgcr8, and dicer mrnas are downregulated in human cells infected with dengue virus 4" - genet. mol. res. 15 (2): gmr.15027891 - drosha, dicer, and trbp mrna are downregulated in vero cells with the 3'utr of dengue virus.dear editor, a recent paper (casseb et al., 2016) published in the journal genetics and molecular research described the interesting concept that dengue virus (denv)-4 infection, in the human cell line a-549, leads to the downregulation of expression of key components of microrna (mirna) biogenesis, such as drosha, dicer, and dgcr8. for this, the authors performed a time course infection of a-549 cells for 5 days. the highest viral load was observed at 3 days post-infection, which corresponded w ...201627706675
drosha, dgcr8, and dicer mrnas are down-regulated in human cells infected with dengue virus 4, and play a role in viral pathogenesis.dengue virus (denv) and its four serotypes (denv1-4) belong to the flavivirus genus of the flaviviridae family. denv infection is a life-threatening disease, which results in up to 20,000 deaths each year. viruses have been shown to encode trans-regulatory small rnas, or micrornas (mirnas), which bind to messenger rna and negatively regulate host or viral gene expression. during denv infections, mirnas interact with proteins in the rnai pathway, and are processed by ribonucleases such as dicer a ...201627173348
insights into the molecular evolution of dengue virus type 4 in puerto rico over two decades of emergence.dengue has emerged globally as a major human health problem since the 1950s and is now the most important arboviral disease of humans, infecting nearly 400 million people annually. while some cases are asymptomatic, others can develop a febrile illness (dengue fever) or even progress to severe and fatal dengue. dengue is caused by any of 4 closely related but distinct viruses, known as dengue virus serotype 1 to 4 (denv-1 to denv-4) which are maintained in endemic transmission to humans in large ...201626569594
priorities and needs for research on urban interventions targeting vector-borne diseases: rapid review of scoping and systematic reviews.this paper highlights the critical importance of evidence on vector-borne diseases (vbd) prevention and control interventions in urban settings when assessing current and future needs, with a view to setting policy priorities that promote inclusive and equitable urban health services. research should produce knowledge about policies and interventions that are intended to control and prevent vbds at the population level and to reduce inequities. such interventions include policy, program, and res ...201627903305
contribution of intertwined loop to membrane association revealed by zika virus full-length ns1 structure.the association of zika virus (zikv) infections with microcephaly and neurological diseases has highlighted an emerging public health concern. here, we report the crystal structure of the full-length zikv nonstructural protein 1 (ns1), a major host-interaction molecule that functions in flaviviral replication, pathogenesis, and immune evasion. of note, a long intertwined loop is observed in the wing domain of zikv ns1, and forms a hydrophobic "spike", which can contribute to cellular membrane as ...201627578809
the epidemiology, virology and clinical findings of dengue virus infections in a cohort of indonesian adults in western java.dengue has emerged as one of the most important infectious diseases in the last five decades. evidence indicates the expansion of dengue virus endemic areas and consequently the exponential increase of dengue virus infections across the subtropics. the clinical manifestations of dengue virus infection include sudden fever, rash, headache, myalgia and in more serious cases, spontaneous bleeding. these manifestations occur in children as well as in adults. defining the epidemiology of dengue in a ...201626872216
spot the difference-development of a syndrome based protein microarray for specific serological detection of multiple flavivirus infections in travelers.the family flaviviridae, genus flavivirus, holds many of the world's most prevalent arboviral diseases that are also considered the most important travel related arboviral infections. in most cases, flavivirus diagnosis in travelers is primarily based on serology as viremia is often low and typically has already been reduced to undetectable levels when symptoms set in and patients seek medical attention. serological differentiation between flaviviruses and the false-positive results caused by va ...201525767876
usutu virus: an emerging flavivirus in europe.usutu virus (usuv) is an african mosquito-borne flavivirus belonging to the japanese encephalitis virus serocomplex. usuv is closely related to murray valley encephalitis virus, japanese encephalitis virus, and west nile virus. usuv was discovered in south africa in 1959. in europe, the first true demonstration of circulation of usuv was reported in austria in 2001 with a significant die-off of eurasian blackbirds. in the subsequent years, usuv expanded to neighboring countries, including italy, ...201525606971
