In vitro culture of equine respiratory mucosa explants.

An in vitro model of the upper respiratory tract of the horse was developed to investigate mechanisms of respiratory diseases. Four tissues of the upper respiratory tract of three horses were collected. Explants were maintained in culture at an air-liquid interface for 96h. At 0, 24, 48, 72 and 96h of cultivation, a morphometric analysis was performed using light microscopy, scanning electron microscopy and transmission electron microscopy. The explants were judged on morphometric changes of epithelium, basement membrane and connective tissue. Viability was evaluated using a fluorescent Terminal deoxynucleotidyl transferase-mediated dUTP Nick End Labelling (TUNEL) staining. No significant changes in morphometry and viability of any of the explants were observed during cultivation. Hence, the in vitro model may be useful to study infectious and non-infectious diseases at the level of the equine respiratory tract, with potential application to the development of vaccines and treatments for diseases of the respiratory tract.

Immune escape of equine herpesvirus 1 and other herpesviruses of veterinary importance.

Equine herpesvirus (EHV)-1 is a pathogen of horses, well known for its ability to induce abortion and nervous system disorders. Clinical signs may occur despite the presence of a virus-specific immune response in the horse. The current review will summarize the research, on how, EHV-1-infected cells can hide from recognition by the immune system. Research findings on immune evasion of EHV-1 will be compared with those of other herpesviruses of veterinary importance.

Absence of viral antigens on the surface of equine herpesvirus-1-infected peripheral blood mononuclear cells: a strategy to avoid complement-mediated lysis.

Equine herpesvirus-1 (EHV-1) may cause abortion in vaccination- and infection-immune horses. EHV-1-infected peripheral blood mononuclear cells (PBMCs) play an important role in virus immune evasion. The mechanisms by which infected PBMCs can avoid destruction by EHV-1-specific antibody and equine complement were examined. The majority of EHV-1-infected PBMCs (68.6 %) lacked surface expression of viral antigens and these cells were not susceptible to complement-mediated lysis. In infected PBMCs with surface expression of viral antigens, 63 % showed focal surface expression, whereas 37 % showed general surface expression. General surface expression rendered infected PBMCs susceptible to lysis by antibody and complement (from 5.4 to 31.2 % lysed cells depending on the concentration of antibody and complement). Infected PBMCs with focal surface expression showed significant lysis only in the presence of high concentrations of antibody and complement. Thus, the absence of surface expression protects infected PBMCs against complement-mediated lysis.

Increased susceptibility of peripheral blood mononuclear cells to equine herpes virus type 1 infection upon mitogen stimulation: a role of the cell cycle and of cell-to-cell transmission of the virus.

Equine herpesvirus-1 (EHV-1) is an important pathogen of horses, causing abortion and nervous system disorders, even in vaccinated animals. During the cell-associated viremia, EHV-1 is carried by peripheral blood mononuclear cells (PBMC), mainly lymphocytes. In vitro, monocytes are the most important fraction of PBMC in which EHV-1 replicates, however, mitogen stimulation prior to EHV-1 infection increases the percentage of infected lymphocytes. The role of the cell cycle in viral replication and the role of cluster formation in cell-to-cell transmission of the virus were examined in mitogen-stimulated PBMC. Involvement of the cell cycle was examined by stimulating PBMC with ionomycin/phorbol dibutyrate (IONO/PDB) during 0, 12, 24 and 36 h prior to inoculation. Cell cycle distribution at the moment of inoculation and the percentage of EHV-1 antigen-positive PBMC at 0, 12 and 24 hours post inoculation (hpi) were determined by flow cytometry and immunofluorescence microscopy, respectively. The role of clusters was examined by immunofluorescence staining within clusters of stimulated PBMC using antibodies against EHV-1. Significant correlations were found between the increase of cells in the S- or G2/M-phase after a certain time interval of prestimulation and the increase of EHV-1 antigen-positive cells. The percentage of clusters with adjacent infected cells significantly increased from 3.3% at 8 hpi to 23.7% at 24 hpi and the maximal number of adjacent infected cells increased from 2 to 7. Addition of anti-EHV-1 hyperimmune serum did not significantly alter these percentages. Mitogen stimulation favours EHV-1 infection in PBMC by: (i) initiating cell proliferation and (ii) inducing formation of clusters, thereby facilitating direct cell-associated transmission of virus.

Mitogen stimulation favours replication of equine herpesvirus-1 in equine blood mononuclear cells by inducing cell proliferation and formation of close intercellular contacts.

In the present study, equine herpesvirus-1 (EHV-1)-infected cells were identified in ionomycin/phorbol dibutyrate (IONO/PDB)-stimulated peripheral blood mononuclear cells (PBMC) and the mechanism by which stimulation increases the percentage of infected cells was examined. In the population of viral antigen-positive PBMC, 38.4+/-4.5% were CD5(+) T-lymphocytes (18.1+/-3.2% CD4(+) 13.6+/-1.8% CD8(+)), 18.1+/-5.4% were B-lymphocytes, 8.5+/-3.9% were monocytes and 35% remained unidentified. The role of the cell cycle in the increased susceptibility to EHV-1 upon stimulation was examined by stimulating PBMC for 0, 12, 24 or 36 h prior to inoculation. A high correlation was found between the increase of cells in the S- (r=0.974) and G(2)/M-phase (r=0.927) at the moment of inoculation and the increase of infected cells at 12 h post-inoculation (p.i.). This suggests that a specific stage of the S-phase or S- and G(2)/M-phase facilitates virus replication. At 24 h p.i. lower correlations were found, suggesting that other effects are involved. From 12 h after addition of IONO/PDB, formation of clusters of PBMC became manifest. We examined whether close intercellular contacts in these clusters facilitated cell-to-cell transmission of EHV-1. Between 8 and 17 h p.i., the percentage of clusters containing adjacent infected cells increased from 1.6 to 13.4% and the maximal number of adjacent infected cells increased from two to four. Confocal microscopy visualized close intercellular contacts between adjacent infected cells. It can be concluded that mitogen stimulation favours EHV-1 infection of PBMC (i) by initiating specific cell cycle events and (ii) by inducing formation of clusters, thereby facilitating transmission of virus between cells.