African swine fever virus infection enhances CD14-dependent phagocytosis of porcine alveolar macrophages to promote bacterial uptake and apoptotic body-mediated viral transmission

African swine fever virus (ASFV) is an economically devastating pathogen that can cause fatal infections in both domestic pigs and wild boar, with monocytes and macrophages as its target cells. For macrophages, phagocytosis is a fundamental and crucial process, which is usually suppressed by the virus, impeding pathogen clearance and antigen presentation. However, it was unexpectedly found that ASFV infection enhances the phagocytic ability of primary porcine alveolar macrophages (PAMs), as evaluated using an EGFP-labeled bacterial phagocytosis model. The phagocytic processes, including cell migration, bacterial adhesion, pseudopod extension, and pattern recognition receptor (PRR) expression, in ASFV-infected PAMs were systematically investigated. In addition, the upregulated PRRs were knocked down to analyze their role in enhanced phagocytosis. CD14, a receptor of LPS and phospholipid, was identified as being upregulated by ASFV, leading to enhanced bacterial uptake. Further exploration revealed that ASFV's genomic nucleic acid in infected PAMs activates the cGAS/STING/NF-κB pathway to increase CD14 expression. Meanwhile, the free ASFV nucleic acid released from infected PAMs can also activate CD14 expression in bystander PAMs via the TLR9 pathway, facilitating ASFV transmission via apoptotic bodies (ApoBDs). Moreover, the boosted bacterial phagocytosis in the early stages of ASFV infection potentially creates a more inflamed environment with more intense cytokine production. Here, it reveals a critical mechanism by which ASFV enhances CD14-dependent bacterial uptake in PAMs via the cGAS/STING/NF-κB and TLR9 pathways, promoting viral transmission through ApoBDs and amplifying inflammatory responses to bacterial co-infections, providing vital insights into ASFV pathogenesis and host immune manipulation.IMPORTANCEPorcine alveolar macrophages (PAMs) are the target cells of African swine fever virus (ASFV), but how ASFV impacts their phagocytic function is less known. Here, it was discovered that the nucleic acids of ASFV can enhance the expression of CD14, a receptor of LPS and phospholipid, in infected PAMs via the cGAS/STING/NF-κB pathway, or in bystander PAMs via the TLR9 pathway. Consequently, enhanced CD14 expression facilitates the uptake of bacteria and apoptotic bodies (ApoBDs), promoting the inflammatory response and ASFV cell-to-cell transmission. It provides new insights into the innate immunity response following ASFV infection and the transmission of ASFV.