tumor necrosis factor-alpha production and disease severity after immunization with enriched major core protein (p26) and/or infection with equine infectious anemia virus.cardinal features of equine infectious anemia (eia) include fever, hemolytic anemia and thrombocytopenia during the acute phase of the disease, and cachexia and anemia seen during the chronic phase. these signs are thought to result from the release of inflammatory cytokines such as tnf-alpha. in order to determine if tnf-alpha has a role in the pathogenesis of acute eia and vaccine-induced disease enhancement, we measured plasma concentrations of tnf-alpha in ponies immunized with virus enriche ...19979239836
equine infectious anemia virus utilizes a yxxl motif within the late assembly domain of the gag p9 protein.we have previously demonstrated that the gag p9 protein of equine infectious anemia virus (eiav) is functionally homologous with rous sarcoma virus (rsv) p2b and human immunodeficiency virus type 1 (hiv-1) p6 in providing a critical late assembly function in rsv gag-mediated budding from transfected cos-1 cells (l. j. parent et al., j. virol. 69:5455-5460, 1995). in light of the absence of amino acid sequence homology between eiav p9 and the functional homologs of rsv and hiv-1, we have now desi ...19979261374
scientists to pursue aids vaccine based on eia model. 19979262653
suppression of megakaryocyte colony growth by plasma from foals infected with equine infectious anemia virus.foals infected with equine infectious anemia virus become thrombocytopenic 7 to 20 days after virus inoculation, and within a few days following the onset of detectable viremia. the thrombocytopenia is associated with suppression of platelet production. possible mediators of suppression of thrombopoiesis include tumor necrosis factor-alpha (tnf-alpha) and transforming growth factor-beta (tgf-beta), cytokines that are released during inflammation. to assess effects of plasma or serum from infecte ...19979310486
dutpase from the retrovirus equine infectious anemia virus: specificity, turnover and inhibition.the kinetic properties of dutpase from equine infectious anemia virus (eiav) were investigated. k(m) (1.1 +/- 0.1 microm) and k(cat) (25 s(-1)) were found to be independent of ph in the neutral ph range. above ph 8.0, k(m) increases slightly. below ph 6.0, the enzyme is rapidly deactivated. detergent was found to enhance activity, leaving k(m) and k(cat) unaffected. compared to the escherichia coli dutpase, the eiav enzyme is equally potent in hydrolyzing dutp, but less specific. inhibition of t ...19979315700
flow cytometric method for detecting thiazole orange-positive (reticulated) platelets in thrombocytopenic evaluate a method for detecting thiazole orange-positive (to+, reticulated) platelets in equine blood, using flow cytometry.19979328660
elevation of cytokines associated with the thrombocytopenia of equine infectious anaemia.thrombocytopenia is a common finding in infection with equine infectious anaemia virus (eiav), a lentivirus with some homology to human immunodeficiency virus (hiv). the thrombocytopenia of eia, like that in some hiv patients, appears to have a multifactorial pathogenesis. to investigate the decreased platelet production seen in experimental eia, the levels of three potential negative regulators of platelet production--tumour necrosis factor-alpha (tnf-alpha), transforming growth factor-beta (tg ...19979349475
north american and french caprine arthritis-encephalitis viruses emerge from ovine maedi-visna viruses.the full extent of genetic diversity among small ruminant lentiviruses (srlvs), i.e., caprine arthritis encephalitis viruses (caevs) and maedi-visna viruses (mvvs), remains unknown. this is due in part to the fact that few sequences of caev are available. to contribute to this knowledge, gag, pol, and env nucleotide sequences from an srlv named ca680 originating from a goat from western france were determined. this analysis revealed that this virus is closely related to the cork and 63 caev amer ...19979356342
novel antimicrobial peptides derived from human immunodeficiency virus type 1 and other lentivirus transmembrane proteins.we have previously described a conserved set of peptides derived from lentiviral envelope transmembrane proteins that are similar to the natural antimicrobial peptides cecropins and magainins in overall structure but bear no sequence homology to them or other members of their class. we describe here an evaluation of the antimicrobial properties of these virally derived peptides, designated lentivirus lytic peptides (llps). the results of this study demonstrate that they are potent and selective ...19979371339
novel and dynamic evolution of equine infectious anemia virus genomic quasispecies associated with sequential disease cycles in an experimentally infected pony.we have investigated the genetic evolution of three functionally distinct regions of the equine infectious anemia virus (eiav) genome (env, rev, and long terminal repeat) during recurring febrile episodes in a pony experimentally infected with a well-characterized reference biological clone designated eiav(pv). viral populations present in the plasma of an eiav(pv)-infected pony during sequential febrile episodes (18, 34, 80, 106, and 337 days postinfection) were amplified from viral rna, analyz ...19979371627
frequency of memory cytotoxic t lymphocytes to equine infectious anemia virus proteins in blood from carrier horses.horses with equine infectious anemia virus (eiav) have episodes of viremia and disease; however, most eventually become inapparent carriers. a possible mechanism of control is cytotoxic t lymphocytes (ctl). to evaluate ctl in inapparent carriers with low viral loads, peripheral blood mononuclear cells (pbmc) were stimulated in vitro with autologous eiav-infected pbmc and human il-2 to detect memory ctl (ctlm). in initial studies, three carriers had ctlm and one of these had low-level effector ct ...19979375012
infection of bone marrow macrophages by equine infectious anemia characterize infection of bone marrow-derived macrophages (bmdm) with equine infectious anemia virus (eiav) by determining virus production, effects on viability, and induction of cytokines.19979401688
disease induction by virus derived from molecular clones of equine infectious anemia virus.equine infectious anemia virus (eiav), a macrophage-tropic lentivirus, causes persistent infections of horses. a number of biologic features, including the rapid development of acute disease, the episodic nature of chronic disease, the propensity for viral genetic variation, and the ability for many infected animals to eventually control virus replication, render eiav a potentially useful model system for the testing of antiretroviral therapies and vaccine strategies. the utility of the eiav sys ...19989420249
development and characterization of an in vivo pathogenic molecular clone of equine infectious anemia infectious nonpathogenic molecular clone (19-2-6a) of equine infectious anemia virus (eiav) was modified by substitution of a 3.3-kbp fragment amplified by pcr techniques from a pathogenic variant (eiav(pv)) of the cell culture-adapted strain of eiav (eiav(pr)). this substitution consisted of coding sequences for 77 amino acids at the carboxyl terminus of the integrase, the s1 (encoding the second exon of tat), s2, and s3 (encoding the second exon of rev) open reading frames, the complete env ...19989445039
