Previous studies have suggested that anti-viral cytotoxic effector cells induced by infection with a sublethal dose of crucian carp hematopoietic necrosis virus (CHNV) correspond to mammalian cytotoxic T-lymphocytes (CTLs), because the mRNA expression patterns of the effector cells are similar to those of mammalian CTLs. To further characterize the effector population in cell-mediated cytotoxic (CMC) activity, we isolated the effector cells using an anti-CD8α monoclonal antibody, a density gradient and plastic adherence. As expected, the purified CD8α-positive cells killed CHNV-infected cells, indicating that the fish CTLs are one of anti-viral effector cells similar those to in mammals. However, it appeared that cytotoxic cells other than CTLs were the dominant effectors, because CTL-depleted peripheral blood leukocytes (PBL) exhibited significant cytotoxic activity against CHNV-infected cells. In addition, the adoptive transfer of CTL-depleted PBL provided as efficient protection against CHNV-infection as the transfer of PBL containing CTLs. Further analyses showed that sIg/CD8α-negative cells and monocyte-enriched effectors possessed activities that were comparable to or were higher than that of CD8α-positive cells, suggesting that natural killer (NK)-like cells and monocytes are among the dominant effector cells. CMC inhibition assays with concanamycin A suggested that CTLs and CD8α-negative lymphocytes lysed virus-infected cells by a perforin-based cytotoxic pathway. These results indicate that CMC induced by viral-infection is executed by not only CTLs but monocytes and CD8/IgM-negative lymphocytes.
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