TitleAbstractYear(sorted descending)
development and validation of a quantitative, one-step, multiplex, real-time reverse transcriptase pcr assay for detection of dengue and chikungunya viruses.dengue virus (denv) and chikungunya virus (chikv) are important human pathogens with common transmission vectors and similar clinical presentations. patient care may be impacted by the misdiagnosis of denv and chikv in areas where both viruses cocirculate. in this study, we have developed and validated a one-step multiplex reverse transcriptase pcr (rt-pcr) to simultaneously detect, quantify, and differentiate between four denv serotypes (pan-denv) and chikungunya virus. the assay uses taqman te ...201627098955
antagonism of the sodium-potassium atpase impairs chikungunya virus infection.chikungunya virus (chikv) is a reemerging alphavirus that has caused epidemics of fever, arthralgia, and rash worldwide. there are currently no licensed vaccines or antiviral therapies available for the prevention or treatment of chikv disease. we conducted a high-throughput, chemical compound screen that identified digoxin, a cardiac glycoside that blocks the sodium-potassium atpase, as a potent inhibitor of chikv infection. treatment of human cells with digoxin or a related cardiac glycoside, ...201627222471
identification of critical residues in hepatitis e virus macro domain involved in its interaction with viral methyltransferase and orf3 proteins.hepatitis e virus (hev) is a major cause of hepatitis in normal and organ transplant individuals. hev open reading frame-1 encodes a polypeptide comprising of the viral nonstructural proteins as well as domains of unknown function such as the macro domain (x-domain), v, duf3729 and y. the macro domain proteins are ubiquitously present from prokaryotes to human and in many positive-strand rna viruses, playing important roles in multiple cellular processes. towards understanding the function of th ...201627113483
vertical transmission of indian ocean lineage of chikungunya virus in aedes aegypti and aedes albopictus mosquitoes.the re-emergence of chikungunya (chik) fever in thailand has been caused by a novel lineage of chikungunya virus (chikv) termed the indian ocean lineage (iol). the aedes albopictus mosquito is thought to be a primary vector of chik fever in thailand, whereas ae. aegypti acts as a secondary vector of the virus. the vertical transmission is believed to be a primary means to maintain chikv in nature and may be associated with an increased risk of outbreak. therefore, the goal of this study was to a ...201627108077
dynamics of chikungunya virus cell entry unraveled by single-virus tracking in living cells.chikungunya virus (chikv) is a rapidly emerging mosquito-borne human pathogen causing major outbreaks in africa, asia, and the americas. the cell entry pathway hijacked by chikv to infect a cell has been studied previously using inhibitory compounds. there has been some debate on the mechanism by which chikv enters the cell: several studies suggest that chikv enters via clathrin-mediated endocytosis, while others show that it enters independently of clathrin. here we applied live-cell microscopy ...201626912616
assessment of productivity of culex spp. larvae (diptera: culicidae) in urban storm water catch basin system in wrocław (sw poland).in urban environments, catch basins serve as major developmental and resting sites for anthropophilic and zoophilic mosquitoes. however, the use of this habitat is inconsistent, with abundance of larvae varying significantly across catch basins at a fine spatial scale. during seasonal summer investigations on mosquito species composition, their spatial and temporal distribution and the environmental characteristic of the breeding sites in the underground storm drain systems of the wrocław urban ...201626809340
rainbow trout (oncorhynchus mykiss) muscle satellite cells are targets of salmonid alphavirus infection.sleeping disease in rainbow trout is characterized by an abnormal swimming behaviour of the fish which stay on their side at the bottom of the tanks. this sign is due to extensive necrosis and atrophy of red skeletal muscle induced by the sleeping disease virus (sdv), also called salmonid alphavirus 2. infections of humans with arthritogenic alphaviruses, such as chikungunya virus (chikv), are global causes of debilitating musculoskeletal diseases. the mechanisms by which the virus causes these ...201626743565
sindbis virus-pseudotyped lentiviral vectors carrying vegfr2-specific nanobody for potential transductional targeting of tumor vasculature.introduction of selectivity/specificity into viral-based gene delivery systems, such as lentiviral vectors (lvs), is crucial in their systemic administration for cancer gene therapy. the pivotal role of tumor-associated endothelial cells (taecs) in tumor angiogenesis and overexpression of vascular endothelial growth factor receptor-2 (vegfr2 or kdr) in taecs makes them a potent target in cancer treatment. herein, we report the development of vegfr2-targeted lvs pseudotyped with chimeric sindbis ...201627647452
sindbis virus can exploit a host antiviral protein to evade immune surveillance.viral infection induces production of type i interferons (ifns), which stimulate the expression of a variety of antiviral factors to inhibit viral replication. to establish effective infection, viruses need to develop strategies to evade the immune responses. a neurovirulent sindbis virus strain with neuroinvasive properties (svni) causes lethal encephalitis in mice, and its replication in cultured cells is inhibited by the zinc finger antiviral protein (zap), a host factor that specifically inh ...201627581990
wide recognition of culex pipiens and lack of detection of culex torrentium through biomolecular differentiation of mosquitoes in the emilia-romagna region, northern italy.the culex pipiens complex includes species with reported differences in vector competence for arthropod-borne viruses, many of which are of significant importance to human health such as the west nile virus and the sindbis virus. this group of mosquitoes is difficult to distinguish morphologically; particularly as adult females. in europe, the two species of the complex, culex pipiens linnaeus 1758 and culex torrentium martini 1925, are often found sympatrically. with the aim to characterize the ...201627477051
interferon gamma modulation of disease manifestation and the local antibody response to alphavirus encephalomyelitis.infection of mice with sindbis virus (sinv) produces encephalomyelitis and provides a model for examination of the central nervous system (cns) immune response to alphavirus infection. clearance of infectious virus is accomplished through a cooperative effort between sinv-specific antibody and ifn-γ, but the regulatory interactions are poorly understood. to determine the effects of ifn-γ on clinical disease and the antiviral immune response, c57bl/6 mice lacking ifn-γ (ifng-/-) or ifn-γ receptor ...201627667782
protective effects of glutamine antagonist 6-diazo-5-oxo-l-norleucine in mice with alphavirus encephalomyelitis.inflammation is a necessary part of the response to infection but can also cause neuronal injury in both infectious and autoimmune diseases of the central nervous system (cns). a neurovirulent strain of sindbis virus (nsv) causes fatal paralysis in adult c57bl/6 mice during clearance of infectious virus from the cns, and the virus-specific immune response is implicated as a mediator of neuronal damage. previous studies have shown that survival is improved in t-cell-deficient mice and in mice wit ...201627489275