genetic variation of envelope gp90 gene of equine infectious anemia virus isolated from an experimentally infected horse.six strains of equine infectious anemia virus (eiav) were recovered from febrile and non-febrile stages of a horse experimentally infected with the p337-v70 strain given once to a horse. the env gp90 genes of the isolates, the p337-v70 and p337-v26, avirulent virus derived from the p337-v70 strain, were sequenced. a comparison of the gp90 gene sequences revealed that amino acid variations among the viruses tested showed as high as 8.2 to 11.5%. in addition, the comparison also indicated that the ...19979450237
application of equine infectious anemia virus core proteins produced in a baculovirus expression system to serological diagnosis.equine infectious anemia virus (eiav) core proteins were obtained from a baculovirus expression system. recombinant baculoviruses (rbvs) highly expressed the gag precursor and p26 antigens in an rbv-infected sf21 cell culture supernatant. enzyme-linked immunosorbent assay (elisa) and agar gel immunodiffusion (agid) were conducted using the expressed proteins to detect antibodies from experimentally infected horses. the expressed antigens showed low background levels, high specificity and sensiti ...19979492183
risk analysis of quarantine station performance: a case study of the importation of equine infectious anemia virus-infected horses into california.we examined the risk of importing and mistakenly releasing equine infectious anemia virus (eiav)-infected horses into california. a computer simulation model was constructed to evaluate current and alternative quarantine station procedures; 150,000 iterations were performed to simulate 15 different scenarios of 10,000 horses imported into the state over a 14-year period. simulation results showed that under current conditions of low eiav prevalence in exporting countries, increasing the quaranti ...19989526854
equine infectious anemia virus transactivator is a homeodomain-type protein.lentiviral transactivator (tat) proteins are essential for viral replication. tat proteins of human immunodeficiency virus type 1 and bovine immunodeficiency virus form complexes with their respective rna targets (tat responsive element, tar), and specific binding of the equine anemia virus (eiav) tat protein to a target tar rna is suggested by mutational analysis of the tar rna. structural data on equine infectious anemia virus tat protein reveal a helix-loop-helix-turn-helix limit structure ve ...19989545368
biological characterization of rev variation in equine infectious anemia virus.sequence analysis identified significant variation in the second exon of equine infectious anemia virus (eiav) rev. functional analysis indicated that limited amino acid variation in rev significantly altered the export activity of the protein but did not affect rev-dependent alternative splicing. eiav rev can mediate export through two independent cis-acting rev-responsive elements (rres), and differences among rev variants were more pronounced when both rres were present. variation in rev may ...19989557734
the aspartic proteinase from equine infectious anaemia virus. 19989561197
endotoxin treatment of equine infectious anaemia virus-infected horse macrophage cultures decreases production of infectious virus.lentiviruses replicate in cells of the immune system, and activation of immune cells has been shown to modulate virus replication. to determine the effects of macrophage activation on replication of equine infectious anaemia virus (eiav), primary horse macrophage cultures (hmcs) were established from 20 different horses, infected with an avirulent strain of eiav, and stimulated with 5 microg/ml of bacterial endotoxin. supernatants collected from hmcs were assayed for the presence of tumour necro ...19989568970
regulation of equine infectious anemia virus expression.equine infectious anemia virus (eiav) is an ungulate lentivirus that is related to human immunodeficiency virus (hiv). much of the understanding of lentiviral gene regulation comes from studies using hiv. hiv studies have provided insights into molecular regulation of eiav expression; however, much of the regulation of eiav expression stands in stark contrast to that of hiv. this review provides an overview of the current state of knowledge of eiav regulation by comparing and contrasting eiav ge ...19989570509
immunization with a recombinant envelope protein (rgp90) of eiav produces a spectrum of vaccine efficacy ranging from lack of clinical disease to severe enhancement.we have previously reported that immunization of ponies with a baculovirus-expressed recombinant surface unit envelope protein (rgp90) for equine infectious anemia virus (eiav) resulted in enhancement of disease symptoms and virus replication in 4 of 4 vaccine recipients subjected to a heterologous virus challenge (rpg90 i vaccine trial) (wang et al., 1994). to extend these studies of eiav vaccine enhancement, two additional and independent rgp90 vaccine trials (rgp90 ii and rgp90 iii) were perf ...19989614876
equine monocyte-derived macrophage cultures and their applications for infectivity and neutralization studies of equine infectious anemia virus.equine infectious anemia virus (eiav) has been shown to infect cells of monocyte/macrophage lineage. these primary cells are intrinsically difficult to obtain, to purify and to culture in vitro for extended periods of time. as a result, most in vitro studies concerning this lentivirus make use of primary equine fibroblasts or transformed canine or feline cell lines. we describe methods that yield reproducibly pure cultures of equine blood monocytes from peripheral blood mononuclear cells. the in ...19989628225
differential requirements for alternative splicing and nuclear export functions of equine infectious anemia virus rev protein.the rev protein of equine infectious anemia virus (erev) exports unspliced and partially spliced viral rnas from the nucleus. like several cellular proteins, erev regulates its own mrna by mediating an alternative splicing event. to determine the requirements for these functions, we have identified erev mutants that affect rna export or both export and alternative splicing. mutants were further characterized for subcellular localization, nuclear-cytoplasmic shuttling, and multimerization. none o ...19989632773
general method for the detection and in vitro expansion of equine cytolytic t lymphocytes.equine immunological research is hindered by the lack of a simple yet reliable general protocol by which to assay ctl activity specific for viral or parasitic antigens. we present here the first comprehensive analysis of the parameters necessary to reliably culture equine t cells and to analyze the antigen specific cytolytic activity of t lymphocytes utilizing the equine infectious anemia virus (eiav) infection of outbred ponies as a source for in vivo primed t lymphocytes. effective long-term i ...19989671126
equine infectious anemia virus is found in tissue macrophages during subclinical infection.the equine infectious anemia virus (eiav) often results in lifelong subclinical infection following early episodes of clinical disease. to identify the cellular reservoirs of eiav during subclinical infection, horses were infected with eiav and allowed to develop subclinical infections. horses with acute disease served as a basis for comparison. the tissue distribution, replication status, location of infected cells, and viral load were characterized by pcr for proviral dna and reverse transcrip ...19989696821
