Insect and fish cell lines support replication and serial passage of covert mortality nodavirus

Covert mortality nodavirus (CMNV) is the pathogen causing viral covert mortality disease (VCMD) in cultured shrimp, and cross-species transmission is an important characteristic of this virus. The lack of stable, continuously passable shrimp cell lines has long been a bottleneck for in-depth studies on CMNV pathogenesis, virus-host interactions, and antiviral strategies. In this study, we systematically evaluated the infectivity, replication kinetics, and serial passage stability of CMNV in eight cell lines of different origins, including three insect cell lines (Sf21, C6/36, and S2) and five fish cell lines (EMRB, EPC, ZF4, TiB, and GiCB). Based on CPE observations and viral RNA replication profiles across three blind passages, cell lines exhibiting both obvious CPE and viral RNA replication in at least two passages were classified as susceptible, including EPC (P1 and P3), TiB (P1 and P3), and S2 (P1-P3). ZF4 (P1-P3) and C6/36 (P1 and P3), despite lacking obvious CPE, supported viral RNA replication across multiple passages and were therefore tentatively designated as atypically susceptible. In contrast, Sf21 and EMRB showed no detectable viral RNA replication in any of the three passages, while GiCB supported replication only at P1; these three cell lines were accordingly classified as non-susceptible. These findings identify multiple non-shrimp cell lines that support stable CMNV replication at the RNA level and serial passage, providing useful experimental systems for studies of viral pathogenesis, host interactions, diagnostic development, and antiviral product screening.