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g3bp1, g3bp2 and caprin1 are required for translation of interferon stimulated mrnas and are targeted by a dengue virus non-coding rna.viral rna-host protein interactions are critical for replication of flaviviruses, a genus of positive-strand rna viruses comprising major vector-borne human pathogens including dengue viruses (denv). we examined three conserved host rna-binding proteins (rbps) g3bp1, g3bp2 and caprin1 in dengue virus (denv-2) infection and found them to be novel regulators of the interferon (ifn) response against denv-2. the three rbps were required for the accumulation of the protein products of several interfe ...201424992036
substitution of ns5 n-terminal domain of dengue virus type 2 rna with type 4 domain caused impaired replication and emergence of adaptive mutants with enhanced fitness.flavivirus ns3 and ns5 are required in viral replication and 5'-capping. ns3 has ns2b-dependent protease, rna helicase, and 5'-rna triphosphatase activities. ns5 has 5'-rna methyltransferase (mt)/guanylyltransferase (gt) activities within the n-terminal 270 amino acids and the rna-dependent rna polymerase (pol) activity within amino acids 271-900. a chimeric ns5 containing the d4mt/d4gt and the d2pol domains in the context of wild-type (wt) d2 rna was constructed. rnas synthesized in vitro were ...201424904061
development, characterization and application of monoclonal antibodies against brazilian dengue virus isolates.dengue is the most prevalent human arboviral disease. the morbidity related to dengue infection supports the need for an early, quick and effective diagnostic test. brazil is a hotspot for dengue, but no serological diagnostic test has been produced using brazilian dengue virus isolates. this study aims to improve the development of immunodiagnostic methods for dengue virus (denv) detection through the production and characterization of 22 monoclonal antibodies (mabs) against brazilian isolates ...201425412181
abrogation of tlr3 inhibition by discrete amino acid changes in the c-terminal half of the west nile virus ns1 protein.west nile virus (wnv) is a mosquito-transmitted pathogen, which causes significant disease in humans. the innate immune system is a first-line defense against invading microorganism and many flaviviruses, including wnv, have evolved multifunctional proteins, which actively suppress its activation and antiviral actions. the wnv non-structural protein 1 (ns1) inhibits signal transduction originating from toll-like receptor 3 (tlr3) and also critically contributes to virus genome replication. in th ...201424889229
ebola virus vaccines: an overview of current approaches.ebola hemorrhagic fever is one of the most fatal viral diseases worldwide affecting humans and nonhuman primates. although infections only occur frequently in central africa, the virus has the potential to spread globally and is classified as a category a pathogen that could be misused as a bioterrorism agent. as of today there is no vaccine or treatment licensed to counteract ebola virus infections. dna, subunit and several viral vector approaches, replicating and non-replicating, have been tes ...201424575870
rna virus reverse genetics and vaccine design.rna viruses are capable of rapid spread and severe or potentially lethal disease in both animals and humans. the development of reverse genetics systems for manipulation and study of rna virus genomes has provided platforms for designing and optimizing viral mutants for vaccine development. here, we review the impact of rna virus reverse genetics systems on past and current efforts to design effective and safe viral therapeutics and vaccines.201424967693
the bioactive lipid 4-hydroxyphenyl retinamide inhibits flavivirus replication.dengue virus (denv), a member of the flaviviridae family, is a mosquito-borne pathogen and the cause of dengue fever. the increasing prevalence of denv worldwide heightens the need for an effective vaccine and specific antivirals. due to the dependence of denv upon the lipid biosynthetic machinery of the host cell, lipid signaling and metabolism present unique opportunities for inhibiting viral replication. we screened a library of bioactive lipids and modulators of lipid metabolism and identifi ...201425313218
whole genome phylogenetic investigation of a west nile virus strain isolated from a tick sampled from livestock in north eastern kenya.west nile virus (wnv) has a wide geographical distribution and has been associated to cause neurological disease in humans and horses. mosquitoes are the traditional vectors for wnv; however, the virus has also been isolated from tick species in north africa and europe which could be a means of introduction and spread of the virus over long distances through migratory birds. although wnv has been isolated in mosquitoes in kenya, paucity of genetic and pathogenicity data exists. we previously rep ...201425430727
evidence of west nile virus infection in nepal.acute febrile illness is common among those seeking medical care and is frequently treated empirically with the underlying illness remaining undiagnosed in resource-poor countries. a febrile illness study was conducted 2009-2010 to identify known and unknown pathogens circulating in nepal.201425427544
two distinct sets of ns2a molecules are responsible for dengue virus rna synthesis and virion assembly.flavivirus nonstructural protein 2a (ns2a) plays important roles in both viral rna synthesis and virion assembly. the molecular details of how the ns2a protein modulates the two distinct events have not been defined. to address this question, we have performed a systematic mutagenesis of ns2a using dengue virus (denv) serotype 2 (denv-2) as a model. we identified two sets of ns2a mutations with distinct defects during a viral infection cycle. one set of ns2a mutations (d125a and g200a) selective ...201425392211
a review of the epidemiological and clinical aspects of west nile virus.the resurgence of west nile virus (wnv) in north america and europe in recent years has raised the concerns of local authorities and highlighted that mosquito-borne disease is not restricted to tropical regions of the world. wnv is maintained in enzootic cycles involving, primarily, culex spp. mosquitoes and avian hosts, with epizootic spread to mammals, including horses and humans. human infection results in symptomatic illness in approximately one-fifth of cases and neuroinvasive disease in le ...201424748813
mechanisms underlying the inhibition of interferon signaling by viruses.a hallmark of the antiviral response is the induction of interferons. first discovered in 1957 by issac and lindeman, interferons are noted for their ability to interfere with viral replication. interferons act via autocrine and paracrine pathways to induce an antiviral state in infected cells and in neighboring cells containing interferon receptors. interferons are the frontline defenders against viral infection and their primary function is to locally restrict viral propagation. viruses have e ...201424504013