leptomycin b inhibits equine infectious anemia virus rev and feline immunodeficiency virus rev function but not the function of the hepatitis b virus posttranscriptional regulatory element.human immunodeficiency virus type 1 rev export depends upon the presence of the nuclear export signal (nes), a leucine-rich stretch of hydrophobic amino acids. recently, the nuclear nes-binding receptor has been identified as crm1 or exportin 1. rev export has been shown to be crm1 dependent. the function of the atypical nes-containing rev-like proteins of equine infectious anemia virus (eiav) and feline immunodeficiency virus (fiv) is inhibited by leptomycin b, a drug that specifically blocks n ...19989696859
hyperglobulinemia and lymphocyte subset changes in naturally infected, inapparent carriers of equine infectious anemia determine blood protein concentration, immunoglobulin concentration, and lymphocyte profiles in equine infectious anemia virus (eiav) seropositive, naturally infected horses without clinical signs of disease.19989706205
analysis of the polymerization kinetics of homodimeric eiav p51/51 reverse transcriptase implies the formation of a polymerase active site identical to heterodimeric eiav p66/51 reverse transcriptase.homodimeric eiav p51/51 and heterodimeric eiav p66/51 reverse transcriptase were purified in order to compare the different modes of dna synthesis supported by the enzymes. analysis of the dimerization behavior of the eiav enzymes indicates that the dimer stability of eiav reverse transcriptase enzymes is higher than that of their hiv-1 reverse transcriptase counterparts. eiav p51/51 polymerizes dna distributively whereas dna synthesis by eiav p66/51 is processive. steady-state and pre-steady-st ...19989724526
the s2 gene of equine infectious anemia virus is dispensable for viral replication in vitro.equine infectious anemia virus (eiav) contains the simplest genome among lentiviruses in that it encodes only three putative regulatory genes (s1, s2, s3) in addition to the canonical gag, pol, and env genes, presumably reflecting its limited tropism to cells of monocyte/macrophage lineage. tat and rev functions have been assigned to s1 and s3, respectively, but the specific function for the s2 gene has yet to be determined. thus, the function of s2 in virus replication in vitro was investigated ...19989733881
the tat protein of equine infectious anemia virus (eiav) activates cellular gene expression by read-through transcription.the tat protein of equine infectious anemia virus, eiav, was shown to augment viral gene expression, presumably through interaction with the tat responsive element, tar. recently, cell-free polyadenylation assays suggested that perturbation of the eiav tar secondary structure diminished polyadenylation efficiency. the present study indicates that the eiav tar regulates the efficiency of the 3'-end processing of viral rna also in transfected cells. moreover, our data suggest that the provision of ...19989756988
equine endothelial cells support productive infection of equine infectious anemia virus.previous cell infectivity studies have demonstrated that the lentivirus equine infectious anemia virus (eiav) infects tissue macrophages in vivo and in vitro. in addition, some strains of eiav replicate to high titer in vitro in equine fibroblasts and fibroblast cell lines. here we report a new cell type, macrovascular endothelial cells, that is infectible with eiav. we tested the ability of eiav to infect purified endothelial cells isolated from equine umbilical cords and renal arteries. infect ...19989765477
cdna excision in stable retroviral cdna transfectants is prevented by r removal.the present study provides evidence on the occurrence of dna rearrangement between the redundant 5' and 3' r domains of equine infectious anemia virus (eiav) tat cdna. this was correlated with a gradual loss of cdna copy number concomitantly with a decrease in gene expression. removal of the 5' ru5 abolished rearrangement and stabilized tat expression in eiav tat cdna transfectants. our data suggest that prior removal of the 5' r from cloned retroviral cdnas can impede dna rearrangement, thus pr ...19989784417
the role of oxygen in the antiviral activity of hypericin and hypocrellin.the light-induced antiviral activity of hypericin and hypocrellin in the presence and absence of oxygen was examined under experimental conditions where the effect of oxygen depletion could be quantified. there was a significant reduction of light-induced antiviral activity of hypericin and hypocrellin under hypoxic conditions. interestingly, antiviral activity of hypocrellin was not observed at low oxygen levels at which hypericin retained measurable virucidal activity. this suggests that addit ...19989796444
gag protein epitopes recognized by ela-a-restricted cytotoxic t lymphocytes from horses with long-term equine infectious anemia virus infection.most equine infectious anemia virus (eiav)-infected horses have acute clinical disease, but they eventually control the disease and become lifelong carriers. cytotoxic t lymphocytes (ctl) are considered an important immune component in the control of infections with lentiviruses including eiav, but definitive evidence for ctl in the control of disease in carrier horses is lacking. by using retroviral vector-transduced target cells expressing different gag proteins and overlapping synthetic pepti ...19989811694
equine infectious anemia virus gag polyprotein late domain specifically recruits cellular ap-2 adapter protein complexes during virion assembly.we have identified an interaction between the equine infectious anemia virus (eiav) late assembly domain and the cellular ap-2 clathrin-associated adapter protein complex. a yxxl motif within the eiav gag late assembly domain was previously characterized as a sequence critical for release of assembling virions. we now show that this yxxl sequence interacts in vitro with the ap-50 subunit of the ap-2 complex, while the functionally interchangeable late assembly domains carried by the rous sarcoma ...19989811764
maturation of immune responses to lentivirus infection: implications for aids vaccine development.the evaluation of attenuated vaccines in the simian immunodeficiency virus and equine infectious anemia virus animal models has demonstrated the ability of this immunization strategy to elicit broad and enduring immune protection from virus exposure. the development of protective immunity by these attenuated virus vaccines, however, has been shown to be time dependent and to be associated with a complex and lengthy maturation of immune responses over the first 6 to 8 months postinoculation. duri ...19989814952
toward a universal inhibitor of retroviral proteases: comparative analysis of the interactions of lp-130 complexed with proteases from hiv-1, fiv, and of the major problems encountered in antiviral therapy against aids is the emergence of viral variants that exhibit drug resistance. the sequences of proteases (prs) from related retroviruses sometimes include, at structurally equivalent positions, amino acids identical to those found in drug-resistant forms of hiv-1 pr. the statine-based inhibitor lp-130 was found to be a universal, nanomolar-range inhibitor against all tested retroviral prs. we solved the crystal structures of lp-130 in co ...19989827997
