expression of the zinc-finger antiviral protein inhibits alphavirus replication.the rat zinc-finger antiviral protein (zap) was recently identified as a host protein conferring resistance to retroviral infection. we analyzed zap's ability to inhibit viruses from other families and found that zap potently inhibits the replication of multiple members of the alphavirus genus within the togaviridae, including sindbis virus, semliki forest virus, ross river virus, and venezuelan equine encephalitis virus. however, expression of zap did not induce a broad-spectrum antiviral state ...200314557641
changes in mumps virus gene sequence associated with variability in neurovirulent phenotype.mumps virus is highly neurotropic and, prior to widespread vaccination programs, was the major cause of viral meningitis in the united states. nonetheless, the genetic basis of mumps virus neurotropism and neurovirulence was until recently not understood, largely due to the lack of an animal model. here, nonneurovirulent (jeryl lynn vaccine) and highly neurovirulent (88-1961 wild type) mumps virus strains were passaged in human neural cells or in chicken fibroblast cells with the goal of neuroad ...200314557647
induction of caspase-dependent apoptosis in cultured rat oligodendrocytes by murine coronavirus is mediated during cell entry and does not require virus replication.murine coronavirus mouse hepatitis virus (mhv) causes demyelination of the central nervous system (cns) in rats and mice. apoptotic oligodendrocytes have been detected in the vicinity of the cns demyelinating lesions in these animals. however, whether mhv can directly induce oligodendrocyte apoptosis has not been documented. here, we established a rat oligodendrocyte culture that is morphologically and phenotypically indistinguishable from the primary rat oligodendrocytes. using this culture, we ...200314581532
dc-sign and l-sign can act as attachment receptors for alphaviruses and distinguish between mosquito cell- and mammalian cell-derived viruses.c-type lectins such as dc-sign and l-sign, which bind mannose-enriched carbohydrate modifications of host and pathogen proteins, have been shown to bind glycoproteins of several viruses and facilitate either cis or trans infection. dc-sign and l-sign are expressed in several early targets of arbovirus infection, including dendritic cells (dcs) and cells of the reticuloendothelial system. in the present study, we show that dc-sign and l-sign can function as attachment receptors for sindbis (sb) v ...200314581539
borna disease virus glycoprotein is required for viral dissemination in neurons.borna disease virus (bdv) is a nonsegmented negative-strand rna virus with a tropism for neurons. infection with bdv causes neurological diseases in a wide variety of animal species. although it is known that the virus spreads from neuron to neuron, assembled viral particles have never been visualized in the brains of infected animals. this has led to the hypothesis that bdv spreads as nonenveloped ribonucleoproteins (rnp) rather than as enveloped viral particles. we assessed whether the viral e ...200314581559
are weak protein-protein interactions the general rule in capsid assembly? 200314585329
female lethality and apoptosis of spermatocytes in mice lacking the ubr2 ubiquitin ligase of the n-end rule pathway.substrates of the ubiquitin-dependent n-end rule pathway include proteins with destabilizing n-terminal residues. ubr1(-/-) mice, which lacked the pathway's ubiquitin ligase e3alpha, were viable and retained the n-end rule pathway. the present work describes the identification and analysis of mouse ubr2, a homolog of ubr1. we demonstrate that the substrate-binding properties of ubr2 are highly similar to those of ubr1, identifying ubr2 as the second e3 of the mammalian n-end rule pathway. ubr2(- ...200314585983
rna interference-mediated knockdown of a gata factor reveals a link to anautogeny in the mosquito aedes aegypti.blood feeding tightly regulates the reproductive cycle in anautogenous mosquitoes. vitellogenesis (the synthesis of yolk protein precursors) is a key event in the mosquito reproductive cycle and is activated in response to a blood meal. before blood feeding, aedes aegypti is in a state of reproductive arrest during which the yolk protein precursor genes (ypps) are repressed. the regulatory region of the major ypp gene vitellogenin (vg) has multiple gata-binding sites required for the high expres ...200314595016
sindbis virus-associated pathology in aedes albopictus (diptera: culicidae).virus dissemination and associated pathology were examined in aedes albopictus after intrathoracic inoculation of sindbis virus (sin), the prototypic alphavirus. at 10 days postinfection, virus rna was detected in all three-body segments of the insect. colocalization of virus antigen with structural pathology was observed in mosquito salivary glands and midgut-associated visceral muscles, representing yet another example of arbovirus-associated pathology in a mosquito host. sin antigen and gross ...200314596286
brain-derived neurotrophic factor signal enhances and maintains the expression of ampa receptor-associated pdz proteins in developing cortical neurons.postsynaptic molecules with pdz domains (pdz proteins) interact with various glutamate receptors and regulate their subcellular trafficking and stability. in rat neocortical development, the protein expression of ampa-type glutamate receptor glur1 lagged behind its mrna expression and rather paralleled an increase in pdz protein levels. one of the neurotrophins, brain-derived neurotrophic factor (bdnf), appeared to contribute to this process, regulating the pdz protein expression. in neocortical ...200314597197
virus concentration using polyethyleneimine-conjugated magnetic beads for improving the sensitivity of nucleic acid amplification enhance the sensitivity of virus detection by polymerase chain reaction (pcr) and reverse-transcriptional (rt)-pcr, we developed a novel virus concentration method using polyethyleneimine (pei)-conjugated magnetic beads. pei-magnetic beads adsorbed efficiently the enveloped viruses sindbis virus and herpes simplex 1 virus, and the nonenveloped virus sv-40, but not the nonenveloped viruses porcine parvovirus (ppv) or poliovirus, based on the pcr detection data. furthermore, the infectivity in ...200314599674
deletions in the transmembrane domain of a sindbis virus glycoprotein alter virus infectivity, stability, and host range.the alphaviruses are composed of two icosahedral protein shells, one nested within the other. a membrane bilayer derived from the host cell is sandwiched between the protein shells. the protein shells are attached to one another by protein domains which extend one of the proteins of the outer shell through the membrane bilayer to attach to the inner shell. we have examined the interaction of the membrane-spanning domain of one of the membrane glycoproteins with the membrane bilayer and with othe ...200314610193
liposomes as target membranes in the study of virus receptor interaction and membrane fusion. 200314610825
mitogenic effect of orphan receptor tr3 and its regulation by mekk1 in lung cancer cells.tr3, also known as ngfi-b or nur77, is an immediate-early response gene and an orphan member of the steroid/thyroid/retinoid receptor superfamily. we previously reported that tr3 expression was induced by apoptotic stimuli and was required for their apoptotic effect in lung cancer cells. here, we present evidence that tr3 was also induced by epidermal growth factor (egf) and serum and was required for their mitogenic effect in lung cancer cells. ectopic expression of tr3 in both h460 and calu-6 ...200314612408