human recq1 helicase-driven dna unwinding, annealing, and branch migration: insights from dna complex structures.recq helicases are a widely conserved family of atp-dependent motors with diverse roles in nearly every aspect of bacterial and eukaryotic genome maintenance. however, the physical mechanisms by which recq helicases recognize and process specific dna replication and repair intermediates are largely unknown. here, we solved crystal structures of the human recq1 helicase in complexes with tailed-duplex dna and ssdna. the structures map the interactions of the ssdna tail and the branch point along ...201525831490
declining transition/transversion ratios through time reveal limitations to the accuracy of nucleotide substitution models.genetic analyses of dna sequences make use of an increasingly complex set of nucleotide substitution models to estimate the divergence between gene sequences. however, there is currently no way to assess the validity of nucleotide substitution models over short time-scales and with limited mutational accumulation.201525886870
sequence and structure analysis of distantly-related viruses reveals extensive gene transfer between viruses and hosts and among viruses.the origin and evolution of viruses is a subject of ongoing debate. in this study, we provide a full account of the evolutionary relationships between proteins of significant sequence and structural similarity found in viruses that belong to different classes according to the baltimore classification. we show that such proteins can be found in viruses from all baltimore classes. for protein families that include these proteins, we observe two patterns of the taxonomic spread. in the first patter ...201526492264
dual mirna targeting restricts host range and attenuates neurovirulence of flaviviruses.mosquito-borne flaviviruses are among the most significant arboviral pathogens worldwide. vaccinations and mosquito population control programs remain the most reliable means for flavivirus disease prevention, and live attenuated viruses remain one of the most attractive flavivirus vaccine platforms. some live attenuated viruses are capable of infecting principle mosquito vectors, as demonstrated in the laboratory, which in combination with their intrinsic genetic instability could potentially l ...201525906260
generation of monoclonal antibodies against dengue virus type 4 and identification of enhancing epitopes on envelope protein.the four serotypes of dengue virus (denv1-4) pose a serious threat to global health. cross-reactive and non-neutralizing antibodies enhance viral infection, thereby exacerbating the disease via antibody-dependent enhancement (ade). studying the epitopes targeted by these enhancing antibodies would improve the immune responses against denv infection. in order to investigate the roles of antibodies in the pathogenesis of dengue, we generated a panel of 16 new monoclonal antibodies (mabs) against d ...201526309127
absolute quantification of dengue virus serotype 4 chimera vaccine candidate in vero cell culture by targeted mass spectrometry.infection by dengue flavivirus is transmitted by mosquitoes and affects tens to hundreds of millions people around the world each year. four serotypes have been described, all of which cause similar disease. currently, there no approved vaccines or specific therapeutics for dengue, although several vaccine prototypes are in different stages of clinical development. among them, a chimeric vaccine, built from the replication machinery of the yellow fever 17d virus, has shown promising results in p ...201526205729
natural transovarial transmission of dengue virus 4 in aedes aegypti from cuiabá, state of mato grosso, brazil.dengue is the most prevalent arboviral disease in tropical areas. in mato grosso, outbreaks are reported every year, but studies on dengue in this state are scarce.201525860459
asian genotypes of dengue virus 4 in brazil.dengue virus, commonly transmitted by mosquitoes, causes a human disease of significant social impact and presents a serious public health problem in brazil. this report describes the unusual emergence of denv-4 in northern brazil after a nearly 30-year-long absence. denv-4 genotype i is of asian origin and was identified in the serum of patients receiving treatment at a hospital serving the salvador area (brazilian state of bahia). the identification of dengue virus serotypes through molecular ...201525703810