"hidden" dutpase sequence in human immunodeficiency virus type 1 gp120.a coding region homologous to the sequence for essential eukaryotic enzyme dutpase has been identified in different genomic regions of several viral lineages. unlike the nonprimate lentiviruses (caprine arthritis- encephalitis virus, equine infectious anemia virus, feline immunodeficiency virus, and visna virus), where dutpase is integrated into the pol coding region, this enzyme has never been demonstrated to be present in the primate lentivirus genomes (human immunodeficiency virus type 1 [hiv ...19999847382
crystal structure of dutpase from equine infectious anaemia virus; active site metal binding in a substrate analogue complex.the x-ray structures of dutpase from equine infectious anaemia virus (eiav) in unliganded and complexed forms have been determined to 1.9 and 2.0 a resolution, respectively. the structures were solved by molecular replacement using escherichia coli dutpase as search model. the exploitation of a relatively novel refinement approach for the initial model, combining maximum likelihood refinement with stereochemically unrestrained updating of the model, proved to be of crucial importance and should ...19999878436
evaluation of equine infectious anemia virus core proteins produced in a baculovirus expression system in agar gel immunodiffusion test and enzyme-linked immunosorbent assay.equine infectious anemia virus (eiav) core proteins (gag and p26) obtained from a baculovirus expression system were used in agar gel immunodiffusion (agid) and enzyme-linked immunosorbent assay (elisa) antigens to test seventy-six horse sera. those sera showed false-positive reaction in agid test using nisseiken antigen. however, none of them showed false-positive reaction with both of the expressed antigens. the 76 horse sera were also tested by elisa. the sera gave a high background in elisa ...19989879541
kinetic properties and stereospecificity of the monomeric dutpase from herpes simplex virus type 1.kinetic properties of the monomeric enzyme dutpase from herpes simplex virus type 1 (hsv) were investigated and compared to those previously determined for homotrimeric dutpases of bacterial and retroviral origins. the hsv and escherichia coli dutpases are equally potent as catalysts towards the native substrate dutp with a kcat/k(m) of about 10(7) m(-1) s(-1) and a k(m) of 0.3 microm. however, the viral enzymes are less specific than the bacterial enzyme. the hsv and e. coli dutpases show the s ...19989883909
a particulate viral protein vaccine reduces viral load and delays progression to disease in immunized ponies challenged with equine infectious anemia virus.immunization regimens that induce a broadly reactive cytolytic t lymphocyte (ctl) response specific for lentiviral antigens have emerged as the leading candidates in efficacy trials conducted in both animal modelshumans. to date, lentivirus vaccination strategies have overlooked one such immunization strategy, namely the use of particulate antigens. to evaluate the efficacy of targeting antigen into the phagocytic pathway to elicit a cell-mediated immune response to lentiviral antigens, we initi ...19999927572
gene transfer vectors derived from equine infectious anemia virus.equine infectious anemia virus (eiav) is a lentivirus in the retrovirus family of viruses. replication-defective eiav vectors have been constructed that encode bacterial puromycin-n-acetyl transferase and e. coli beta-galactosidase. these vectors could be prepared with titers greater than 10(5) infectious units/ml and were able to act as vehicles to carry genes into cultured human cells. in addition, stable helper cell lines were created by modifying human 293 cells to express eiav proteins. unl ...19989930301
model for lentivirus capsid core assembly based on crystal dimers of eiav p26.two crystal forms of recombinant p26 capsid protein (ca) from the equine infectious anemia virus (eiav) have in common an antiparallel four-helix bundle dimer interface between n-terminal domains (ntds). the dimer interface provides a lenient scaffold to accommodate the wide sequence variation in these helices within lentivirus ca. pairs of dimers weakly associate to form exact or approximate d2 symmetry tetramers. in one of the two crystal forms, the tetramers are linked via dimerization of c-t ...19999931251
identification of retroviral late domains as determinants of particle size.retroviral gag proteins, in the absence of any other viral products, induce budding and release of spherical, virus-like particles from the plasma membrane. gag-produced particles, like those of authentic retrovirions, are not uniform in diameter but nevertheless fall within a fairly narrow distribution of sizes. for the human immunodeficiency virus type 1 (hiv-1) gag protein, we recently reported that elements important for controlling particle size are contained within the c-terminal region of ...19999971814
photoactivated virucidal properties of tridentate 2,2'-dihydroxyazobenzene and 2-salicylideneaminophenol platinum pyridine complexes.the potent photoactivated virucidal activity of tridentate 2,2'-dihydroxyazobenzene- and 2-salicylideneaminophenol platinum pyridine complexes 1, 2, 4, 6, 7, 9, and 10 against enveloped viruses (e.g., eiav, hiv, and hsv) is described.199910021936
detection and induction of equine infectious anemia virus-specific cytotoxic t-lymphocyte responses by use of recombinant retroviral vectors.cytotoxic t lymphocytes (ctl) appear to be critical in resolving or reducing the severity of lentivirus infections. retroviral vectors expressing the gag/pr or su protein of the lentivirus equine infectious anemia virus (eiav) were constructed and used to evaluate eiav-specific ctl responses in horses. three promoters, cytomegalovirus, simian virus sv40, and moloney murine sarcoma virus (momsv) long terminal repeat (ltr), were used, and there was considerable variation in their ability to direct ...199910074123
reduction of cd4+ and cd8+ t lymphocytes during febrile periods in horses experimentally infected with equine infectious anemia virus.three horses were experimentally infected with equine infectious anemia virus (eiav). all horses were febrile after inoculation with eiav and then developed chronic symptoms with intermittent fever. the febrile period was characterized by a rise in body temperature with reduced pbl and erythrocyte counts. flow cytometric analysis showed that the reduced number of lymphocytes was due to significant decreases in cd4+ and cd8+ t cells in the absence of any change in b cell number. at the end of the ...199910077419
increased interleukin-6 activity in the serum of ponies acutely infected with equine infectious anaemia ponies were infected with the virulent wild-type wyoming strain of equine infectious anaemia virus (eiav). infection status was monitored by serum reverse transcriptase activity, rectal temperature, and complete blood count. preinfection serum and serum obtained during the initial febrile episode following infection were assayed for interleukin 6 (il-6) activity. postinfection il-6 activity was significantly increased as compared to preinfection values. the magnitude of increase in il-6 wa ...199910088717