inhibition of hsp70 reduces porcine reproductive and respiratory syndrome virus replication in vitro.successful viral infection requires the involvement of host cellular factors in their life cycle. heat shock protein 70 (hsp70) can be recruited by numerous viruses to promote the folding, maturation, or assembly of viral proteins. we have previously shown that hsp70 is significantly elevated in porcine reproductive and respiratory syndrome virus (prrsv)-infected lungs, suggesting hsp70 may play a potential role during prrsv infection. in this study, we tried to investigate the role of hsp70 dur ...201424625230
the role of viral persistence in flavivirus biology.in nature, vector-borne flaviviruses are persistently cycled between either the tick or mosquito vector and small mammals such as rodents, skunks, and swine. these viruses account for considerable human morbidity and mortality worldwide. increasing and substantial evidence of viral persistence in humans, which includes the isolation of rna by rt-pcr and infectious virus by culture, continues to be reported. viral persistence can also be established in vitro in various human, animal, arachnid, an ...201424737600
vector competence of culex neavei and culex quinquefasciatus (diptera: culicidae) from senegal for lineages 1, 2, koutango and a putative new lineage of west nile virus.west nile virus (wn virus) is one of the most widespread arbovirus and exhibits a great genetic diversity with 8 lineages, at least 4 (1, 2, koutango, and putative new) are present in africa. in west africa, culex neavei and culex quinquefasciatus are considered as potential vectors for wn virus transmission in sylvatic or urban context. we analyzed the vector competence of these culex species from senegal for african lineages and envelope proteins sequences of viral strains used. we showed that ...201424567319
host competence and helicase activity differences exhibited by west nile viral variants expressing ns3-249 amino acid polymorphisms.a single helicase amino acid substitution, ns3-t249p, has been shown to increase viremia magnitude/mortality in american crows (amcrs) following west nile virus (wnv) infection. lineage/intra-lineage geographic variants exhibit consistent amino acid polymorphisms at this locus; however, the majority of wnv isolates associated with recent outbreaks reported worldwide have a proline at the ns3-249 residue. in order to evaluate the impact of ns3-249 variants on avian and mammalian virulence, multip ...201424971589
infection with non-lethal west nile virus eg101 strain induces immunity that protects mice against the lethal west nile virus ny99 strain.herein we demonstrate that infection of mice with west nile virus (wnv) eg101 provides protective immunity against lethal challenge with wnv ny99. our data demonstrated that wnv eg101 is largely non-virulent in adult mice when compared to wnv ny99. by day 6 after infection, wnv-specific igm and igg antibodies, and neutralizing antibodies were detected in the serum of all wnv eg101 infected mice. plaque reduction neutralization test data demonstrated that serum from wnv eg101 infected mice neutra ...201424915459
a novel t-cell receptor mimic defines dendritic cells that present an immunodominant west nile virus epitope in mice.we used a newly generated t-cell receptor mimic monoclonal antibody (tcrm mab) that recognizes a known nonself immunodominant peptide epitope from west nile virus (wnv) ns4b protein to investigate epitope presentation after virus infection in c57bl/6 mice. previous studies suggested that peptides of different length, either ssvwnattai (10-mer) or ssvwnatta (9-mer) in complex with class i mhc antigen h-2d(b) , were immunodominant after wnv infection. our data establish that both peptides are pres ...201424723377
ultrastructural characterization and three-dimensional architecture of replication sites in dengue virus-infected mosquito cells.during dengue virus infection of host cells, intracellular membranes are rearranged into distinct subcellular structures such as double-membrane vesicles, convoluted membranes, and tubular structures. recent electron tomographic studies have provided a detailed three-dimensional architecture of the double-membrane vesicles, representing the sites of dengue virus replication, but temporal and spatial evidence linking membrane morphogenesis with viral rna synthesis is lacking. integrating techniqu ...201424522909
experimental infection of rhesus macaques and common marmosets with a european strain of west nile virus.west nile virus (wnv) is a mosquito-borne flavivirus that infects humans and other mammals. in some cases wnv causes severe neurological disease. during recent years, outbreaks of wnv are increasing in worldwide distribution and novel genetic variants of the virus have been detected. although a substantial amount of data exists on wnv infections in rodent models, little is known about early events during wnv infection in primates, including humans. to gain a deeper understanding of this process, ...201424743302
the nairovirus nairobi sheep disease virus/ganjam virus induces the translocation of protein disulphide isomerase-like oxidoreductases from the endoplasmic reticulum to the cell surface and the extracellular space.nairobi sheep disease virus (nsdv) of the genus nairovirus causes a haemorrhagic gastroenteritis in sheep and goats with mortality up to 90%; the virus is found in east and central africa, and in india, where the virus is called ganjam virus. nsdv is closely related to the human pathogen crimean-congo haemorrhagic fever virus, which also causes a haemorrhagic disease. as with other nairoviruses, replication of nsdv takes place in the cytoplasm and the new virus particles bud into the golgi appar ...201424714576
rna structures that resist degradation by xrn1 produce a pathogenic dengue virus rna.dengue virus is a growing global health threat. dengue and other flaviviruses commandeer the host cell's rna degradation machinery to generate the small flaviviral rna (sfrna), a noncoding rna that induces cytopathicity and pathogenesis. host cell exonuclease xrn1 likely loads on the 5' end of viral genomic rna and degrades processively through ∼10 kb of rna, halting near the 3' end of the viral rna. the surviving rna is the sfrna. we interrogated the architecture of the complete dengue 2 sfrna, ...201424692447
effect of dengue-2 virus infection on protein expression in the salivary glands of aedes aegypti mosquitoes.dengue virus (denv) is the most important mosquito-transmitted flavivirus that is transmitted throughout the tropical and subtropical regions of the world. the primary mosquito vector of denv in urban locations is aedes aegypti. key to understanding the transmission of denv is the relationship between pathogen and vector. accordingly, we report our preliminary characterization of the differentially expressed proteins from ae. aegypti mosquitoes after denv infection. we investigated the virus-vec ...201424445208