translational recoding signals between gag and pol in diverse ltr retrotransposons.because of their compact genomes, retroelements (including retrotransposons and retroviruses) employ a variety of translational recoding mechanisms to express gag and pol. to assess the diversity of recoding strategies, we surveyed gag/pol gene organization among retroelements from diverse host species, including elements exhaustively recovered from the genome sequences of caenorhabditis elegans, drosophila melanogaster, schizosaccharomyces pombe, candida albicans, and arabidopsis thaliana. in c ...200314623998
detection of antibodies to alphaviruses and discrimination between antibodies to eastern and western equine encephalitis viruses in rabbit sera using a recombinant antigen and virus-specific monoclonal antibodies.three arthropod-borne alphaviruses, western equine encephalitis viruses (weev), eastern equine encephalitis viruses (eeev) and venezuelan equine encephalitis viruses are the aetiological agents of a sometimes severe encephalomyelitis in equines and humans in the new world. with regard to the different ecology and epidemiology of these viruses, a method applied in serological screening should be able to distinguish between them as well as other related members of the genus alphavirus in the ameri ...200314628996
promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene.malignant cells often display defects in autophagy, an evolutionarily conserved pathway for degrading long-lived proteins and cytoplasmic organelles. however, as yet, there is no genetic evidence for a role of autophagy genes in tumor suppression. the beclin 1 autophagy gene is monoallelically deleted in 40-75% of cases of human sporadic breast, ovarian, and prostate cancer. therefore, we used a targeted mutant mouse model to test the hypothesis that monoallelic deletion of beclin 1 promotes tum ...200314638851
a comparison of the properties of a bcl-xl variant to the wild-type anti-apoptosis inhibitor in mammalian cell cultures.the overexpression of bcl-2 and its homologues is a widely used strategy to inhibit apoptosis in mammalian cell culture systems. in this study, we have evaluated the bcl-2 homologue, bcl-x(l) and compared its effectiveness to a bcl-x(l) mutant lacking most of the non-conserved unstructured loop domain, bcl-x(l)delta (deletion of amino acids 26 through 83). the cell line, chinese hamster ovary (cho), was genetically modified to express constitutively bcl-x(l) or the bcl-x(l) variant and subjected ...200314642351
vesicular stomatitis virus expressing a chimeric sindbis glycoprotein containing an fc antibody binding domain targets to her2/neu overexpressing breast cancer cells.vesicular stomatitis virus (vsv) is a candidate for development for cancer therapy. it is an oncolytic virus that is safe in humans. recombinant virus can be made directly from plasmid components. we attempted to create a virus that targeted specifically to breast cancer cells. nonreplicating and replicating pseudotype vsv were created whose only surface glycoprotein (gp) was a sindbis gp, called sindbis-zz, modified to severely reduce its native binding function and to contain the fc-binding do ...200314644615
molecular dynamics simulations of the e1/e2 transmembrane domain of the semliki forest virus.transmembrane (tm) helix-helix interactions are important for virus budding and fusion. we have developed a simulation strategy that reveals the main features of the helical packing between the tm domains of the two glycoproteins e1 and e2 of the alpha-virus semliki forest virus and that can be extrapolated to sketch tm helical packing in other alpha-viruses. molecular dynamics simulations were performed in wild-type and mutant peptides, both isolated and forming e1/e2 complexes. the simulations ...200314645057
targeting of adenovirus via genetic modification of the viral capsid combined with a protein bridge.a potential barrier to the development of genetically targeted adenovirus (ad) vectors for cell-specific delivery of gene therapeutics lies in the fact that several types of targeting protein ligands require posttranslational modifications, such as the formation of disulfide bonds, which are not available to ad capsid proteins due to their nuclear localization during assembly of the virion. to overcome this problem, we developed a new targeting strategy, which combines genetic modifications of t ...200314645549
functional analysis of nsp3 phosphoprotein mutants of sindbis virus.alphavirus nsp3 phosphoprotein is essential for virus replication and functions initially within polyprotein p123 or p23 components of the short-lived minus-strand replicase, and upon polyprotein cleavage, mature nsp3 likely functions also in plus-strand synthesis. we report the identification of a second nsp3 mutant from among the a complementation group of sindbis virus (sin) heat-resistant strain, ts rna-negative mutants. the ts138 mutant possessed a change of g4303 to c, predicting an ala68- ...200314645567
infection and injury of neurons by west nile encephalitis virus.west nile virus (wnv) infects neurons and leads to encephalitis, paralysis, and death in humans, animals, and birds. we investigated the mechanism by which neuronal injury occurs after wnv infection. neurons in the anterior horn of the spinal cords of paralyzed mice exhibited a high degree of wnv infection, leukocyte infiltration, and degeneration. because it was difficult to distinguish whether neuronal injury was caused by viral infection or by the immune system response, a novel tissue cultur ...200314645577
systemic tumor targeting and killing by sindbis viral vectors.successful cancer gene therapy requires a vector that systemically and specifically targets tumor cells throughout the body. although several vectors have been developed to express cytotoxic genes via tumor-specific promoters or to selectively replicate in tumor cells, most are taken up and expressed by just a few targeted tumor cells. by contrast, we show here that blood-borne sindbis viral vectors systemically and specifically infect tumor cells. a single intraperitoneal treatment allows the v ...200414647305
use of sindbis virus-mediated rna interference to demonstrate a conserved role of broad-complex in insect metamorphosis.the transcription factor broad-complex (br-c) is required for differentiation of adult structures as well as for the programmed death of obsolete larval organs during metamorphosis of the fruit fly drosophila melanogaster. whether br-c has a similar role in other holometabolous insects could not be proven without a loss-of-function genetic test, performed in a non-drosophilid species. here we use a recombinant sindbis virus as a tool to silence br-c expression in the silkmoth bombyx mori. the vi ...200314668449
generation and characterization of closely related epizootic and enzootic infectious cdna clones for studying interferon sensitivity and emergence mechanisms of venezuelan equine encephalitis virus.venezuelan equine encephalitis virus (veev) is a reemerging pathogen and a continuing threat to humans and equines in the americas. identification of the genetic determinants that enable epizootic veev strains to arise and exploit equines as amplification hosts to cause widespread human disease is pivotal to understanding vee emergence. the sensitivity to murine alpha/beta interferon-mediated antiviral activity was previously correlated to the epizootic phenotype of several veev strains. infecti ...200414671082
