Domestic cats enrolled in a recent clinical trial comparing three treatments for naturally-acquired ocular FHV-1 (12) were utilized in the present study. All procedures were performed in accordance with an approved Louisiana State University Institutional Animal Care and Use Committee protocol (LSU 19–034). All cats were housed in animal shelter facilities at the time of sampling and had ocular surface disease consistent with FHV-1 infection. Quantitative ocular disease scoring was performed by a trained observer, as previously described (12). All cats were confirmed to be actively shedding FHV-1 using quantitative polymerase chain reaction (qPCR) (Texas A&M Veterinary Medical Diagnostic Laboratory). On entry to the shelter, cats were screened for feline immunodeficiency virus and feline leukemia virus (SNAP, Idexx). Cats were randomly allocated to receive one of four treatment protocols: ophthalmic placebo solution (eyewash) + oral lactulose placebo, cidofovir 0.5% ophthalmic solution + oral lactulose placebo, ophthalmic placebo solution (eyewash) + famciclovir oral suspension (250 mg/mL at 90 mg/kg), or ganciclovir 0.15% ophthalmic solution + oral lactulose placebo. Shelter staff were masked to treatment group for all animals. All cats received one ocular preparation and one oral preparation twice daily regardless of group. All medications and placebo were acquired from compounding (503A and 503B) facilities (Stokes Pharmacy and Epicur Pharma) with drug concentrations validated at an independent external laboratory. All cats were sampled before (day 1) and after (day 8) 7 days of treatment using swabs (sterile foam tipped swab, Puritan) rolled against the oropharyngeal and conjunctival surfaces. Following sample acquisition, each swab was immediately placed into viral transport media (composed of Hank’s balanced salt solution, heat-inactivated fetal bovine serum, gentamicin sulfate and amphotericin B, Thermo Fisher) and placed on ice for transportation.

On the same day as sample collection, viral isolation was performed using Crandell Rees feline kidney cells (CRFK) (ATCC), as previously described (1, 3). Cells were cultured in Dulbecco’s modified Eagle medium (DMEM; Life Technologies) with 10% fetal bovine serum (VWR) and 1% penicillin and streptomycin sulfate (Life Technologies) at 37°C/5% CO2. A 0.5 mL aliquot of each sample was added to individual T25 culture flasks containing maximally confluent CRFK cells along with 2 mL of DMEM containing 2% fetal bovine serum and 1% penicillin and streptomycin before being placed on a rocker for 60 min. An additional 4 mL of DMEM was then added to each flask before being incubated at 37°C with 5% CO₂ and checked daily until 100% cytopathic effect (CPE) was observed. The infected cells and media in the flask were then frozen at −80°C and thawed at room temperature for 3 cycles. The sample was centrifuged at 1,000 rpm for 5 min (ThermoScientific Heraeus Megafuge 8R centrifuge) to acquire supernatant which was stored at −80°C.

Viral DNA extracted from the frozen supernatant was prepared using a commercial kit according to the manufacturer’s instructions (PureLink Viral RNA/DNA Mini Kit), as previously described (3). DNA purity and concentration were assessed using a Qubit dsDNA HS Assay Kit (Life Technologies). Samples were confirmed to contain FHV-1 DNA prior to sequencing using qPCR (ABI 7900-2) with FHV-1 thymidine kinase primers (22) (IDT).

Viral genome sequencing was performed as previously described (3, 23). DNA samples were diluted to 1 ng DNA in 5 mL solution and libraries were constructed according to the Nextera XT DNA Library Preparation Kit protocol (FC-131-1096; Illumina Inc.). Libraries were amplified and indexed using Nextera XT Index Kit v2 (Set A; Illumina Inc.). Quality and quantity of finished libraries were assessed using a Fragment Analyzer Instrument (Advanced Analytical) and dsDNA HS Assay Kit. Indexed libraries were pooled and paired-end sequenced using an Illumina MiSeq 500 bp (v2) sequencing kit (MS-102-2003).

Reference-based assembly was performed using Geneious Prime ver 2022.2.2. Paired-end reads were trimmed using BBDuk adapter/quality trimmer ver 38.84 (right end, Kmer length = 27, maximum substitutions = 1, minimum quality = 20, minimum overlap = 20, minimum length = 20). Trimmed paired-end reads were then assembled to FHV-1 reference sequence C-27 (GenBank accession NC_013590). A consensus sequence was extracted from aligned reads with gaps filled with “N’s.” Genomes were then annotated and submitted to GenBank using Geneious Prime ver 2022.2.2. Genbank accession numbers for each isolate are shown in Table 1.