characterization of (+) strand initiation and termination sequences located at the center of the equine infectious anemia virus genome.permeabilized preparations of equine infectious anemia virus (eiav) are shown here to support efficient and accurate synthesis of full-length double-stranded proviral dna. when (-) and (+) strand products were analyzed by southern blotting, a discontinuity, mapping approximately to the center of the eiav genome, could be demonstrated for the (+) strand, predicting a second site for initiation of dna synthesis and a specific mechanism of (+) strand termination. precise localization of this (+) st ...199910090753
long terminal repeat sequences of equine infectious anaemia virus are a major determinant of cell tropism.the wyoming strain of equine infectious anaemia virus (eiav) is a highly virulent field strain that replicates to high titre in vitro only in primary equine monocyte-derived macrophages. in contrast, wyoming-derived fibroblast-adapted eiav strains (malmquist virus) replicate in primary foetal equine kidney and equine dermis cells as well as in the cell lines fea and cf2th. wyoming and malmquist viruses differ extensively both in long terminal repeat (ltr) and envelope region sequences. we have c ...199910092016
gag protein epitopes recognized by cd4(+) t-helper lymphocytes from equine infectious anemia virus-infected carrier horses.antigen-specific t-helper (th) lymphocytes are critical for the development of antiviral humoral responses and the expansion of cytotoxic t lymphocytes (ctl). identification of relevant th lymphocyte epitopes remains an important step in the development of an efficacious subunit peptide vaccine against equine infectious anemia virus (eiav), a naturally occurring lentivirus of horses. this study describes th lymphocyte reactivity in eiav carrier horses to two proteins, p26 and p15, encoded by the ...199910196322
platelets from thrombocytopenic ponies acutely infected with equine infectious anemia virus are activated in vivo and hypofunctional.thrombocytopenia is a consistent finding and one of the earliest hematological abnormalities in horses acutely infected with equine infectious anemia virus (eiav), a lentivirus closely related to human immunodeficiency virus. multifactorial mechanisms, including immune-mediated platelet destruction and impaired platelet production, are implicated in the pathogenesis of eiav-associated thrombocytopenia. this study was undertaken to investigate whether regenerative thrombopoiesis and platelet dest ...199910364485
highly divergent lentiviral tat proteins activate viral gene expression by a common mechanism.the human immunodeficiency virus type 1 (hiv-1) tat protein (htat) activates transcription initiated at the viral long terminal repeat (ltr) promoter by a unique mechanism requiring recruitment of the human cyclin t1 (hcyct1) cofactor to the viral tar rna target element. while activation of equine infectious anemia virus (eiav) gene expression by the eiav tat (etat) protein appears similar in that the target element is a promoter proximal rna, etat shows little sequence homology to htat, does no ...199910373508
in vitro antibody-dependent enhancement assays are insensitive indicators of in vivo vaccine enhancement of equine infectious anemia virus.we have previously demonstrated a high propensity for enhancement of virus replication and disease resulting from experimental immunization of ponies with a baculovirus recombinant envelope (rgp90) vaccine from equine infectious anemia virus (eiav). the current studies were undertaken to examine the correlation between the observed in vivo vaccine enhancement and in vitro assays for antibody-dependent enhancement (ade) of eiav replication. toward this goal an optimized eiav in vitro enhancement ...199910388665
solution structure of the capsid protein from the human t-cell leukemia virus type-i.the solution structure of the capsid protein (ca) from the human t-cell leukemia virus type one (htlv-i), a retrovirus that causes t-cell leukemia and htlv-i-associated myelopathy in humans, has been determined by nmr methods. the protein consists of independent n and c-terminal domains connected by a flexible linker. the domains are structurally similar to the n-terminal "core" and c-terminal "dimerization" domains, respectively, of the human immunodeficiency virus type one (hiv-1) and equine i ...199910438634
effect of substrate residues on the p2' preference of retroviral proteinases.the substrate sequence requirements for preference toward p2' glu residue by human immunodeficiency virus type 1 (hiv-1) proteinase were studied in both the matrix protein/ capsid protein (ma/ca) and ca/p2 cleavage site sequence contexts. these sequences represent typical type 1 (-aromatic*pro-) and type 2 (-hydrophobic* hydrophobic-) cleavage site sequences, respectively. while in the type 1 sequence context, the preference for p2' glu over ile or gln was found to be strongly dependent on the i ...199910491141
natural variation of equine infectious anemia virus gag protein cytotoxic t lymphocyte epitopes.two defined cytotoxic t lymphocyte (ctl) epitopes from equine infectious anemia virus (eiav)-infected horses, equine leukocyte alloantigen (ela)-a5.1-restricted epitope 18a, and ela-a9-restricted epitope 28b-1 were evaluated for conservation among three wild-type eiav strains. epitope 18a variation occurred in all three wild-type eiav strains, while epitope 28b-1 varied in one strain. further, 12% amino acid changes occurred in the gag proteins of a recently isolated wild-type strain, documentin ...199910497109
endothelial cell infection in vivo by equine infectious anaemia virus.equine infectious anaemia virus (eiav) infection of horses is characterized clinically by recurrent episodes of fever, thrombocytopenia and anaemia. in vivo, the only site of virus replication that has been previously demonstrated for eiav is the tissue macrophage. in this study, in situ hybridization for eiav was combined with immunohistochemistry for cell-type-specific markers to identify infected endothelial cells. eiav-infected endothelial cells and macrophages were detected in horses infect ...199910501492
evaluation of antibody parameters as potential correlates of protection or enhancement by experimental vaccines to equine infectious anemia virus.we previously demonstrated in trials of a variety of experimental vaccines to equine infectious anemia virus (eiav) a remarkable spectrum of efficacy ranging from sterilizing protection to severe enhancement of virus replication and disease, depending on the immunization strategy used. this range of vaccine efficacy observed in vivo offers a unique opportunity for evaluating potential in vitro immune correlates of protection and enhancement. we describe here a comprehensive analysis and comparis ...199910502520
effects of long terminal repeat sequence variation on equine infectious anemia virus replication in vitro and in vivo.the long terminal repeat (ltr) is reported to be one of the most variable portions of the equine infectious anemia virus (eiav) genome. to date, however, no information is available on the effects of observed sequence variations on viral replication properties, despite a widespread assumption of the biological importance of eiav ltr variation. eiav ltr sequence variability is confined mostly to a small portion of the enhancer within the u3 segment of the ltr. analysis of published eiav ltr seque ...199910544113