selection of functional 5' cis-acting elements promoting efficient sindbis virus genome replication.the 5' portion of the sindbis virus (sin) genome rna is multifunctional. besides initiating translation of the nonstructural polyprotein, rna elements in the 5' 200 bases of the sin genome rna, or its complement at the 3' end of the negative-strand intermediate, play key roles in the synthesis of both negative- and positive-strand rnas. we used here a combination of genetic and biochemical approaches to further dissect the functions of this sequence. replacement of the sin 5' end in defective-in ...200414671088
a jc virus-induced signal is required for infection of glial cells by a clathrin- and eps15-dependent pathway.infectious entry of jc virus (jcv) into human glial cells occurs by receptor-mediated clathrin-dependent endocytosis. in this report we demonstrate that the tyrosine kinase inhibitor genistein blocks virus entry and inhibits infection. transient expression of dominant-negative eps15 mutants, including a phosphorylation-defective mutant, inhibited both virus entry and infection. we also show that the jcv-induced signal activates the mitogen-activated protein kinases erk1 and erk2. these data demo ...200414671106
the orf2 protein of hepatitis e virus binds the 5' region of viral rna.hepatitis e virus (hev) is a major human pathogen in much of the developing world. it is a plus-strand rna virus with a 7.2-kb polyadenylated genome consisting of three open reading frames, orf1, orf2, and orf3. of these, orf2 encodes the major capsid protein of the virus and orf3 encodes a small protein of unknown function. using the yeast three-hybrid system and traditional biochemical techniques, we have studied the rna binding activities of orf2 and orf3, two proteins encoded in the 3' struc ...200414671114
tetracycline-inducible packaging cell line for production of flavivirus replicon particles.we have previously developed replicon vectors derived from the australian flavivirus kunjin that have a unique noncytopathic nature and have been shown to direct prolonged high-level expression of encoded heterologous genes in vitro and in vivo and to induce strong and long-lasting immune responses to encoded immunogens in mice. to facilitate further applications of these vectors in the form of virus-like particles (vlps), we have now generated a stable bhk packaging cell line, tetkuncprme, carr ...200414671135
systematic, genome-wide identification of host genes affecting replication of a positive-strand rna virus.positive-strand rna viruses are the largest virus class and include many pathogens such as hepatitis c virus and the severe acute respiratory syndrome coronavirus (sars). brome mosaic virus (bmv) is a representative positive-strand rna virus whose rna replication, gene expression, and encapsidation have been reproduced in the yeast saccharomyces cerevisiae. by using traditional yeast genetics, host genes have been identified that function in controlling bmv translation, selecting bmv rnas as rep ...200314671320
sindbis virus nucleocapsid assembly: rna folding promotes capsid protein sindbis virus, initiation of nucleocapsid core assembly begins with recognition of the encapsidation signal of the viral rna genome by capsid protein. this nucleation event drives the recruitment of additional capsid proteins to fully encapsidate the genome, generating an icosahedral nucleocapsid core. the encapsidation signal of the sindbis virus genomic rna has previously been localized to a 132-nucleotide region of the genome within the coding region of the nsp1 protein, and the rna-bindin ...200414681591
synthesis and antiviral evaluation of benzimidazoles, quinoxalines and indoles from dehydroabietic acid.several heterocycles, such as benzimidazoles, quinoxalines and indoles incorporated into a hydrophenanthrene and naphthalene skeleton, were synthesised from two useful ortho-bromonitro precursors derived from dehydroabietic acid: methyl 12-bromo-13-nitro-deisopropyldehydroabietate and methyl 12-bromo-13,14-dinitro-deisopropyldehydroabietate. the new heterocycles were evaluated for their activity in vitro against several rna and dna viruses.200414697775
flavivirus isolations from mosquitoes collected from western cape york peninsula, australia, 1999-2000.after the 1st appearance of japanese encephalitis virus (je) on mainland australia in 1998, a study was undertaken to investigate whether je had become established in enzootic transmission cycles on western cape york peninsula. adult mosquitoes were collected during the late wet season from kowanyama and pormpuraaw in april 1999, and pormpuraaw and barr's yard in april 2000. despite processing 269,270 mosquitoes for virus isolation, no isolates of je were obtained. however, other flaviviruses co ...200314710742
sucrose density gradient centrifugation and cross-flow filtration methods for the production of arbovirus antigens inactivated by binary ethylenimine.sucrose density gradient centrifugation and cross-flow filtration methods have been developed and standardised for the safe and reproducible production of inactivated arbovirus antigens which are appropriate for use in diagnostic serological applications.200414720306
borna disease virus multiplication in mouse organotypic slice cultures is site-specifically inhibited by gamma interferon but not by interleukin-12.borna disease virus (bdv) induces a nonpurulent cd4- and cd8-t-cell-dependent meningoencephalitis in susceptible animals. upon intracerebral infection, bdv replicates in the mouse central nervous system (cns), but only a few mouse strains develop neurological disorder. the antiviral t cells appear to suppress bdv replication by a noncytolytic mechanism. since bdv does not replicate in standard mouse cell cultures, the putative role of gamma interferon (ifn-gamma) in virus control could not be te ...200414722276
critical residues for gtp methylation and formation of the covalent m7gmp-enzyme intermediate in the capping enzyme domain of bamboo mosaic reading frame 1 of bamboo mosaic virus (bamv), a potexvirus in the alphavirus-like superfamily, encodes a 155-kda replicase responsible for the formation of the 5' cap structure and replication of the viral rna genome. the n-terminal domain of the viral replicase functions as an mrna capping enzyme, which exhibits both gtp methyltransferase and s-adenosylmethionine (adomet)-dependent guanylyltransferase activities. we mutated each of the four conserved amino acids among the capping enzymes ...200414722282
human immunodeficiency virus type 1-derived lentivirus vectors pseudotyped with envelope glycoproteins derived from ross river virus and semliki forest virus.ross river virus (rrv) and semliki forest virus (sfv) are two alphaviruses that have a high degree of amino acid homology, as well as a very broad host range. we show here that envelope glycoproteins derived from both viruses can pseudotype human immunodeficiency virus type 1 (hiv-1)-derived lentivirus vectors. both rrv and sfv glycoproteins considerably expand the host range of the lentivirus vector, and vectors can be efficiently concentrated by ultracentrifugation. a systematic analysis compa ...200414722297
