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development of reverse transcription-pcr assays specific for detection of equine encephalitis viruses.specific and sensitive reverse transcription-pcr (rt-pcr) assays were developed for the detection of eastern, western, and venezuelan equine encephalitis viruses (eee, wee, and vee, respectively). tests for specificity included all known alphavirus species. the eee-specific rt-pcr amplified a 464-bp region of the e2 gene exclusively from 10 different eee strains from south and north america with a sensitivity of about 3,000 rna molecules. in a subsequent nested pcr, the specificity was confirmed ...200010747138
identification of a novel function of the alphavirus capping apparatus. rna 5'-triphosphatase activity of nsp2.both genomic and subgenomic rnas of the alphavirus have m(7)g(5')ppp(5')n (cap0 structure) at their 5' end. previously it has been shown that alphavirus-specific nonstructural protein nsp1 has guanine-7n-methyltransferase and guanylyltransferase activities needed in the synthesis of the cap structure. during normal cap synthesis the 5' gamma-phosphate of the nascent viral rna chain is removed by a specific rna 5'-triphosphatase before condensation with gmp, delivered by the guanylyltransferase. ...200010748213
clinical isolates of measles virus use cd46 as a cellular receptor.laboratory strains of measles viruses (mv), such as edmonston and halle, use the complement regulatory protein cd46 as a cell surface receptor. the receptor usage of clinical isolates of mv, however, remains unclear. receptor usage by primary patient isolates of mv was compared to isolates that had been passaged on a variety of tissue culture cell lines. all of the isolates could infect cells in a cd46-dependent manner, but their tropism was restricted according to cell type (e.g., lymphocytes v ...200010756008
subgenomic negative-strand rna function during mouse hepatitis virus infection.mouse hepatitis virus (mhv)-infected cells contain full-length and subgenomic-length positive- and negative-strand rnas. the origin and function of the subgenomic negative-strand rnas is controversial. in this report we demonstrate that the synthesis and molar ratios of subgenomic negative strands are similar in alternative host cells, suggesting that these rnas function as important mediators of positive-strand synthesis. using kinetic labeling experiments, we show that the full-length and subg ...200010756015
a single amino acid change in nsp1 attenuates neurovirulence of the sindbis-group alphavirus s.a.ar86.s.a.ar86, a member of the sindbis group of alphaviruses, is neurovirulent in adult mice and has a unique threonine at position 538 of nsp1; nonneurovirulent members of this group of alphaviruses encode isoleucine. isoleucine was introduced at position 538 in the wild-type s.a.ar86 infectious clone, ps55, and virus derived from this mutant clone, ps51, was significantly attenuated for neurovirulence compared to that derived from ps55. intracranial (i.c. ) s55 infection resulted in severe disease, ...200010756033
frequent homologous recombination events between molecules of one rna component in a multipartite rna virus.brome mosaic bromovirus (bmv), a tripartite plus-sense rna virus, has been used as a model system to study homologous rna recombination among molecules of the same rna component. pairs of bmv rna3 variants carrying marker mutations at different locations were coinoculated on a local lesion host, and the progeny rna3 in a large number of lesions was analyzed. the majority of doubly infected lesions accumulated the rna3 recombinants. the distribution of the recombinant types was relatively even, i ...200010756034
a single deletion in the membrane-proximal region of the sindbis virus glycoprotein e2 endodomain blocks virus assembly.the envelopment of the sindbis virus nucleocapsid in the modified cell plasma membrane involves a highly specific interaction between the capsid (c) protein and the endodomain of the e2 glycoprotein. we have previously identified a domain of the sindbis virus c protein involved in binding to the e2 endodomain (h. lee and d. t. brown, virology 202:390-400, 1994). the c-e2 binding domain resides in a hydrophobic cleft with c y180 and w247 on opposing sides of the cleft. structural modeling studies ...200010756035
the use of chimeric venezuelan equine encephalitis viruses as an approach for the molecular identification of natural virulence determinants.venezuelan equine encephalitis (vee) virus antigenic subtypes and varieties are considered either epidemic/epizootic or enzootic. in addition to epidemiological differences between the epidemic and enzootic viruses, several in vitro and in vivo laboratory markers distinguishing the viruses have been identified, including differential plaque size, sensitivity to interferon (ifn), and virulence for guinea pigs. these observations have been shown to be useful predictors of natural, equine virulence ...200010756040
nucleic acid-dependent cross-linking of the nucleocapsid protein of sindbis virus.the assembly of the alphavirus nucleocapsid core is a multistep event requiring the association of the nucleocapsid protein with nucleic acid and the subsequent oligomerization of capsid proteins into an assembled core particle. although the mechanism of assembly has been investigated extensively both in vivo and in vitro, no intermediates in the core assembly pathway have been identified. through the use of both truncated and mutant sindbis virus nucleocapsid proteins and a variety of cross-lin ...200010756045
stable high-level expression of heterologous genes in vitro and in vivo by noncytopathic dna-based kunjin virus replicon vectors.primary features of the flavivirus kunjin (kun) subgenomic replicons include continuous noncytopathic replication in host cell cytoplasm and the ability to be encapsidated into secreted virus-like particles (vlps). previously we reported preparation of rna-based kun replicon vectors and expression of heterologous genes (hg) in cell culture after rna transfection or after infection with recombinant kun vlps (a. n. varnavski and a. a. khromykh, virology 255:366-375, 1999). in this study we describ ...200010756054
evaluation of recombinant alphaviruses as vectors in gene therapy.alphavirus vectors based on sindbis virus and semliki forest virus (sfv) were characterized as potential gene transfer vectors. initial studies were performed using vectors engineered to transfer either lacz or green fluorescent protein (gfp). high levels of gene transfer were achieved in human primary fibroblasts, bhk and 293t cells, with low levels of transduction observed in more than 20 other target cells. alphavirus-based expression was generally very high, but transient in every cell type. ...200010757020
antiviral effect of nitric oxide during japanese encephalitis virus infection.the ability of japanese encephalitis virus (jev) and jev-induced macrophage derived neutrophil chemotactic factor (mdf) to produce nitric oxide (no), and the possible antiviral effect of no during jev infection, was investigated. splenic macrophages of jev infected mice produced maximum no in vivo at day 7 post infection, and in vitro at 24 h after jev stimulation. mdf-induced no production was dose dependent and maximal at 60 min after mdf treatment. the response was sensitive to anti-mdf antib ...200010762444