the s2 gene of equine infectious anemia virus is a highly conserved determinant of viral replication and virulence properties in experimentally infected ponies.equine infectious anemia virus (eiav) is genetically one of the simplest lentiviruses in that the viral genome encodes only three accessory genes, tat, rev, and s2. although serological analyses demonstrate the expression of the s2 protein in persistently infected horses, the role of this viral gene remains undefined. we recently reported that the s2 gene is not essential for eiav replication in primary equine macrophages, as eiav mutants lacking the s2 gene replicate to levels similar to those ...200010590152
stable gene transfer to the nervous system using a non-primate lentiviral vector.we have constructed a non-primate lentiviral vector system based on the equine infectious anaemia virus (eiav). this system is able to transduce both dividing and non-dividing cells, including primary cultured hippocampal neurons and neurons and glia in the adult rat central nervous system (cns), at efficiencies comparable with hiv-based vectors. we demonstrate that the only eiav proteins required for this activity are gag/pol and that the only accessory protein required for vector production is ...199910602376
replication ability in vitro and in vivo of equine infectious anemia virus avirulent japanese attenuated equine infectious anemia virus (eiav), v26, was previously prepared by 50 passages of the japanese virulent strain v70 in primary horse macrophage culture. the horses inoculated with this v26 virus were shown to raise neutralizing antibodies against v70 without any viremia. here, we investigated the in vitro and in vivo replication ability of v26. comparison of the long-terminal repeat (ltr) sequences between v26 and v70 revealed a large insertion within the ltr u3 hypervariable re ...200010612667
interactions between equine cyclin t1, tat, and tar are disrupted by a leucine-to-valine substitution found in human cyclin t1.transcriptional transactivators (tat) from human immunodeficiency and equine infectious anemia viruses (hiv and eiav) interact with their transactivation response elements (tar) to increase the rates of viral transcription. whereas the human cyclin t1 is required for the binding of tat to tar from hiv, it is unknown how tat from eiav interacts with its tar. furthermore, tat from eiav functions in equine and canine cells but not in human cells. in this study, we present sequences of cyclins t1 fr ...200010623752
temperature inducible beta-sheet structure in the transactivation domains of retroviral regulatory proteins of the rev family.the interaction of the human immunodeficiency virus type 1 (hiv-1) regulatory protein rev with cellular cofactors is crucial for the viral life cycle. the hiv-1 rev transactivation domain is functionally interchangeable with analog regions of rev proteins of other retroviruses suggesting common folding patterns. in order to obtain experimental evidence for similar structural features mediating protein-protein contacts we investigated activation domain peptides from hiv-1, hiv-2, visna virus, fel ...199910629982
phenanthrenequinone antiretroviral agents.compounds 3 and 5 are the first phenanthrenequinones to exhibit significant virucidal activity against the retrovirus equine infectious anemia virus. they differ from hypericin in that their virucidal activity is not light dependent.200010636231
cell specificity of the transcription-factor repertoire used by a lentivirus: motifs important for expression of equine infectious anemia virus in nonmonocytic cells.the equine infectious anemia virus (eiav) long-terminal repeat (ltr) has been identified as highly variable, both in infected horses and in cell culture. this nucleotide hypervariation is localized to the ltr enhancer region. the eiav ltr has been implicated in controlling both the cell tropism and virulence of the virus and it is postulated that the enhancer-region hypervariation may be responsible for the ltr effects. our previous studies have demonstrated that the presence of dna motifs bound ...200010662622
binding of equine infectious anemia virus matrix protein to membrane bilayers involves multiple interactions.human immunodeficiency virus (hiv) and equine infectious anemia virus (eiav) are closely related lentiviruses that infect immune cells, but their pathogenesis differ. localization to the cytosolic leaflet of the plasma membrane is critical for replication of both viruses. this localization is accomplished through the matrix (ma) domain of the gag precursor protein. in hiv-1, association of ma to anionic membranes appears to be primarily driven by a linear cluster of basic residues in the ma doma ...200010677289
canine cyclin t1 rescues equine infectious anemia virus tat trans-activation in human cells.human immunodeficiency virus-1 tat protein and human cyclin t1 mediate transcriptional activation by enhancing the elongation efficiency of rna polymerase ii. activation of transcription of the related equine infectious anemia virus (eiav) requires a similar protein known as etat, which does not function in human cells. expression of equine cyclin t1 in human cells rescues etat function, suggesting a general mechanism of transcription activation among lentiviruses. here we present the cloning of ...200010683321
tissue sites of persistent infection and active replication of equine infectious anemia virus during acute disease and asymptomatic infection in experimentally infected equids.equine infectious anemia virus (eiav) infection of horses is characterized by recurring cycles of disease and viremia that typically progress to an inapparent infection in which clinical symptoms are absent as host immune responses maintain control of virus replication indefinitely. the dynamics of eiav viremia and its association with disease cycles have been well characterized, but there has been to date no comprehensive quantitative analyses of the specific tissue sites of eiav infection and ...200010708426
structural and biochemical studies of retroviral proteases.retroviral proteases form a unique subclass of the family of aspartic proteases. these homodimeric enzymes from a number of viral sources have by now been extensively characterized, both structurally and biochemically. the importance of such knowledge to the development of new drugs against aids has been, to a large extent, the driving force behind this progress. high-resolution structures are now available for enzymes from human immunodeficiency virus types 1 and 2, simian immunodeficiency viru ...200010708846
aminoglycoside-arginine conjugates that bind tar rna: synthesis, characterization, and antiviral activity.regulation of hiv gene expression is crucially dependent on binding of the trans-activator protein, tat, to the trans-activation response rna element, tar, found at the 5' end of all hiv-1 transcripts. tat-tar interaction is mediated by a short arginine-rich domain of the protein. disruption of this interaction could, in theory, create a state of complete viral latency. a new class of small-molecule peptidomimetic tar rna binders, conjugates of aminoglycosides and arginine, was recently designed ...200010715103