development of a new sindbis virus transducing system and its characterization in three culicine mosquitoes and two lepidopteran species.alphavirus transducing systems (atss) are alphavirus-based tools for expressing genes in insects. here we describe an ats (5'dsmre16ic) based entirely on sindbis mre16 virus. gfp expression was used to characterize alimentary tract infections and dissemination in three culicine and two lepidopteran species. following per os infection, 5'dsmre16ic-egfp efficiently infected aedes aegypti and culex tritaeniorhynchus, but not culex pipiens pipiens. ae. aegypti clearly showed accumulation of green fl ...200414728670
reactive oxygen species formation by uv-a irradiation of urocanic acid and the role of trace metals in this chemistry.we have extended our study of the decomposition of urocanic acid (uca) with ultraviolet a radiation (uv-a) by the self-sensitized generation of singlet oxygen (see photochem. photobiol. 75, 565 [2002]). the chemistry has been found to be partially dependent on the presence of trace metal, most likely iron. rigorous removal of metal impurities from the reaction mixture, using chelex, retarded (but did not eliminate) the uv-a-initiated uca degradation. the addition of small amounts of ferric chlor ...200314743864
sindbis vector sinrep(nsp2s726): a tool for rapid heterologous expression with attenuated cytotoxicity in neurons.sindbis virus-based vectors have been successfully used for transient heterologous protein expression in neurons. their main limitation arises from infection-associated cytotoxicity, attributed largely to a progressive shut down of host cell protein synthesis. here we evaluated a modified sindbis vector, based on a viral strain containing a point mutation in the second nonstructural protein, nsp2 p726s, described to delay inhibition of protein synthesis in bhk cells [virology 228 (1997) 74], for ...200414757348
alterations of pr-m cleavage and virus export in pr-m junction chimeric dengue viruses.during the export of flavivirus particles through the secretory pathway, a viral envelope glycoprotein, prm, is cleaved by the proprotein convertase furin; this cleavage is required for the subsequent rearrangement of receptor-binding e glycoprotein and for virus infectivity. similar to many furin substrates, prm in vector-borne flaviviruses contains basic residues at positions p1, p2, and p4 proximal to the cleavage site; in addition, a number of charged residues are found at position p3 and be ...200414963133
analysis of the 3' cis-acting elements of rubella virus by using replicons expressing a puromycin resistance gene.a rubella virus (rub) replicon, rubrep/pac, was constructed and used to map the 3' cis-acting elements (3' cse) of the rub genome required for rub replication. the rubrep/pac replicon had the structural protein open reading frame partially replaced by a puromycin acetyltransferase (pac) gene. cells transfected with rubrep/pac transcripts expressed the pac gene from the subgenomic rna, were rendered resistant to puromycin, and thus survived selection with this drug. the relative survival followin ...200414963158
structure of a flavivirus envelope glycoprotein in its low-ph-induced membrane fusion conformation.enveloped viruses enter cells via a membrane fusion reaction driven by conformational changes of specific viral envelope proteins. we report here the structure of the ectodomain of the tick-borne encephalitis virus envelope glycoprotein, e, a prototypical class ii fusion protein, in its trimeric low-ph-induced conformation. we show that, in the conformational transition, the three domains of the neutral-ph form are maintained but their relative orientation is altered. similar to the postfusion c ...200414963486
aven and bcl-xl enhance protection against apoptosis for mammalian cells exposed to various culture conditions.a balance between proliferation and cell death is critical for achieving desirable high cell densities in mammalian cell culture. in this study, we evaluate a recently discovered anti-apoptotic gene, aven, and examine its effectiveness alone and in combination with a member of the bcl-2 family, bcl-xl. the commercially popular cell line, chinese hamster ovary (cho), was genetically modified to constitutively express aven, bcl-xl, and the two genes in combination. cells were exposed to several mo ...200414966800
genes encoding pir51, beclin 1, rbap48 and aldolase b are up or down-regulated in human primary hepatocellular reveal new tumor markers and target genes from differentially expressed genes of primary tumor samples using cdna microarray.200414966907
secondary structure and function of the 5'-proximal region of the equine arteritis virus rna genome.nidoviruses produce an extensive 3'-coterminal nested set of subgenomic mrnas, which are used to express their structural proteins. in addition, arterivirus and coronavirus mrnas contain a common 5' leader sequence, derived from the genomic 5' end. the joining of this leader sequence to different segments (mrna bodies) from the genomic 3'-proximal region presumably involves a unique mechanism of discontinuous minus-strand rna synthesis. key elements in this process are the so-called transcriptio ...200414970388
comparison of the transmission potential of two genetically distinct sindbis viruses after oral infection of aedes aegypti (diptera: culicidae).within mosquitoes, arboviruses encounter barriers to infection and dissemination that are critical determinants of vector competence. the molecular mechanisms responsible for these barriers have yet to be elucidated. the prototype sindbis (sin) strain, ar339, and viruses derived from this strain, such as tr339 virus, have limited infection and transmission potential in the medically important arthropod vector, aedes aegypti (l.). however, the malaysian sin virus strain, mre16, disseminates in ne ...200414989352
requirements for ceacams and cholesterol during murine coronavirus cell entry.previous reports have documented that cholesterol supplementations increase cytopathic effects in tissue culture and also intensify in vivo pathogenicities during infection by the enveloped coronavirus murine hepatitis virus (mhv). to move toward a mechanistic understanding of these phenomena, we used growth media enriched with methyl-beta-cyclodextrin or cholesterol to reduce or elevate cellular membrane sterols, respectively. cholesterol depletions reduced plaque development 2- to 20-fold, dep ...200414990688
bluetongue virus outer capsid proteins are sufficient to trigger apoptosis in mammalian cells.bluetongue virus (btv) is transmitted by culicoides sp. biting midges to livestock, causing severe hemorrhagic disease in sheep, but is asymptomatic in the insect host. similarly, btv causes rapid cell death in infected mammalian cells in culture, whereas infections of insect cells are long-term and unapparent, despite productive virus replication. to assess whether apoptosis plays any role in these two distinct cell responses, we have investigated apoptosis in cultured insect and mammalian cell ...200414990706
cholesterol is required for endocytosis and endosomal escape of adenovirus type 2.the species c adenovirus type 2 (ad2) and ad5 bind the coxsackievirus b ad receptor and alphav integrin coreceptors and enter epithelial cells by clathrin-mediated endocytosis. this pathway is rapid and efficient. it leads to cell activation and the cholesterol-dependent formation of macropinosomes. macropinosomes are triggered to release their contents when incoming ad2 escapes from endosomes. here, we show that cholesterol extraction of epithelial cells by methyl-beta-cyclodextrin (mbetacd) tr ...200414990728