linking pig-tailed macaque major histocompatibility complex class i haplotypes and cytotoxic t lymphocyte escape mutations in simian immunodeficiency virus infection.the influence of major histocompatibility complex class i (mhc-i) alleles on human immunodeficiency virus (hiv) diversity in humans has been well characterized at the population level. mhc-i alleles likely affect viral diversity in the simian immunodeficiency virus (siv)-infected pig-tailed macaque (macaca nemestrina) model, but this is poorly characterized. we studied the evolution of siv in pig-tailed macaques with a range of mhc-i haplotypes. siv(mac251) genomes were amplified from the plasma ...201425275134
interferon regulatory factor 5-dependent immune responses in the draining lymph node protect against west nile virus infection.upon activation of toll-like and rig-i-like receptor signaling pathways, the transcription factor irf5 translocates to the nucleus and induces antiviral immune programs. the recent discovery of a homozygous mutation in the immunoregulatory gene guanine exchange factor dedicator of cytokinesis 2 (dock2mu/mu) in several irf5-/- mouse colonies has complicated interpretation of immune functions previously ascribed to irf5. to define the antiviral functions of irf5 in vivo, we infected backcrossed ir ...201425031348
monomeric nature of dengue virus ns3 helicase and thermodynamic analysis of the interaction with single-stranded rna.dengue virus nonstructural protein 3 (ns3) is a multifunctional protein formed by a superfamily-2 rna helicase linked to a protease domain. in this work, we report results from in vitro experiments designed to determine the oligomeric state of dengue virus ns3 helicase (ns3h) and to characterize fundamental properties of the interaction with single-stranded (ss)rna. pulsed field gradient-nmr spectroscopy was used to determine the effective hydrodynamic radius of ns3h, which was constant over a w ...201425223789
the west nile virus-like flavivirus koutango is highly virulent in mice due to delayed viral clearance and the induction of a poor neutralizing antibody response.the mosquito-borne west nile virus (wnv) is responsible for outbreaks of viral encephalitis in humans, horses, and birds, with particularly virulent strains causing recent outbreaks of disease in eastern europe, the middle east, north america, and australia. previous studies have phylogenetically separated wnv strains into two main genetic lineages (i and ii) containing virulent strains associated with neurological disease. several wnv-like strains clustering outside these lineages have been ide ...201424942584
modulation of α-enolase post-translational modifications by dengue virus: increased secretion of the basic isoforms in infected hepatic cells.hepatic cells are major sites of dengue virus (denv) replication and liver injury constitutes a characteristic of severe forms of dengue. the role of hepatic cells in dengue pathogenesis is not well established, but since hepatocytes are the major source of plasma proteins, changes in protein secretion by these cells during infection might contribute to disease progression. previously, we showed that denv infection alters the secretion pattern of hepatic hepg2 cells, with α-enolase appearing as ...201425171719
in vitro and in vivo characterization of a west nile virus mad78 infectious clone.the viral determinants governing the varied neuropathogenicity of different west nile virus (wnv) strains are poorly understood. here, we generated an infectious clone (wnv-mad(ic)) of the non-pathogenic strain wnv-mad78 and compared its replication to that of parental wnv-mad78 and a wnv-mad78 infectious clone (wnv-mad(tx-utrs)) containing the 5' and 3' untranslated regions (utrs) of the pathogenic strain wnv-tx. all three viruses replicated at similar rates and caused similar lethality in mice ...201425023336
applications of a sugar-based surveillance system to track arboviruses in wild mosquito populations.effective arbovirus surveillance is essential to ensure the implementation of control strategies, such as mosquito suppression, vaccination, or dissemination of public warnings. traditional strategies employed for arbovirus surveillance, such as detection of virus or virus-specific antibodies in sentinel animals, or detection of virus in hematophagous arthropods, have limitations as an early-warning system. a system was recently developed that involves collecting mosquitoes in co2-baited traps, ...201424359415
comparison of the neuropathology induced by two west nile virus strains.some strains of west nile virus (wnv) are neuroinvasive and may induce fatal encephalitis/meningitis in a variety of animal species including humans. whether, however, there is a strain-specific signature in the brain is as yet unknown. here we investigated the neuropathogenesis induced by two phylogenetically distant wnv strains of lineage 1, wnv(is98) and wnv(kun35 911). while four-week old c57bl/6j mice were susceptible to both strains and succumbed rapidly after intraperitoneal inoculation, ...201324367664
comparison of the live attenuated yellow fever vaccine 17d-204 strain to its virulent parental strain asibi by deep sequencing.the first comparison of a live rna viral vaccine strain to its wild-type parental strain by deep sequencing is presented using as a model the yellow fever virus (yfv) live vaccine strain 17d-204 and its wild-type parental strain, asibi.201324141982
molecular epidemiology and evolution of west nile virus in north america.west nile virus (wnv) was introduced to new york in 1999 and rapidly spread throughout north america and into parts of central and south america. displacement of the original new york (ny99) genotype by the north america/west nile 2002 (na/wn02) genotype occurred in 2002 with subsequent identification of a novel genotype in 2003 in isolates collected from the southwestern unites states region (sw/wn03 genotype). both genotypes co-circulate to date. subsequent wnv surveillance studies have confir ...201324135819
characterization of the mouse neuroinvasiveness of selected european strains of west nile virus.west nile virus (wnv) has caused outbreaks and sporadic infections in central, eastern and mediterranean europe for over 45 years. most strains responsible for the european and mediterranean basin outbreaks are classified as lineage 1. in recent years, wnv strains belonging to lineage 1 and 2 have been causing outbreaks of neuroinvasive disease in humans in countries such as italy, hungary and greece, while mass mortality among birds was not reported. this study characterizes three european stra ...201324058590
regulation of flavivirus rna synthesis and capping.rna viruses, such as flaviviruses, are able to efficiently replicate and cap their rna genomes in vertebrate and invertebrate cells. flaviviruses use several specialized proteins to first make an uncapped negative strand copy of the viral genome that is used as a template for the synthesis of large numbers of capped genomic rnas. despite using relatively simple mechanisms to replicate their rna genomes, there are significant gaps in our understanding of how flaviviruses switch between negative a ...201323929625