isolation and complete nucleotide sequence of a chinese sindbis-like virus.infection with alphaviruses is common in the chinese population. here we report the isolation of a sindbis-like virus from a pool of anopheles mosquitoes collected in xinjiang, china during an arbovirus survey. this virus, designated xj-160, rapidly produced cytopathic effects on mosquito and hamster cells. in addition, it was lethal to neonatal mice if inoculated intracerebrally. serologically, xj-160 reacted with and was neutralized by an anti-sindbis antibody. anti-xj-160 antibodies were foun ...200010769078
maintenance of cd8(+) t-cell memory following infection with recombinant sindbis and vaccinia viruses.cd8(+) t-cell memory is critical for protection against pathogens poorly controlled by humoral immunity. to characterize two distinct vaccine vectors, the acute and memory cd8(+) t-cell responses to an hiv-1 epitope (p18) expressed by recombinant vaccinia (vp18) and sindbis (sinp18) viruses were compared. whereas 9 to 13% of cd8(+) splenocytes were p18 specific during the acute response to vp18, 4% were induced by sinp18 as revealed by class i tetramer staining. increased t-cell activation by vp ...200010772979
replicase complex genes of semliki forest virus confer lethal neurovirulence.semliki forest virus (sfv) is a mosquito-transmitted pathogen of small rodents, and infection of adult mice with sfv4, a neurovirulent strain of sfv, leads to lethal encephalitis in a few days, whereas mice infected with the avirulent a7(74) strain remain asymptomatic. in adult neurons, a7(74) is unable to form virions and hence does not reach a critical threshold of neuronal damage. to elucidate the molecular mechanisms of neurovirulence, we have cloned and sequenced the entire 11,758-nucleotid ...200010775594
alkylation of cysteine-containing peptides to mimic palmitoylation.numerous proteins that are involved in cell signaling and viral replication require post-translational modification by palmitoylation to function properly. the molecular details by which this palmitoyl modification affects protein function remain poorly understood. to facilitate in vitro biochemical and structural studies of the role of palmitoylation on protein function, a method was developed for alkylating peptides with saturated c16 groups at cysteine residues and demonstrated using peptides ...200010784030
standardization of immunoglobulin m capture enzyme-linked immunosorbent assays for routine diagnosis of arboviral infections.immunoglobulin m antibody-capture enzyme-linked immunosorbent assay (mac-elisa) is a rapid and versatile diagnostic method that readily permits the combination of multiple assays. test consolidation is especially important for arthropod-borne viruses (arboviruses) which belong to at least three virus families: the togaviridae, flaviviridae, and bunyaviridae. using prototype viruses from each of these families and a panel of well-characterized human sera, we have evaluated and standardized a comb ...200010790107
modulation of dengue virus infection in human cells by alpha, beta, and gamma interferons.a role for interferon (ifn) in modulating infection by dengue virus (dv) has been suggested by studies in dv-infected patients and ifn receptor-deficient mice. to address how ifn modulates dv type 2 infection, we have assayed ifn-alpha, -beta, and -gamma for the ability to enhance or diminish antibody-independent and antibody-dependent cell infection using a competitive, asymmetric reverse transcriptase-mediated pcr (rt-pcr) assay that quantitates positive and negative strands of viral rna, a fl ...200010799569
assembly of the coronavirus envelope: homotypic interactions between the m proteins.the viral membrane proteins m and e are the minimal requirements for the budding of coronavirus particles. since the e protein occurs in particles only in trace amounts, the lateral interactions between the m proteins apparently generate the major driving force for envelope formation. by using coimmunoprecipitation and envelope incorporation assays, we provide extensive evidence for the existence of such m-m interactions. in addition, we determined which domains of the m protein are involved in ...200010799570
antiapoptotic herpesvirus bcl-2 homologs escape caspase-mediated conversion to proapoptotic proteins.the antiapoptotic bcl-2 and bcl-x(l) proteins of mammals are converted into potent proapoptotic factors when they are cleaved by caspases, a family of apoptosis-inducing proteases (e. h.-y. cheng, d. g. kirsch, r. j. clem, r. ravi, m. b. kastan, a. bedi, k. ueno, and j. m. hardwick, science 278:1966-1968, 1997; r. j. clem, e. h.-y. cheng, c. l. karp, d. g. kirsch, k. ueno, a. takahashi, m. b. kastan, d. e. griffin, w. c. earnshaw, m. a. veliuona, and j. m. hardwick, proc. natl. acad. sci. usa 95 ...200010799576
host protein interactions with the 3' end of bovine coronavirus rna and the requirement of the poly(a) tail for coronavirus defective genome replication.rna viruses have 5' and 3' untranslated regions (utrs) that contain specific signals for rna synthesis. the coronavirus genome is capped at the 5' end and has a 3' utr that consists of 300 to 500 nucleotides (nt) plus a poly(a) tail. to further our understanding of coronavirus replication, we have begun to examine the involvement of host factors in this process for two group ii viruses, bovine coronavirus (bcv) and mouse hepatitis coronavirus (mhv). specific host protein interactions with the bc ...200010799579
cell fusion activity of hepatitis c virus envelope proteins.to examine the cell fusion activity of hepatitis c virus (hcv) envelope proteins (e1 and e2), we have established a sensitive cell fusion assay based on the activation of a reporter gene as described previously (o. nussbaum, c. c. broder, and e. a. berger, j. virol. 68:5411-5422, 1994). the chimeric hcv e1 and e2 proteins, each consisting of the ectodomain of the e1 and e2 envelope protein and the transmembrane and cytoplasmic domains of the vesicular stomatitis virus g glycoprotein, were expres ...200010799580
mov34 protein from mouse brain interacts with the 3' noncoding region of japanese encephalitis virus.the plus-sense rna genome of japanese encephalitis virus (jev) contains noncoding regions (ncrs) of 95 and 585 bases at its 5' and 3' ends, respectively. the last 83 nucleotides of the 3'-ncr are predicted to form stable stem-loop (sl) structures. the shape of this 3'-sl structure is highly conserved among divergent flaviviruses even though only small stretches of nucleotide sequence contained within these structures are conserved. these sl structures have been predicted to function as cis-actin ...200010799585
mutational analysis of the rubella virus nonstructural polyprotein and its cleavage products in virus replication and rna synthesis.rubella virus nonstructural proteins, translated from input genomic rna as a p200 polyprotein and subsequently processed into p150 and p90 by an intrinsic papain-like thiol protease, are responsible for virus replication. to examine the effect of p200 processing on virus replication and to study the roles of nonstructural proteins in viral rna synthesis, we introduced into a rubella virus infectious cdna clone a panel of mutations that had variable defective effects on p200 processing. the virus ...200010799588
activation of divergent neuronal cell death pathways in different target cell populations during neuroadapted sindbis virus infection of mice.infection of adult mice with neuroadapted sindbis virus (nsv) results in a severe encephalomyelitis accompanied by prominent hindlimb paralysis. we find that the onset of paralysis parallels morphologic changes in motor neuron cell bodies in the lumbar spinal cord and in motor neuron axons in ventral nerve roots, many of which are eventually lost over time. however, unlike nsv-induced neuronal cell death found in the brain of infected animals, the loss of motor neurons does not appear to be apop ...200010799613
