Establishment of a Novel Primary Cell Culture Model for the Continuous Propagation of Carp Edema Virus In Vitro

Koi sleepy disease (KSD), also called carp edema virus disease (CEVD), is a widespread viral infection affecting common carp (Cyprinus carpio) and its ornamental varieties, leading to substantial economic losses in global aquaculture. The development of effective prophylactic and therapeutic measures against CEVD has been hampered by the lack of a robust system for in vitro propagation of its causative agent, carp edema virus (CEV). To address this constraint, a method was established for the isolation and continuous culture of CEV using primary head kidney cells derived from Songpu mirror carp. A highly pathogenic CEV strain, L-3, isolated from an outbreak in Liaoning, China, was successfully propagated in vitro through progressive adaptation during serial passage. Pronounced cytopathic effects (CPE) were observed over 17 consecutive passages, with CEV presence confirmed in each passage by nested PCR. Viral replication dynamics, assessed across 20 successive passages, demonstrated a progressive increase in viral copy numbers from very low levels at initial passage to peak at passage 18, confirming successful adaptation to primary head kidney cells. Challenge trials with the 20th-passage virus induced clinical signs characteristic of KSD in carp. Moreover, examination of gill tissues from infected fish by transmission electron microscopy revealed viral particles with morphology typical of CEV. The present work reveals a novel approach for the propagation of CEV in vitro. These findings are expected to provide a critical tool for advancing fundamental research on viral pathogenesis and supporting the systematic development and evaluation of vaccines against CEVD.