full inactivation of alphaviruses in single particle and crystallized forms.inherent in the study of viruses is the risk of pathogenic exposure, which necessitates appropriate levels of biosafety containment. unfortunately, this also limits the availability of useful research instruments that are located at facilities not equipped to handle infectious pathogens. abrogation of viral infectivity can be accomplished without severely disrupting the physical structure of the virus particle. virus samples that are verifiably intact but not infectious may be enabled for study ...201627465218
interferon regulatory factor 2 protects mice from lethal viral neuroinvasion.the host responds to virus infection by activating type i interferon (ifn) signaling leading to expression of ifn-stimulated genes (isgs). dysregulation of the ifn response results in inflammatory diseases and chronic infections. in this study, we demonstrate that ifn regulatory factor 2 (irf2), an isg and a negative regulator of ifn signaling, influences alphavirus neuroinvasion and pathogenesis. a sindbis virus strain that in wild-type (wt) mice only causes disease when injected into the brain ...201627899441
inhibition of host protein synthesis by sindbis virus: correlation with viral rna replication and release of nuclear proteins to the cytoplasm.infection of mammalian cells by sindbis virus (sinv) profoundly blocks cellular mrna translation. experimental evidence points to viral non-structural proteins (nsps), in particular nsp2, as the mediator of this inhibition. however, individual expression of nsp1, nsp2, nsp3 or nsp1-4 does not block cellular protein synthesis in bhk cells. trans-complementation of a defective sinv replicon lacking most of the coding region for nsps by the co-expression of nsp1-4 propitiates viral rna replication ...201525329362
model-driven engineering of gene expression from rna replicons.rna replicons are an emerging platform for engineering synthetic biological systems. replicons self-amplify, can provide persistent high-level expression of proteins even from a small initial dose, and, unlike dna vectors, pose minimal risk of chromosomal integration. however, no quantitative model sufficient for engineering levels of protein expression from such replicon systems currently exists. here, we aim to enable the engineering of multigene expression from more than one species of replic ...201524877739
cryo-em structures elucidate neutralizing mechanisms of anti-chikungunya human monoclonal antibodies with therapeutic activity.chikungunya virus (chikv) is a mosquito-transmitted alphavirus that causes severe acute and chronic disease in humans. although highly inhibitory murine and human monoclonal antibodies (mabs) have been generated, the structural basis of their neutralizing activity remains poorly characterized. here, we determined the cryo-em structures of chikungunya virus-like particles complexed with antibody fragments (fab) of two highly protective human mabs, 4j21 and 5m16, that block virus fusion with host ...201526504196
myeloid cell arg1 inhibits control of arthritogenic alphavirus infection by suppressing antiviral t cells.arthritogenic alphaviruses, including ross river virus (rrv) and chikungunya virus (chikv), are responsible for explosive epidemics involving millions of cases. these mosquito-transmitted viruses cause inflammation and injury in skeletal muscle and joint tissues that results in debilitating pain. we previously showed that arginase 1 (arg1) was highly expressed in myeloid cells in the infected and inflamed musculoskeletal tissues of rrv- and chikv-infected mice, and specific deletion of arg1 from ...201526436766
la crosse virus field detection and vector competence of culex crosse virus (lacv), a leading cause of arboviral pediatric encephalitis in the united states, is emerging in appalachia. here, we report field and laboratory evidence that suggest lacv may be using culex mosquitoes as additional vectors in this region. this bunyavirus was detected by reverse-transcriptase polymerase chain reaction in two pools of culex mosquitoes in southwestern virginia and in six pools in west virginia. to assess vector competence, we offered lacv blood meals to field-coll ...201526175029
viral polymerase-helicase complexes regulate replication fidelity to overcome intracellular nucleotide date, the majority of work on rna virus replication fidelity has focused on the viral rna polymerase, while the potential role of other viral replicase proteins in this process is poorly understood. previous studies used resistance to broad-spectrum rna mutagens, such as ribavirin, to identify polymerases with increased fidelity that avoid misincorporation of such base analogues. we identified a novel variant in the alphavirus viral helicase/protease, nonstructural protein 2 (nsp2) that opera ...201526311883
a potent neutralizing igm mab targeting the n218 epitope on e2 protein protects against chikungunya virus pathogenesis.chikungunya virus (chikv) is a medically important human viral pathogen that causes chikungunya fever accompanied with debilitating and persistent joint pain. host-elicited or passively-transferred monoclonal antibodies (mab) are essential mediators of chikv clearance. therefore, this study aimed to generate and characterize a panel of mabs for their neutralization efficacy against chikv infection in a cell-based and murine model. to evaluate their antigenicity and neutralization profile, indire ...201526305993
serial femtosecond x-ray diffraction of enveloped virus microcrystals.serial femtosecond crystallography (sfx) using x-ray free-electron lasers has produced high-resolution, room temperature, time-resolved protein structures. we report preliminary sfx of sindbis virus, an enveloped icosahedral rna virus with ∼700 å diameter. microcrystals delivered in viscous agarose medium diffracted to ∼40 å resolution. small-angle diffuse x-ray scattering overlaid bragg peaks and analysis suggests this results from molecular transforms of individual particles. viral proteins un ...201526798819
sec14l2 enables pan-genotype hcv replication in cell culture.since its discovery in 1989, efforts to grow clinical isolates of the hepatitis c virus (hcv) in cell culture have met with limited success. only the jfh-1 isolate has the capacity to replicate efficiently in cultured hepatoma cells without cell culture-adaptive mutations. we hypothesized that cultured cells lack one or more factors required for the replication of clinical isolates. to identify the missing factors, we transduced huh-7.5 human hepatoma cells with a pooled lentivirus-based human c ...201526266980
early events in chikungunya virus infection-from virus cell binding to membrane fusion.chikungunya virus (chikv) is a rapidly emerging mosquito-borne alphavirus causing millions of infections in the tropical and subtropical regions of the world. chikv infection often leads to an acute self-limited febrile illness with debilitating myalgia and arthralgia. a potential long-term complication of chikv infection is severe joint pain, which can last for months to years. there are no vaccines or specific therapeutics available to prevent or treat infection. this review describes the crit ...201526198242
