Characterization of the infection of equine fibroblasts by equine infectious anemia virus

Summary Equine dermal fibroblasts persistently infected with equine infectious anemia virus (EIAV) show no alterations in cell morphology or growth kinetics when compared to uninfected cells. The percentage of cells immunofluorescent positive for viral proteins fluctuated, depending upon the stage of the cell cycle, while production of extracellular virus was uniform throughout the cell cycle, increasing only as the cell number increased. This was shown in log versus stationary phase cultures as well as in cultures synchronized by serum starvation. The establishment of productive infection did not require host cell DNA synthesis. Normal levels of progeny virus were produced in cultures pretreated with mitomycin C and placed in serum-containing medium. Serum-starved cultures, however, did not support EIAV replication as well as other cultures, presumably because synthesis of provirus was inhibited.With 3 Figures     

Characterization of BPV-like DNA in equine sarcoids

Summary The DNA from equine sarcoid samples from New York State and Switzerland was isolated and probed with bovine papillomavirus type 1 (BPV-1) to determine if BPV genomes were present. Twelve of 13 sarcoids from New York State and 17/20 sarcoids from Switzerland contained DNA that hybridized to the BPV-1 probe. Restriction enzyme analysis of the positive samples demonstrated restriction fragment profiles characteristic of BPV-1 in 22 sarcoids and restriction fragment profiles characteristic of bovine papillomavirus type 2 (BPV-2) in 7 sarcoids. In addition, three tissues histologically diagnosed as pyogranulomatous dermatitis, fibropapilloma, and fibrosarcoma contained BPV-like DNA. Tissues with BPV-1-like and BPV-2-like DNA contained an average of 285.7 (21 to 808) and 125.8 (2 to 762) BPV-like genomes per cell, respectively. Minor differences in the restriction fragment profiles of the BPV-like DNA and evidence for partial BPV-like genomes were found in some sarcoids. BPV-like DNA was not detected in lymphocyte DNA from sarcoid-affected horses. These results confirm previous observations and support the hypothesis that bovine papillomavirus, or a very similar virus, is linked to the cause of equine sarcoid.     

Characterization of equine humoral antibody response to the nonstructural proteins of equine arteritis virus

Equine arteritis virus (EAV) replicase consists of two polyproteins (pp1a and pp1ab) that are encoded by open reading frames (ORFs) 1a and 1b of the viral genome. These two replicase polyproteins are posttranslationally processed by three ORF 1a-encoded proteinases to yield at least 13 nonstructural proteins (nsp1 to nsp12, including nsp7α and 7β). These nsps are expressed in EAV-infected cells, but the equine immune response they induce has not been studied. Therefore, the primary purpose of this study was to evaluate the humoral immune response of horses to each of the nsps following EAV infection. Individual nsp coding regions were cloned and expressed in both mammalian and bacterial expression systems. Each recombinant protein was used in an immunoprecipitation assay with equine serum samples from horses (n = 3) that were experimentally infected with three different EAV strains (VB, KY77, and KY84), from stallions (n = 4) that were persistently infected with EAV, and from horses (n = 4) that were vaccinated with the modified live-virus (MLV) vaccine strain. Subsequently, protein-antibody complexes were subjected to Western immunoblotting analysis with individual nsp-specific rabbit antisera, mouse anti-His antibody, or anti-FLAG tag antibody. Nsp2, nsp4, nsp5, and nsp12 were immunoprecipitated by most of the sera from experimentally or persistently infected horses, while sera from vaccinated horses did not react with nsp5 and reacted weakly with nsp4. However, serum samples from vaccinated horses were able to immunoprecipitate nsp2 and nsp12 proteins consistently. Information from this study will assist ongoing efforts to develop improved methods for the serologic diagnosis of EAV infection in horses.     

