Hemagglutination by equine infectious anemia virus.

Equine infectious anemia (EIA) virus which was propagated on an equine dermal cell line agglutinated guinea pig erythrocytes. Viral fluids containing about 10(7.5) mean tissue culture infective doses/ml showed hemagglutinating (HA) titers ranging from 16 to 32 units/0.05 ml. Results of cesium chloride equilibrium density gradient centrifugation revealed that the hemagglutinin was inseparable from the virus particles. The hemagglutination reaction persisted over a wide range of temperature and pH, and the absence of divalent cations did not decrease its activity. The HA activity was stable at 4 degrees C but not at 56 degreesC. The activity was destroyed by virus-disrupting lipid solvents and moderately sensitive to a proteolytic enzyme. Neuraminidase enhanced HA activity slightly. Phospholipase C had no effect on HA titer, although it completely inactivated infectivity. It was relatively stable to ultraviolet irradiation. Thus, the hemagglutinin appears to be closely associated with virus particles, and its activity is dependent on the presence of its lipids and proteins. Hemagglutination was inhibited by sera from horses infected with EIA virus. Hemagglutinin receptors on the erythrocytes were inactivated by a proteolytic enzyme and formaldehyde but were not influenced by neuraminidase, sodium deoxycholate, or KIO4.     

Antigenic variation of equine infectious anemia virus as detected by virus neutralization

Summary The antigenic structure of 16 viruses isolated from four horses which were inoculated with a clone of equine infectious anemia (EIA) virus was compared by the neutralization test. The antigenic structure of viruses isolated after development of neutralizing antibody differed from virus to virus. Back mutation of the antigenic structure was also demonstrated by serial passage of the virus in horses. These results suggest that EIA virus is subject to multidirectional antigenic variation. The possibility that the variants originated in the heterologous virus population in the inoculum seems to be unlikely since the virus used for the primary inoculation was cloned by three repeated high-limiting dilutions.     

Purification of equine infectious anemia virus antigen by affinity chromatography.

Affinity chromatography was performed to obtain highly purified antigen from equine infectious anemia (EIA) virus. After crude antigen was concentrated by polyethylene glycol precipitation of culture fluids from equine dermal cells persistently infected with EIA virus, and after the virus was disrupted with ether, it was added to a column of cyanogen bromide-activated Sepharose 4B to which EIA-specific antibody had been conjugated. The antigen was effectively released from the column with 5M MgCl2 and proved to be highly purified. Passive hemagglutination tests on sera from EAI infections were carried out, using the purified antigen. Results indicated that the passive hemagglutination test with the antigen was a specific laboratory test with high sensitivity for EIA infection.     

Hemagglutination by several strains of equine infectious anemia virus

Summary Six strains of equine infectious anemia (EIA) virus propagated in equine leukocyte cultures were found to agglutinate horse erythrocytes. Concentrated virus material containing about 20 units of complement fixation (CF) titer showed hemagglutinating (HA) titers ranging from 4 to 8 units. The HA activity remained stable after ether treatment and was reduced by trypsin, formaldehyde and KIO₄. Cesium chloride equilibrium density gradient centrifugation revealed two populations of hemagglutinin, one in the density range of 1.15–1.16 g/ml coinciding with a peak of CF antigen and the other at round 1.27 g/ml. However, after the antigen was treated with ether, hemagglutinin showed a single peak at about 1.27 g/ml. Hemagglutinin receptors on the erythrocytes were inactivated by trypsin and formaldehyde but their activity was enhanced by neuraminidase. Hemagglutination was inhibited by sera from horses infected with homologous strain for EIA virus. The hemagglutinin showed immunological properties similar to those of the hemagglutinin of guinea pig erythrocytes as reported in our previous paper.With 2 Figures     

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     

Physicochemical studies of equine infectious anemia virus

Summary A purification procedure for equine infectious anemia (EIA) virus has been developed by combining ultracentrifugation, DEAE cellulose column chromatography and cesium chloride equilibrium density gradient centrifugation. Recovery and purity of the virus were determined at each step of the purification procedure. Using this combined method, an amount of purified and concentrated virus was prepared from a large volume of virus material. Such specimens showed an infectivity of 10^8.25 TCID₅₀/0.5 ml, a complement fixing antigenicity of 80 density of 1.146 g/ml, and proved to be suitable for electron microscopic observation of negatively stained preparations.Most of the virus particles had a spherical shape and sized between 90 and 140 m, in diameter. A well-defined outer envelope was observed in spontaneously disrupted particles, but no organized internal component could be resolved.     

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 assay was developed using primary equine macrophage cells and used to evaluate the enhancement properties of immune serum taken from rgp90 immunized ponies that displayed various levels of vaccine enhancement after experimental challenge with EIAV. For comparison, we analyzed in parallel immune serum samples from a group of ponies immunized with a viral envelope subunit vaccine (LL-gp) that produced sterile protection from EIAV challenge. The results of these assays demonstrated that the rgp90 immune serum had a greater propensity for in vitro enhancement of EIAV replication than serum from the protected LL-gp immunized ponies; in vitro enhancement levels for the rgp90 immune sera averaged about 1.5, with a maximum enhancement value of about 2.0. While distinguishing between immune serum produced by the rgp90 and LL-gp immunizations, the in vitro enhancement assay failed to reliably correlate with the severity of in vivo enhancement observed among the rgp90 vaccine recipients. Vaccinated ponies that experienced moderate to no disease signs displayed levels of in vitro enhancement similar to those of ponies that experienced severe and fatal enhancement of disease after viral challenge. The observed in vitro enhancement was demonstrated to be dependent on serum immunoglobulin, but independent of complement. These studies demonstrate in the EIAV system that in vitro ADE assays appear to be relatively insensitive indicators of the severity of in vivo enhancement and that relatively low levels of in vitro ADE can be associated with severe to fatal enhancement of virus replication and disease in vivo. These observations suggest that relatively low levels of serum ADE observed in other lentivirus systems, including HIV-1, may have more profound effects on in vivo virus replication and disease than previously recognized.     