the chikungunya virus capsid protein contains linear b cell epitopes in the n- and c-terminal regions that are dependent on an intact c-terminus for antibody recognition.chikungunya virus (chikv) is an arthropod-borne agent that causes severe arthritic disease in humans and is considered a serious health threat in areas where competent mosquito vectors are prevalent. chikv has recently been responsible for several millions of cases of disease, involving over 40 countries. the recent re-emergence of chikv and its potential threat to human health has stimulated interest in better understanding of the biology and pathogenesis of the virus, and requirement for impro ...201526061335
ability of the encephalitic arbovirus semliki forest virus to cross the blood-brain barrier is determined by the charge of the e2 glycoprotein.semliki forest virus (sfv) provides a well-characterized model system to study the pathogenesis of virus encephalitis. several studies have used virus derived from the molecular clone sfv4. sfv4 virus does not have the same phenotype as the closely related l10 or the prototype virus from which its molecular clone was derived. in mice, l10 generates a high-titer plasma viremia, is efficiently neuroinvasive, and produces a fatal panencephalitis, whereas low-dose sfv4 produces a low-titer viremia, ...201525972559
alphaviruses in gene therapy.alphavirus vectors present an attractive approach for gene therapy applications due to the rapid and simple recombinant virus particle production and their broad range of mammalian host cell transduction. mainly three types of alphavirus vectors, namely naked rna, recombinant particles and dna/rna layered vectors, have been subjected to preclinical studies with the goal of achieving prophylactic or therapeutic efficacy, particularly in oncology. in this context, immunization with alphavirus vect ...201525961488
native wolbachia from aedes albopictus blocks chikungunya virus infection in cellulo.wolbachia, a widespread endosymbiont of terrestrial arthropods, can protect its host against viral and parasitic infections, a phenotype called "pathogen blocking". however, in some cases wolbachia may have no effect or even enhance pathogen infection, depending on the host-wolbachia-pathogen combination. the tiger mosquito aedes albopictus is naturally infected by two strains of wolbachia, walba and walbb, and is a competent vector for different arboviruses such as dengue virus (denv) and chiku ...201525923352
noncapped alphavirus genomic rnas and their role during infection.alphaviruses are enveloped positive-sense rna viruses that exhibit a wide host range consisting of vertebrate and invertebrate species. previously we have reported that the infectivity of sindbis virus (sinv), the model alphavirus, was largely a function of the cell line producing the viral particles. mammalian-cell-derived sinv particles, on average, exhibit a higher particle-to-pfu ratio than mosquito cell-derived sinv particles. nevertheless, the outcome of nonproductive infection, the molecu ...201525833042
nuclear proteins hijacked by mammalian cytoplasmic plus strand rna strand rna viruses that replicate in the cytoplasm face challenges in supporting the numerous biosynthetic functions required for replication and propagation. most of these viruses are genetically simple and rely heavily on co-opting cellular proteins, particularly cellular rna-binding proteins, into new roles for support of virus infection at the level of virus-specific translation, and building rna replication complexes. in the course of infectious cycles many nuclear-cytoplasmic shuttlin ...201525818028
fighting arbovirus transmission: natural and engineered control of vector competence in aedes mosquitoes.control of aedine mosquito vectors, either by mosquito population reduction or replacement with refractory mosquitoes, may play an essential role in the fight against arboviral diseases. in this review, we will focus on the development and application of biological approaches, both natural or engineered, to limit mosquito vector competence for arboviruses. the study of mosquito antiviral immunity has led to the identification of a number of host response mechanisms and proteins that are required ...201526463078
viral rna intermediates as targets for detection and discovery of novel and emerging mosquito-borne viruses.mosquito-borne viruses encompass a range of virus families, comprising a number of significant human pathogens (e.g., dengue viruses, west nile virus, chikungunya virus). virulent strains of these viruses are continually evolving and expanding their geographic range, thus rapid and sensitive screening assays are required to detect emerging viruses and monitor their prevalence and spread in mosquito populations. double-stranded rna (dsrna) is produced during the replication of many of these virus ...201525799391
dissecting the role of e2 protein domains in alphavirus pathogenicity.alphaviruses represent a diverse set of arboviruses, many of which are important pathogens. chikungunya virus (chikv), an arthritis-inducing alphavirus, is the cause of a massive ongoing outbreak in the caribbean and south america. in contrast to chikv, other related alphaviruses, such as venezuelan equine encephalitis virus (veev) and semliki forest virus (sfv), can cause encephalitic disease. e2, the receptor binding protein, has been implicated as a determinant in cell tropism, host range, pa ...201526676771
loss of glycosaminoglycan receptor binding after mosquito cell passage reduces chikungunya virus infectivity.chikungunya virus (chikv) is a mosquito-transmitted alphavirus that can cause fever and chronic arthritis in humans. chikv that is generated in mosquito or mammalian cells differs in glycosylation patterns of viral proteins, which may affect its replication and virulence. herein, we compare replication, pathogenicity, and receptor binding of chikv generated in vero cells (mammal) or c6/36 cells (mosquito) through a single passage. we demonstrate that mosquito cell-derived chikv (chikv mos) has s ...201526484530
eastern equine encephalitis virus in mice ii: pathogenesis is dependent on route of exposure.eastern equine encephalitis virus (eeev) is an alphavirus with a case fatality rate estimated to be as high as 75 % in humans and 90 % in horses. surviving patients often have long-lasting and severe neurological sequelae. at present, there is no licensed vaccine or therapeutic for eeev infection. this study completes the clinical and pathological analysis of mice infected with a north american strain of eeev by three different routes: aerosol, intranasal, and subcutaneous. such an understanding ...201526423229
the virus-host interplay: biogenesis of +rna replication complexes.positive-strand rna (+rna) viruses are an important group of human and animal pathogens that have significant global health and economic impacts. notable members include west nile virus, dengue virus, chikungunya, severe acute respiratory syndrome (sars) coronavirus and enteroviruses of the picornaviridae family.unfortunately, prophylactic and therapeutic treatments against these pathogens are limited. +rna viruses have limited coding capacity and thus rely extensively on host factors for succes ...201526287230
production of a sindbis/eastern equine encephalitis chimeric virus inactivated cell culture antigen.eastern equine encephalitis virus (eeev) is a medically important pathogen that can cause severe encephalitis in humans, with mortality rates ranging from 30 to 80%. unfortunately there are no antivirals or licensed vaccines available for human use, and laboratory diagnosis is essential to differentiate eeev infection from other pathogens with similar clinical manifestations. the arboviral diseases branch (adb) reference laboratory at the cdc division of vector-borne diseases (dvbd) produces ref ...201526205552