altered activity, social behavior, and spatial memory in mice lacking the ntan1p amidase and the asparagine branch of the n-end rule pathway.the n-end rule relates the in vivo half-life of a protein to the identity of its n-terminal residue. n-terminal asparagine and glutamine are tertiary destabilizing residues, in that they are enzymatically deamidated to yield secondary destabilizing residues aspartate and glutamate, which are conjugated to arginine, a primary destabilizing residue. n-terminal arginine of a substrate protein is bound by the ubr1-encoded e3alpha, the e3 component of the ubiquitin-proteasome-dependent n-end rule pat ...200010805755
a novel membrane-anchored rab5 interacting protein required for homotypic endosome fusion.the ras-related gtpase rab5 is rate-limiting for homotypic early endosome fusion. we used a yeast two-hybrid screen to identify a rab5 interacting protein, rab5ip. the cdna sequence encodes a ubiquitous 75-kda protein with an n-terminal transmembrane domain (tm), a central coiled-coil structure, and a c-terminal region homologous to several centrosome-associated proteins. rab5ip lacking the transmembrane domain (rab5iptm(-)) had a greater affinity in vitro for rab5-guanosine 5'-o-2-(thio)diphosp ...200010818110
rubella virus nonstructural protein protease domains involved in trans- and cis-cleavage activities.rubella virus (rv) genomic rna contains two large open reading frames (orfs): a 5'-proximal orf encoding nonstructural proteins (nsps) that function primarily in viral rna replication and a 3'-proximal orf encoding the viral structural proteins. proteolytic processing of the rv nsp orf translation product p200 is essential for viral replication. processing of p200 to two mature products (p150 and p90) in the order nh(2)-p150-p90-cooh is carried out by an rv-encoded protease residing in the c-ter ...200010823845
the surface conformation of sindbis virus glycoproteins e1 and e2 at neutral and low ph, as determined by mass spectrometry-based mapping.sindbis virus contains two membrane glycoproteins, e1 and e2, which are organized into 80 trimers of heterodimers (spikes). these trimers form a precise t=4 icosahedral protein lattice on the surface of the virus. very little is known about the organization of the e1 and e2 glycoproteins within the spike trimer. to gain a better understanding of how the proteins e1 and e2 are arranged in the virus membrane, we have used the techniques of limited proteolysis and amino acid chemical modification i ...200010823875
self-amplifying vectors for gene delivery.current methods of gene transfer have a rather low efficiency, especially in vivo. therefore, one tries to achieve the highest possible levels of expression in the few cells that do take up foreign dna. one approach is to use self-amplifying expression vectors. these vectors are based on the (+)-strand rna viruses (alphaviruses) sindbis virus and semliki forest virus. in these vectors, the viral capsid protein coding sequences are replaced with the gene of interest. after introduction into the t ...199710837547
transplacental drug delivery: gene and virus delivery to the trophoblast.the development of successful strategies for delivering genes to the placenta may provide new opportunities for modifying trophoblast function in order to learn more about trophoblast physiology and to offer novel therapeutic options for complications of pregnancy that result from placental dysfunction. replication-deficient recombinant viral vectors are useful vehicles for introducing genes into cells in vitro and in vivo. recombinant adenovirus and herpes simplex virus vectors are unable to ef ...199910837747
characterization of the zinc binding activity of the rubella virus nonstructural protease.the rubella virus (rub) nonstructural (ns) protein (nsp) orf encodes a protease that cleaves the nsp precursor (240 kda) at a single site to produce two products. a cleavage site mutation was introduced into a rub infectious cdna clone and found to be lethal, demonstrating that cleavage of the nsp precursor is necessary for rub replication. based on computer alignments, the rub ns protease was predicted to be a papain-like cysteine protease (pcp) with the residues cys1152 and his1273 as the cata ...200010846076
a positive-strand rna virus with three very different subgenomic rna promoters.numerous rna viruses generate subgenomic mrnas (sgrnas) for expression of their 3'-proximal genes. a major step in control of viral gene expression is the regulation of sgrna synthesis by specific promoter elements. we used barley yellow dwarf virus (bydv) as a model system to study transcriptional control in a virus with multiple sgrnas. bydv generates three sgrnas during infection. the sgrna1 promoter has been mapped previously to a 98-nucleotide (nt) region which forms two stem-loop structure ...200010846080
the role of cd8(+) t cells and major histocompatibility complex class i expression in the central nervous system of mice infected with neurovirulent sindbis virus.little is known about the role of cd8(+) t cells infiltrating the neural parenchyma during encephalitis induced by neurovirulent sindbis virus (nsv). nsv preferentially infects neurons in the mouse brain and spinal cord; however, it is generally accepted that neurons can express few if any major histocompatibility complex (mhc) class i molecules. we evaluated the possible roles and interactions of cd8(+) t cells during nsv encephalitis and demonstrated that mhc class i antigen (h2k/d) was expres ...200010846095
differences between c57bl/6 and balb/cby mice in mortality and virus replication after intranasal infection with neuroadapted sindbis virus.neuroadapted sindbis virus (nsv), given intranasally, caused fatal encephalitis in 100% of adult c57bl/6 mice and 0% of balb/cby mice. most c57bl/6 mice developed severe kyphoscoliosis followed by hind-limb paralysis, while balb/cby mice did not. in situ hybridization for detecting nsv rna and immunohistochemistry for detecting nsv antigen indicated that virus delivered by this route infected neurons of the olfactory region and spread caudally without infection of ependymal cells. virus antigen ...200010846099
generation and characterization of a hepatitis c virus ns3 protease-dependent bovine viral diarrhea virus.unique to pestiviruses, the n-terminal protein encoded by the bovine viral diarrhea virus (bvdv) genome is a cysteine protease (npro) responsible for a self-cleavage that releases the n terminus of the core protein (c). this unique protease is dispensable for viral replication, and its coding region can be replaced by a ubiquitin gene directly fused in frame to the core. to develop an antiviral assay that allows the assessment of anti-hepatitis c virus (hcv) ns3 protease inhibitors, a chimeric b ...200010864644
selective membrane permeabilization by the rotavirus vp5* protein is abrogated by mutations in an internal hydrophobic domain.rotavirus infectivity is dependent on the proteolytic cleavage of the vp4 spike protein into vp8* and vp5* proteins. proteolytically activated virus, as well as expressed vp5*, permeabilizes membranes, suggesting that cleavage exposes a membrane-interactive domain of vp5* which effects rapid viral entry. the vp5* protein contains a single long hydrophobic domain (vp5*-hd, residues 385 to 404) at an internal site. in order to address the role of the vp5*-hd in permeabilizing cellular membranes, w ...200010864647