development of improved sindbis virus-based dna expression vector.we have constructed an improved dna expression vector based on the sindbis virus. several dna-based sindbis virus vectors were constructed to investigate the efficiency of transgene expression. these vectors, when transfected into mammalian cells, have been used to express heterologous genes. a recombinant genome of sindbis plasmid dna, in which the structural genes were replaced by a polylinker cassette to allow for insertion of heterologous genes, was placed under the control of a simian virus ...200415000747
alphavirus vectors for cancer gene therapy (review).alphaviruses have several characteristics that make them attractive as gene therapy vectors such as transient and high-level expression of a heterologous gene. alphavirus vectors, semliki forest virus (sfv), sindbis virus (sin) and venezuelan equine encephalitis virus (vee) have been developed as gene expression vectors. alphaviruses are positive-strand rna viruses that can mediate efficient cytoplasmic gene expression in mammalian cells. the alphavirus rna replication machinery has been enginee ...200415010830
transgenic mice expressing the nucleoprotein of borna disease virus in either neurons or astrocytes: decreased susceptibility to homotypic infection and disease.the nucleoprotein (n) of borna disease virus (bdv) is the major target of the disease-inducing antiviral cd8 t-cell response in the central nervous system of mice. we established two transgenic mouse lines which express bdv-n in either neurons (neuro-n) or astrocytes (astro-n). despite strong transgene expression, neurological disease or gross behavioral abnormalities were not observed in these animals. when neuro-n mice were infected as adults, replication of bdv was severely impaired and was r ...200415016883
a peptide from autoantigen la blocks poliovirus and hepatitis c virus cap-independent translation and reveals a single tyrosine critical for la rna binding and translation, a 52-kda autoantigen in patients with systemic lupus erythematosus, was one of the first cellular proteins identified to interact with viral internal ribosome entry site (ires) elements and stimulate poliovirus (pv) and hepatitis c virus (hcv) ires-mediated translation. previous results from our laboratory have shown that a small, yeast rna (irna) could selectively inhibit pv and hcv ires-mediated translation by sequestering the la protein. here we have identified an 18-amino-acid-long seque ...200415016896
rubella virus capsid protein modulates viral genome replication and virus infectivity.the structural proteins (sp) of the togaviridae can be deleted in defective interfering rnas. the dispensability of viral sp has allowed construction of noninfectious viral expression vectors and replicons from viruses of the alphavirus and rubivirus genera. nevertheless, in this study, we found that the sp of rubella virus (rub) could enhance expression of reporter genes from rub replicons in trans. sp enhancement required capsid protein (cp) expression and was not due to rna-rna recombination. ...200415047844
glutamate receptor antagonists protect from virus-induced neural degeneration.neuronal damage during acute viral encephalomyelitis can result directly from virus infection or indirectly from the host immune response to infection. in neurodegenerative diseases and stroke, neuronal death also can result from excess release of excitatory amino acid neurotransmitters, such as glutamate. to determine the role of glutamate excitotoxicity in fatal alphavirus-induced paralytic encephalomyelitis, we treated mice infected with neuroadapted sindbis virus (nsv) with antagonists of n- ...200415048893
attenuation of sindbis virus variants incorporating uncleaved pe2 glycoprotein is correlated with attachment to cell-surface heparan sulfate.sindbis virus virions incorporating uncleaved precursor envelope protein pe2 bind efficiently to cell-surface heparan sulfate (hs) because the furin cleavage site (a consensus hs-binding domain) is retained in the mature virus particle. however, they are essentially nonviable. resuscitating mutations selected in the e3 or e2 protein preserve the pe2 noncleaving phenotype and hs binding, but facilitate fusion, and thereby restore wild-type infectivity on cultured cells. here, we have demonstrated ...200415063111
apoptosis is essential for the increased efficacy of alphaviral replicase-based dna vaccines.alphaviral replicons can increase the efficacy and immunogenicity of naked nucleic acid vaccines. to study the impact of apoptosis on this increased effectiveness, we co-delivered an anti-apoptotic gene (bcl-x(l)) with the melanocyte/melanoma differentiation antigen trp-1. although cells co-transfected with bcl-x(l) lived longer, produced more antigen and elicited increased antibody production in vivo, co-delivery of pro-survival bcl-x(l) with antigen significantly reduced the ability of the rep ...200415063579
rna interference, arthropod-borne viruses, and mosquitoes.rna interference (rnai) probably functions as an antiviral mechanism in most eukaryotic organisms. variations in the activity of this antiviral pathway in mosquitoes could explain, in part, why some mosquitoes are competent vectors of medically important, arthropod-borne viruses (arboviruses) and others are not. there are three lines of evidence that show the rnai pathway exists in aedes species that transmit arboviruses. the first is that recombinant sindbis viruses expressing a rna fragment fr ...200415068882
the human membrane cofactor cd46 is a receptor for species b adenovirus serotype 3.many human adenovirus (ad) serotypes use the coxsackie b virus-ad receptor (car). recently, cd46 was suggested to be a receptor of species b ad serotype 11 (ad11), ad14, ad16, ad21, ad35, and ad50. using sindbis virus-mediated cdna library expression, we identify here the membrane cofactor protein cd46 as a surface receptor of species b ad3. all four major cd46 transcripts and one minor cd46 transcript expressed in nucleated human cells were isolated. rodent bhk cells stably expressing the bc1 f ...200415078926
the hypervariable region 1 of the e2 glycoprotein of hepatitis c virus binds to glycosaminoglycans, but this binding does not lead to infection in a pseudotype system.the hypervariable region 1 (hvr1) of hepatitis c virus (hcv) e2 envelope glycoprotein is a 27-amino-acid sequence located at its n terminus. in this study, we investigated the functional role of hvr1 for interaction with the mammalian cell surface. the c-terminal truncated e2 glycoprotein was appended to a transmembrane domain and cytoplasmic tail of vesicular stomatitis virus (vsv) g protein for generation of the chimeric e2-g gene construct. a deletion of the hvr1 sequence from e2 was created ...200415078928
genomic analysis of the host response to hepatitis b virus infection.previous studies in hepatitis b virus (hbv)-infected humans and chimpanzees suggest that control of hbv infection involves the cells, effector functions, and molecular mediators of the immune response. the objective of the current study was to identify, in the liver of acutely hbv-infected chimpanzees, the spectrum of virus-induced and immune response-related genes that regulate the infection. the results demonstrate that hbv does not induce any genes during entry and expansion, suggesting it is ...200415100412