tissue barriers to arbovirus infection in mosquitoes.arthropod-borne viruses (arboviruses) circulate in nature between arthropod vectors and vertebrate hosts. arboviruses often cause devastating diseases in vertebrate hosts, but they typically do not cause significant pathology in their arthropod vectors. following oral acquisition of a viremic bloodmeal from a vertebrate host, the arbovirus disease cycle requires replication in the cellular environment of the arthropod vector. once the vector has become systemically and persistently infected, the ...201526184281
an alphavirus temperature-sensitive capsid mutant reveals stages of nucleocapsid assembly.alphaviruses have a nucleocapsid core composed of the rna genome surrounded by an icosahedral lattice of capsid protein. an insertion after position 186 in the capsid protein produced a strongly temperature-sensitive growth phenotype. even when the structural proteins were synthesized at the permissive temperature (28°c), subsequent incubation of the cells at the non-permissive temperature (37°c) dramatically decreased mutant capsid protein stability and particle assembly. electron microscopy co ...201526051211
do we need a vaccine against chikungunya?during the last decade, the chikungunya (chikv) virus has expanded its range of activity, conquering new territories and becoming an important global health threat. in particular, the challenge represented by the recent emergence of chikv in the americas has strengthened the need of a safe and effective vaccine. although research on vaccines against chikv has been slow, a few vaccine candidates have been tested over the years. inactivated and attenuated vaccine candidates have shown promising re ...201525971340
global emergence of alphaviruses that cause arthritis in humans.arthropod-borne viruses (arboviruses) may cause severe emerging and re-emerging infectious diseases, which pose a significant threat to human and animal health in the world today. these infectious diseases range from mild febrile illnesses, arthritis, and encephalitis to haemorrhagic fevers. it is postulated that certain environmental factors, vector competence, and host susceptibility have a major impact on the ecology of arboviral diseases. presently, there is a great interest in the emergence ...201526689654
sindbis virus interferes with dengue 4 virus replication and its potential transmission by aedes albopictus.mosquitoes transmit a number of arboviruses associated with disease outbreaks in humans and other animals. the majority of medically important arboviruses belong to three families: togaviridae, flaviviridae and bunyaviridae. several members of these families have overlapping distributions and share common vectors, increasing the potential for arboviral coinfections. this study examined how two model viruses: sindbis virus (sinv, togaviridae: alphavirus) and dengue-4 virus (denv-4, flaviviridae: ...201525633862
genetic dissection of the host tropism of human-tropic pathogens.infectious diseases are the second leading cause of death worldwide. although the host multitropism of some pathogens has rendered their manipulation possible in animal models, the human-restricted tropism of numerous viruses, bacteria, fungi, and parasites has seriously hampered our understanding of these pathogens. hence, uncovering the genetic basis underlying the narrow tropism of such pathogens is critical for understanding their mechanisms of infection and pathogenesis. moreover, such gene ...201526407032
syndromic approach to arboviral diagnostics for global travelers as a basis for infectious disease surveillance.arboviruses have overlapping geographical distributions and can cause symptoms that coincide with more common infections. therefore, arbovirus infections are often neglected by travel diagnostics. here, we assessed the potential of syndrome-based approaches for diagnosis and surveillance of neglected arboviral diseases in returning travelers.201526372010
mammalian synthetic biology: emerging medical this review, we discuss new emerging medical applications of the rapidly evolving field of mammalian synthetic biology. we start with simple mammalian synthetic biological components and move towards more complex and therapy-oriented gene circuits. a comprehensive list of on-off switches, categorized into transcriptional, post-transcriptional, translational and post-translational, is presented in the first sections. subsequently, boolean logic gates, synthetic mammalian oscillators and toggle ...201525808341
a fast cross-validation method for alignment of electron tomography images based on beer-lambert electron tomography, accurate alignment of tilt series is an essential step in attaining high-resolution 3d reconstructions. nevertheless, quantitative assessment of alignment quality has remained a challenging issue, even though many alignment methods have been reported. here, we report a fast and accurate method, tomoaligneval, based on the beer-lambert law, for the evaluation of alignment quality. our method is able to globally estimate the alignment accuracy by measuring the goodness of l ...201526455556
rna degradation in antiviral immunity and control determines the fate of cellular rna molecules. nonsense-mediated decay (nmd) provides quality control of mrna, targeting faulty cellular transcripts for degradation by multiple nucleases including the rna exosome. recent findings have revealed a role for nmd in targeting viral rna molecules, thereby restricting virus infection. interestingly, nmd is also linked to immune responses at another level: mutations affecting the nmd or rna exosome machineries cause chronic ...201525709093
emerging roles for rna degradation in viral replication and antiviral defense.viral replication significantly alters the gene expression landscape of infected cells. many of these changes are driven by viral manipulation of host transcription or translation machinery. several mammalian viruses encode factors that broadly dampen gene expression by directly targeting messenger rna (mrna). here, we highlight how these factors promote mrna degradation to globally regulate both host and viral gene expression. although these viral factors are not homologous and use distinct mec ...201525721579
the role of rna interference (rnai) in arbovirus-vector interactions.rna interference (rnai) was shown over 18 years ago to be a mechanism by which arbovirus replication and transmission could be controlled in arthropod vectors. during the intervening period, research on rnai has defined many of the components and mechanisms of this antiviral pathway in arthropods, yet a number of unexplored questions remain. rnai refers to rna-mediated regulation of gene expression. originally, the term described silencing of endogenous genes by introduction of exogenous double- ...201525690800
viral and cellular proteins containing fgdf motifs bind g3bp to block stress granule formation.the ras-gap sh3 domain-binding proteins (g3bp) are essential regulators of the formation of stress granules (sg), cytosolic aggregates of proteins and rna that are induced upon cellular stress, such as virus infection. many viruses, including semliki forest virus (sfv), block sg induction by targeting g3bp. in this work, we demonstrate that the g3bp-binding motif of sfv nsp3 consists of two fgdf motifs, in which both phenylalanine and the glycine residue are essential for binding. in addition, w ...201525658430
