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Objective: To determine the replication kinetics and cytopathic effect (CPE) of feline calicivirus (FCV) in feline corneal epithelial cells (FCEC)., Animals Studied: Seven archived FCV isolates and one archived feline herpesvirus type 1 (FHV-1) isolate, previously obtained from eight domestic short hair cats., Procedures: FCV RNA was extracted for sequencing using Illumina MiSeq, to identify three genomically diverse isolates for further testing. Following reference-based assembly, viral genomes were annotated and assessed. Superficial keratectomies were performed to isolate the corneal epithelium of cats and the cells were cultured in vitro. FCEC were infected with the three chosen FCV isolates and one FHV-1 isolate at two different multiplicity of infection ratios (MOIs, 0.1 and 0.01 PFU/cell) and virus titration was assessed at 0, 2, 6, 12, 24, and 48 h post-infection (hpi). Viral identity was confirmed by RT-qPCR., Results: Three genomically diverse FCV isolates were chosen for further assessment in the FCEC model. All infections of FCEC with FCV led to visible CPE, characterized by epithelial cell rounding and detachment from the plate by 24 hpi, while FHV-1 led to visible CPE within 48 hpi. All three of the FCV isolates replicated effectively in FCEC at both 0.1 and 0.01 MOI, with a peak increase in titer approximately 12-24 hpi., Conclusions: The results support the possible role of FCV as a primary pathogen of the feline ocular surface. FCV replicates in FCEC in vitro, leading to profound CPE., (© 2024 American College of Veterinary Ophthalmologists.)