west nile virus. 201324179475
west nile virus genome with glycosylated envelope protein and deletion of alpha helices 1, 2, and 4 in the capsid protein is noninfectious and efficiently secretes subviral particles.flavivirus genomes with deletions in the capsid (c) gene are attractive vaccine candidates, as they secrete highly immunogenic subviral particles (svps) without generating infectious virus. here, we report that cytomegalovirus promoter-driven cdna of west nile virus kunjin (kunv) containing a glycosylation motif in the envelope (e) gene and a combined deletion of alpha helices 1, 2, and 4 in c produces significantly more svps than kunv cdnas with nonglycosylated e and various other deletions in ...201324049184
vaccines in development against west nile virus.west nile encephalitis emerged in 1999 in the united states, then rapidly spread through the north american continent causing severe disease in human and horses. since then, outbreaks appeared in europe, and in 2012, the united states experienced a new severe outbreak reporting a total of 5,387 cases of west nile virus (wnv) disease in humans, including 243 deaths. so far, no human vaccine is available to control new wnv outbreaks and to avoid worldwide spreading. in this review, we discuss the ...201324084235
natural exposure of horses to mosquito-borne flaviviruses in south-east queensland, australia.in 2011 an unprecedented epidemic of equine encephalitis occurred in south-eastern (se) australia following heavy rainfall and severe flooding in the preceding 2-4 months. less than 6% of the documented cases occurred in queensland, prompting the question of pre-existing immunity in queensland horses. a small-scale serological survey was conducted on horses residing in one of the severely flood-affected areas of se-queensland. using a flavivirus-specific blocking-elisa we found that 63% (39/62) ...201324048209
membrane topology and function of dengue virus ns2a protein.flavivirus nonstructural protein 2a (ns2a) is a component of the viral replication complex that functions in virion assembly and antagonizes the host immune response. although flavivirus ns2a is known to associate with the endoplasmic reticulum (er) membrane, the detailed topology of this protein has not been determined. here we report the first topology model of flavivirus ns2a on the er membrane. using dengue virus (denv) ns2a as a model, we show that (i) the n-terminal 68 amino acids are loca ...201323408612
s-adenosyl-homocysteine is a weakly bound inhibitor for a flaviviral methyltransferase.the methyltransferase enzyme (mtase), which catalyzes the transfer of a methyl group from s-adenosyl-methionine (adomet) to viral rna, and generates s-adenosyl-homocysteine (adohcy) as a by-product, is essential for the life cycle of many significant human pathogen flaviviruses. here we investigated inhibition of the flavivirus mtase by several adohcy-derivatives. unexpectedly we found that adohcy itself barely inhibits the flavivirus mtase activities, even at high concentrations. adohcy was als ...201324130807
increased early rna replication by chimeric west nile virus w956ic leads to ips-1-mediated activation of nf-κb and insufficient virus-mediated counteraction of the resulting canonical type i interferon signaling.although infections with "natural" west nile virus (wnv) and the chimeric w956ic wnv infectious clone virus produce comparable peak virus yields in type i interferon (ifn) response-deficient bhk cells, w956ic infection produces higher levels of "unprotected" viral rna at early times after infection. analysis of infections with these two viruses in ifn-competent cells showed that w956ic activated nf-κb, induced higher levels of ifn-β, and produced lower virus yields than wnv strain eg101. ips-1 w ...201323678179
identification of conserved motifs in the west nile virus envelope essential for particle secretion.enveloped viruses utilize cellular membranes to bud from infected cells. the process of virion assembly and budding is often facilitated by the presence of certain conserved motifs within viral proteins in conjunction with cellular factors. we hence examined the west nile virus (wnv) envelope protein for the presence of any such motifs and their functional characterization.201324007503
west nile viral infection of equids.west nile virus (wnv) is a flavivirus transmitted between certain species of birds and mosquito vectors. tangential infections of equids and subsequent equine epizootics have occurred historically. although the attack rate has been estimated to be below 10%, mortality rates can approach 50% in horses that present clinical disease. symptoms are most commonly presenting in the form of encephalitis with ataxia as well as limb weakness, recumbency and muscle fasciculation. the most effective strateg ...201324035480
the effect of vaccination coverage and climate on japanese encephalitis in sarawak, malaysia.japanese encephalitis (je) is the leading cause of viral encephalitis across asia with approximately 70,000 cases a year and 10,000 to 15,000 deaths. because je incidence varies widely over time, partly due to inter-annual climate variability effects on mosquito vector abundance, it becomes more complex to assess the effects of a vaccination programme since more or less climatically favourable years could also contribute to a change in incidence post-vaccination. therefore, the objective of this ...201323951373
clinical and radiological predictors of outcome for murray valley encephalitis.a review of the laboratory-confirmed cases of murray valley encephalitis (mve) from western australia between 2009 and 2011 was conducted to describe the clinical, laboratory, and radiological features of the disease. the nine encephalitis patients presented with altered mental state and seizures, tremor, weakness, or paralysis. all patients developed a raised c-reactive protein, whereas most developed acute liver injury, neutrophilia, and thrombocytosis. all patients with encephalitis developed ...201323296449
development and characterization of the replicon system of japanese encephalitis live vaccine virus sa14-14-2.viral self-replicating sub-genomic replicons represent a powerful tool for studying viral genome replication, antiviral screening and chimeric vaccine development. many kinds of flavivirus replicons have been developed with broad applications.201323442449
west nile virus in the transfusion setting with a special focus on italian preventive measures adopted in 2008-2012 and their impact on blood safety. 201324120610
altered protein networks and cellular pathways in severe west nile disease in mice.the recent west nile virus (wnv) outbreaks in developed countries, including europe and the united states, have been associated with significantly higher neuropathology incidence and mortality rate than previously documented. the changing epidemiology, the constant risk of (re-)emergence of more virulent wnv strains, and the lack of effective human antiviral therapy or vaccines makes understanding the pathogenesis of severe disease a priority. thus, to gain insight into the pathophysiological pr ...201323874584