stress granule components g3bp1 and g3bp2 play a proviral role early in chikungunya virus replication.stress granules (sgs) are protein-mrna aggregates that are formed in response to environmental stresses, resulting in translational inhibition. sgs are generally believed to play an antiviral role and are manipulated by many viruses, including various alphaviruses. gtpase-activating protein (sh3 domain)-binding protein 1 (g3bp1) is a key component and commonly used marker of sgs. its homolog g3bp2 is a less extensively studied sg component. here, we demonstrate that chikungunya virus (chikv) inf ...201525653451
atp1a1-mediated src signaling inhibits coronavirus entry into host addition to transporting ions, the multisubunit na(+),k(+)-atpase also functions by relaying cardiotonic steroid (cts)-binding-induced signals into cells. in this study, we analyzed the role of na(+),k(+)-atpase and, in particular, of its atp1a1 α subunit during coronavirus (cov) infection. as controls, the vesicular stomatitis virus (vsv) and influenza a virus (iav) were included. using gene silencing, the atp1a1 protein was shown to be critical for infection of cells with murine hepatitis v ...201525653449
bovine lactoferrin inhibits toscana virus infection by binding to heparan sulphate.toscana virus is an emerging sandfly-borne bunyavirus in mediterranean europe responsible for neurological diseases in humans. it accounts for about 80% of paediatric meningitis cases during the summer. despite the important impact of toscana virus infection-associated disease on human health, currently approved vaccines or effective antiviral treatments are not available. in this research, we have analyzed the effect of bovine lactoferrin, a bi-globular iron-binding glycoprotein with potent ant ...201525643293
chikungunya virus (chikv) inhibitors from natural sources: a medicinal chemistry perspective.chikungunya virus (chikv) is one of the re-emerging "neglected" tropical diseases whose recent outbreak affected not only africa and south-east asia but also several parts of america and europe. to date, despite its serious nature, no antivirals or vaccines were developed in order to counter this resurgent infectious disease. the recent advancement in crystallography and availability of crystal structures of certain domains of chikv initiates the development of anti-chikv agents using structure- ...201525921858
sequence analysis reveals a conserved extension in the capping enzyme of the alphavirus supergroup, and a homologous domain in nodaviruses.members of the alphavirus supergroup include human pathogens such as chikungunya virus, hepatitis e virus and rubella virus. they encode a capping enzyme with methyltransferase-guanylyltransferase (mtase-gtase) activity, which is an attractive drug target owing to its unique mechanism. however, its experimental study has proven very difficult.201525886938
arbovirus-mosquito interactions: rnai pathway.arthropod-borne (arbo) viruses infect hematophagous arthropods (vectors) to maintain virus transmission between vertebrate hosts. the mosquito vector actively controls arbovirus infection to minimize its fitness costs. the rna interference (rnai) pathway is the major antiviral response vectors use to restrict arbovirus infections. we know this because depleting rnai gene products profoundly impacts arbovirus replication, the antiviral rnai pathway genes undergo positive, diversifying selection a ...201526629932
fluorescent protein-tagged sindbis virus e2 glycoprotein allows single particle analysis of virus budding from live cells.sindbis virus (sinv) is an enveloped, mosquito-borne alphavirus. here we generated and characterized a fluorescent protein-tagged (fp-tagged) sinv and found that the presence of the fp-tag (mcherry) affected glycoprotein transport to the plasma membrane whereas the specific infectivity of the virus was not affected. we examined the virions by transmission electron cryo-microscopy and determined the arrangement of the fp-tag on the surface of the virion. the fluorescent proteins are arranged icos ...201526633461
battle between influenza a virus and a newly identified antiviral activity of the parp-containing zapl protein.previous studies showed that zapl (parp-13.1) exerts its antiviral activity via its n-terminal zinc fingers that bind the mrnas of some viruses, leading to mrna degradation. here we identify a different antiviral activity of zapl that is directed against influenza a virus. this zapl antiviral activity involves its c-terminal parp domain, which binds the viral pb2 and pa polymerase proteins, leading to their proteasomal degradation. after the pb2 and pa proteins are poly(adp-ribosylated), they ar ...201526504237
sphingosine kinase 2 is a chikungunya virus host factor co-localized with the viral replication complex.chikungunya virus (chikv) is a re-emerging alphavirus which causes severe and prolonged arthralgic febrile illness. the recent global spread of the virus and lack of approved therapeutic options makes it imperative to gain greater insight into the molecular mechanisms underlying chikv pathogenesis, in particular host factors recruited by the virus. in the current study, we identify sphingosine kinase 2 (sk2) as a chikv host factor co-localized with the viral replication complex (vrc) during infe ...201526576339
immunoproteomic profiling of antiviral antibodies in new-onset type 1 diabetes using protein arrays.the rapid rise in the incidence of type 1 diabetes (t1d) suggests the involvement of environmental factors including viral infections. we evaluated the association between viral infections and t1d by profiling antiviral antibodies using a high-throughput immunoproteomics approach in patients with new-onset t1d. we constructed a viral protein array comprising the complete proteomes of seven viruses associated with t1d and open reading frames from other common viruses. antibody responses to 646 vi ...201526450993
interactions involved in ph protection of the alphavirus fusion protein.the alphavirus membrane protein e1 mediates low ph-triggered fusion of the viral and endosome membranes during virus entry. during virus biogenesis e1 associates as a heterodimer with the transmembrane protein p62. late in the secretory pathway, cellular furin cleaves p62 to the mature e2 protein and a peripheral protein e3. e3 remains bound to e2 at low ph, stabilizing the heterodimer and thus protecting e1 from the acidic ph of the secretory pathway. release of e3 at neutral ph then primes the ...201526433749
mosquito rasputin interacts with chikungunya virus nsp3 and determines the infection rate in aedes albopictus.chikungunya virus (chikv) is an arthritogenic alphavirus (family togaviridae), transmitted by aedes species mosquitoes. chikv re-emerged in 2004 with multiple outbreaks worldwide and recently reached the americas where it has infected over a million individuals in a rapidly expanding epidemic. while alphavirus replication is well understood in general, the specific function (s) of non-structural protein nsp3 remain elusive. chikv nsp3 modulates the mammalian stress response by preventing stress ...201526384002
japanese encephalitis virus replicon-based vaccine expressing enterovirus-71 epitope confers dual protection from lethal construct safer recombinant flavivirus vaccine, we exploited japanese encephalitis virus (jev) replicon-based platform to generate single-round infectious particles (srips) that expressed heterologous neutralizing epitope sp70 derived from enterovirus-71 (ev71). such pseudo-infectious virus particles, named srip-sp70, although are not genuine viable viruses, closely mimic live virus infection to elicit immune responses within one round of viral life cycle.201526362772