glycosphingolipids promote entry of a broad range of human immunodeficiency virus type 1 isolates into cell lines expressing cd4, cxcr4, and/or ccr5.treatment of human osteosarcoma cells, expressing cd4 and various chemokine receptors, with the glucosylceramide synthase inhibitor 1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol (ppmp), blocked target membrane glycosphingolipid (gsl) biosynthesis and reduced the susceptibility of cells to infection and fusion mediated by envelope glycoproteins from a variety of human immunodeficiency virus type 1 (hiv-1) isolates that utilize cxcr4 and/or ccr5. ppmp treatment of the cell lines did not sig ...200010864648
sindbis virus entry into cells triggers apoptosis by activating sphingomyelinase, leading to the release of ceramide.sindbis virus (sv) causes acute encephalomyelitis by infecting and inducing the death of neurons. induction of apoptosis occurs during virus entry and involves acid-induced conformational changes in the viral surface glycoproteins and sphingomyelin (sm)-dependent fusion of the virus envelope with the endosomal membrane. we have studied neuroblastoma cells to determine how this entry process triggers cell death. acidic sphingomyelinase was activated during entry followed by activation of neutral ...200010864654
the fusion glycoprotein of human respiratory syncytial virus facilitates virus attachment and infectivity via an interaction with cellular heparan sulfate.human respiratory syncytial virus (rsv) f glycoprotein (rsv-f) can independently interact with immobilized heparin and facilitate both attachment to and infection of cells via an interaction with cellular heparan sulfate. rsv-glycosaminoglycan (gag) interactions were evaluated using heparin-agarose affinity chromatography. rsv-f from a2- and b1/cp-52 (cp-52)-infected cell lysates, rsv-f derived from a recombinant vaccinia virus, and affinity-purified f protein all bound to and were specifically ...200010864656
trypsin-induced structural transformation in aquareovirus.aquareovirus, a member of the family reoviridae, is a large virus with multiple capsid layers surrounding a genome composed of 11 segments of double-stranded rna. biochemical studies have shown that treatment with the proteolytic agent trypsin significantly alters the infectivity of the virus. the most infectious stage of the virus is produced by a 5-min treatment with trypsin. however, prolonged trypsin treatment almost completely abolishes the infectivity. we have used three-dimensional electr ...200010864668
a cytoplasmic rna vector derived from nontransmissible sendai virus with efficient gene transfer and expression.we have recovered a virion from defective cdna of sendai virus (sev) that is capable of self-replication but incapable of transmissible-virion production. this virion delivers and expresses foreign genes in infected cells, and this is the first report of a gene expression vector derived from a defective viral genome of the paramyxoviridae. first, functional ribonucleoprotein complexes (rnps) were recovered from sev cloned cdna defective in the f (envelope fusion protein) gene, in the presence of ...200010864670
construction and characterization of monoclonal antibodies against western equine encephalitis virus.a repertoire of mouse monoclonal antibodies (mabs) against western equine encephalitis virus (wee) was constructed and characterized. anti-wee antibodies were expressed from hybridomas and purified by protein g chromatography. each of the antibodies was functionally assessed by indirect enzyme-linked immunosorbent assays (elisas), western blotting, and immunoprecipitations. all antibodies bound to wee antigen in elisas, whereas only a subgroup of antibodies was found to be active in western blot ...200010868791
a mutant of sindbis virus that is resistant to pyrazofurin encodes an altered rna polymerase.pyrazofurin (pzf), a cytidine analog and an inhibitor of orotate monophosphate decarboxylase, has been shown to decrease the levels of utp and ctp in treated cells. when sindbis virus (sv)-infected aedes albopictus cells were treated with pzf, the yield of virus was reduced 100- to 1000-fold. by serial passage of our standard sv(std) in ae. albopictus cells in the presence of increasing concentrations of pzf, a mutant, sv(pzf), was derived, which was not inhibited by pzf. sv(pzf) is also resista ...200010873749
effects of palmitoylation of replicase protein nsp1 on alphavirus infection.the membrane-associated alphavirus rna replication complex contains four virus-encoded subunits, the nonstructural proteins nsp1 to nsp4. semliki forest virus (sfv) nsp1 is hydrophobically modified by palmitoylation of cysteines 418 to 420. here we show that sindbis virus nsp1 is also palmitoylated on the same site (cysteine 420). when mutations preventing nsp1 palmitoylation were introduced into the genomes of these two alphaviruses, the mutant viruses remained viable and replicated to high tit ...200010888610
measles virus matrix protein specifies apical virus release and glycoprotein sorting in epithelial cells.in polarized epithelial cells measles virus (mv) is predominantly released at the apical cell surface, irrespective of the sorting of its two envelope glycoproteins f and h. it has been reported previously that the viral matrix (m) protein modulates the fusogenic capacity of the viral envelope glycoproteins. here, extant mv mutants and chimeras were used to determine the role of m protein in the transport of viral glycoproteins and release of progeny virions in polarized epithelial caco2 cells. ...200010899112
sindbis virus vector system for functional analysis of apoptosis regulators. 200010914042
ns3 serine protease of bovine viral diarrhea virus: characterization of active site residues, ns4a cofactor domain, and protease-cofactor interactions.the gene expression of bovine viral diarrhea virus (bvdv), a pestivirus, occurs via translation of a hypothetical polyprotein that is processed cotranslationally and posttranslationally by viral and cellular enzymes. a protease located in the n-terminal region of nonstructural (ns) protein ns3 catalyzes the cleavages, leading to the release of ns4a, ns4b, ns5a, and ns5b. our study provides experimental evidence that histidine at position 1658 and aspartic acid at position 1686 constitute togethe ...200010915606
semliki forest virus budding: assay, mechanisms, and cholesterol requirement.all enveloped viruses must bud through a cellular membrane in order to acquire their lipid bilayer, but little is known about this important stage in virus biogenesis. we have developed a quantitative biochemical assay to monitor the budding of semliki forest virus (sfv), an enveloped alphavirus that buds from the plasma membrane in a reaction requiring both viral spike proteins and nucleocapsid. the assay was based on cell surface biotinylation of newly synthesized virus spike proteins and retr ...200010933676
infection of human cells by dengue virus is modulated by different cell types and viral strains.although prior studies have investigated cellular infection by dengue virus (dv), many have used highly passaged strains. we have reassessed cellular infection by dv type 2 (dv2) using prototype and low-passage isolates representing genotypes from different geographic areas. we observed marked variation in the susceptibility to infection among cell types by different dv2 strains. hepg2 hepatoma cells were susceptible to infection by all dv2 strains assayed. although the prototype strain generate ...200010933688
