Genotype-Dependent Modulation of Macrophage Apoptosis and T Lymphocyte Responses by Duck Tembusu Virus Potentially Contributes to Genotype-Specific Immunopathogenesis.

Duck Tembusu virus (DTMUV) is an emerging avian pathogenic flavivirus that causes neurological disorders and acute egg drop syndrome in ducks. Multiple genotypes of DTMUV, including clusters 1, 2, and 3, have been identified, and recent evidence suggests a correlation between viral genotype and disease severity. Our recent study demonstrated that DTMUV cluster 2.1 induces more severe clinical disease in ducks than cluster 1; however, the underlying mechanisms responsible for this difference remain unclear. Macrophages are key target cells for DTMUV replication and play a crucial role during the early stage of viral pathogenesis. Therefore, we hypothesized that distinct DTMUV genotypes may differentially regulate macrophage apoptosis, thereby influencing viral replication and modulating T lymphocyte responses. To investigate this, we developed an in vitro protocol to generate duck monocyte-derived macrophages (MDMs) and assessed their response to infection with two different DTMUV genotypes: cluster 1 and cluster 2.1. Our results showed that DTMUV cluster 1 infection significantly increased macrophage apoptosis and reduced cell viability. In contrast, DTMUV cluster 2.1-infected MDM exhibited higher cell viability and fewer apoptotic cells. Correspondingly, viral titers in supernatants from DTMUV cluster 2.1-infected cultures were significantly higher than those from cluster 1. Furthermore, culture with DTMUV cluster 2.1 resulted in enhanced proliferation of Th (CD3+CD4+BrdU+) and cytotoxic T lymphocyte (CTL; CD3+CD8+BrdU+) lymphocytes in both naïve peripheral blood mononuclear cells (PBMCs) and macrophage-peripheral blood lymphocytes (PBLs) co-culture systems. These results suggest that the inhibition of apoptosis in DTMUV cluster 2.1-infected macrophages may enhance antigen-specific T lymphocyte responses. Overall, our findings reveal a genotype-dependent mechanism by which DTMUV modulates macrophage survival, thereby influencing both viral replication and T cell activation, and ultimately contributing to genotype-specific immunopathogenesis. These insights deepen our understanding of DTMUV immunopathogenesis and may inform the development of more effective genotype-targeted vaccination and disease control strategies.