Characterization of the equine influenza virus H 3 with monoclonal antibodies

Summary Mouse monoclonal antibodies specific for the hemagglutinin of the prototype equine-2 virus, A/equine/Miami/1/63 (H 3, N 8) demonstrated two related antigenic sites on the H 3. One of the sites was detected on 14 H 3 viruses isolated over a 22 year period (1963–1985). Variant viruses were selected with these antibodies at frequencies similar to those decribed for H 3 hemagglutinins from human isolates.With 1 Figure     

Characterization of equine arteritis virus particles and demonstration of their hemolytic activity

Equine arteritis virus (EAV), a member of the newly established family Arteriviridae, is a small, positive-stranded RNA virus. It carries two protein complexes in its envelope, gp5/M and the recently described gp2b/gp3/gp4 complex. We report here on several basic features of EAV replication in cell culture and on the protein composition of virus particles. We have also characterized gp2b, gp3, and gp4 expressed using a baculovirus system in insect cells. Finally, we provide evidence that EAV possess hemagglutinating and hemolytic activity. The hemolysis assay might be useful for determining which of the surface proteins carries the receptor-binding and membrane fusion activity of EAV.     

Characterization of three species of nucleocapsids of equine herpesvirus type-1 (EHV-1).

Three distinct species of nucleocapsids of equine herpesvirus type-1 (EHV-1) were isolated from infected L-M cell nuclei. The particles were classified on the basis of their densities in Renografin gradients as Light (L; ? = 1.237 g/cc), Intermediate (I; ? = 1.244 g/cc), and Heavy (H; ? = 1.258 g/cc). Analysis of the three nucleocapsid species, radioactively labeled with an ³H-labeled or ¹⁴C-labeled amino acid mixture, by discontinuous SDS-polyacrylamide-gel electrophoresis revealed significant and reproducible differences in their structural protein compositions. H nucleocapsids were comprised of six major proteins (I, II, III, IV, IVa, and V) with respective molecular weights of 148,000, 59,000, 46,000, 37,000, 30,000, and 18,000; these proteins comprised 63.7, 9.3, 6.0, 10.8, 5.3, and 4.1%, respectively, of total protein. These six proteins were also present in I nucleocapsids, however nucleocapsid protein IVa (MW, 30,000) was present only as a minor component and comprised less than 1% of total protein. L nucleocapsids were comprised of only four of these major structural proteins, as proteins III (MW, 46,000) and IVa (MW, 30,000) were absent. In addition, several minor proteins, each comprising less than 1% of total nucleocapsid protein, were present in each nucleocapsid species.Electrophoretic analysis of nucleocapsids labeled with [³H]arginine indicated that proteins extremely rich in arginine are not present in these three species. Phosphoproteins both of enveloped virions and of nucleocapsids were identified by electrophoretic analysis of particles radioactively labeled with inorganic phosphate (H₃³²PO₄). Twelve virion structural proteins were phosphorylated in vivo; five of these, including the major virion phosphoprotein VP 10 (MW, 127,000), were envelope specific proteins. Four of these 12 viral proteins were also phosphorylated in nucleocapsids, however the pattern of phosphorylation of nucleocapsid proteins varied among the three species. The two major phosphoproteins of nucleocapsids were proteins III and IVa which are absent in L nucleocapsids; protein V is phosphorylated only in H nucleocapsids.Analysis of the DNA content of the three nucleocapsid species indicated that preparations of H nucleocapsids contain more DNA than do those of the I and L species. Electron microscopic analysis of the nucleocapsids supported these results, as L and I nucleocapsids lack a dense inner nucleoid structure characteristic of the H species.     

Characterization of the antiphagocytic activity of equine fibrinogen for Streptococcus equi subsp. equi.

