A novel corneal explant model system to evaluate antiviral drugs against feline herpesvirus type 1 (FHV-1)

Abstract

Feline herpesvirus type-1 (FHV-1) is the most common viral cause of ocular surface disease in cats. Many antiviral drugs are used to treat FHV-1, but require frequent topical application and most lack well-controlled in vivo studies to justify their clinical use. Therefore, better validation of current and novel treatment options are urgently needed. Here, we report on the development of a feline whole corneal explant model that supports FHV-1 replication and thus can be used as a novel model system to evaluate the efficacy of antiviral drugs. The anti-herpes nucleoside analogues cidofovir and acyclovir, which are used clinically to treat ocular herpesvirus infection in cats and have previously been evaluated in traditional two-dimensional feline cell cultures in vitro, were evaluated in this explant model. Both drugs suppressed FHV-1 replication when given every 12 h, with cidofovir showing greater efficacy. In addition, the potential efficacy of the retroviral integrase inhibitor raltegravir against FHV-1 was evaluated in cell culture as well as in the explant model. Raltegravir was not toxic to feline cells or corneas, and most significantly, inhibited FHV-1 replication at 500 µM in both systems. Importantly, this drug was effective when given only once every 24 h. Taken together, our data indicate that the feline whole corneal explant model is a useful tool for the evaluation of antiviral drugs and, furthermore, that raltegravir appears a promising novel antiviral drug to treat ocular herpesvirus infection in cats.

Electric Cell-Substrate Impedance Sensing To Monitor Viral Growth and Study Cellular Responses to Infection with Alphaherpesviruses in Real Time

“…For these experiments, we decided to use cidofovir, which is a topical nucleoside analogue commonly used to treat FHV-1-induced ocular disease with reported clinical efficacy based on a controlled in vivo experimental study (12, 24). We and others have previously determined the EC 50 of cidofovir for FHV-1 by using traditional plaque reduction assays and found the EC 50 to range between 7.9 and 21.5 µM (25–27). To determine the EC 50 by ECIS, CRFK cells were infected with FHV-1 at an MOI of 0.01 and treated with decreasing concentrations of cidofovir at the time of infection.…”

“…WT FHV-infected CRFK cells and isotype control antibodies (Abcam, Inc., Cambridge, United Kingdom) were included as controls. Cells were imaged exactly as previously described (27). …”

“…Single-step (MOI of 3) and multistep (MOI of 0.01) growth kinetic assays were performed, also exactly as previously described (39), except that the adsorption period lasted 2 h. Samples were collected at the intervals indicated with cell-free supernatant samples used for standard plaque assays (27) and cell lysates used for quantitative PCR (qPCR). For the latter, a standard curve with primers targeting a region in the US7 gene homology arm was created with the linearized donor plasmid used to create FHV-1-gD-DsRed as the template (Table 1).…”

The HIV Integrase Inhibitor Raltegravir Inhibits Felid Alphaherpesvirus 1 Replication by Targeting both DNA Replication and Late Gene Expression

“…For flow cytometry, a recombinant FH2CS FeHV-1 strain (FeHV-1-gD-DsRed) was used, which we generated in house using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 genome engineering to express DsRed Express 2 fused to the C-terminal end of glycoprotein D (48). All viral stocks were grown and their titers were determined on CRFK cells, as previously described (26). Raltegravir (ChemieTek, Indianapolis, IN) and acyclovir (Sigma-Aldrich, St. Louis, MO) were diluted in DMSO.…”

“…Like HHV-1, FeHV-1 has similar challenges for successful treatment (24,25). Our laboratory has shown previously that raltegravir can inhibit replication of FeHV-1, both in cell culture and in an ex vivo corneal explant model, comparably to the currently utilized antivirals (26). Furthermore, we recently demonstrated that raltegravir reduces FeHV-1 shedding duration and improves clinical outcomes in experimentally infected cats (C. B. Spertus, M. R. Pennington, G. R. Van de Walle, Z. I. Badanes, B. E. Judd, H. O. Mohammed, and E. C. Ledbetter, submitted for publication).…”