japanese b encephalitis: an overview of the disease and use of chimerivax-je as a preventative vaccine.the japanese encephalitis virus (jev) is endemic in many countries in southern asia and the western pacific rim, with new spread to previously unrecognized countries. it is an important cause of childhood neurological disease associated with permanent neurological sequelae and death. fortunately, je is a vaccine-preventable disease. the chimerivax™-je (sanofi pasteur, lyon, france) is a live-attenuated chimeric vaccine derived from the live-attenuated yellow fever virus, yf17d, which expresses t ...201325134477
exploring the spatio-temporal dynamics of reservoir hosts, vectors, and human hosts of west nile virus: a review of the recent literature.over the last two decades west nile virus (wnv) has been responsible for significant disease outbreaks in humans and animals in many parts of the world. its extremely rapid global diffusion argues for a better understanding of its geographic extent. the purpose of this inquiry was to explore spatio-temporal patterns of wnv using geospatial technologies to study populations of the reservoir hosts, vectors, and human hosts, in addition to the spatio-temporal interactions among these populations. r ...201324284356
circulation of japanese encephalitis virus in pigs and mosquito vectors within can tho city, vietnam.japanese encephalitis virus (jev) is a mosquito-borne, zoonotic flavivirus causing encephalitis in humans and reproductive disorder in pigs. jev is present in large parts of asia, where urbanization is high. households within and outside can tho city, south vietnam, were selected to monitor circulation of jev. a nested rt-pcr was established to detect the presence of jev in mosquitoes whereas sera from pigs belonging to households within the province were analyzed for the presence of antibodies ...201323593520
ecology of west nile virus in north america.the introduction, dispersal and establishment of west nile virus in north america were reviewed, focusing on factors that may have enhanced receptivity and enabled the invasion process. the overwintering persistence of this tropical virus within temperate latitudes was unexpected, but was key in the transition from invasion to endemic establishment. the cascade of temporal events allowing sporadic amplification to outbreak levels was discussed within a future perspective.201324008376
increased pathogenicity of west nile virus (wnv) by glycosylation of envelope protein and seroprevalence of wnv in wild birds in far eastern russia.in this review, we discuss the possibility that the glycosylation of west nile (wn) virus e-protein may be associated with enhanced pathogenicity and higher replication of wn virus. the results indicate that e-protein glycosylation allows the virus to multiply in a heat-stable manner and therefore, has a critical role in enhanced viremic levels and virulence of wn virus in young-chick infection model. the effect of the glycosylation of the e protein on the pathogenicity of wn virus in young chic ...201324351738
three-dimensional architecture of tick-borne encephalitis virus replication sites and trafficking of the replicated rna.flavivirus replication is accompanied by the rearrangement of cellular membranes that may facilitate viral genome replication and protect viral components from host cell responses. the topological organization of viral replication sites and the fate of replicated viral rna are not fully understood. we exploited electron microscopy to map the organization of tick-borne encephalitis virus (tbev) replication compartments in infected cells and in cells transfected with a replicon. under both conditi ...201323552408
case definitions, diagnostic algorithms, and priorities in encephalitis: consensus statement of the international encephalitis consortium.encephalitis continues to result in substantial morbidity and mortality worldwide. advances in diagnosis and management have been limited, in part, by a lack of consensus on case definitions, standardized diagnostic approaches, and priorities for research.201323861361
structural complexity of dengue virus untranslated regions: cis-acting rna motifs and pseudoknot interactions modulating functionality of the viral genome.the dengue virus (denv) genome contains multiple cis-acting elements required for translation and replication. previous studies indicated that a 719-nt subgenomic minigenome (denv-mini) is an efficient template for translation and (-) strand rna synthesis in vitro. we performed a detailed structural analysis of denv-mini rna, combining chemical acylation techniques, pb(2+) ion-induced hydrolysis and site-directed mutagenesis. our results highlight protein-independent 5'-3' terminal interactions ...201323531545
non-structural protein-1 is required for west nile virus replication complex formation and viral rna synthesis.flavivirus ns1 is a non-structural glycoprotein that is expressed on the cell surface and secreted into the extracellular space, where it acts as an antagonist of complement pathway activation. despite its transit through the secretory pathway and intracellular localization in the lumen of the endoplasmic reticulum and golgi vesicles, ns1 is as an essential gene for flavivirus replication. how ns1 modulates infection remains uncertain given that the viral rna replication complex localizes to the ...201324245822
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
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
zoonotic encephalitides caused by arboviruses: transmission and epidemiology of alphaviruses and flaviviruses.in this review, we mainly focus on zoonotic encephalitides caused by arthropod-borne viruses (arboviruses) of the families flaviviridae (genus flavivirus) and togaviridae (genus alphavirus) that are important in both humans and domestic animals. specifically, we will focus on alphaviruses (eastern equine encephalitis virus, western equine encephalitis virus, venezuelan equine encephalitis virus) and flaviviruses (japanese encephalitis virus and west nile virus). most of these viruses were origin ...201324427764
cytoplasmic viruses: rage against the (cellular rna decay) machine. 201324339774
european surveillance for west nile virus in mosquito populations.a wide range of arthropod-borne viruses threaten both human and animal health either through their presence in europe or through risk of introduction. prominent among these is west nile virus (wnv), primarily an avian virus, which has caused multiple outbreaks associated with human and equine mortality. endemic outbreaks of west nile fever have been reported in italy, greece, france, romania, hungary, russia and spain, with further spread expected. most outbreaks in western europe have been due ...201324157510