dengue virus 4 (denv-4) re-emerges after 30 years in brazil: cocirculation of denv-2, denv-3, and denv-4 in bahia.dengue fever (df) is a mosquito-borne viral disease of great concern in tropical and subtropical regions of the world. one important cause of the increase in df is rapid development and urbanization has led to proliferation of the aedes aegypti mosquito, the vector responsible for transmission of the illness. surveillance of dengue virus (denv) infection in brazil shows the predominance of denv-1, denv-2, and denv-3 until 2010. this study reports the reappearance of denv-4 in brazil for the firs ...201525420650
a dengue virus type 4 model of disseminated lethal infection in ag129 mice.dengue is a mosquito-borne disease of global public health significance that is caused by four serologically and genetically related viruses (denv-1 to denv-4). most human denv infections are asymptomatic, but clinical cases can range in severity from a relatively mild self-limiting illness to a severe life-threatening disease. infection with one serotype of denv results in life-long homotypic immunity but only short term heterotypic protection. there are no licensed vaccines or antivirals for d ...201525938762
characterization of lethal dengue virus type 4 (denv-4) tvp-376 infection in mice lacking both ifn-α/β and ifn-γ receptors (ag129) and comparison with the denv-2 ag129 mouse model.dengue is a mosquito-borne disease caused by four related but distinct dengue viruses, denv-1 to denv-4. dengue is endemic in most tropical countries, and over a third of the world's population is at risk of being infected. although the global burden is high, no vaccine or antiviral is licensed to combat this disease. an obstacle complicating dengue research is the lack of animal challenge models that mimic human disease. advances in immunocompromised murine infection models resulted in developm ...201526296350
delayed antibody dependent enhancement of low passage dengue virus 4 isolates.the concept of antibody dependent enhancement (ade) of dengue virus (denv) infection is a cornerstone of our current understanding of dengue pathogenesis, although some questions as to the mechanism remain, particularly in regards to the behavior of low and high passage virus isolates. this study utilized two low passage denv 4 isolates and a laboratory adapted denv 4 isolate to investigate the potential of low passage isolates to undergo ade.201526328895
characterization of the ectodomain of the envelope protein of dengue virus type 4: expression, membrane association, secretion and particle formation in the absence of precursor membrane protein.the envelope (e) of dengue virus (denv) is the major target of neutralizing antibodies and vaccine development. after biosynthesis e protein forms a heterodimer with precursor membrane (prm) protein. recent reports of infection enhancement by anti-prm monoclonal antibodies (mabs) suggest anti-prm responses could be potentially harmful. previously, we studied a series of c-terminal truncation constructs expressing denv type 4 prm/e or e proteins and found the ectodomain of e protein alone could b ...201424950216
assurance of neuroattenuation of a live vaccine against west nile virus: a comprehensive study of neuropathogenesis after infection with chimeric wn/den4δ30 vaccine in comparison to two parental viruses and a surrogate flavivirus reference vaccine.the upsurge of west nile virus (wnv) human infections in 2012 suggests that the us can expect periodic wnv outbreaks in the future. availability of safe and effective vaccines against wnv in endemic areas, particularly for aging populations that are at high risk of west nile neuroinvasive disease (wnnd), could be beneficial. wn/den4δ30 is a live, attenuated chimeric vaccine against wnv produced by replacement of the genes encoding the pre-membrane and envelope protein genes of the vaccine virus ...201424736001
japanese encephalitis virus vaccine candidates generated by chimerization with dengue virus type 4.japanese encephalitis virus (jev) is a leading cause of viral encephalitis worldwide and vaccination is one of the most effective ways to prevent disease. a suitable live-attenuated jev vaccine could be formulated with a live-attenuated tetravalent dengue vaccine for the control of these viruses in endemic areas. toward this goal, we generated chimeric virus vaccine candidates by replacing the precursor membrane (prm) and envelope (e) protein structural genes of recombinant dengue virus type 4 ( ...201424699473
molecular detection of mayaro virus during a dengue outbreak in the state of mato grosso, central-west brazil.mayaro virus (mayv) is frequently reported in pan-amazonia. the aim of this study was to investigate the circulation of alphaviruses during a dengue outbreak in the state of mato grosso, brazil. serum samples from dengue-suspected patients were subjected to multiplex semi-nested reverse transcriptase polymerase chain reaction for 11 flaviviruses and five alphaviruses, to nucleotide sequencing and to viral isolation. mayv was detected in 15 (2.5%) of 604 patients. twelve were co-infected with den ...201425141284
probable importation of dengue virus type 4 to angola from brazil. 201425271974