The antiphagocytic property of equine fibrinogen for Streptococcus equi subsp. equi strain CF32 was examined in vitro. The results of bactericidal assays demonstrated that the presence of fibrinogen enhanced the ability of overnight and early log phase cultures of strain CF32 to resist killing by equine neutrophils by 12-fold and seven-fold, respectively (p>0.01). In addition, fibrinogen-coated bacteria treated with fibrinogen specific F(ab′)₂ fragments were 32% more susceptible to killing by equine neutrophils after opsonization in serum (p>0.05), indicating that specific epitopes on fibrinogen may be important for its antiphagocytic effect. Since complement deposition is inhibited on subsp. equi (Boschwitz JS, Timoney JF, Infect Immun 1994; 42, 3515-20, we examined the effect of fibrinogen on complement deposition by using colloidal gold labeling of surface-bound C3. No significant differences were detected in the quantity of C3 deposited on the cell surface after opsonization with serum, serum plus fibrinogen, or plasma. These results suggest that the antiphagocytic property of fibrinogen is not related to the inhibition of complement deposition on the bacterial surface. Pretreatment of CF32 with M protein specific antibody inhibited fibrinogen binding by 72%, and a strain of subsp. equi expressing low levels of M protein bound 64% less fibrinogen than CF32, suggesting that the some of the fibrinogen deposited on the surface of subsp. equi is bound to M protein.     

Characterization of monoclonal antibodies specific for equine homologues of CD3 and CD5.

Two monoclonal antibodies (mAb), UC F6G-3 and UC F13C-5, were characterized as being specific for the apparent equine homologues of CD3 and CD5, respectively. Both antibodies exhibited characteristics of pan-T-lymphocyte markers based upon immunohistology and two-colour flow cytometry. UC F6G-3 precipitated a complex of proteins (up to seven) with molecular weights ranging from 18,000 to 42,000, similar to the human and murine CD3 complex. Upon further dissociation of the precipitated complex, two proteins were identified with molecular weights of 22,000 and 27,000. Immobilized UC F6G-3 was effective at inducing interleukin-2 receptor (IL-2R) expression on T lymphocytes, a feature consistent with antibodies specific for the epsilon chain of human and murine CD3. Three populations of cells in the thymus were distinguishable by UC F6G-3 target antigen density, suggesting increasing stages of T-cell maturation. UC F13C-5 precipitated a 67,000 MW protein, consistent with reported values for CD5 in multiple species. While this antibody exhibited characteristics of a pan-T-cell marker, low numbers of B lymphocytes also expressed the target antigen. Phorbol esters induced variable increases in target antigen density on B lymphocytes. These two antibodies, taken together with the few equine CD markers currently available, represent a substantial resource for further defining the equine immune system in health and disease.     

Characterization of equine natural killer and IL-2 stimulated lymphokine activated killer cell populations.

Natural killer (NK) cells are an important component of the innate immune system. Though intensively studied in humans and rodents. NK cells remain less well characterized in other species. Studies are often limited by the lack of specific cell markers; however, the mAb NK-5C6 has been suggested to recognize an evolutionarily conserved molecule on NK cells and reacts with cells from several species. This mAb was used in the current investigation to identify and characterize equine NK cells, and was found to label approximately 10% of peripheral blood lymphocytes (PBL). Two-color flow cytometry analysis identified the NK-5C6+ cell population as being CD3-CD4- and CD8-, but positive for MHC class I and LFA-1 expression. Depletion of CD3+ T cells increased the percent NK-5C6+ cells in PBL; this enriched population demonstrated a specific cytotoxic response against a major histocompatibility complex (MHC) deficient NK target cell line (K-562), but not MHC+ target cells (EqT8888). These results provide evidence for an equine NK cell population, which exhibits endogenous lytic activity and a phenotype similar to that of human and mouse NK cells. Stimulation of peripheral blood mononuclear cells (PBMC) with IL-2 promoted the development of LAK cells. These cells were predominantly CD3+ T cells, demonstrated intracellular perforin expression, and effectively lysed both K-562 and EqT8888 target cells. Hence, equine NK cells can be identified by the NK-5C6 mAb and distinguished from IL-2 stimulated LAK cells by their cytotoxic response to specific target cell lines.     

Characterization of a G14 equine rotavirus (strain CH3) isolated in Japan

Summary Antigenic and genomic properties of equine rotavirus strain CH3 isolated in Japan were studied by cross-neutralization tests and nucleotide sequence determination of the VP4 and VP7 genes. It was shown that the strain CH3 belongs to G14 and shares VP4 genotype with strain H2.The nucleotide sequence data reported in this paper appear in the DDBJ, EMBL and GeneBank nucleotide sequence detabases under the accession numbers D25228 (VP4 of strain CH3) and D25229 (VP7 of strain CH3).     