surveillance for arboviral zoonoses in new zealand birds.given the significant burden that emerging infectious diseases place on global economies and public health, the monitoring and mitigation of, and early response to, potential infectious diseases are of the highest priority. the objective of this study was to survey for known and other potential arboviral zoonoses in multiple bird species at four locations in new zealand.201324478919
enhanced arbovirus surveillance with deep sequencing: identification of novel rhabdoviruses and bunyaviruses in australian mosquitoes.viral metagenomics characterizes known and identifies unknown viruses based on sequence similarities to any previously sequenced viral genomes. a metagenomics approach was used to identify virus sequences in australian mosquitoes causing cytopathic effects in inoculated mammalian cell cultures. sequence comparisons revealed strains of liao ning virus (reovirus, seadornavirus), previously detected only in china, livestock-infecting stretch lagoon virus (reovirus, orbivirus), two novel dimarhabdov ...201324314645
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
chloroquine inhibits dengue virus type 2 replication in vero cells but not in c6/36 cells.dengue viruses are the most important arthropod-borne viruses in terms of morbidity and mortality in the world. since there is no dengue vaccine available for human use, we have set out to investigate the use of chloroquine as an antiviral drug against dengue. chloroquine, an amine acidotropic drug known to affect intracellular exocytic pathways by increasing endosomal ph, was used in the in vitro treatment of vero and c6/36 cells infected with dengue virus type 2 (denv-2). real-time rt-pcr and ...201323431254
the changing epidemiology of kunjin virus in australia.west nile virus (wnv) is a mosquito-borne virus responsible for outbreaks of viral encephalitis in humans and horses, with particularly virulent strains causing recent outbreaks of disease in eastern europe, the middle east and north america. a strain of wnv, kunjin (wnvkun), is endemic in northern australia and infection with this virus is generally asymptomatic. however in early 2011, an unprecedented outbreak of encephalitis in horses occurred in south-eastern australia, resulting in mortalit ...201324287851
multi-gene detection and identification of mosquito-borne rna viruses using an oligonucleotide microarray.arthropod-borne viruses are important emerging pathogens world-wide. viruses transmitted by mosquitoes, such as dengue, yellow fever, and japanese encephalitis viruses, infect hundreds of millions of people and animals each year. global surveillance of these viruses in mosquito vectors using molecular based assays is critical for prevention and control of the associated diseases. here, we report an oligonucleotide dna microarray design, termed arbochip5.1, for multi-gene detection and identifica ...201323967358
viruses and antiviral immunity in drosophila.viral pathogens present many challenges to organisms, driving the evolution of a myriad of antiviral strategies to combat infections. a wide variety of viruses infect invertebrates, including both natural pathogens that are insect-restricted, and viruses that are transmitted to vertebrates. studies using the powerful tools in the model organism drosophila have expanded our understanding of antiviral defenses against diverse viruses. in this review, we will cover three major areas. first, we will ...201323680639
west nile virus in the united states - a historical perspective.prior to 1999, west nile virus (wnv) was a bit player in the screenplay of global vector-borne viral diseases. first discovered in the west nile district of uganda in 1937, this culex sp.-transmitted virus was known for causing small human febrile outbreaks in africa and the middle east. prior to 1995, the last major human wnv outbreak was in the 1950s in israel. the epidemiology and ecology of wnv began to change in the mid-1990s when an epidemic of human encephalitis occurred in romania. the i ...201324335779
a new insect-specific flavivirus from northern australia suppresses replication of west nile virus and murray valley encephalitis virus in co-infected mosquito cells.recent reports of a novel group of flaviviruses that replicate only in mosquitoes and appear to spread through insect populations via vertical transmission have emerged from around the globe. to date, there is no information on the presence or prevalence of these insect-specific flaviviruses (isfs) in australian mosquito species. to assess whether such viruses occur locally, we used reverse transcription-polymerase chain reaction (rt-pcr) and flavivirus universal primers that are specific to the ...201323460804
vector-virus interactions and transmission dynamics of west nile virus.west nile virus (wnv; flavivirus; flaviviridae) is the cause of the most widespread arthropod-borne viral disease in the world and the largest outbreak of neuroinvasive disease ever observed. mosquito-borne outbreaks are influenced by intrinsic (e.g., vector and viral genetics, vector and host competence, vector life-history traits) and extrinsic (e.g., temperature, rainfall, human land use) factors that affect virus activity and mosquito biology in complex ways. the concept of vectorial capacit ...201324351794
a novel platform for virus-like particle-display of flaviviral envelope domain iii: induction of dengue and west nile virus neutralizing antibodies.cd16-rige is a chimeric human membrane glycoprotein consisting of the cd16 ectodomain fused to the transmembrane domain and cytoplasmic tail of the gamma chain of the high affinity receptor of ige (rige). coexpression of cd16-rige and hiv-1 pr55gag polyprotein precursor (pr55gaghiv) in insect cells resulted in the incorporation of cd16-rige glycoprotein into the envelope of extracellular virus-like particles (vlps), a phenomenon known as pseudotyping. taking advantage of this property, we replac ...201323617954
simultaneous detection of west nile and japanese encephalitis virus rna by duplex taqman rt-pcr.west nile virus (wnv) and japanese encephalitis virus (jev) are important mosquito-borne viruses of the flaviviridae family, associated with encephalitis, mainly in humans and horses. wnv is also pathogen for many bird species. the incidence of human and animal wnv infections in europe has risen, mostly in recent years, and jev was detected in 2011 in mosquitoes collected in italy and may emerge in europe in the same way as other flaviviruses had emerged recently (usutu and bagaza virus) and sho ...201323892127
low seroprevalence of murray valley encephalitis and kunjin viruses in an opportunistic serosurvey, victoria 2011.to assess evidence of recent and past exposure to murray valley encephalitis virus (mvev) and west nile clade kunjin virus (kunv) in residents of the murray valley, victoria, during a period of demonstrated activity of both viruses in early 2011.201324090325
