Cyprinid herpesvirus 3 does not replicate productively in EPC cells but induces S-phase cell cycle arrest.

EPC (Epithelioma Papulosum Cyprini) cell line is widely used for culturing various aquatic animal viruses. However, prior studies reported that EPC cells were non-permissive to CyHV-3 replication. In this study, CyHV-3 were inoculated to EPC cells and conducted cytopathic effect (CPE) observation, PCR and quantitative real-time PCR for viral gene detection and load quantification, electron microscopy to visualize virion formation, transcriptome sequencing (RNA-seq) to explore infection mechanisms, and flow cytometry to assess cell cycle alterations. Results demonstrated that CyHV-3-infected EPC cells exhibited no CPE at 24 h, 48 h, 72 h, 96 h, 7d, and 14d post-infection. While viral DNA was detected in cell pellets, neither viral genes nor cDNA were found in the supernatant. RT-qPCR confirmed low viral loads (<103 copies/5 ng) at all time-points, and no virions were observed via transmission electron microscopy, collectively indicating the establishment of non-productive infection. Transcriptomic analysis of EPC cells at 96 h post-infection identified 607 differentially expressed genes (DEGs). GO enrichment categorized these DEGs into biological processes (BP), molecular functions (MF), and cellular components (CC), primarily associated with cellular compartments, binding, metabolic processes, catalytic activity, and biological regulation. KEGG pathway analysis revealed enrichment in sterol biosynthesis, cell cycle, terpenoid backbone synthesis, and DNA replication. Flow cytometry showed that CyHV-3 infection induced S-phase arrest in EPC cells, with the arrested population increasing over time. These findings suggest that CyHV-3 does not replicate productively in EPC cells but induces S-phase cell cycle arrest, which may help virus establish non-productive persistent infection.