human cytomegalovirus causes endothelial injury through the ataxia telangiectasia mutant and p53 dna damage signaling pathways.atherosclerosis is the leading cause of death in the united states, and human cytomegalovirus (hcmv), a member of the herpes virus family, may play a role in the development of the disease. we previously showed that hcmv regulated endothelial apoptosis. in this study, we investigated the induction of apoptosis and signal transduction pathways regulating this process in hcmv-infected endothelial cells. as observed previously, hcmv induced a typical cytopathic effect in human aortic endothelial ce ...200415105295
repelling class discrimination: ephrin-a5 binds to and activates ephb2 receptor signaling.the interactions between eph receptor tyrosine kinases and their ephrin ligands regulate cell migration and axon pathfinding. the epha receptors are generally thought to become activated by ephrin-a ligands, whereas the ephb receptors interact with ephrin-b ligands. here we show that two of the most widely studied of these molecules, ephb2 and ephrin-a5, which have never been described to interact with each other, do in fact bind one another with high affinity. exposure of ephb2-expressing cells ...200415107857
changes of the secondary structure of the 5' end of the sindbis virus genome inhibit virus growth in mosquito cells and lead to accumulation of adaptive mutations.both the 5' end of the sindbis virus (sin) genome and its complement in the 3' end of the minus-strand rna synthesized during virus replication serve as parts of the promoters recognized by the enzymes that comprise the replication complex (rdrp). in addition to the 5' untranslated region (utr), which was shown to be critical for the initiation of replication, another 5' sequence element, the 51-nucleotide (nt) conserved sequence element (cse), was postulated to be important for virus replicatio ...200415113874
regional differences in viral growth and central nervous system injury correlate with apoptosis.infection of neonatal mice with reovirus t3 dearing (t3d), the prototypic neurotropic reovirus, causes fatal encephalitis associated with neuronal injury and virus-induced apoptosis throughout the brain. t3d variant k (vark) is an antigenic variant that has a nearly 1 million-fold reduction in neurovirulence following intracerebral (i.c.) inoculation compared to t3d and a restricted pattern of central nervous system injury with damage limited to the hippocampus, sparing other brain regions. we w ...200415113925
receptor-dependent coronavirus infection of dendritic several mammalian species, including humans, coronavirus infection can modulate the host immune response. we show a potential role of dendritic cells (dc) in murine coronavirus-induced immune modulation and pathogenesis by demonstrating that the jaw sii dc line and primary dc from balb/c mice and p/p mice with reduced expression of the murine coronavirus receptor, murine ceacam1a, are susceptible to murine coronavirus infection by a receptor-dependent pathway.200415113927
human immunodeficiency virus type 1 envelope glycoproteins that lack cytoplasmic domain cysteines: impact on association with membrane lipid rafts and incorporation onto budding virus particles.the human immunodeficiency virus type 1 (hiv-1) envelope comprises a surface gp120 and a transmembrane gp41. the cytoplasmic domain of gp41 contains cysteine residues (c764 and c837) which are targets for palmitoylation and were reported to be required for envelope association with lipid rafts and assembly on budding virions (i. rousso, m. b. mixon, b. k. chen, and p. s. kim, proc. natl. acad. sci. usa 97:13523-13525, 2000). several infectious hiv-1 clones contain envelopes that have no gp41 cyt ...200415113929
axonal growth of embryonic stem cell-derived motoneurons in vitro and in motoneuron-injured adult rats.we generated spinal motoneurons from embryonic stem (es) cells to determine the developmental potential of these cells in vitro and their capacity to replace motoneurons in the adult mammalian spinal cord. es cell-derived motoneurons extended long axons, formed neuromuscular junctions, and induced muscle contraction when cocultured with myoblasts. we transplanted motoneuron-committed es cells into the spinal cords of adult rats with motoneuron injury and found that approximately 3,000 es cell-de ...200415118094
persistent infection and suppression of host response by alphaviruses.alphaviruses cause chronic noncytopathic infection in mosquito cells and develop a highly cytopathic infection in a wide variety of cells of vertebrate origin. upon infection, alphaviruses modify cellular processes to meet the virus needs for propagation. downregulation of translation and transcription caused by viral infection appears to reduce interferon (ifn) and cytokine gene expression and allows more efficient dissemination of infection. alphaviruses with mutations in nonstructural protein ...200415119769
photodynamic therapy: a new antimicrobial approach to infectious disease?photodynamic therapy (pdt) employs a non-toxic dye, termed a photosensitizer (ps), and low intensity visible light which, in the presence of oxygen, combine to produce cytotoxic species. pdt has the advantage of dual selectivity, in that the ps can be targeted to its destination cell or tissue and, in addition, the illumination can be spatially directed to the lesion. pdt has previously been used to kill pathogenic microorganisms in vitro, but its use to treat infections in animal models or pati ...200415122361
poliovirus rna-dependent rna polymerase (3dpol): pre-steady-state kinetic analysis of ribonucleotide incorporation in the presence of mg2+.we have solved the complete kinetic mechanism for correct nucleotide incorporation catalyzed by the rna-dependent rna polymerase from poliovirus, 3d(pol). the phosphoryl-transfer step is flanked by two isomerization steps. the first conformational change may be related to reorientation of the triphosphate moiety of the bound nucleotide, and the second conformational change may be translocation of the enzyme into position for the next round of nucleotide incorporation. the observed rate constant ...200415122878
processing of a pestivirus protein by a cellular protease specific for light chain 3 of microtubule-associated proteins.the genome of the cytopathogenic (cp) bovine viral diarrhea virus (bvdv) jacp contains a cellular insertion coding for light chain 3 (lc3) of microtubule-associated proteins, the mammalian homologue of yeast aut7p/apg8p. the cellular insertion induces cp bvdv-specific processing of the viral polyprotein by a cellular cysteine protease homologous to the known yeast protease aut2p/apg4p. three candidate bovine protease genes were identified on the basis of the sequence similarity of their products ...200415140988
virus inactivation and protein recovery in a novel ultraviolet-c reactor.ultraviolet-c (uvc) irradiation is a viral-inactivation method that was dismissed by many plasma fractionators as a result of the potential for protein damage and the difficulty in delivering uniform doses. a reactor with novel spiral flow hydraulic mixing was recently designed for uniform and controlled uvc treatment. the objective of this study was to investigate virus inactivation and protein recovery after treatment through the new reactor.200415144527