mutations in the 5' nontranslated region of bovine viral diarrhea virus result in altered growth characteristics.the 5' nontranslated region (ntr) of pestiviruses functions as an internal ribosome entry site (ires) that mediates cap-independent translation of the viral polyprotein and probably contains additional cis-acting rna signals involved in crucial processes of the viral life cycle. computer modeling suggests that the 5'-terminal 75 nucleotides preceding the ires element form two stable hairpins, ia and ib. spontaneous and engineered mutations located in the genomic region comprising ia and ib were ...200010933696
identification of mouse hepatitis virus papain-like proteinase 2 activity.mouse hepatitis virus (mhv) is a 31-kb positive-strand rna virus that is replicated in the cytoplasm of infected cells by a viral rna-dependent rna polymerase, termed the replicase. the replicase is encoded in the 5'-most 22 kb of the genomic rna, which is translated to produce a polyprotein of >800 kda. the replicase polyprotein is extensively processed by viral and perhaps cellular proteinases to give rise to a functional replicase complex. to date, two of the mhv replicase-encoded proteinases ...200010933699
membrane interface-interacting sequences within the ectodomain of the human immunodeficiency virus type 1 envelope glycoprotein: putative role during viral fusion.we have identified a region within the ectodomain of the fusogenic human immunodeficiency virus type 1 (hiv-1) gp41, different from the fusion peptide, that interacts strongly with membranes. this conserved sequence, which immediately precedes the transmembrane anchor, is not highly hydrophobic according to the kyte-doolittle hydropathy prediction algorithm, yet it shows a high tendency to partition into the membrane interface, as revealed by the wimley-white interfacial hydrophobicity scale. we ...200010933713
chimeric yellow fever/dengue virus as a candidate dengue vaccine: quantitation of the dengue virus-specific cd8 t-cell response.we have constructed a chimeric yellow fever/dengue (yf/den) virus, which expresses the premembrane (prm) and envelope (e) genes from den type 2 (den-2) virus in a yf virus (yfv-17d) genetic background. immunization of balb/c mice with this chimeric virus induced a cd8 t-cell response specific for the den-2 virus prm and e proteins. this response protected yf/den virus-immunized mice against lethal dengue encephalitis. control mice immunized with the parental yfv-17d were not protected against de ...200010933719
characterization of the coronavirus m protein and nucleocapsid interaction in infected cells.coronavirus contains three envelope proteins, m, e and s, and a nucleocapsid, which consists of genomic rna and n protein, within the viral envelope. we studied the macromolecular interactions involved in coronavirus assembly in cells infected with a murine coronavirus, mouse hepatitis virus (mhv). coimmunoprecipitation analyses demonstrated an interaction between n protein and m protein in infected cells. pulse-labeling experiments showed that newly synthesized, unglycosylated m protein interac ...200010933723
sex differences in seoul virus infection are not related to adult sex steroid concentrations in norway rats.field studies of hantavirus infection in rodents report that a higher percentage of infected individuals are males than females. to determine whether males were more susceptible to hantavirus infection than females, adult male and female long evans rats (rattus norvegicus) were inoculated with doses of seoul virus ranging from 10(-4) to 10(6) pfu. the 50% infective doses (id(50)) were not significantly different for male and female rats (10(0.05) and 10(0.8) pfu, respectively). to determine whet ...200010933735
densovirinae as gene transfer vehicles.densoviruses present an attractive opportunity to develop expression vectors for insects. they exhibit several features that are beneficial for such vectors. the genomes of densoviruses are among the smallest of animal dna viruses, and, therefore are easier to use for cloning and transfection procedures. the fact that cloned densovirus genomes are infectious greatly simplifies their applications. it seems that both densovirus promoters have high constitutive activity and they can be further tran ...200010941569
specific ablation of antiviral gene expression in macrophages by antibody-dependent enhancement of ross river virus infection.ross river virus (rrv) is an indigenous australian arthropod-borne alphavirus responsible for epidemic polyarthritis (epa), myalgia, and lethargy in humans. macrophages and monocytes have been associated with human rrv disease, and previous studies have shown that rrv is capable of infecting macrophages via both a natural virus receptor and by fc receptor-mediated antibody-dependent enhancement (ade). similar to other viruses, such as human immunodeficiency virus and dengue virus, ade infection ...200010954537
potential dengue virus-triggered apoptotic pathway in human neuroblastoma cells: arachidonic acid, superoxide anion, and nf-kappab are sequentially involved.direct in vivo evidence for the susceptibility of human neuronal cells to dengue virus has not been reported. in this study, we demonstrated that type 2 dengue (den-2) virus infection induced extensive apoptosis in the human neuroblastoma cell line sk-n-sh. phospholipase a(2) (pla(2)) was activated by den-2 infection, which led to the generation of arachidonic acid (aa). inhibition of pla(2) activity by the pla(2) inhibitors, aacocf(3) and ono-rs-082, diminished den-2 virus-induced apoptosis. in ...200010954569
influenza virus assembly: effect of influenza virus glycoproteins on the membrane association of m1 protein.influenza virus matrix protein (m1), a critical protein required for virus assembly and budding, is presumed to interact with viral glycoproteins on the outer side and viral ribonucleoprotein on the inner side. however, because of the inherent membrane-binding ability of m1 protein, it has been difficult to demonstrate the specific interaction of m1 protein with hemagglutinin (ha) or neuraminidase (na), the influenza virus envelope glycoproteins. using triton x-100 (tx-100) detergent treatment o ...200010954572
inactivation and clearance of viruses during the manufacture of high purity factor ix.haemophilia is a bleeding disorder characterised by a deficiency in factor ix. replacement therapy in the form of a factor ix concentrate is a widely accepted practice. in this paper we describe a double virus inactivated chromatographic process for producing a high purity factor ix product, monofix((r))-vf. the process involves separation of the prothrombin complex by cryoprecipitation, fraction i precipitation and deae-cellulose adsorption, further ion-exchange chromatography of crude factor i ...200010964439
activation-dependent degradation of protein kinase c eta.prolonged activation of protein kinase cs (pkcs) by long-term treatment of cells with phorbol ester tumor promoters down-regulates the expression of many pkcs. to investigate the molecular mechanisms involved in the down-regulation of pkc eta, we expressed the novel pkcs eta and θ and various mutant forms in baby hamster kidney cells. upon overexpression, constitutively active pkc eta, but not wild type or kinase-dead pkc eta, underwent rapid degradation to generate several lower molecular ...200010980600