differential phosphatidylinositol-3-kinase-akt-mtor activation by semliki forest and chikungunya viruses is dependent on nsp3 and connected to replication complex internalization.many viruses affect or exploit the phosphatidylinositol-3-kinase (pi3k)-akt-mammalian target of rapamycin (mtor) pathway, a crucial prosurvival signaling cascade. we report that this pathway was strongly activated in cells upon infection with the old world alphavirus semliki forest virus (sfv), even under conditions of complete nutrient starvation. we mapped this activation to the hyperphosphorylated/acidic domain in the c-terminal tail of sfv nonstructural protein nsp3. viruses with a deletion ...201526339054
alphavirus rna synthesis and non-structural protein functions.the members of the genus alphavirus are positive-sense rna viruses, which are predominantly transmitted to vertebrates by a mosquito vector. alphavirus disease in humans can be severely debilitating, and depending on the particular viral species, infection may result in encephalitis and possibly death. in recent years, alphaviruses have received significant attention from public health authorities as a consequence of the dramatic emergence of chikungunya virus in the indian ocean islands and the ...201526219641
dna-launched alphavirus replicons encoding a fusion of mycobacterial antigens acr and ag85b are immunogenic and protective in a murine model of tb infection.there is an urgent need for effective prophylactic measures against mycobacterium tuberculosis (mtb) infection, particularly given the highly variable efficacy of bacille calmette-guerin (bcg), the only licensed vaccine against tuberculosis (tb). most studies indicate that cell-mediated immune responses involving both cd4+ and cd8+ t cells are necessary for effective immunity against mtb. genetic vaccination induces humoral and cellular immune responses, including cd4+ and cd8+ t-cell responses, ...201526317509
differences in processing determinants of nonstructural polyprotein and in the sequence of nonstructural protein 3 affect neurovirulence of semliki forest virus.the a7(74) strain of semliki forest virus (sfv; genus alphavirus) is avirulent in adult mice, while the l10 strain is virulent in mice of all ages. it has been previously demonstrated that this phenotypic difference is associated with nonstructural protein 3 (nsp3). consensus clones of l10 (designated sfv6) and a7(74) (designated a774wt) were used to construct a panel of recombinant viruses. the insertion of nsp3 from a774wt into the sfv6 backbone had a minor effect on the virulence of the resul ...201526311875
microrna-attenuated clone of virulent semliki forest virus overcomes antiviral type i interferon in resistant mouse ct-2a glioma.glioblastoma is a terminal disease with no effective treatment currently available. among the new therapy candidates are oncolytic viruses capable of selectively replicating in cancer cells, causing tumor lysis and inducing adaptive immune responses against the tumor. however, tumor antiviral responses, primarily mediated by type i interferon (ifn-i), remain a key problem that severely restricts viral replication and oncolysis. we show here that the semliki forest virus (sfv) strain sfv4, which ...201526269187
flavivirus rna synthesis in vitro.establishment of in vitro systems to study mechanisms of rna synthesis for positive strand rna viruses have been very useful in the past and have shed light on the composition of protein and rna components, optimum conditions, the nature of the products formed, cis-acting rna elements and trans-acting protein factors required for efficient synthesis. in this review, we summarize our current understanding regarding the requirements for flavivirus rna synthesis in vitro. we describe details of rea ...201526272247
haploid genetic screen reveals a profound and direct dependence on cholesterol for hantavirus membrane fusion.hantaviruses cause hemorrhagic fever with renal syndrome (hfrs) in the old world and a highly fatal hantavirus cardiopulmonary syndrome (hcps) in the new world. no vaccines or antiviral therapies are currently available to prevent or treat hantavirus disease, and gaps in our understanding of how hantaviruses enter cells challenge the search for therapeutics. we performed a haploid genetic screen in human cells to identify host factors required for entry by andes virus, a highly virulent new worl ...201526126854
a novel system for visualizing alphavirus assembly.alphaviruses are small, enveloped rna viruses that form infectious particles by budding through the cellular plasma membrane. to help visualize and understand the intracellular assembly of alphavirus virions we have developed a bimolecular fluorescence complementation-based system (bifc) that allows visualization of capsid and e2 subcellular localization and association in live cells. in this system, n- or c-terminal venus fluorescent protein fragments (vn- and vc-) are fused to the n-terminus o ...201526122073
distinct sets of piwi proteins produce arbovirus and transposon-derived pirnas in aedes aegypti mosquito cells.the piwi-interacting rna (pirna) pathway is essential for transposon silencing in many model organisms. its remarkable efficiency relies on a sophisticated amplification mechanism known as the ping-pong loop. in alphavirus-infected aedes mosquitoes, pirnas with sequence features that suggest ping-pong-dependent biogenesis are produced from viral rna. the piwi family in aedes mosquitoes is expanded when compared to other model organisms, raising the possibility that individual piwi proteins have ...201526068474
mrna capping by venezuelan equine encephalitis virus nsp1: functional characterization and implications for antiviral research.alphaviruses are known to possess a unique viral mrna capping mechanism involving the viral nonstructural protein nsp1. this enzyme harbors methyltransferase (mtase) and nsp1 guanylylation (gt) activities catalyzing the transfer of the methyl group from s-adenosylmethionine (adomet) to the n7 position of a gtp molecule followed by the formation of an m(7)gmp-nsp1 adduct. subsequent transfer of m(7)gmp onto the 5' end of the viral mrna has not been demonstrated in vitro yet. here we report the bi ...201526041283
conformational changes required for reovirus cell entry are sensitive to ph.during cell entry, reovirus particles disassemble to generate isvps. isvps undergo conformational changes to form isvp(*) and this conversion is required for membrane penetration. in tissues where isvp formation occurs within endosomes, isvp-to-isvp(*) conversion occurs at low ph. in contrast, in tissues where isvp formation occurs extracellularly, isvp-to-isvp(*) transition occurs at neutral ph. whether these two distinct ph environments influence the efficiency of cell entry is not known. in t ...201526004253
the broad-spectrum antiviral protein zap restricts human retrotransposition.intrinsic immunity describes the set of recently discovered but poorly understood cellular mechanisms that specifically target viral pathogens. their discovery derives in large part from intensive studies of hiv and siv that revealed restriction factors acting at various stages of the retroviral life cycle. recent studies indicate that some factors restrict both retroviruses and retrotransposons but surprisingly in ways that may differ. we screened known interferon-stimulated antiviral proteins ...201526001115
