Replication characteristics of equine herpesvirus 1 and equine herpesvirus 3: comparative analysis using ex vivo tissue cultures

Abstract

Replication kinetics and invasion characteristics of equine herpesvirus-1 and -3 (EHV-1/-3) in nasal and vaginal mucosae were compared using explants. The explants were cultured during 96 h with little change in viability. The tissues were inoculated with EHV-1 03P37 (neuropathogenic), 97P70 (abortigenic) and EHV-3 04P57, collected at 0, 24, 48 and 72 h post inoculation (pi) and stained for viral antigens. Both EHV-1 and EHV-3 replicated in a plaquewise manner. The plaques were already observed at 24 h pi, their size increased over time and did not directly cross the basement membrane (BM). However, EHV-1 infected the monocytic cells (MC) and hijacked these cells to invade the lamina propria. In contrast, EHV-3 replication was fully restricted to epithelial cells; the virus did not breach the BM via a direct cell-to-cell spread nor used infected MC. EHV-1-induced plaques were larger in nasal mucosa compared to vaginal mucosa. The opposite was found for EHV-3-induced plaques. Both EHV-1 strains replicated with comparable kinetics in nasal mucosa. However, the extent of replication of the abortigenic strain in vaginal mucosa was significantly higher than that of the neuropathogenic strain. Two-to-five-fold lower numbers of EHV-1-infected MC underneath the BM were found in vaginal mucosa than in nasal mucosa. Our study has shown that (i) EHV-1 has developed in evolution a predisposition for respiratory mucosa and EHV-3 for vaginal mucosa, (ii) abortigenic EHV-1 replicates better in vaginal mucosa than neuropathogenic EHV-1 and (iii) EHV-3 demonstrated a strict epithelial tropism whereas EHV-1 in addition hijacked MC to invade the lamina propria.

Figure 1

Confocal photomicrographs showing EHV-1-induced plaques in nasal and vaginal explants inoculated with EHV-1 03P37 and EHV-1 97P70. The BM is visualized using mouse anti-collagen VII and goat anti-mouse Texas Red^® antibodies. The viral antigens are detected using biotinylated equine polyclonal anti-EHV-1 IgG antibodies and striptavidin-FITC^®. Scale bar 100 µm.

Figure 2

Confocal photomicrographs showing EHV-3-induced plaques in the nasal and vaginal mucosa infected with EHV-3 04P57. The BM is visualized using mouse anti-collagen VII and goat anti-mouse Texas Red^® antibodies. The viral antigens are detected using biotinylated rabbit polyclonal anti-EHV-3 IgG antibodies and streptavidin-FITC^®. Scale bar 100 µm.

Figure 3

Replication kinetics of EHV-1 (A and B) and EHV-3 (C) in the nasal and vaginal explants. The number of plaques/8 mm² explants and the plaque size are shown. Data represent mean ± SD of triplicate independent experiments. Significant differences are indicated by the use of different letters.

Figure 4

Representative confocal microscope images of marker positive-EHV-1-infected cells in the vaginal mucosa. The explants were sectioned (10 µm) and co-immunostained for EHV-1 infected cells (green) and marker positive cells (red). White dotted line indicates the BM. White arrowheads shows double positive cells.

Figure 5

Percentage of EHV-1-infected individual cells. The cells were identified as monocytic cells (CD172a⁺), pan T-lymphocytes (CD3⁺) and B-lymphocytes (IgM⁺) per 20 sections of the nasal and vaginal mucosal explant infected with EHV-1 03P37 (A) and EHV-1 97P70 (B). Lines show the mean ± SD of three independent experiments.