molecular characterization and phylogenetic analysis of murray valley encephalitis virus and west nile virus (kunjin subtype) from an arbovirus disease outbreak in horses in victoria, australia, in 2011.virus was detected in the central nervous system (cns) tissue of 11 horses from victoria that died displaying neurological symptoms during an outbreak of disease in australia in 2011. five horses were identified as being infected with murray valley encephalitis virus (mvev) and 6 as being infected with west nile virus subtype kunjin (wnv(kun)). analysis of partial sequence information from the ns5 and e genes indicated that the mvevs within the samples were highly homogenous and all belonged to ...201323345269
the human and animal health impacts of introduction and spread of an exotic strain of west nile virus in australia.vector-borne diseases can have substantial impacts on human and animal health, including major epidemics. west nile virus (wnv) is of particular international importance due to its recent emergence and impact in the western hemisphere. despite the presence of a sub-type of wnv (kunjin virus, kun) in australia, a potential ecological niche could be occupied by an exotic strain of wnv of the north american type. this study assesses the probability an exotic strain of wnv enters australia via an in ...201323098914
arboviral diseases and malaria in australia, 2010-11: annual report of the national arbovirus and malaria advisory committee.the national notifiable diseases surveillance system (nndss) received notification of 9,291 cases of disease transmitted by mosquitoes during the 2010-11 season (1 july 2010 to 30 june 2011). the alphaviruses barmah forest virus and ross river virus accounted for 7,515 (81%) of these. there were 133 notifications of dengue virus infection acquired in australia and 1,133 cases that were acquired overseas, while for 10 cases, the place of acquisition was unknown. the number of overseas acquired ca ...201323692155
arboviral diseases and malaria in australia, 2009-10: annual report of the national arbovirus and malaria advisory committee.the national notifiable diseases surveillance system received 7,609 notified cases of disease transmitted by mosquitoes for the season 1 july 2009 to 30 june 2010. the alphaviruses barmah forest virus and ross river virus, accounted for 6,546 (79%) of these notifications during the 2009-10 season. there were 37 notifications of dengue virus infection locally-acquired from north queensland and 581 notified cases in australia that resulted from overseas travel. this number of overseas acquired cas ...201223153083
kunjin flaviviral encephalomyelitis in an arabian gelding in new south wales, australia.flaviviruses, including kunjin virus, are arboviruses that cause encephalomyelitis in humans and horses. this case report describes an arabian gelding exhibiting neurological signs of flavivirus encephalomyelitis, the diagnostic investigation and confirmation of an unreported case of kunjin virus equine encephalomyelitis in australia.201222827627
chemoproteomic profiling identifies changes in dna-pk as markers of early dengue virus infection.many cellular factors are regulated via mechanisms affecting protein conformation, localization, and function that may be undetected by most commonly used rna- and protein-based profiling methods that monitor steady-state gene expression. mass-spectrometry-based chemoproteomic profiling provides alternatives for interrogating changes in the functional properties of proteins that occur in response to biological stimuli, such as viral infection. taking dengue virus 2 (dv2) infection as a model sys ...201222999307
cell culture-adaptive mutations promote viral protein-protein interactions and morphogenesis of infectious hepatitis c virus.recent genetic studies suggested that viral nonstructural (ns) proteins play important roles in morphogenesis of flaviviruses, particularly hepatitis c virus (hcv). adaptive and compensatory mutations occurring in different ns proteins were demonstrated to promote hcv production in cell culture. however, the underlying molecular mechanism of ns proteins in hcv morphogenesis is poorly understood. we have isolated a cell culture-adapted hcv of genotype 2a (jfh1) which grew to an infectious titer 3 ...201222674987
genetic characterization of an insect-specific flavivirus isolated from culex theileri mosquitoes collected in southern portugal.we describe the full genetic characterization of an insect-specific flavivirus (isf) from culex theileri (theobald) mosquitoes collected in portugal. this represents the first isolation and full characterization of an isf from portuguese mosquitoes. the virus, designated ctfv, for culex theileri flavivirus, was isolated in the c6/36 stegomyia albopicta (=aedes albopictus) cell line, and failed to replicate in vertebrate (vero) cells in common with other isfs. the ctfv genome encodes a single pol ...201222579596
proteomic profiling of the traf3 interactome network reveals a new role for the er-to-golgi transport compartments in innate immunity.tumor necrosis factor receptor-associated factor-3 (traf3) is a central mediator important for inducing type i interferon (ifn) production in response to intracellular double-stranded rna (dsrna). here, we report the identification of sec16a and p115, two proteins of the er-to-golgi vesicular transport system, as novel components of the traf3 interactome network. notably, in non-infected cells, traf3 was found associated with markers of the er-exit-sites (eres), er-to-golgi intermediate compartm ...201222792062
visualizing coronavirus rna synthesis in time by using click chemistry.coronaviruses induce in infected cells the formation of replicative structures, consisting of double-membrane vesicles (dmvs) and convoluted membranes, where viral rna synthesis supposedly takes place and to which the nonstructural proteins (nsp's) localize. double-stranded rna (dsrna), the presumed intermediate in rna synthesis, is localized to the dmv interior. however, as pores connecting the dmv interior with the cytoplasm have not been detected, it is unclear whether rna synthesis occurs at ...201222438542
small-molecule inhibitors of dengue-virus entry.flavivirus envelope protein (e) mediates membrane fusion and viral entry from endosomes. a low-ph induced, dimer-to-trimer rearrangement and reconfiguration of the membrane-proximal "stem" of the e ectodomain draw together the viral and cellular membranes. we found stem-derived peptides from dengue virus (dv) bind stem-less e trimer and mimic the stem-reconfiguration step in the fusion pathway. we adapted this experiment as a high-throughput screen for small molecules that block peptide binding ...201222496653
novel vaccine against venezuelan equine encephalitis combines advantages of dna immunization and a live attenuated vaccine.dna vaccines combine remarkable genetic and chemical stability with proven safety and efficacy in animal models, while remaining less immunogenic in humans. in contrast, live-attenuated vaccines have the advantage of inducing rapid, robust, long-term immunity after a single-dose vaccination. here we describe novel idna vaccine technology that is based on an infectious dna platform and combines advantages of dna and live attenuated vaccines. we applied this technology for vaccination against infe ...201223287629