antiviral antibody profiling by high-density protein arrays.viral infections elicit antiviral antibodies and have been associated with various chronic diseases. detection of these antibodies can facilitate diagnosis, treatment of infection, and understanding of the mechanisms of virus-associated diseases. in this work, we assayed antiviral antibodies using a novel high-density nucleic acid programmable protein array (hd-nappa) platform. individual viral proteins were expressed in situ directly from plasmids encoding proteins in an array of microscopic re ...201525758251
shp-1-dependent macrophage differentiation exacerbates virus-induced myositis.virus-induced myositis is an emerging global affliction that remains poorly characterized with few treatment options. moreover, muscle-tropic viruses often spread to the cns, causing dramatically increased morbidity. therefore, there is an urgent need to explore genetic factors involved in this class of human disease. this report investigates critical innate immune pathways affecting murine virus-induced myositis. of particular importance, the key immune regulator src homology region 2 domain-co ...201525681345
the 5' and 3' ends of alphavirus rnas--non-coding is not non-functional.the non-coding regions found at the 5' and 3' ends of alphavirus genomes regulate viral gene expression, replication, translation and virus-host interactions, which have significant implications for viral evolution, host range, and pathogenesis. the functions of these non-coding regions are mediated by a combination of linear sequence and structural elements. the capped 5' untranslated region (utr) contains promoter elements, translational regulatory sequences that modulate dependence on cellula ...201525630058
rnai and antiviral defense in the honey bee.honey bees play an important agricultural and ecological role as pollinators of numerous agricultural crops and other plant species. therefore, investigating the factors associated with high annual losses of honey bee colonies in the us is an important and active area of research. pathogen incidence and abundance correlate with colony collapse disorder- (ccd-) affected colonies in the us and colony losses in the us and in some european countries. honey bees are readily infected by single-strande ...201526798663
the phosphatidylserine and phosphatidylethanolamine receptor cd300a binds dengue virus and enhances infection.dengue virus (denv) is the etiological agent of the major human arboviral disease. we previously demonstrated that the tim and tam families of phosphatidylserine (ptdser) receptors involved in the phagocytosis of apoptotic cells mediate denv entry into target cells. we show here that human cd300a, a recently identified phospholipid receptor, also binds directly denv particles and enhances viral entry. cd300a facilitates infection of the four denv serotypes, as well as of other mosquito-borne vir ...201526468529
aberrant regulation of synchronous network activity by the attention-deficit/hyperactivity disorder-associated human dopamine d4 receptor variant d4.7 in the prefrontal cortex.the hd4.7 variant has been linked to attention-deficit/hyperactivity disorder (adhd); however, the underlying mechanism is unknown. we found that activation of hd4.7 induced over-suppression of glutamatergic excitatory network bursts and under-suppression of gabaergic inhibitory network bursts in the prefrontal cortex (pfc) circuitry. methylphenidate, a psychostimulant drug used to treat adhd, normalized the effects of hd4.7 on synchronous network bursts in pfc pyramidal neurons. the findings of ...201526541360
direct pcr of indigenous and invasive mosquito species: a time- and cost-effective technique of mosquito barcoding.millions of people die each year as a result of pathogens transmitted by mosquitoes. however, the morphological identification of mosquito species can be difficult even for experts. the identification of morphologically indistinguishable species, such as members of the anopheles maculipennis complex (diptera: culicidae), and possible hybrids, such as culex pipiens pipiens/culex pipiens molestus (diptera: culicidae), presents a major problem. in addition, the detection and discrimination of newly ...201526663040
characterization of a novel human-specific sting agonist that elicits antiviral activity against emerging alphaviruses.pharmacologic stimulation of innate immune processes represents an attractive strategy to achieve multiple therapeutic outcomes including inhibition of virus replication, boosting antitumor immunity, and enhancing vaccine immunogenicity. in light of this we sought to identify small molecules capable of activating the type i interferon (ifn) response by way of the transcription factor ifn regulatory factor 3 (irf3). a high throughput in vitro screen yielded 4-(2-chloro-6-fluorobenzyl)-n-(furan-2- ...201526646986
mtor is essential for corticosteroid effects on hippocampal ampa receptor function and fear memory.glucocorticoid hormones, via activation of their receptors, promote memory consolidation, but the exact underlying mechanisms remain elusive. we examined how corticosterone regulates ampa receptors (ampars), which are crucial for synaptic plasticity and memory formation. combining a live imaging fluorescent recovery after photobleaching approach with the use of the ph-sensitive gfp-ampar tagging revealed that corticosterone enhances the ampar mobile fraction and increases synaptic trapping of am ...201526572647
self-assembling protein nanoparticles in the design of vaccines.for over 100 years, vaccines have been one of the most effective medical interventions for reducing infectious disease, and are estimated to save millions of lives globally each year. nevertheless, many diseases are not yet preventable by vaccination. this large unmet medical need demands further research and the development of novel vaccines with high efficacy and safety. compared to the 19th and early 20th century vaccines that were made of killed, inactivated, or live-attenuated pathogens, mo ...201526862374
chikungunya nsp2 protease is not a papain-like cysteine protease and the catalytic dyad cysteine is interchangeable with a proximal serine.chikungunya virus is the pathogenic alphavirus that causes chikungunya fever in humans. in the last decade millions of cases have been reported around the world from africa to asia to the americas. the alphavirus nsp2 protein is multifunctional and is considered to be pivotal to viral replication, as the nsp2 protease activity is critical for proteolytic processing of the viral polyprotein during replication. classically the alphavirus nsp2 protease is thought to be papain-like with the enzyme r ...201526597768
conformational ensemble of the poliovirus 3cd precursor observed by md simulations and confirmed by saxs: a strategy to expand the viral proteome?the genomes of rna viruses are relatively small. to overcome the small-size limitation, rna viruses assign distinct functions to the processed viral proteins and their precursors. this is exemplified by poliovirus 3cd protein. 3c protein is a protease and rna-binding protein. 3d protein is an rna-dependent rna polymerase (rdrp). 3cd exhibits unique protease and rna-binding activities relative to 3c and is devoid of rdrp activity. the origin of these differences is unclear, since crystal structur ...201526610545