Viral genomes were aligned using MAFFT ver 7.490 within Geneious Prime ver 2022.2.2 (24). Default parameters were used for all alignments (scoring matrix of 1PAM/k = 2, gap penalty of 1.53 and offset value of 0.123). Alignments of FHV-1 genomes were created using a canine herpesvirus type 1 (CHV-1) outgroup (0194, GenBank Accession NC_030117.1).

Analysis of variants was performed as previously described using the Geneious variant finder within Geneious Prime ver 2022.2.2 (3, 23). Each day 8 isolate was individually compared to the corresponding day 1 isolate to identify variants which had developed over the course of the 7-day treatment period. Variant totals were compared between treatment groups using one way ANOVA with significance set at p < 0.05.

Alignments were analyzed (1,000 bootstrap replicates) using ModelFinder (25) via IQ-Tree 2 ver 1.6.12 (26), which described the best-fit model (TVM + F + R4/5). The resultant maximum likelihood tree was visualized using Splitstree ver 4.16.1 (27).

In-vitro IC₅₀ analysis was performed as previously described (14) on viral pairs where day 8 isolates demonstrated non-synonymous mutations detected by genome variant analysis. IC₅₀ in the present study was defined as the concentration of antiviral drug at which the number of observed viral plaques is reduced by 50%, relative to untreated infected cells. CRFK cells were cultured in Dulbecco’s modified Eagle medium (DMEM; Life Technologies) with 10% fetal bovine serum (VWR) and 1% penicillin and streptomycin sulfate (Life Technologies) at 37°C/5% CO2 on 24 well plates until maximally confluent. Fifty viral plaque-forming units of each FHV-1 isolate were absorbed by the cells in each well over 1 h (on a rocker at room temperature). Based on the antiviral drug each individual host cat received, the supernatant in each well was then replaced with DMEM containing 1% methylcellulose, 2% FBS and 1% penicillin–streptomycin ± ganciclovir (Acros Organics), cidofovir (Apexbio) or penciclovir, the active metabolite of famciclovir (TCI). Plates were incubated at 37°C/5% CO2 for 48 h, then fixed with methanol and stained with 0.5% crystal violet solution (Fisher Scientific). Viral plaque numbers were then counted using a light microscope. Concentrations of each antiviral assessed were based on previously published mean IC₅₀ values for FHV-1 (7): 8.9 μM for ganciclovir, 19 μM for cidofovir and 14 μM for penciclovir. Multiple concentrations of each antiviral were assessed in order to determine IC₅₀ values for each day 1 and day 8 isolate; 0.25 X IC_50, 0.5 X IC_50, 1 X IC_50, 2 X IC₅₀ and 5 X IC_50. Positive (no antiviral) and negative controls (no virus) were included and all wells were repeated in triplicate. IC₅₀ values were determined for each day 1 and day 8 isolate and values were compared for matched pairs. Antiviral resistance was considered to have developed if a 3-fold increase in IC50 was present (28) in day 8 isolates, relative to day 1 isolates.

To ensure that reduction in viral plaque number was unrelated to CRFK cellular viability, a cellular cytotoxicity assay was utilized (CellTiter-Glo^® kit, Promega) according to manufacturer recommendations. Trypsinized CRFK cells were transferred to 96-well tissue culture plates (Corning Costar) and incubated in DMEM +10% FBS/1% PS until confluent as outlined above. Growth medium was decanted and replaced with DMEM +2% FBS/1% PS containing 0.25X, 0.5X, 1X, 2X, or 5X the IC₅₀ values for each antiviral compound (cidofovir, ganciclovir, and penciclovir), as utilized for the plaque reduction assay. Negative control wells containing CRFK cells in media without added antiviral compounds and separate blank wells containing only liquid media without cells were included on each plate, to allow for creation of corrected controls. Plates were incubated at 37°C/5% CO2 for 48 h and were then transferred to room temperature for 30 min. CellTiter-Glo^® reagent was added to each well and cell lysis was induced using an orbital shaker at room temperature for 2 min. Plates were then incubated at room temperature for 10 min before luminescence was measured with a microplate reader (Synergy HTX, BioTek). Four replicates were assessed for each antiviral concentration. Cytotoxicity was expressed as relative luminescence, calculated as the mean luminescence of a given antiviral concentration divided by the mean luminescence of the corrected plate negative control.

