ZBP1-mediated macrophage necroptosis inhibits ASFV replication

African swine fever (ASF) is an infectious disease characterized by hemorrhagic fever, highly pathogenic, and severe mortality in domestic pigs caused by the African swine fever virus (ASFV). ASFV is a large DNA virus and primarily infects porcine monocyte macrophages. The interaction between ASFV and host macrophages is the major reason for gross pathological lesions caused by ASFV. Necroptosis is an inflammatory programmed cell death and plays an important immune role during virus infection. However, whether and how ASFV induces macrophage necroptosis and what effect of necroptosis signaling on host immunity and ASFV infection remains unknown. This study uncovered that ASFV activates the necroptosis signaling in the spleen, lung, liver, and kidney from ASFV-infected pigs. And macrophage necroptosis also was induced by ASFV infection in vitro. Further evidence showed that the macrophage necroptosis is independent of TNF--RIPK1 or TLR-TRIF pathway but depends on Z-DNA binding protein 1 (ZBP1). ASFV infection upregulates the expression of ZBP1 and RIPK3 to consist of the ZBP1-RIPK3-MLKL necrosome and further activates macrophage necroptosis. Subsequently, multiple Z-DNA sequences were predicted to be present in the ASFV genome. And the Z-DNA signals were further confirmed to be present and colocalized with ZBP1 in the cytoplasm and nucleus of ASFV-infected cells. Moreover, ZBP1-mediated macrophage necroptosis caused the extracellular release of proinflammatory cytokines TNF- and IL-1{beta} induced by ASFV infection. Finally, we demonstrated that ZBP1-mediated necroptosis signaling significantly inhibits ASFV replication in host macrophages. Our findings uncovered a novel mechanism by which ASFV induces macrophage necroptosis by facilitating Z-DNA accumulation and ZBP1 necrosome assembly, providing significant insights into the pathogenesis of ASFV infection. ImportanceASFV infection causes an acute, febrile, hemorrhagic, and severely lethal disease in swine, which seriously threatens the global porcine industry. Understanding the interaction mechanism between ASFV and host macrophages during infection is essential for elucidating the pathogenesis of ASFV. To our knowledge, this study revealed the interaction mechanism between ASFV infection and host macrophage necroptosis. The results showed that ASFV infection induces macrophage necroptosis through ZBP1 activation instead of the TNF--RIPK1 or TLR-TRIF pathway. ASFV infection promotes Z-DNA accumulation, which causes ZBP1-RIPK3-MLKL necrosome assembly and macrophage necroptosis. The ZBP1-mediated necroptosis signaling facilitates the extracellular release of proinflammatory cytokines, inhibiting ASFV replication in host macrophages. This study found a new interaction mechanism between ASFV and host macrophages, which may help understand the pathogenesis caused by ASFV infection.