dna bioassay-on-chip using sers detection for dengue diagnosis.a novel dna bioassay-on-chip using surface-enhanced raman scattering (sers) on a bimetallic nanowave chip is presented. in this bioassay, sers signals were measured after a single reaction on the chip's surface without any washing step, making it simple-to-use and reducing the reagent cost. using the technique, specific oligonucleotide sequences of the dengue virus 4 were detected.201425248522
introduction of the dengue virus type 4 in the state of mato grosso do sul, brazil.we report on the first isolation of dengue virus serotype 4 (denv-4) in the state of mato grosso do sul, brazil, in february, 2012. the cases were isolated in the city of campo grande, the state capital, and presented the classic signs and symptoms of dengue fever. denv-4 was primarily identified through viral isolation in c6/36 clone lineage of aedes albopictus cells; followed by indirect immunofluorescence, using type-specific monoclonal antibodies. the results were subsequently confirmed by n ...201425210917
construction and preliminary investigation of a novel dengue serotype 4 chimeric virus using japanese encephalitis vaccine strain sa14-14-2 as the backbone.for the purpose of developing a novel dengue vaccine candidate, recombinant plasmids were constructed which contained the full length cdna clone of japanese encephalitis (je) vaccine strain sa14-14-2 with its premembrane (prem) and envelope (e) genes replaced by the counterparts of dengue virus type 4 (denv4). by transfecting the in vitro transcription products of the recombinant plasmids into bhk-21 cells, a chimeric virus jev/denv4 was successfully recovered. the chimeric virus was identified ...201425091563
construction of self-replicating subgenomic dengue virus 4 (denv4) replicon.dengue virus serotypes 1-4 are members of mosquito-borne flavivirus genus of flaviviridae family that encode one long open reading frame (orf) that is translated to a polyprotein. both host and virally encoded proteases function in the processing of the polyprotein by co-translational and posttranslational mechanisms to yield 10 mature proteins prior to viral rna replication. to study cis- and trans-acting factors involved in viral rna replication, many groups [1-8] have constructed cdnas encodi ...201424696335
dna microarray for detection of gastrointestinal viruses.gastroenteritis is a clinical illness of humans and other animals that is characterized by vomiting and diarrhea and caused by a variety of pathogens, including viruses. an increasing number of viral species have been associated with gastroenteritis or have been found in stool samples as new molecular tools have been developed. in this work, a dna microarray capable in theory of parallel detection of more than 100 viral species was developed and tested. initial validation was done with 10 differ ...201425355758
the association of cytokines with severe dengue in children.dengue virus infection is a major public health problem. a hypothesis put forward for severe dengue is the cytokine storm, a sudden increase in cytokines that induces vascular permeability. previous studies and our recent meta-analysis showed that il-6, il-8, ifnγ, tnfα, vegf-a and vcam-1 are associated with dengue shock syndrome. therefore, in this study we aim to validate the association of these cytokines with severe dengue.201425589878
characterization of a novel insect-specific flavivirus from brazil: potential for inhibition of infection of arthropod cells with medically important flaviviruses.in the past decade, there has been an upsurge in the number of newly described insect-specific flaviviruses isolated pan-globally. we recently described the isolation of a novel flavivirus (tentatively designated 'nhumirim virus'; nhuv) that represents an example of a unique subset of apparently insect-specific viruses that phylogenetically affiliate with dual-host mosquito-borne flaviviruses despite appearing to be limited to replication in mosquito cells. we characterized the in vitro growth p ...201425146007
serological evidence of widespread circulation of west nile virus and other flaviviruses in equines of the pantanal, brazil.a recent study reported neutralizing antibodies to west nile virus (wnv) in horses from four ranches of southern pantanal. to extend that study, a serosurvey for wnv and 11 brazilian flaviviruses was conducted with 760 equines, 238 sheep and 61 caimans from 17 local cattle ranches. among the tested equines, 32 were collected from a ranch where a neurologic disorder outbreak had been recently reported. the sera were initially screened by using a blocking elisa and then titrated by 90% plaque-redu ...201424551266
