Optimisation of a VSV-based ASFV pseudovirus platform enabling safe evaluation of envelope protein-mediated entry and host innate immune responses.

African swine fever virus (ASFV) remains a global threat to the swine industry, yet research is hampered by the requirement for high-containment biosafety facilities. To facilitate investigation into ASFV entry mechanisms under Biosafety Level 2 conditions, we engineered a robust vesicular stomatitis virus (VSV)-based pseudotype system. Five envelope-associated proteins p17 (D117L), p54 (E183L), pE248R (E248R), p22 (KP177R), and p30 (CP204L) were systematically evaluated. Through optimization of plasmid transfection ratios and packaging kinetics, we demonstrated that dual-envelope combinations particularly those involving E248R and KP177R significantly enhanced entry efficiency compared to single-protein pseudotypes, suggesting a synergistic role in viral attachment and membrane fusion. Functional assays across diverse cell lines including PAMs, Vero, and BHK-21 revealed distinct species and cell-line specific tropism dictated by specific envelope compositions. Notably, while the system exhibited minimal cytotoxicity in permissive cells, pseudovirus entry triggered early modulation of the host innate immune landscape. Specifically, we observed a consistent upregulation of IL-10 and cell-type-dependent regulation of IFN-γ, indicating that envelope-mediated attachment and internalization can transiently alter early cytokine secretion prior to viral replication. Ultimately, this multivalent platform provides a high-titer, safe experimental alternative for dissecting ASFV entry mechanisms and potentially serves as a foundational model that could likely assist in the future preliminary evaluation of entry-targeted antivirals and neutralizing antibodies.