design, synthesis, and antiviral activity of novel ribonucleosides of 1,2,3-triazolylbenzyl-aminophosphonates.a novel series of ribonucleosides of 1,2,3-triazolylbenzyl-aminophosphonates was synthesized through the kabachnik-fields reaction using i2 as catalyst followed by copper-catalyzed cycloaddition of the azide-alkyne reaction (cuaac). all structures of the newly prepared compounds were characterized by (1) h nmr, (13) c nmr, and hrms spectra. the structures of 2e, 2f, 3d, and 3g were further confirmed by x-ray diffraction analysis. these compounds were tested against various strains of dna and rna ...201526575425
chikungunya virus replication in salivary glands of the mosquito aedes albopictus.chikungunya virus (chikv) is an emerging arbovirus transmitted to humans by mosquitoes such as aedes albopictus. to be transmitted, chikv must replicate in the mosquito midgut, then disseminate in the hemocele and infect the salivary glands before being released in saliva. we have developed a standardized protocol to visualize viral particles in the mosquito salivary glands using transmission electron microscopy. here we provide direct evidence for chikv replication and storage in ae. albopictus ...201526593936
pros and cons of antigen-presenting cell targeted tumor therapeutic antitumor vaccination, dendritic cells play the leading role since they decide if, how, when, and where a potent antitumor immune response will take place. since the disentanglement of the complexity and merit of different antigen-presenting cell subtypes, antitumor immunotherapeutic research started to investigate the potential benefit of targeting these subtypes in situ. this review will discuss which antigen-presenting cell subtypes are at play and how they have been targeted a ...201526583156
viral interference and persistence in mosquito-borne flaviviruses.mosquito-borne flaviviruses are important pathogens for humans, and the detection of two or more flaviviruses cocirculating in the same geographic area has often been reported. however, the epidemiological impact remains to be determined. mosquito-borne flaviviruses are primarily transmitted through aedes and culex mosquitoes; these viruses establish a life-long or persistent infection without apparent pathological effects. this establishment requires a balance between virus replication and the ...201526583158
hiv vaccine: recent advances, current roadblocks, and future is a leading cause of mortality and morbidity worldwide. in spite of successful interventions and treatment protocols, an hiv vaccine would be the ultimate prevention and control strategy. ever since identification of hiv/aids, there have been meticulous efforts for vaccine development. the specific aim of this paper is to review recent vaccine efficacy trials and associated advancements and discuss the current challenges and future directions. recombinant dna technologies greatly facil ...201526579546
parp9-dtx3l ubiquitin ligase targets host histone h2bj and viral 3c protease to enhance interferon signaling and control viral infection.enhancing the response to interferon could offer an immunological advantage to the host. in support of this concept, we used a modified form of the transcription factor stat1 to achieve hyper-responsiveness to interferon without toxicity and markedly improve antiviral function in transgenic mice and transduced human cells. we found that the improvement depended on expression of a parp9-dtx3l complex with distinct domains for interaction with stat1 and for activity as an e3 ubiquitin ligase that ...201526479788
serological evidence of chikungunya virus among acute febrile patients in southern the last two decades, chikungunya virus (chikv) has rapidly expanded to several geographical areas, causing frequent outbreaks in sub-saharan africa, south east asia, south america, and europe. therefore, the disease remains heavily neglected in mozambique, and no recent study has been conducted.201526473605
mouse superkiller-2-like helicase ddx60 is dispensable for type i ifn induction and immunity to multiple viruses.ifn-α/β allow cells to fight virus infection by inducing the expression of many genes that encode effectors of antiviral defense. one of these, the ski2-like dexh-box helicase ddx60, was recently implicated in resistance of human cells to hepatitis c virus, as well as in induction of ifn-α/β by retinoic acid inducible gene 1-like receptors (rlrs) that detect the presence of rna viruses in a cell-intrinsic manner. here, we sought to investigate the role of ddx60 in ifn-α/β induction and in resist ...201526457795
simultaneous determination of sample thickness, tilt, and electron mean free path using tomographic tilt images based on beer-lambert law.cryo-electron tomography (cryo-et) is an emerging technique that can elucidate the architecture of macromolecular complexes and cellular ultrastructure in a near-native state. some important sample parameters, such as thickness and tilt, are needed for 3-d reconstruction. however, these parameters can currently only be determined using trial 3-d reconstructions. accurate electron mean free path plays a significant role in modeling image formation process essential for simulation of electron micr ...201526433027
hur and ago2 bind the internal ribosome entry site of enterovirus 71 and promote virus translation and replication.ev71 (enterovirus 71) rna contains an internal ribosomal entry site (ires) that directs cap-independent initiation of translation. ires-dependent translation requires the host's translation initiation factors and ires-associated trans-acting factors (itafs). we reported recently that mrna decay factor auf1 is a negative-acting itaf that binds ires stem-loop ii. we also reported that the small rna-processing enzyme dicer produces at least four small rnas (vsrnas) from the ev71 ires. one of these, ...201526451954
serum levels of inflammatory cytokines in rift valley fever patients are indicative of severe disease.rift valley fever (rvf) is a mosquito-borne viral zoonosis affecting domestic and wild ruminants, camels and humans. outbreaks of rvf are characterized by a sudden onset of abortions and high mortality amongst domestic ruminants. humans develop disease ranging from a mild flu-like illness to more severe complications including hemorrhagic syndrome, ocular and neurological lesions and death. during the rvf outbreak in south africa in 2010/11, a total of 278 human cases were laboratory confirmed, ...201526437779
virus-based nanoparticles as versatile nanomachines.nanoscale engineering is revolutionizing the way we prevent, detect, and treat diseases. viruses have played a special role in these developments because they can function as prefabricated nanoscaffolds that have unique properties and are easily modified. the interiors of virus particles can encapsulate and protect sensitive compounds, while the exteriors can be altered to display large and small molecules in precisely defined arrays. these properties of viruses, along with their innate biocompa ...201526958921
intracellular mono-adp-ribosylation in signaling and disease.a key process in the regulation of protein activities and thus cellular signaling pathways is the modification of proteins by post-translational mechanisms. knowledge about the enzymes (writers and erasers) that attach and remove post-translational modifications, the targets that are modified and the functional consequences elicited by specific modifications, is crucial for understanding cell biological processes. moreover detailed knowledge about these mechanisms and pathways helps to elucidate ...201526426055
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