independent functions of hsp90 in neurotransmitter release and in the continuous synaptic cycling of ampa receptors.the delivery of neurotransmitter receptors into synapses is essential for synaptic function and plasticity. in particular, ampa-type glutamate receptors (ampa receptors) reach excitatory synapses according to two distinct routes: a regulated pathway, which operates transiently during synaptic plasticity, and a constitutive pathway, which maintains synaptic function under conditions of basal transmission. however, the specific mechanisms that distinguish these two trafficking pathways are essenti ...200415152036
inhibition of reovirus by mycophenolic acid is associated with the m1 genome segment.mycophenolic acid (mpa), an inhibitor of imp dehydrogenase, inhibits reovirus replication and viral rna and protein production. in mouse l929 cells, antiviral effects were greatest at 30 microg of mpa/ml. at this dosage, mpa inhibited replication of reovirus strain t3d more than 1,000-fold and inhibited replication of reovirus strain t1l nearly 100-fold, compared to non-drug-treated controls. genetic reassortant analysis indicated the primary determinant of strain-specific differences in sensiti ...200415163710
mcp-1 and ccr2 contribute to non-lymphocyte-mediated brain disease induced by fr98 polytropic retrovirus infection in mice: role for astrocytes in retroviral neuropathogenesis.virus infection of the central nervous system (cns) often results in chemokine upregulation. although often associated with lymphocyte recruitment, increased chemokine expression is also associated with non-lymphocyte-mediated cns disease. in these instances, the effect of chemokine upregulation on neurological disease is unclear. in vitro, several chemokines including monocyte chemotactic protein 1 (mcp-1) protect neurons from apoptosis. therefore, in vivo, chemokine upregulation may be a prote ...200415163738
regulation of semliki forest virus rna replication: a model for the control of alphavirus pathogenesis in invertebrate hosts.alphavirus nonstructural proteins are translated as a polyprotein that is ultimately cleaved into four mature proteins called nsp1, nsp2, nsp3, and nsp4 from their order in the polyprotein. the role of this nonstructural polyprotein, of cleavage intermediates, and of mature proteins in synthesis of semliki forest virus (sfv) rna has been studied using mutants unable to cleave one or more of the sites in the nonstructural polyprotein or that had the arginine sense codon between nsp3 and nsp4 chan ...200415165827
neuronal apoptosis pathways in sindbis virus encephalitis.sindbis virus infects neurons of the brain and spinal cord leading to neuronal apoptosis and encephalitis in mice. during postnatal development, neurons of mice remain susceptible to infection but become refractory to sv-induced programmed cell death. failure to undergo programmed cell death results in a persistent infection. however, some neurovirulent strains of sindbis virus overcome the age-dependent protective function in neurons, leading to enhanced apoptotic cell death in the central nerv ...200415171608
identification of sympathetic premotor neurons in medullary raphe regions mediating fever and other thermoregulatory functions.sympathetic premotor neurons directly control sympathetic preganglionic neurons (spns) in the intermediolateral cell column (iml) of the thoracic spinal cord, and many of these premotor neurons are localized in the medulla oblongata. the rostral ventrolateral medulla contains premotor neurons controlling the cardiovascular conditions, whereas rostral medullary raphe regions are a candidate source of sympathetic premotor neurons for thermoregulatory functions. here, we show that these medullary r ...200415190110
regulation of synaptic strength and ampa receptor subunit composition by pick1.pick1 (protein interacting with c kinase-1) regulates the surface expression of the ampa receptor (ampar) glur2 subunit, however, the functional consequences of this interaction are not well understood. previous work has suggested that pick1 promotes the internalization of ampars. however, we found that when pick1 is virally expressed in the ca1 region of hippocampal slices, it causes an increase in ampar-mediated epsc amplitude. this effect is associated with increased ampar rectification and s ...200415190111
identification of the amino acid sequence in sindbis virus nsp4 that binds to the promoter for the synthesis of the subgenomic rna.a gel mobility-shift assay was used to demonstrate the binding of the sindbis virus transcriptase to the promoter for the synthesis of subgenomic (sg) rna. the assay made use of a p15 fraction (the cell fraction that is pelleted at 15,000 x g) from cells infected with recombinant vaccinia virions expressing various sindbis virus nonstructural proteins (nsps) and a (32)p-labeled 24-mer oligoribonucleotide representing the minimal sequence with sg promoter activity. by itself, nsp4, the viral rna- ...200415197279
endemic venezuelan equine encephalitis in northern peru.since venezuelan equine encephalitis virus (veev) was isolated in peru in 1942, >70 isolates have been obtained from mosquitoes, humans, and sylvatic mammals primarily in the amazon region. to investigate genetic relationships among the peru veev isolates and between the peru isolates and other veev strains, a fragment of the pe2 gene was amplified and analyzed by single-stranded conformation polymorphism. representatives of seven genotypes underwent sequencing and phylogenetic analysis. the res ...200415200823
causative agent of pogosta disease isolated from blood and skin lesions.pogosta disease is a mosquito-borne viral disease in finland, which is clinically manifested by rash and arthritis; larger outbreaks occur in 7-year intervals. the causative agent of the disease has been suspected of being closely related to sindbis virus (sinv). we isolated sinv from five patients with acute pogosta disease during an outbreak in fall 2002 in finland. one virus strain was recovered from a whole blood sample and four other strains from skin lesions. the etiology of pogosta diseas ...200415200824
yellow fever outbreak, imatong, southern may 2003, the world health organization received reports about a possible outbreak of a hemorrhagic disease of unknown cause in the imatong mountains of southern sudan. laboratory investigations were conducted on 28 serum samples collected from patients in the imatong region. serum samples from 13 patients were positive for immunoglobulin m antibody to flavivirus, and serum samples from 5 patients were positive by reverse transcription-polymerase chain reaction with both the genus flavivirus- ...200415207058
conformational changes in sindbis virions resulting from exposure to low ph and interactions with cells suggest that cell penetration may occur at the cell surface in the absence of membrane fusion.alphaviruses have the ability to induce cell-cell fusion after exposure to acid ph. this observation has served as an article of proof that these membrane-containing viruses infect cells by fusion of the virus membrane with a host cell membrane upon exposure to acid ph after incorporation into a cell endosome. we have investigated the requirements for the induction of virus-mediated, low ph-induced cell-cell fusion and cell-virus fusion. we have correlated the ph requirements for this process to ...200415207623
doxorubicin and paclitaxel enhance the antitumor efficacy of vaccines directed against her 2/neu in a murine mammary carcinoma model.the purpose of the present study was to determine whether cytotoxic chemotherapeutic agents administered prior to immunotherapy with gene vaccines could augment the efficacy of the vaccines.200415217493