helicase and capping enzyme active site mutations in brome mosaic virus protein 1a cause defects in template recruitment, negative-strand rna synthesis, and viral rna capping.brome mosaic virus (bmv) encodes two rna replication proteins: 1a, which contains rna capping and helicase-like domains, and 2a, which is related to polymerases. bmv 1a and 2a can direct virus-specific rna replication in the yeast saccharomyces cerevisiae, which reproduces the known features of bmv replication in plant cells. we constructed single amino acid point mutations at the predicted capping and helicase active sites of 1a and analyzed their effects on bmv rna3 replication in yeast. the h ...200010982322
substitutions at the putative receptor-binding site of an encephalitic flavivirus alter virulence and host cell tropism and reveal a role for glycosaminoglycans in entry.the flavivirus receptor-binding domain has been putatively assigned to a hydrophilic region (fg loop) in the envelope (e) protein. in some flaviviruses this domain harbors the integrin-binding motif arg-gly-asp (rgd). one of us has shown earlier that host cell adaptation of murray valley encephalitis virus (mve) can result in the selection of attenuated variants altered at e protein residue asp(390), which is part of an rgd motif. here, a full-length, infectious cdna clone of mve was constructed ...200010982329
remodeling the endoplasmic reticulum by poliovirus infection and by individual viral proteins: an autophagy-like origin for virus-induced vesicles.all positive-strand rna viruses of eukaryotes studied assemble rna replication complexes on the surfaces of cytoplasmic membranes. infection of mammalian cells with poliovirus and other picornaviruses results in the accumulation of dramatically rearranged and vesiculated membranes. poliovirus-induced membranes did not cofractionate with endoplasmic reticulum (er), lysosomes, mitochondria, or the majority of golgi-derived or endosomal membranes in buoyant density gradients, although changes in io ...200010982339
efficient homologous rna recombination and requirement for an open reading frame during replication of equine arteritis virus defective interfering rnas.equine arteritis virus (eav), the prototype arterivirus, is an enveloped plus-strand rna virus with a genome of approximately 13 kb. based on similarities in genome organization and protein expression, the arteriviruses have recently been grouped together with the coronaviruses and toroviruses in the newly established order nidovirales. previously, we reported the construction of pedi, a full-length cdna copy of eav di-b, a natural defective interfering (di) rna of 5.6 kb (r. molenkamp et al., j ...200010982351
pathogenicity of hantaan virus in newborn mice: genetic reassortant study demonstrating that a single amino acid change in glycoprotein g1 is related to virulence.two hantaan virus strains, clone 1 (cl-1), which is virulent in newborn mice, and its attenuated mutant (mu11e10), were used to examine the pathogenesis of hantaan virus infection in a mouse model and identify virus factors relating to virulence. after subcutaneous inoculation of newborn balb/c mice, cl-1 caused fatal disease with high viral multiplication in peripheral organs, but mu11e10 produced nonfatal infection with a low level of virus multiplication. intracerebral inoculation of either s ...200010982372
sindbis-group alphavirus replication in periosteum and endosteum of long bones in adult mice.several alphaviruses, including the sindbis-group viruses, ross river virus, o'nyong-nyong virus, and chikungunya virus, are associated with outbreaks of acute and persistent arthralgia and arthritis in humans. mechanisms underlying alphavirus-induced arthralgia and arthritis are not clearly understood, though direct viral replication within or around the affected joints is thought to contribute to disease. s.a.ar86 is a sindbis-group alphavirus closely related to the arthralgia-associated ockel ...200010982376
sindbis virus glycoprotein e1 is divided into two discrete domains at amino acid 129 by disulfide bridge connections.the e1 membrane glycoprotein of sindbis virus contains structural and functional domains, which are conformationally dependent on the presence of intramolecular disulfide bridges (b. a. abell and d. t. brown, j. virol. 67:5496-5501, 1993; r. p. anthony, a. m. paredes, and d. t. brown, virology 190:330-336, 1992). we have examined the disulfide bonds in e1 and have determined that the e1 membrane glycoprotein contains two separate sets of interconnecting disulfide linkages, which divide the prote ...200010982379
mutagenesis reveals a role for abp/grip binding to glur2 in synaptic surface accumulation of the ampa receptor.we studied the role of pdz proteins grip, abp, and pick1 in glur2 ampa receptor trafficking. an epitope-tagged mycglur2 subunit, when expressed in hippocampal cultured neurons, was specifically targeted to the synaptic surface. with the mutant mycglur2delta1-10, which lacks the pdz binding site, the overall dendritic intracellular transport and the synaptic surface targeting were not affected. however, over time, myc-glur2delta1-10 accumulated at synapses significantly less than mycglur2. notabl ...200010985351
fitting atomic models into electron-microscopy maps.combining x-ray crystallographically determined atomic structures of component domains or subunits with cryo-electron microscopic three-dimensional images at around 22 a resolution can produce structural information that is accurate to about 2.2 a resolution. in an initial step, it is necessary to determine accurately the absolute scale and absolute hand of the cryo-electron microscopy map, the former of which can be off by up to 5%. it is also necessary to determine the relative height of densi ...200010998631
adeno-associated virus type 2 rep78 induces apoptosis through caspase activation independently of p53.adeno-associated virus (aav) type 2 rep78 is a multifunctional protein required for aav dna replication, integration, and gene regulation. the biochemical activities of rep78 have been described, but the effects of rep proteins on the cell have not been characterized. we have analyzed rep-mediated cytotoxicity. we demonstrated that rep78 expression is sufficient to induce cell death and disruption of the cell cycle. cell death was found to be mediated by apoptosis. rep78 expression resulted in t ...200011000213
passage of classical swine fever virus in cultured swine kidney cells selects virus variants that bind to heparan sulfate due to a single amino acid change in envelope protein e(rns).infection of cells with classical swine fever virus (csfv) is mediated by the interaction of envelope glycoprotein e(rns) and e2 with the cell surface. in this report we studied the role of the cell surface glycoaminoglycans (gags), chondroitin sulfates a, b, and c (cs-a, -b, and -c), and heparan sulfate (hs) in the initial binding of csfv strain brescia to cells. removal of hs from the surface of swine kidney cells (sk6) by heparinase i treatment almost completely abolished infection of these c ...200011000226
biochemical characterization of the equine arteritis virus helicase suggests a close functional relationship between arterivirus and coronavirus helicases.the arterivirus equine arteritis virus nonstructural protein 10 (nsp10) has previously been predicted to contain a zn finger structure linked to a superfamily 1 (sf1) helicase domain. a recombinant form of nsp10, mbp-nsp10, was produced in escherichia coli as a fusion protein with the maltose-binding protein. the protein was partially purified by affinity chromatography and shown to have atpase activity that was strongly stimulated by poly(dt), poly(u), and poly(da) but not by poly(g). the prote ...200011000230