A total of 14 host animals actively shedding FHV-1 were sampled to obtain 28 isolates of FHV-1 (Table 1). The median age of the host cats was 3 months with a range of 2–24 months. A total of six females and eight male animals were sampled. Three animals received placebo medications, three received cidofovir 0.5% ophthalmic solution, five received famciclovir oral suspension (250 mg/mL at 90 mg/kg) and three received ganciclovir 0.15% ophthalmic solution. Five of the 14 animals experienced worsened ocular clinical signs following treatment, 8/14 animals improved and 1/14 had no change in ocular clinical signs (Table 1). Of the five animals which experienced worsened ocular clinical signs, 3/5 were receiving famciclovir, 1/5 was receiving ganciclovir and 1/5 was receiving placebo. All enrolled animals were seronegative for feline immunodeficiency virus and feline leukemia virus.

A total of 28 FHV-1 isolates (14 pairs) were fully sequenced using Illumina Miseq. The total number of reads ranged from 251,648 to 4,643,990 and the number of reads mapped to the reference genome ranged from 34,056 to 2,993,610. The mean coverage of the FHV-1 genome ranged from 56.4 to 5114.1. The GC content of the FHV-1 genomes sequenced ranged from 44.7 to 46% and genome lengths ranged from 135,996 to 140,625, which is consistent with previous reports of FHV-1 (1, 3). Individual isolate sequencing information is shown in Supplementary Table S1.

Variants were detected in all day 8 isolates when compared with the paired day 1 isolates and are summarized in Table 2. All detected variants in each day eight isolate are shown in Supplementary Table S2. No variants were detected in viral genes associated with viral replication; thymidine kinase (UL23) and DNA polymerase (UL30 and UL42). The median (range) number of genome variants was 10 (10-38) in FHV-1 from hosts receiving cidofovir, 9 (4-9) in FHV-1 from hosts receiving ganciclovir, 13 (2-27) in FHV-1 from hosts receiving famciclovir and 12 (2-15) from hosts receiving placebo. There was no significant different in variant totals by host treatment (p = 0.4997). The vast majority (171/174) of variants led to synonymous amino acid changes, whereas only three were non-synonymous. These were found in the day eight isolate from three separate animals receiving either famciclovir (host animal 12, LOU-23/24), cidofovir (host animal 2, LOU-3/4) or ganciclovir (host animal 14, LOU-27/28). In all three cases, the single non-synonymous variant was located in the FHV-1 ICP4 gene.

Phylogenetic tree analysis was utilized to visualize and estimate distances between pairs of isolates with a CHV-1 outgroup (Figure 1). For the majority of isolates (10/14), pairs clustered closely together. Four pairs of isolates were not clustered immediately together (LOU-3/4, LOU-7/8, LOU-23/24, and LOU-27/28). In each of these four cases, the day 8 isolate contained a relatively high total of variants (LOU-8), at least 1 non-synonymous variant (LOU-24, LOU-28) or both (LOU-4) (Table 2).

Three pairs of isolates (LOU-3/4, LOU-23/24, and LOU-27/28) which demonstrated non-synonymous variants were chosen for in-vitro antiviral plaque reduction assay analysis (Table 2). These isolates were obtained from host animal 2 (treated with cidofovir), host animal 12 (treated with famciclovir) and host animal 14 (treated with ganciclovir). In summary, no evidence of antiviral drug resistance (>3 fold increase in IC₅₀ values) was detected in any of the 3 day 8 isolates assessed. The IC₅₀ values were 49.5 μM (day 1, LOU-3) and 45.8 μM (day 8, LOU-4) for isolates from host animal 2 (cidofovir), 50.2 μM (day 1, LOU-23) and 52.1 μM (day 8, LOU-24) for isolates from host animal 12 (penciclovir) and 15.2 μM (day 1, LOU-27) and 17.5 μM (day 8, LOU-28) for isolates from host animal 14 (ganciclovir) (Figure 2). CRFK cellular viability was maintained at all concentrations of antiviral which were assessed and visual observation of cellular monolayers confirmed this finding.