positive effect of the hepatitis c virus nonstructural 5a protein on viral multiplication.hepatitis c virus infection (hcv), is a major cause of liver disease worldwide, and are frequently resistant to the interferon alpha treatment. the nonstructural (ns) 5a protein of hcv has been proposed to be involved in this resistance. additional studies have pointed out a role for ns5a in several other cellular interactions as well as an important role of its adaptative mutations in hcv genome replication. however, no infectious system is available to assess the role of ns5a in the hcv life c ...200415221536
bad is a pro-survival factor prior to activation of its pro-apoptotic function.the mammalian bad protein belongs to the bh3-only subgroup of the bcl-2 family. in contrast to its known pro-apoptotic function, we found that endogenous and overexpressed bad(l) can inhibit cell death in neurons and other cell types. several mechanisms regulate the conversion of bad from an anti-death to a pro-death factor, including alternative splicing that produces the n-terminally truncated bad(s). in addition, caspases convert bad(l) into a pro-death fragment that resembles the short splic ...200415231831
input- and subunit-specific ampa receptor trafficking underlying long-term potentiation at hippocampal ca3 synapses.hippocampal ca3 pyramidal neurons receive synaptic inputs from both mossy fibres (mfs) and associational fibres (afs). long-term potentiation (ltp) at these synapses differs in its induction sites and n-methyl-d-aspartate receptor (nmdar) dependence. most evidence favours the presynaptic and postsynaptic mechanisms for induction of mf ltp and af ltp, respectively. this implies that molecular and functional properties differ between mf and af synapses at both presynaptic and postsynaptic sites. i ...200415245483
the pdz domain of pick1 differentially accepts protein kinase c-alpha and glur2 as interacting ligands.the c terminus (ct) of protein kinase c-alpha (pkcalpha) has a type i pdz binding motif, whereas glur2 has a type ii pdz binding motif. both motifs are recognized by the pdz domain of protein interacting with protein kinase c (pick1), and pick1-pkcalpha-controlled phosphorylation regulates the synaptic expression and function of glur2. here, we show that a specific mutation within the carboxylate-binding loop of the pdz domain of pick1 (k27e; pick1-ke) results in a loss of interaction with glur2 ...200415247289
cytokine-mediated downregulation of coxsackievirus-adenovirus receptor in endothelial cells.endothelial cells have the ability to change their complement of cell surface proteins in response to inflammatory cytokines. we hypothesized that the expression of the coxsackievirus-adenovirus receptor (car), a viral receptor and putative cell-cell adhesion molecule, may be altered during the response of endothelial cells to inflammation. to test this hypothesis, we evaluated car protein and mrna levels in human umbilical vein endothelial cells after they were exposed to tumor necrosis factor ...200415254176
role of cd8+ t cells in control of west nile virus infection.infection with west nile virus (wnv) causes fatal encephalitis more frequently in immunocompromised humans than in those with a healthy immune system. although a complete understanding of this increased risk remains unclear, experiments with mice have begun to define how different components of the adaptive and innate immune response function to limit infection. previously, we demonstrated that components of humoral immunity, particularly immunoglobulin m (igm) and igg, have critical roles in pr ...200415254203
inactivation of west nile virus, vaccinia virus and viral surrogates for relevant and emergent viral pathogens in plasma-derived products.human plasma is the source of a wide variety of therapeutic proteins, yet it is also a potential source of viral contamination. recent outbreaks of emergent viral pathogens, such as west nile virus, and the use of live vaccinia virus as a vaccine have prompted a reassessment of the viral safety of plasma-derived products. the purpose of this study was to evaluate the efficacy of current viral inactivation methods for west nile and vaccinia viruses and to reassess the use of model viruses to pred ...200415260817
venezuelan equine encephalitis emergence: enhanced vector infection from a single amino acid substitution in the envelope 1993 and 1996, subtype ie venezuelan equine encephalitis (vee) virus caused epizootics in the mexican states of chiapas and oaxaca. previously, only subtype iab and ic vee virus strains had been associated with major outbreaks of equine and human disease. the iab and ic epizootics are believed to emerge via adaptation of enzootic (sylvatic, equine-avirulent) strains for high titer equine viremia that results in efficient infection of mosquito vectors. however, experimental equine infections w ...200415277679
pkr-dependent and -independent mechanisms are involved in translational shutoff during sindbis virus infection.the replication of sindbis virus (sin) profoundly affects the metabolism of infected vertebrate cells. one of the main events during sin infection is the strong inhibition of translation of cellular mrnas. in this study, we used a combination of approaches, including the study of sin replication in pkr(-/-) mouse embryo fibroblasts or in the presence of an excess of catalytically inactive pkr. we show that the pkr-dependent inhibition of translation is not the only and most likely not the major ...200415280454
heparan sulfate-independent infection attenuates high-neurovirulence gdvii virus-induced encephalitis.the high-neurovirulence theiler's murine encephalomyelitis virus (tmev) strain gdvii uses heparan sulfate (hs) as a coreceptor to enter target cells. we report here that gdvii virus adapted to growth in hs-deficient cells exhibited two amino acid substitutions (r3126l and n1051s) in the capsid and no longer used hs as a coreceptor. infectious-virus yields in cho cells were 25-fold higher for the adapted virus than for the parental gdvii virus, and the neurovirulence of the adapted virus in intra ...200415280499
rapid preparative purification of west nile and sindbis virus pcr products utilizing a microbore anion-exchange column.analysis and purification of specific pcr products from pcr reactions can be problematic due to several issues relating to amplification and low product yield. the use of hplc as a preparative tool in pcr product analysis is common but has not replaced traditional electrophoretic techniques for purifying dna to be used in subsequent experiments. gel purification of pcr products can result in a net loss greater than 50% of the starting dna amount. thus, this method of recovery can become the limi ...200415288956
attenuation of dengue virus infection by adeno-associated virus-mediated sirna delivery.background: the need for safe and effective treatment of dengue virus (den), a class a agent that causes dengue hemorrhagic fever/dengue shock syndrome, has been a critical global priority. an effective vaccine for den is not yet available. in this study the possibility of attenuating den infection using adeno-associated virus (aav)-encoded short interfering rnas (sirna) was examined in vero cells and human dendritic cells (dcs). methods: a cassette encoding sirna targeted to a 3' untranslated s ...200415301687
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