alphavirus rna genome repair and evolution: molecular characterization of infectious sindbis virus isolates lacking a known conserved motif at the 3' end of the genome.the 3' nontranslated region of the genomes of sindbis virus (sin) and other alphaviruses carries several repeat sequence elements (rses) as well as a 19-nucleotide (nt) conserved sequence element (3'cse). the 3'cse and the adjoining poly(a) tail of the sin genome are thought to act as viral promoters for negative-sense rna synthesis and genome replication. eight different sin isolates that carry altered 3'cses were studied in detail to evaluate the role of the 3'cse in genome replication. the sa ...200011000254
replicon vectors derived from sindbis virus and semliki forest virus that establish persistent replication in host cells.alphavirus replicon vectors are well suited for applications where transient, high-level expression of a heterologous gene is required. replicon vector expression in cells leads to inhibition of host macromolecular synthesis, culminating in eventual cell death by an apoptotic mechanism. for many applications, including gene expression studies in cultured cells, a longer duration of transgene expression without resulting cytopathic effects is useful. recently, noncytopathic sindbis virus (sin) va ...200011000258
membrane proteins organize a symmetrical virus.alphaviruses are enveloped icosahedral viruses that mature by budding at the plasma membrane. according to a prevailing model maturation is driven by binding of membrane protein spikes to a preformed nucleocapsid (nc). the t = 4 geometry of the membrane is thought to be imposed by the nc through one-to-one interactions between spike protomers and capsid proteins (cps). this model is challenged here by a semliki forest virus capsid gene mutant. its cps cannot assemble into ncs, or its intermediat ...200011013211
suppressor mutations that allow sindbis virus rna polymerase to function with nonaromatic amino acids at the n-terminus: evidence for interaction between nsp1 and nsp4 in minus-strand rna synthesis.the alphavirus rna polymerase, nsp4, invariably has a tyr residue at the n-terminus. previously we reported that the n-terminal tyr residue of nsp4 of sindbis virus, the type species of the genus alphavirus, can be substituted with phe, trp, or his without altering the wild-type phenotype in cultured cells but that other substitutions tested, except for met, were lethal or quasilethal. here we report the identification of two suppressor mutations in nsp4 (glu-191 to leu and glu-315 to gly, val, ...200011022003
rubella virus replication and links to teratogenicity.rubella virus (rv) is the causative agent of the disease known more popularly as german measles. rubella is predominantly a childhood disease and is endemic throughout the world. natural infections of rubella occur only in humans and are generally mild. complications of rubella infection, most commonly polyarthralgia in adult women, do exist; occasionally more serious sequelae occur. however, the primary public health concern of rv infection is its teratogenicity. rv infection of women during th ...200011023958
molecular strategies for interrupting arthropod-borne virus transmission by mosquitoes.arthropod-borne virus (arbovirus) infections cause a number of emerging and resurgent human and veterinary infectious diseases. traditional means of controlling arbovirus diseases include vaccination of susceptible vertebrates and mosquito control, but in many cases these have been unavailable or ineffective, and so novel strategies for disease control are needed. one possibility is genetic manipulation of mosquito vectors to render them unable to transmit arboviruses. this review describes rece ...200011023962
differential regulation of bcl-2 and bax expression in cells infected with virulent and nonvirulent strains of sindbis virus.sindbis virus is an alphavirus that infects cells in either lytic or persistent infection. in this study we examined the effects of sindbis virus on cell apoptosis and on the expression of bcl-2 and bax. of the two strains studied, sva and svni, only the neurovirulent strain, svni, induced apoptosis of astrocytes and pc-12 cells. sva, which infects cells in a persistent manner, induced up-regulation of bcl-2 mrna and bcl-2 protein, whereas svni induced an increase in bax levels. our results indi ...200011040115
ecotropic murine leukemia virus receptor is physically associated with caveolin and membrane rafts.we used a sindbis virus expression system to stably express a chimeric ecotropic murine leukemia virus (mlv) receptor gene, cat1, fused to green fluorescent protein (gfp) in bhk cells. the chimeric gene was expressed on the cell surface and functioned as an mlv receptor. using gfp as an epitope tag, we found that cat1 cross-immunoprecipitated with caveolin, a cellular protein associated non-clathrin-coated endocytic vesicles. biochemical studies showed that cat1 copurified with caveolin in a det ...200011040117
generation of subgenomic rna directed by a satellite rna associated with bamboo mosaic potexvirus: analyses of potexvirus subgenomic rna promoter.satellite rna of bamboo mosaic potexvirus (satbamv), a single-stranded positive-sense rna encoding a nonstructural protein of 20 kda (p20), depends on bamboo mosaic potexvirus (bamv) for replication and encapsidation. a full-length cdna clone of satbamv was used to examine the sequences required for the synthesis of potexvirus subgenomic rnas (sgrnas). subgenomic promoter-like sequences (sgps), 107 nucleotides (nt) upstream from the capsid protein (cp) gene of bamv-v, were inserted upstream of t ...200011044078
glycosaminoglycan sulfation requirements for respiratory syncytial virus infection.glycosaminoglycans (gags) on the surface of cultured cells are important in the first step of efficient respiratory syncytial virus (rsv) infection. we evaluated the importance of sulfation, the major biosynthetic modification of gags, using an improved recombinant green fluorescent protein-expressing rsv (rgrsv) to assay infection. pretreatment of hep-2 cells with 50 mm sodium chlorate, a selective inhibitor of sulfation, for 48 h prior to inoculation reduced the efficiency of rgrsv infection t ...200011044095
in vivo selection of protease cleavage sites by using chimeric sindbis virus libraries.identifying protease cleavage sites contributes to our understanding of their specificity and biochemical properties and can help in designing specific inhibitors. one route to this end is the generation and screening of random libraries of cleavage sites. both synthetic and phage-displayed libraries have been extensively used in vitro. we describe a novel system based on recombinant sindbis virus which can be used to identify cleavage sites in vivo, thus eliminating the need for a purified enzy ...200011044100
strategy for systematic assembly of large rna and dna genomes: transmissible gastroenteritis virus model.a systematic method was developed to assemble functional full-length genomes of large rna and dna viruses. coronaviruses contain the largest single-stranded positive-polarity rna genome in nature. the approximately 30-kb genome, coupled with regions of genomic instability, has hindered the development of a full-length infectious cdna construct. we have assembled a full-length infectious construct of transmissible gastroenteritis virus (tgev), an important pathogen in swine. using a novel approac ...200011044104