west nile virus state of the art report of malwest project.during the last three years greece is experiencing the emergence of west nile virus (wnv) epidemics. within this framework, an integrated surveillance and control programme (malwest project) with thirteen associate partners was launched aiming to investigate the disease and suggest appropriate interventions. one out of seven work packages of the project is dedicated to the state of the art report for wnv. three expert working groups on humans, animals and mosquitoes were established. medical dat ...201324317379
a review of vaccine approaches for west nile virus.the west nile virus (wnc) first appeared in north america in 1999. the north american lineages of wnv were characterized by the presence of neuroinvasive and neurovirulent strains causing disease and death in humans, birds and horses. the 2012 wnv season in the united states saw a massive spike in the number of neuroinvasive cases and deaths similar to what was seen in the 2002-2003 season, according to the west nile virus disease cases and deaths reported to the cdc by year and clinical present ...201324025396
mosquito saliva serine protease enhances dissemination of dengue virus into the mammalian host.dengue virus (denv), a flavivirus of global importance, is transmitted to humans by mosquitoes. in this study, we developed in vitro and in vivo models of saliva-mediated enhancement of denv infectivity. serine protease activity in aedes aegypti saliva augmented virus infectivity in vitro by proteolyzing extracellular matrix proteins, thereby increasing viral attachment to heparan sulfate proteoglycans and inducing cell migration. a serine protease inhibitor reduced saliva-mediated enhancement o ...201324131723
the eukaryotic translation initiation factor 3 subunit l protein interacts with flavivirus ns5 and may modulate yellow fever virus replication.yellow fever virus (yfv) belongs to the flavivirus genus and causes an important disease. an alarming resurgence of viral circulation and the expansion of yfv-endemic zones have been detected in africa and south america in recent years. ns5 is a viral protein that contains methyltransferase and rna-dependent rna polymerase (rdrp) domains, which are essential for viral replication, and the interactions between ns5 and cellular proteins have been studied to better understand viral replication. the ...201323800076
development of one-step quantitative reverse transcription pcr for the rapid detection of flaviviruses.the genus flavivirus includes several pathogenic agents that cause severe illness in humans. re-emergence of west nile virus in europe and continuous spread of certain flaviviruses such as dengue, yellow fever and japanese encephalitis viruses represent a global danger to public health. therefore, a rapid and accurate molecular method is required for diagnostics and epidemiological surveillance of flaviviruses.201323410000
apropos "clinico-genetic characterisation of an encephalitic dengue virus 4 associated with multi-organ involvement". 201323770056
clinico-genetic characterisation of an encephalitic dengue virus 4 associated with multi-organ involvement.neurological manifestations due to dengue virus (denv) infection are atypical and uncommon. genomic information of clinically characterised, neurotrophic denv in humans is extremely limited albeit their importance in deciphering the pathogenicity is substantial. here, we report a rare case of fatal denv-4 infection complicated with encephalitis and multi-organ failure. the clinical presentation was unusual due to its rapid onset of encephalitis despite a very low virus titre. full genomes of ser ...201323415634
surface proteins of c6/36 cells involved in dengue virus 4 binding and entry.dengue virus (denv) is the causative agent of the most important mosquito-borne viral disease, which is endemic to over 100 countries in tropical and subtropical areas of the world. it is transmitted to humans by aedes mosquitoes. the first step in the viral infection of host cells is virion attachment to the plasma membrane, which is mediated by specific surface molecules. there are several molecules that participate in denv infection of mosquitoes, but only a few have been identified. in this ...201323344777
emergence of dengue virus 4 genotypes ii b and i in the city of rio de janeiro. 201323127562
emergence potential of sylvatic dengue virus type 4 in the urban transmission cycle is restrained by vaccination and homotypic immunity.sylvatic dengue viruses (denv) are both evolutionarily and ecologically distinct from human denv and are maintained in an enzootic transmission cycle. evidence of sylvatic human infections from west africa and southeast asia suggests that sylvatic denv come into regular contact with humans. thus, this potential of emergence into the human transmission cycle could limit the potential for eradicating this cycle with vaccines currently in late stages of development. we assessed the likelihood of sy ...201323485373
