Identification of <i>Escherichia coli</i> 166 isolate as an effective inhibitor of African swine fever virus replication

African swine fever is a lethal disease with mortality rates approaching 100% in both domestic pigs and wild boars. With no effective vaccines or treatments available, there is an urgent need for new biologics to combat the African swine fever virus (ASFV). In this study, we isolated bacteria from the intestinal contents of wild boar using culture-based methods and identified them through 16S ribosomal DNA (rDNA) sequencing. These isolates underwent high-throughput screening to evaluate their immunomodulatory effects on J774-Dual cells and their ability to inhibit ASFV replication <i>in vitro</i>. Among them, an <i>Escherichia coli</i> strain, designated as <i>E. coli</i> 166, exhibited strong inhibitory effects on various ASFV strains' replication, including three genotype II strains: virulent strain HLJ/18, moderately virulent strain HLJ/HRB1/20, genetically modified low-virulent strain HLJ/18-6GD, and one genotype I low-virulent strain SD/DY-I/21. Notably, this inhibition did not require direct interaction between the bacteria and porcine alveolar macrophages (PAMs). Both live and heat-inactivated <i>E. coli</i> 166 demonstrated a strong inhibitory effect on ASFV replication. Genetic modification of <i>E. coli</i> 166 did not alter its inhibitory phenotype. Further analysis revealed that PAMs pretreated with <i>E. coli</i> 166 showed upregulation of NF-κB and downregulation of CD163 at different time points post-infection, whereas PAMs only infected with ASFV exhibited the opposite trend. These findings suggest that <i>E. coli</i> 166 holds promise as a biological agent for controlling ASFV infection, through indirect mechanisms involving bacterial metabolites or lysis products. Future studies should focus on identifying the specific components responsible for its antiviral effects.IMPORTANCEThe emergence of the African swine fever virus (ASFV) as a devastating pathogen in swine populations necessitates the development of novel strategies for its control. In this study, <i>Escherichia coli</i> strain 166 (<i>E. coli</i> 166) demonstrated a remarkable ability to inhibit the replication of multiple ASFV strains in porcine alveolar macrophages (PAMs), even without direct bacterial contact. Both live and heat-inactivated <i>E. coli</i> 166 retained this inhibitory effect, suggesting that secreted metabolites or lysis products may play a key role. Furthermore, pretreatment of PAMs with <i>E. coli</i> 166 resulted in upregulated NF-κB activity and downregulated expression of the ASFV entry receptor CD163, presenting an immune-modulatory mechanism distinct from PAMs solely infected with ASFV. These findings highlight the potential of <i>E. coli</i> 166 as a biological agent to combat ASFV, offering a promising alternative or complementary approach to traditional antiviral strategies.