Strain and cell type dependent induction of tunnelling nanotube-like projections by African swine fever virus

African swine fever (ASF) is a devastating viral disease of pigs, causing mortality rates of up to 100%. The causative agent, African swine fever virus (ASFV), preferentially targets cells of the mononuclear phagocytic system (MPS). Despite its severe economic and animal health impact, effective antiviral treatments remain unavailable in Europe. We previously showed that ASFV strain Belgium 2018/01 induces tunnelling nanotube (TNT)-like projections in monocyte-derived macrophages (MDMs), enabling physical connections between distant cells. Here, we investigated the broader relevance of this phenomenon across porcine myeloid cell populations and ASFV strains. We assessed TNT-like projection formation in monocytes, pulmonary alveolar macrophages (PAMs), splenic macrophages, polarized and non-polarized MDMs and Vero cells following ASFV infection. We further compared 20 ASFV strains representing diverse genotypes and virulence levels. Interestingly, luminal monocytes and PAMs did not develop ASFV-induced projections, whereas projection formation increased during monocyte differentiation into MDMs. Splenic macrophages also formed projections, suggesting that interstitial tissue macrophages acquire the ability to induce protrusions. Polarized MDMs exhibited reduced projection formation compared with non-polarized MDMs. Similar structures were induced in Ba71V-infected Vero cells, providing a standardized model for mechanistic studies. Importantly, all 20 ASFV strains induced TNT-like projections in MDMs, although the extent varied substantially and was independent of viral genotype or virulence. Our findings demonstrate that ASFV-induced TNT-like projection formation is a conserved, cell-state-dependent phenotype across diverse ASFV strains. These findings provide a foundation for identifying the cellular and viral determinants of TNT formation and defining its potential contribution to ASFV dissemination and pathogenesis.