suboptimal splice sites of equine infectious anaemia virus control rev responsiveness.the rev protein of equine infectious anaemia virus (eiav) was shown previously to stimulate the expression of a heterologous cat reporter gene when the 3' half of the eiav genome was present downstream in cis. however, computer analysis could not reveal the existence of a stable rna secondary structure that could be analogous to the rev-responsive element of other lentiviruses. in the present study, the inhibitory rna element designated the cis-acting repressing sequence (crs) has been localized ...200010769069
binding of equine infectious anemia virus rev to an exon splicing enhancer mediates alternative splicing and nuclear export of viral addition to facilitating the nuclear export of incompletely spliced viral mrnas, equine infectious anemia virus (eiav) rev regulates alternative splicing of the third exon of the tat/rev mrna. in the presence of rev, this exon of the bicistronic rna is skipped in a fraction of the spliced mrnas. in this report, the cis-acting requirements for exon 3 usage were correlated with sequences necessary for rev binding and transport of incompletely spliced rna. the presence of a purine-rich exon spli ...200010779344
transient kinetics of ligand binding and role of the c-terminus in the dutpase from equine infectious anemia virus.transient kinetics of the equine infectious anemia virus deoxyuridine 5'-triphosphate nucleotide hydrolase were characterized by monitoring the fluorescence of the protein. rate constants for the association and dissociation of substrate and inhibitors were determined and found to be consistent with a one-step mechanism for substrate binding. a c-terminal part of the enzyme presumed to be flexible was removed by limited trypsinolysis. as a result, the activity of the dutpase was completely quenc ...200010788633
development of a fluorescence polarization-based diagnostic assay for equine infectious anemia virus.the control of equine infectious anemia virus (eiav) infections of horses has been over the past 20 years based primarily on the identification and elimination of seropositive horses, predominantly by a standardized agar gel immunodiffusion (agid) assay in centralized reference laboratories. this screening for eiav-seropositive horses has been to date hindered by the lack of a rapid diagnostic format that can be easily employed in the field. we describe here the development of a rapid solution-p ...200010790112
immune responses and viral replication in long-term inapparent carrier ponies inoculated with equine infectious anemia virus.persistent infection of equids by equine infectious anemia virus (eiav) is typically characterized by a progression during the first year postinfection from chronic disease with recurring disease cycles to a long-term asymptomatic infection that is maintained indefinitely. the goal of the current study was to perform a comprehensive longitudinal analysis of the course of virus infection and development of host immunity in experimentally infected horses as they progressed from chronic disease to ...200010846078
mutations occurring during serial passage of japanese equine infectious anemia virus in primary horse attenuated equine infectious anemia virus (eiav), named v26, was previously obtained after 50 passages of the japanese virulent strain v70 in primary macrophage culture. to clarify the differences between both viruses, their full-length sequences were determined. there were higher mutations in s2 (6.15% amino acid difference) and ltr (10.7% nucleotide difference). the presumed initiation codon of the s2 gene was absent from the sequence of v26. there was a large insertion within the long-term ...200010930666
characterization of the equine infectious anaemia virus s2 protein.s2 is an accessory protein of equine infectious anaemia virus (eiav), the function of which is unknown. in order to gain insight into the function of s2, the intracellular localization of the protein, its interaction with viral proteins and its incorporation into viral particles have been investigated. immunolocalization of s2 revealed punctate staining in the cytoplasm and the s2 protein co-precipitated with the eiav gag precursor. despite overexpression of s2 through the use of a codon-optimiz ...200010950976
general effect of sam68 on rev/rex regulated expression of complex retroviruses.we have previously demonstrated that overexpression of sam68 functionally substitutes for, as well as synergizes with, hiv-1 rev in rre-mediated gene expression and virus replication. in addition, c-terminal deletion mutants of sam68 exhibit a transdominant negative phenotype in hiv replication. we now report that sam68 also enhances the activities of rev-like proteins of other complex retroviruses (e.g. htlv-1 and eiav) on their respective rna targets. furthermore, we demonstrate that sam68 can ...200010962565
an in vitro transcription system that recapitulates equine infectious anemia virus tat-mediated inhibition of human immunodeficiency virus type 1 tat activity demonstrates a role for positive transcription elongation factor b and associated proteins in the mechanism of tat activation.equine infectious anemia virus (eiav) activates transcription via a tat protein, a tar element, and the equine elongation factor positive transcription elongation factor b (p-tefb). in human cells, eiav tat (etat) can inhibit the ability of human immunodeficiency virus type 1 (hiv-1) tat (htat) to activate transcription from the hiv-1 long terminal repeat, demonstrating that eiav tat can interact nonproductively with human p-tefb. to study the mechanism of eiav tat and hiv-1 tat activation, we d ...200010964778
antigenic drift of viruses within a host: a finite site model with demographic stochasticity.we theoretically study the antigenic drift of viruses within an infected host, as observed in human immunodeficiency virus (hiv) and equine infectious anemia virus (eiav) infections, assuming that a finite number of antigen-determining sites at the viral envelop gene are responsible for the specific immune response. the pattern of antigen evolution becomes more complex than that predicted from the previous one-dimensional antigen space models. if the viral growth rate is sufficiently large, the ...200011029069
equine infectious anaemia virus proteins with epitopes most frequently recognized by cytotoxic t lymphocytes from infected horses.efficacious lentiviral vaccines designed to induce cytotoxic t lymphocytes (ctl) in outbred populations with a diverse repertoire of mhc class i molecules should contain or express multiple viral proteins. to determine the equine infectious anaemia virus (eiav) proteins with epitopes most frequently recognized by ctl from seven horses infected for 0.5 to 7 years, retroviral vector-transduced target cells expressing viral proteins were used in ctl assays. gag p15 was recognized by ctl from 100% o ...200011038386
developments in preclinical aids vaccine efficacy models. 200011086857
genetic and biological variation in equine infectious anemia virus rev correlates with variable stages of clinical disease in an experimentally infected pony.genetic and biological variation in the regulatory protein rev of equine infectious anemia virus (eiav) were examined throughout a clinically dynamic disease course of an experimentally infected pony. following infection with the virulent eiav(wyo), the pony underwent a variable disease course, including an acute fever episode at 12 days postinfection (dpi), multiple recurrent fever episodes until 135 dpi, a prolonged subclinical period, and two late fever episodes. viral rna was isolated from t ...200111145901