Phosphorylation of surface E-selectin and the effect of soluble ligand (Sialyl Lewisx) on the half-life of E-selectin

E-selectin (ELAM-1) is an adhesion molecule for leukocytes that is transiently expressed on endothelial cells. Following cell surface expression of E-selectin on human umbilical vein endothelial cells (HUVEC) stimulated with tumor necrosis factor, the induced E-selectin molecules are rapidly degraded. The kinetics of turnover of surface disposed E-selectin were investigated. The rapid disappearance of surface E-selectin is temperature dependent and sensitive to the lysosomotropic agent chloroquine. The half-life of E-selectin is not affected by inclusion of soluble sialyl Lewis x (sLe^x) ligands in the medium. Surface E-selectin is phosphorylated on one or more serine residues, but this modification is not obviously related to internalization.     

Epidemiology of equine herpesvirus 2 (equine cytomegalovirus).

The epidemiology of equine herpesvirus 2 was examined by using restriction endonuclease DNA fingerprints to distinguish viruses isolated from two groups of horses. The first group consisted of three yearlings isolated from other horses but in contact with each other for 418 days, whereas the second comprised seven mares and their foals, which were sampled at monthly intervals from parturition until the foals were about 180 days old. There was a complex pattern of transmission, with 15 different viruses isolated from both groups. Four distinguishable viruses were isolated from the three yearlings by day 16 of quarantine, and by day 141 an additional two viruses were isolated. Up to five different viruses were isolated from one yearling. Although four repeat isolations of one virus from the nasal cavity of one yearling over 54 days indicated that equine herpesvirus 2 established persistent infection with constant shedding, most repeat isolations yielded distinguishable viruses. Identical viruses were isolated from the nasal cavity and leukocytes of one yearling and the nasal cavity and vagina of another, indicating that a particular equine herpesvirus 2 strain was not site specific. Although seven different viruses were isolated from the three yearlings throughout the quarantine period, two appeared to establish latent infections; one virus was not isolated until 141 days after quarantine, whereas the second was first isolated 16 days after quarantine and then for the second time, from the same horse, 402 days later. Multiple concurrent local infections were demonstrated by the isolation of two or more viruses from the same nasal swab.     

Cyclosporin A enhances T cell-mediated induction of E-selectin

In a fetal intestinal organ culture model of intestinal inflammation, activation of T cells by a lectin, superantigen or anti-CD3 antibodies induces E-selectin expression which peaks 6-10 h after activation and disappears by 24 h. We now show that addition of cyclosporin A (Cys A) increases the number of E-selectin-expressing endothelial cells and the intensity of expression, and it prolongs expression up to at least 60 h after stimulation. The dynamics of E-selectin expression on tumor necrosis factor activated human umbilical-vein endothelial cell cultures is not affected by Cys A. Furthermore, other immunosuppressive agents FK 506 and dexamethasone inhibit E-selectin expression, indicating that the enhancement observed in the presence of Cys A is not a consequence of general immunosuppression.     

E-选择素在生殖道沙眼衣原体感染中的作用研究

目的 研究E-选择素在生殖道沙眼衣原体感染中的作用.方法 将64只小鼠生殖道沙眼衣原体感染模型随机分成2组,实验组中的小鼠用人工合成E-选择素干预,对照组中的小鼠用生理盐水进行干预,统计两组在1w,2w,3w,4w不同时间点上的感染小鼠阴道脱落菌量以及沙眼衣原体感染率.结果 成功建立生殖道沙眼衣原体感染的小鼠模型:与对照组相比较,人工合成E-选择素干预后1w和2w的生殖道沙眼衣原体感染率(90.2％vs 77.4％,85.3％vs 70.1％)显著增高,而阴道脱落菌量显著增高(4.758 ±0.225 vs 3.210 ±0.315,2.698±0.177 vs 1.809±0.203),两组间差异具有统计学.在干预后3w两组的沙眼衣原体感染率分别为34％和24％,阴道脱落菌量分别为1.412±0.134和1.201±0.189,两组间差异无统计学意义;两组在干预后4w的沙眼衣原体感染率均为0且无阴道脱落菌量.结论 E-选择素能促进生殖道沙眼衣原体感染的发生率以及增加沙眼衣原体的毒力,本文为进一步阐明生殖道沙眼衣原体感染的发病机制提供实验依据.     