functional analysis of antibodies against dengue virus type 4 reveals strain-dependent epitope exposure that impacts neutralization and protection.although prior studies have characterized the neutralizing activities of monoclonal antibodies (mabs) against dengue virus (denv) serotypes 1, 2, and 3 (denv-1, denv-2, and denv-3), few reports have assessed the activity of mabs against denv-4. here, we evaluated the inhibitory activity of 81 new mouse anti-denv-4 mabs. we observed strain- and genotype-dependent differences in neutralization of denv-4 by mabs mapping to epitopes on domain ii (dii) and diii of the envelope (e) protein. several an ...201323785205
optimization of dengue virus genome assembling using gsflx 454 pyrosequencing data: evaluation of assembling strategies.currently assembling genomes without reference is one of the most important challenges for bioinformaticists all over the world in an attempt to characterize new organisms. the current study has used two dengue virus type 4 (denv-4) strains recently isolated in brazil, which have its genomes sequenced using the gsflx 454 sequencer (roche, life science) by the pyrosequencing method. the gsflx 454 data were used for testing different genome assembling strategies. we described a pipeline that was a ...201222930429
first isolation of dengue 4 in the state of são paulo, brazil, 2011.we report the first isolation of dengue virus 4 (denv-4) in the state of são paulo, from two patients - one living in são josé do rio preto and the other one in paulo de faria, both cities located in the northwest region of the state. the virus isolations were accomplished in the clone c6/36 aedes albopictus cell line, followed by indirect immunofluorescence assays, performed with type-specific monoclonal antibodies that showed positive reactions for denv-4. the results were confirmed by nested ...201222370754
emergence of dengue virus 4 genotype ii in guangzhou, china, 2010: survey and molecular epidemiology of one community outbreak.the re-emergence of dengue virus 4 (denv-4) has become a public health concern in south america, southeast asia and south asia. however, it has not been known to have caused a local outbreak in china for the past 20 years. the purpose of this study was to elucidate the epidemiology of one local community outbreak caused by denv-4 in guangzhou city, china, in 2010; and to determine the molecular characteristics of the genotype ii virus involved.201222497881
dengue virus type 4 in niterói, rio de janeiro: the role of molecular techniques in laboratory diagnosis and entomological surveillance.in niterói, state of rio de janeiro, dengue virus type 4 (denv-4) was isolated for the first time in march 2011. we analysed the laboratory findings of the first cases and evaluated the use of molecular techniques for the detection of denv-4 in aedes aegypti that were field-caught. conventional reverse transcriptase-polymerase chain reaction (rt-pcr) and simplexa™ dengue real-time rt-pcr confirmed denv-4 infection in all cases. additionally, denv-4 was confirmed in a female ae. aegypti with 1.08 ...201223147153
c-terminal helical domains of dengue virus type 4 e protein affect the expression/stability of prm protein and conformation of prm and e proteins.the envelope (e) protein of dengue virus (denv) is the major immunogen for dengue vaccine development. at the c-terminus are two α-helices (eh1 and eh2) and two transmembrane domains (et1 and et2). after synthesis, e protein forms a heterodimer with the precursor membrane (prm) protein, which has been shown as a chaperone for e protein and could prevent premature fusion of e protein during maturation. recent reports of enhancement of denv infectivity by anti-prm monoclonal antibodies (mabs) sugg ...201223300717
detection and typing of viruses using broadly sensitive cocktail-pcr and mass spectrometric cataloging: demonstration with dengue virus.virus detection and taxonomic identification of serotypes, strains, or genotypes provide important information relevant for diagnosis, and for the epidemiological characterization and tracking of new strains in an endemic region. in the specific case of dengue virus, rapid serotype identification can also be useful in the treatment of secondary infections that may cause the more severe dengue hemorrhagic fever and dengue shock syndrome. in this work, dengue virus was used as a model to test a ne ...201222579629
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
dengue virus type 4 arrives in the state of rio de janeiro: a challenge for epidemiological surveillance and control. 201121655810
genotype v japanese encephalitis virus is emerging.japanese encephalitis (je) is a global public health issue that has spread widely to more than 20 countries in asia and has extended its geographic range to the south pacific region including australia. je has become the most important cause of viral encephalitis in the world. japanese encephalitis viruses (jev) are divided into five genotypes, based on the nucleotide sequence of the envelope (e) gene. the muar strain, isolated from patient in malaya in 1952, is the sole example of genotype v je ...201121750744