specificity of retroviral proteinases based on substrates containing tyrosine and proline at the site of cleavage.the retroviral proteinase (pr) plays crucial roles in the viral life cycle, therefore it is a target for chemotherapy. however, resistance rapidly develops due to frequent mutations. studies to determine the common features of the specificity of different retroviral prs may help to design broad spectrum inhibitors and reduce the possibility of viable mutants. we have studied the specificity of various retroviral proteinases including those the pr of hiv-1, hiv-2, equine infectious anemia virus a ...199711173643
paq: partition analysis of quasispecies.motivation: the complexities of genetic data may not be accurately described by any single analytical tool. phylogenetic analysis is often used to study the genetic relationship among different sequences. evolutionary models and assumptions are invoked to reconstruct trees that describe the phylogenetic relationship among sequences. genetic databases are rapidly accumulating large amounts of sequences. newly acquired sequences, which have not yet been characterized, may require preliminary genet ...200111222259
differential responses of equus caballus and equus asinus to infection with two pathogenic strains of equine infectious anemia virus.most in vivo studies with equine infectious anemia virus (eiav) have been performed in horses and ponies (equus caballus) with little published information available detailing the clinical responses of donkeys (equus asinus) to infection with this virus. consequently, donkeys were inoculated with two strains of eiav (eiav(pv) and eiav(wy)) which have been documented to produce disease in e. caballus. four ponies, 561, 562, 564 and 567 and two donkeys, 3 and 5 were infected with eiav(pv) and one ...200111230932
design and validation of an elisa for equine infectious anemia (eia) diagnosis using synthetic peptides.three peptides derived from the equine infectious anemia virus (eiav) surface proteins were synthesized to design and validate an elisa for eia diagnosis. peptides identified as gp90-i and gp90-ii correspond to the n- and c-terminal part of the surface glycoprotein gp90. peptide gp45-1 overlaps the immunodominant epitope cierthvfc of the transmembrane glycoprotein gp45, and includes a hydrophilic chain close to the n-terminal end of this nonapeptide loop. serum samples from 140 naturally infecte ...200111230933
the c-terminus of dutpase: observation on flexibility using nmr.the dynamics of the c-terminus of the dutpases from escherichia coli and equine infectious anaemia virus (eiav) were studied by 1h-(15)n nuclear magnetic resonance spectroscopy. the two enzymes differ with regard to flexibility in the backbone of the 15 most c-terminal amino acid residues, some of which are conserved and essential for enzymic activity. in the bacterial enzyme, the residues closest to the c-terminus are highly flexible and display a correlation time in the nanosecond time range. ...200111257499
cross reaction of recombinant equine infectious anemia virus antigen to heterologous strains and application for serological survey among horses in the field.cross reactivity of equine infectious anemia virus (eiav) antigen prepared using a recombinant baculovirus containing the p26 gene of strain p337-v70 was examined by the agar gel immunodiffusion (agid) test and enzyme-linked immunosorbent assay (elisa). serum samples serially collected from 13 horses experimentally infected with six different eiav strains (two or three horses per strain) were subjected to the test. positive reactions were observed in the agid test and elisa before or soon after ...200111270606
binding sites for rev and asf/sf2 map to a 55-nucleotide purine-rich exonic element in equine infectious anemia virus rna.the equine infectious anemia virus (eiav) rev protein (erev) negatively regulates its own synthesis by inducing alternative splicing of its mrna. this bicistronic mrna contains four exons; exons 1 and 2 encode tat, and exons 3 and 4 encode rev. when rev is expressed, exon 3 is skipped to produce an mrna that contains only exons 1, 2, and 4. the interaction of erev with its cis-acting rna response element, the rre, is also essential for nuclear export of intron-containing viral mrnas that encode ...200111278454
equine infectious anemia virus genomic evolution in progressor and nonprogressor ponies.a primary mechanism of lentivirus persistence is the ability of these viruses to evolve in response to biological and immunological selective pressures with a remarkable array of genetic and antigenic variations that constitute a perpetual natural experiment in genetic engineering. a widely accepted paradigm of lentivirus evolution is that the rate of genetic variation is correlated directly with the levels of virus replication: the greater the viral replication, the more opportunities that exis ...200111312327
functional correction of fanconi anemia group c hematopoietic cells by the use of a novel lentiviral vector.lentiviral vectors transduce nondividing hematopoietic cells more efficiently than other currently available vector systems. here we report the results of human hematopoietic cell gene transfer using lentiviral vectors based upon human immunodeficiency virus (hiv-1) and equine infectious anemia virus (eiav). eiav is a nonprimate lentivirus and is severely restricted in its host range to horses and closely related equines. we employed the eiav vector system to test for gene transfer to human fanc ...200111319908
detection of horses infected naturally with equine infectious anemia virus by nested polymerase chain reaction.a nested polymerase chain reaction (pcr) amplifying a region of the gag gene of equine infectious anemia virus (eiav) was developed for the rapid and direct detection of proviral dna from the peripheral blood of naturally infected horses and was compared with the coggins test. dna prepared from white blood cells of 122 field horses from 15 stables with reported cases of eiav and one seronegative stable were analysed. amplifications of expected size fragments were obtained by nested pcr for 88 ho ...200111337044
dh82 cells: a macrophage cell line for the replication and study of equine infectious anemia vivo, tissue macrophages have been implicated as an important cell for the replication of equine infectious anemia virus (eiav). laboratory investigations of eiav/macrophage interactions, however, have been hampered by the laborious blood monocyte isolation procedures. in addition, adherent equine macrophage cultures generally have poor long-term viability and are resistant to transfection. this report describes an adherent canine macrophage-like cell line, dh82, that supports the replication ...200111377712
functional roles of equine infectious anemia virus gag p9 in viral budding and infection.previous studies utilizing gag polyprotein budding assays with transfected cells reveal that the equine infectious anemia virus (eiav) gag p9 protein provides a late assembly function mediated by a critical y(23)p(24)d(25)l(26) motif (l-domain) to release viral particles from the plasma membrane. to elucidate further the role of eiav p9 in virus assembly and replication, we have examined the replication properties of a defined series of p9 truncation and site-directed mutations in the context of ...200111559809
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