expression of the two major envelope proteins of equine arteritis virus as a heterodimer is necessary for induction of neutralizing antibodies in mice immunized with recombinant venezuelan equine encephalitis virus replicon particles.rna replicon particles derived from a vaccine strain of venezuelan equine encephalitis virus (vee) were used as a vector for expression of the major envelope proteins (g(l) and m) of equine arteritis virus (eav), both individually and in heterodimer form (g(l)/m). open reading frame 5 (orf5) encodes the g(l) protein, which expresses the known neutralizing determinants of eav (u. b. r. balasuriya, j. f. patton, p. v. rossitto, p. j. timoney, w. h. mccollum, and n. j. maclachlan, virology 232:114- ...200011044106
development of a rubella virus vaccine expression vector: use of a picornavirus internal ribosome entry site increases stability of expression.rubella virus (rub) is a small plus-strand rna virus classified in the rubivirus genus of the family togaviridae. live, attenuated rub vaccines have been successfully used in vaccination programs for over 25 years, making rub an attractive vaccine vector. in this study, such a vector was constructed using a recently developed rub infectious cdna clone (robo). using a standard strategy employed to produce expression and vaccine vectors with other togaviruses, the subgenomic promoter was duplicate ...200011044128
nmda but not non-nmda excitotoxicity is mediated by poly(adp-ribose) polymerase.poly(adp-ribose) polymerase (parp-1), a nuclear enzyme that facilitates dna repair, may be instrumental in acute neuronal cell death in a variety of insults including, cerebral ischemia, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced parkinsonism, and cns trauma. excitotoxicity is thought to underlie these and other toxic models of neuronal death. different glutamate agonists may trigger different downstream pathways toward neurotoxicity. we examine the role of parp-1 in nmda- and non-nmda ...200011050121
sindbis virus-based vectors for the study of class i antigen presentation in vitro and in vivo. 200111068754
a mutant allele of essential, general translation initiation factor ded1 selectively inhibits translation of a viral mrna.positive-strand rna virus genomes are substrates for translation, rna replication, and encapsidation. to identify host factors involved in these functions, we used the ability of brome mosaic virus (bmv) rna to replicate in yeast. we report herein identification of a mutation in the essential yeast gene ded1 that inhibited bmv rna replication but not yeast growth. ded1 encodes a dead (asp-glu-ala-asp)-box rna helicase required for translation initiation of all yeast mrnas. inhibition of bmv rna ...200011069307
the host range phenotype displayed by a sindbis virus glycoprotein variant results from virion aggregation and retention on the surface of mosquito cells.the sindbis virus variant ne2g216 is a pe2-containing host range mutant that is growth restricted in cultured mosquito cells (c6/36) due to inefficient release of virions from this cell type. the maturation defect of ne2g216 has been linked to the structures of n-linked oligosaccharides synthesized by arthropod cells. analysis of c6/36 cells infected with ne2g216 by transmission electron microscopy revealed the presence of dense virus aggregates within cytoplasmic vacuoles and virus aggregates a ...200011070041
survival motor neuron protein modulates neuron-specific apoptosis.spinal muscular atrophy (sma) is attributed to mutations in the smn1 gene, leading to loss of spinal cord motor neurons. the neurotropic sindbis virus vector system was used to investigate a role for the survival motor neuron (smn) protein in regulating neuronal apoptosis. here we show that smn protects primary neurons and differentiated neuron-like stem cells, but not cultured cell lines from virus-induced apoptotic death. smn also protects neurons in vivo and increases survival of virus-infect ...200011078511
broad antiviral activity in tissues of crustaceans.innate antiviral substances occur in vertebrates and may function as host defenses. virus infections are common among invertebrates, but little is known about the ability of invertebrates to control viral infections. pre-existing antiviral substances may be particularly important, since invertebrates lack the antiviral defense conferred by specific immunity. in our study, we found that tissue extracts of blue crab (callinectes sapidus), shrimp (penaeus setiferus), and crayfish (procambarus clark ...200011080539
correlation of susceptibility of immature mice to fungal infection (blastomycosis) and effector cell function.immature mice are highly susceptible to blastomycosis, which is similar to other mycoses and has parallels in humans. the murine susceptibility is noteworthy in that it persists beyond the development of resistance to other, nonfungal pathogens and the maturation of most immune functions. as the susceptibility to blastomycosis appeared to be related to an early event after infection, primary effector cell function was studied. we found that peritoneal inflammatory cells, enriched for neutrophils ...200011083802
a universal 'one-tube' rt-pcr protocol for amplifying isolates of bovine viral diarrhoea virus.the increase in the knowledge of the genetic variability of bvdv and the identification of some of the genetic determinants of its pathogenicity require robust and practical tools for rapid molecular characterization of the various genotypes of this virus. this study was undertaken to develop a standard protocol for rt-pcr that allows the amplification of various parts of the genome of bvdv without the need for optimizing each individual reaction. the reaction set-up is very flexible because it ...200011085469
prolonged arthritis associated with sindbis-related (pogosta) virus infection.a follow-up study of musculoskeletal symptoms after pogosta virus infection.200011085809
interaction between complement receptor gc1qr and hepatitis c virus core protein inhibits t-lymphocyte proliferation.hepatitis c virus (hcv) is an important human pathogen that is remarkably efficient at establishing persistent infection. the hcv core protein is the first protein expressed during the early phase of hcv infection. our previous work demonstrated that the hcv core protein suppresses host immune responses, including anti-viral cytotoxic t-lymphocyte responses in a murine model. to investigate the mechanism of hcv core-mediated immunosuppression, we searched for host proteins capable of associating ...200011086025
enhancing b- and t-cell immune response to a hepatitis c virus e2 dna vaccine by intramuscular electrical gene transfer.we describe an improved genetic immunization strategy for eliciting a full spectrum of anti-hepatitis c virus (hcv) envelope 2 (e2) glycoprotein responses in mammals through electrical gene transfer (egt) of plasmid dna into muscle fibers. intramuscular injection of a plasmid encoding a cross-reactive hypervariable region 1 (hvr1) peptide mimic fused at the n terminus of the e2 ectodomain, followed by electrical stimulation treatment in the form of high-frequency, low-voltage electric pulses, in ...200011090158
infection of human dendritic cells by a sindbis virus replicon vector is determined by a single amino acid substitution in the e2 glycoprotein.the ability to target antigen-presenting cells with vectors encoding desired antigens holds the promise of potent prophylactic and therapeutic vaccines for infectious diseases and cancer. toward this goal, we derived variants of the prototype alphavirus, sindbis virus (sin), with differential abilities to infect human dendritic cells. cloning and sequencing of the sin variant genomes revealed that the genetic determinant for human dendritic cell (dc) tropism mapped to a single amino acid substit ...200011090185
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