detection of dengue virus type 4 in easter island, chile.we report the detection of dengue virus type 4 (denv-4) for the first time in easter island, chile. the virus was detected in serum samples of two patients treated at the hospital in easter island. the two samples were igm positive, and the infection was confirmed by rt-pcr and genetic sequencing; viral isolation was possible with one of them. the easter island isolates were most closely related to genotype ii of dengue type 4.201121691853
productive dengue virus infection of human endothelial cells is directed by heparan sulfate-containing proteoglycan receptors.dengue virus causes leakage of the vascular endothelium, resulting in dengue hemorrhagic fever and dengue shock syndrome. the endothelial cell lining of the vasculature regulates capillary permeability and is altered by immune and chemokine responses which affect fluid barrier functions of the endothelium. our findings indicate that human endothelial cells are highly susceptible to infection by dengue virus (type 4). we found that dengue virus productively infects ôê+80% of primary human endothe ...201121734047
genetic and phenotypic characterization of sylvatic dengue virus type 4 strains.four serotypes of dengue virus (denv 1-4) currently circulate between humans and domestic/peridomestic aedes mosquitoes, resulting in 100million infections per year. all four serotypes emerged, independently, from sylvatic progenitors transmitted among non-human primates by arboreal aedes mosquitoes. this study investigated the genetic and phenotypic changes associated with emergence of human denv-4 from its sylvatic ancestors. analysis of complete genomes of 3 sylvatic and 4 human strains revea ...201122178263
Dengue virus serotype 4, Roraima State, Brazil. 201122000396
dengue virus type 4 phylogenetics in brazil 2011: looking beyond the veil.dengue fever and dengue hemorrhagic fever are diseases affecting approximately 100 million people/year and are a major concern in developing countries. in the present study, the phylogenetic relationship of six strains of the first autochthonous cases of denv-4 infection occurred in sao paulo state, parana state and rio grande do sul state, brazil, 2011 were studied. nucleotide sequences of the envelope gene were determined and compared with sequences representative of the genotypes i, ii, iii a ...201122216365
[new evidence in the susceptibility to dengue infection associated to hh polymorphism of fcgammariia receptor].polymorphic variants of fcgammariia receptor have been associated to susceptibility to develop several infectious diseases. the relationship between the polymorphism of this receptor and the susceptibility to dengue hemorrhagic fever was recently reported.201123444610
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
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
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
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
vipr: a probabilistic algorithm for analysis of microbial detection microarrays.all infectious disease oriented clinical diagnostic assays in use today focus on detecting the presence of a single, well defined target agent or a set of agents. in recent years, microarray-based diagnostics have been developed that greatly facilitate the highly parallel detection of multiple microbes that may be present in a given clinical specimen. while several algorithms have been described for interpretation of diagnostic microarrays, none of the existing approaches is capable of incorpora ...201020646301
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
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
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
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
predicting the sensitivity and specificity of published real-time pcr assays.in recent years real-time pcr has become a leading technique for nucleic acid detection and quantification. these assays have the potential to greatly enhance efficiency in the clinical laboratory. choice of primer and probe sequences is critical for accurate diagnosis in the clinic, yet current primer/probe signature design strategies are limited, and signature evaluation methods are lacking.200818817537
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 monkeys.to 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
bacterially expressed and refolded envelope protein (domain iii) of dengue virus type-4 binds heparan sulfate.an 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
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
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 diseases.to 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
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
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
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