Assessment of Cidofovir for Treatment of Ocular Bovine Herpesvirus-1 Infection in Cattle Using an Ex-Vivo Model

Abstract

Bovine herpesvirus-1 (BoHV-1) infection contributes to keratoconjunctivitis, respiratory disease, and reproductive losses in cattle. The objective of this study was to determine the most appropriate ophthalmic antiviral agent for BoHV-1 inhibition using in-vitro culture and novel ex-vivo bovine corneal modeling. Half-maximal inhibitory concentrations of BoHV-1 were determined for cidofovir, ganciclovir, idoxuridine, and trifluridine via in-vitro plaque reduction assays. In-vitro cytotoxicity was compared amongst these compounds via luciferase assays. Trifluridine and cidofovir were the most potent BoHV-1 inhibitors in vitro, while trifluridine and idoxuridine were the most cytotoxic agents. Therefore, cidofovir was the most potent non-cytotoxic agent and was employed in the ex-vivo corneal assay. Corneoscleral rings (n = 36) from fresh cadaver bovine globes were harvested and equally divided into an uninfected, untreated control group; a BoHV-1-infected, untreated group; and a BoHV-1-infected, cidofovir-treated group. Virus isolation for BoHV-1 titers was performed from corneal tissue and liquid media. Histologic measurements of corneal thickness, epithelial cell density, and tissue organization were compared between groups. Substantial BoHV-1 replication was observed in infected, untreated corneas, but BoHV-1 titer was significantly reduced in cidofovir-treated (1.69 ± 0.08 × 10³ PFU/mL) versus untreated (8.25 ± 0.25 × 10⁵ PFU/mL, p < 0.0001) tissues by day 2 of culture. No significant differences in histologic criteria were observed between groups. In conclusion, cidofovir warrants further investigation as treatment for BoHV-1 keratoconjunctivitis, with future studies needed to assess in-vivo tolerability and efficacy.

Keywords: Bovine herpesvirus-1; cidofovir; ex-vivo corneal culture; keratoconjunctivitis; nucleotide analogue antiviral agent.

Figure 1

Culture plate for ex-vivo corneal assay featuring plastic pedestal corneal conformers.

Figure 2

Semiquantitative histopathology grading scheme for bovine corneal sections. Epithelial stratification:0 = clear distinction of basal, wing, and squamous cell layers; 1 = weak disorganization with minor squamous separation; 2 = moderate disorganization with moderate squamous separation; 3 = marked disorganization with significant squamous separation and degenerate basal cell attachments. Basal (B), wing (W), and squamous (S) cell layers are indicated in the Grade 0 image. Stromal organization: 0 = no stromal disorganization; 1 = weak stromal disorganization; 2 = moderate stromal disorganization; 3 = marked stromal disorganization. All images are photomicrographs magnified to 400X.

Figure 3

In-vitro comparison of nucleoside analogue antiviral agent cytotoxicity to Madin–Darby Bovine Kidney (MDBK) cells via CellTiter-Glo^® assay. Relative luminescence (RL) is correlated with cellular ATP concentration; lower relative luminescence therefore indicates greater cytotoxicity. Lowercase letter labels “a–d” refer to statistical comparisons of RL amongst antiviral compounds—columns connected by the same letter labels are not significantly different (p > 0.05).

Figure 4

Homogenized corneal button (a) and liquid EpiLife^® cell media (b) BoHV-1 titers for ex vivo bovine corneal culture. I = BoHV-1-infected, untreated group; I+T = BoHV-1-infected, cidofovir-treated group. Columns connected by the same letter labels are not significantly different (p > 0.05). Uppercase letters correspond to within-day comparisons. Lowercase letters correspond to within-treatment comparisons. (c) Representative photomicrographs from histologic specimens of the I and I+T groups on days 0 and 3 depicting corneal epithelial changes over the duration of culture. In the I group, attenuation of the epithelium with cellular condensation, disorganization of epithelial stratification, heterogenous nuclear chromatin, and exfoliation of the squamous epithelium are seen on day 3. In the I+T group, attenuation of the epithelium, cellular condensation, and multiple eosinophilic cytoplasmic inclusions (suggesting cell death) are seen.