Enzyme-linked immunosorbent assay for detection of equine infectious anemia virus p26 antigen and antibody.

A sensitive specific enzyme-linked immunosorbent assay utilizing purified p26 antigen was developed for the detection of antibodies to equine infectious anemia virus in naturally and experimentally infected horses. Generally, antibodies to the virus could be detected by the enzyme-linked immunosorbent assay 3 to 4 days earlier than by the standard agar gel immunodiffusion test, and they could be detected more reliably in horses with weak or equivocal agar gel immunodiffusion test reactions. The enzyme-linked immunosorbent assay was also successfully applied to the detection of p26 antigen in tissue culture fluids.     

Immunodiffusion studies of purified equine infectious anemia virus

Antigenicity of purified equine infectious anemia (EIA) virus was examined by immunodiffusion against sera obtained from horses experimentally infected with EIA virus. The purified virus reacted with the infected horse serum, and virus-specific precipitating antibody was demonstrated. Furthermore, it was found that purified EIA virus reacted against the serum of horses infected with all strains of EIA virus which were antigenically different from one another. From the result, group-specific components of the virus rather than strain-specific ones were considered to be involved in the reaction. Serological reactivity was lost by adding antiserum from the infected horse to the antigen. The precipitating antibody usually appeared in the serum 1 to 2 weeks after the first febrile attack of EIA and remained for a longer period. Some characteristics of the purified antigen and specificity of the reaction for EIA are described.     

Purification and characterization of equine infectious anemia virus

Summary EIA virus was purified from equine fetal kidney cell cultures by PEG-precipitation, two sucrose-gradient sedimentations (5–30 per cent) and (25 to 60 per cent) centrifugation, using the immunodiffusion test to follow the procedure. Purified EIA virus had a density (20° C) of 1.162 and a sedimentation constant of S_20W=656. Electron microscopy revealed a particle of about 100 nm in diameter with a very flexible but usually spherical shape. The dense core may be at various locations inside the membrane bound particle.With 6 Figures     

Mechanisms of equine infectious anemia virus escape from neutralizing antibody responses define epitope specificity

Abstract Determining mechanisms of viral escape to particular epitopes recognized by virus-neutralizing antibody can facilitate characterization of host-neutralizing antibody responses as type- versus group-specific, and provides necessary information for vaccine development. Our study reveals that a single N-glycan located in the 5' region of the Wyoming wild-type equine infectious anemia virus (EIAV) principal neutralizing domain (PND) accounts for the differences in neutralization phenotype observed between PND variants, while variations in charged amino acids within the PND do not appear to play a key role in viral escape. Site-directed mutagenesis and peptide mapping of a conserved epitope to neutralizing antibody in the 3' region of the PND showed rapid selective pressure for acquisition of a 5' PND N-glycan responsible for defining the specificity of the neutralizing-antibody response.     

Transient suppression of equine immune responses by equine infectious anemia virus (EIAV).

Suppression of the immune system is a common aspect of the disease pathogenesis associated with retroviral infections in both man and animals. We have measured transient suppression of the equine immune system as a loss or decrease in antigen-specific and polyclonal lymphocyte proliferation following experimental infection of ponies with three variants of equine infectious anemia virus (EIAV) with difference virulence characteristics. The transient suppression of proliferative responses was temporally associated with recurrent febrile episodes, which are the hallmark symptom of EIAV-induced disease. Decreased proliferative responses occurred at all times when EIAV viremia was identified, based on the detection of an infectious virus in plasma or viral proteins on peripheral blood mononuclear cells. The immunosuppression was observed most frequently in ponies infected with virulent variants of EIAV which suggested that this effect may contribute to disease pathogenesis. Suppression of polyclonal proliferative responses was induced in vitro by the addition of either infectious or heat-inactivated EIAV to cultures, demonstrating that the viral structural proteins were immunosuppressive in the absence of infection. These studies indicated that EIAV is similar to other retroviruses in that it has the ability to suppress the immune system.     

Monocyte activation in horses persistently infected with equine infectious anemia virus.

The monocytes of horses infected with equine infectious anemia virus were shown by their failure to migrate from capillary tubes and their increased adherence to erythrocytes to be activated.     

Serological comparison among various strains of equine infectious anemia virus

Summary The serological relationship between 8 strains of equine infectious anemia (EIA) virus was investigated by means of complement fixation and neutralization tests. All strains had a common complement fixing antigen, although there were some differences in the intensity of CF reaction in certain antigen-antiserum combinations.Cross neutralization tests revealed that all strains were only neutralized by homologous antisera. The respective antibody titers recorded ranged from 32 to 512. When horses were infected with the virus strains, neutralizing antibodies could be detected as early as 32 to 87 days after inoculation and maximum titers were demonstrable after 42 to 148 days. Of 34 horses infected with 6 strains of EIA virus, 33 horses had neutralizing antibody when they were tested 52 to 127 days after inoculation. Antibodies were only produced against the virus strains inoculated, and not against heterologous strains.