Intratracheal administration of endotoxin and cytokines: VIII. LPS induces E-selectin expression; anti-E-selectin and soluble E-selectin inhibit acute inflammation

E-selectin is an inducible endothelial adhesion molecule that binds neutrophils. E-selectin mRNA is not constitutively detectable in the lungs of rats. Intratracheal injection of LPS induces pulmonary E-selectin mRNA expression at 2–4 h. Intratracheal injection of LPS followed at 2 and 4 h by intravenous injection of mouse F(ab′)₂ or F(ab′)) anti-E-selectin monoclonal antibody inhibits the emigration of neutrophils into the bronchoalveolar space at 6 h by 50–70%. TNF and IL-6 bioactivity are not decreased in bronchoalveolar lavage fluid after treatment with anti-E-selectin antibody as compared to controls, suggesting that the anti-E-selectin does not affect the magnitude of the LPS-initiated cytokine cascade. Intratracheal injection of LPS followed at 2 and 4 h by intravenous injection of soluble E-selectin inhibits neutrophilic emigration at 6 h by 64%, suggesting that endogenous soluble E-selectin shed from activated endothelium may play a role in the endogenous down-regulation of acute inflammation. E-selectin-mediated adhesion of neutrophils to endothelium appears crucial to the full development of the acute inflammation response.     

E-selectin polymorphism and atherosclerosis: an association study

Adhesion molecules like the members of the selectin family participateIn the interaction between leukocytes and the endothellum. Theyare also involved in the pathogenesis of atherosclerotic processes.To contribute to the analysis of the genetic background of atherosclerosiswe searched for DNA polymorphisms in the genes encoding adhesionmolecules especially E-selectin which seems to be expressedonly In activated endothelium. An adenlne to cytoslne substitutionfor cDNA position 561 resulting In an amlno acid exchange fromserine to arglnlne (position 128) was detected in the epidermalgrowth factor like domain. A significantly higher mutation frequency(P = 0.02) was observed in 97 patients aged 50 years or lesswith angiographically proven severe atherosclerosis (allelefrequency of arginine 0.155) compared with an unselected population(allele frequency of arglnlne 0.088) as well as in 40 patientsaged 40 years or less (allele frequency of arginine 0.21, P=0.0025). These data suggest that the 128-serine/arginine polymorphismis associated with a higher risk for early severe atherosclerosis.     

Phase-dependent roles of E-selectin during chronic contact hypersensitivity responses

Chronic contact hypersensitivity (CH) models induced by repeated hapten exposure exhibit chronic dermatitis and immunological abnormalities resembling atopic dermatitis. To assess the contribution of endothelial selectins (P- and E-selectins) to cutaneous chronic inflammation, chronic CH responses were assessed in mice lacking P- or E-selectin. Elicitation with oxazolone on the ears of P-selectin(-/-) mice 7 days after the sensitization induced a typical delayed-type hypersensitivity response similar to that found in wild-type mice. By contrast, a significant increase in ear swelling was observed in E-selectin(-/-) mice 36 to 48 hours after first elicitation. E-selectin(-/-) mice showed augmented P-selectin up-regulation, and administration of anti-P-selectin monoclonal antibody significantly inhibited the enhanced ear response, suggesting that the enhanced ear-swelling response in E-selectin(-/-) mice resulted from compensatory increase in P-selectin expression. In the late phase of chronic CH, acceleration of ear swelling was significantly reduced in both E- and P-selectin(-/-) mice relative to wild-type littermates. Thus, the loss of P- or E-selectin suppressed inflammatory responses during the chronic phase of the chronic models, whereas early-phase inflammatory responses were exacerbated by E-selectin blockade. Collectively, P- and E-selectins cooperatively regulate CH response, although their roles may be different depending on the phase of the reaction.