highly similar structural frames link the template tunnel and ntp entry tunnel to the exterior surface in rna-dependent rna polymerases.rna-dependent rna polymerase (rdrp) is essential to viral replication and is therefore one of the primary targets of countermeasures against these dangerous infectious agents. development of broad-spectrum therapeutics targeting polymerases has been hampered by the extreme sequence variability of these sequences. rdrps range in length from 400-800 residues, yet contain only ∼20 residues that are conserved in most species. in this study, we made structure-based comparisons that are independent of ...201223275546
a three-dimensional comparison of tick-borne flavivirus infection in mammalian and tick cell lines.tick-borne flaviviruses (tbfv) are sustained in nature through cycling between mammalian and tick hosts. in this study, we used african green monkey kidney cells (vero) and ixodes scapularis tick cells (ise6) to compare virus-induced changes in mammalian and arthropod cells. using confocal microscopy, transmission electron microscopy (tem), and electron tomography (et), we examined viral protein distribution and the ultrastructural changes that occur during tbfv infection. within host cells, fla ...201223112871
new insights into control of arbovirus replication and spread by insect rna interference pathways.arthropod-borne (arbo) viruses are transmitted by vectors, such as mosquitoes, to susceptible vertebrates. recent research has shown that arbovirus replication and spread in mosquitoes is not passively tolerated but induces host responses to control these pathogens. small rna-mediated host responses are key players among these antiviral immune strategies. studies into one such small rna-mediated antiviral response, the exogenous rna interference (rnai) pathway, have generated a wealth of informa ...201226466541
west nile virus neurologic disease in humans, south africa, september 2008-may 2009.we investigated west nile virus (wnv) as a possible disease etiology in persons hospitalized in south africa. of 206 specimens tested, 36 had wnv neutralizing antibodies, significantly more than in similar earlier serosurveys. seven probable acute wnv cases were identified, suggesting wnv may be overlooked as an etiology of severe disease in south africa.201223171668
amp-activated kinase restricts rift valley fever virus infection by inhibiting fatty acid synthesis.the cell intrinsic innate immune responses provide a first line of defense against viral infection, and often function by targeting cellular pathways usurped by the virus during infection. in particular, many viruses manipulate cellular lipids to form complex structures required for viral replication, many of which are dependent on de novo fatty acid synthesis. we found that the energy regulator ampk, which potently inhibits fatty acid synthesis, restricts infection of the bunyavirus, rift valle ...201222532801
interplay between viruses and host mrna degradation.messenger rna degradation is a fundamental cellular process that plays a critical role in regulating gene expression by controlling both the quality and the abundance of mrnas in cells. naturally, viruses must successfully interface with the robust cellular rna degradation machinery to achieve an optimal balance between viral and cellular gene expression and establish a productive infection in the host. in the past several years, studies have discovered many elegant strategies that viruses have ...201223274304
west nile virus noncoding subgenomic rna contributes to viral evasion of the type i interferon-mediated antiviral response.we previously showed that a noncoding subgenomic flavivirus rna (sfrna) is required for viral pathogenicity, as a mutant west nile virus (wnv) deficient in sfrna production replicated poorly in wild-type mice. to investigate the possible immunomodulatory or immune evasive functions of sfrna, we utilized mice and cells deficient in elements of the type i interferon (ifn) response. replication of the sfrna mutant wnv was rescued in mice and cells lacking interferon regulatory factor 3 (irf-3) and ...201222379089
biogenesis and dynamics of the coronavirus replicative structures.coronaviruses are positive-strand rna viruses that are important infectious agents of both animals and humans. a common feature among positive-strand rna viruses is their assembly of replication-transcription complexes in association with cytoplasmic membranes. upon infection, coronaviruses extensively rearrange cellular membranes into organelle-like replicative structures that consist of double-membrane vesicles and convoluted membranes to which the nonstructural proteins involved in rna synthe ...201223202524
descriptive overview of the 2011 epidemic of arboviral disease in horses in australia.to provide an overview and descriptive analysis of the 2011 arboviral disease epidemic in horses that involved three important australian mosquito-borne viruses: murray valley encephalitis virus, west nile virus (kunjin strain) and ross river virus.201223356366
evolution of mosquito-based arbovirus surveillance systems in australia.control of arboviral disease is dependent on the sensitive and timely detection of elevated virus activity or the identification of emergent or exotic viruses. the emergence of japanese encephalitis virus (jev) in northern australia revealed numerous problems with performing arbovirus surveillance in remote locations. a sentinel pig programme detected jev activity, although there were a number of financial, logistical, diagnostic and ethical limitations. a system was developed which detected vir ...201222505808
etiology of acute, non-malaria, febrile illnesses in jayapura, northeastern papua, indonesia.abstract. we conducted a prospective, inpatient fever study in malaria-endemic papua, indonesia to determine non-malaria fever etiologies. investigations included malaria blood films, blood culture, paired serologic samples analysis for dengue, japanese encephalitis, leptospirosis, scrub typhus, murine typhus, and spotted fever group rickettsia. during 1997-2000, 226 patients (127 males and 99 females) 1-80 years of age (median age = 25 years) were enrolled. positive blood cultures (n = 34, 15%) ...201222232450
Uncoupling cis-Acting RNA Elements from Coding Sequences Revealed a Requirement of the N-Terminal Region of Dengue Virus Capsid Protein in Virus Particle Formation.Little is known about the mechanism of flavivirus genome encapsidation. Here, functional elements of the dengue virus (DENV) capsid (C) protein were investigated. Study of the N-terminal region of DENV C has been limited by the presence of overlapping cis-acting RNA elements within the protein-coding region. To dissociate these two functions, we used a recombinant DENV RNA with a duplication of essential RNA structures outside the C coding sequence. By the use of this system, the highly conserve ...201222072762
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