Abstract
Since its first incursion in 2019, African swine fever (ASF) has remained a constant threat to the Vietnamese swine industry. The nationwide commercialization and implementation of live-attenuated vaccines (LAVs), alongside depopulation-repopulation efforts, necessitate a critical assessment of whether these strategies have driven inadvertent changes in circulating African swine fever virus (ASFV) strains. In this study, molecular surveillance of ASFV core genes was conducted in pigs from non-vaccinated farms across Central and Southern Vietnam, comparing strains before and after LAVs' introduction. The results confirmed spillover and ongoing field circulation of LAV-related strains and revealed a marked increase in genetic diversity, encompassing both vaccine-like and novel wild-type microvariants in the post-vaccine commercialization phase. Notably, these microvariants harbored non-synonymous single-nucleotide polymorphisms (SNPs) and large segmental deletions within the EP153R-EP402R gene, suggesting potential alterations in viral function. Most compellingly, a unique 15-AGACCAGCAACAAAC-nucleotide repeat in the E183L gene resulted in a five-amino-acid insertion (Arg-Pro-Ala-Thr-Asn) in the p54 protein, serving as a key marker for Vietnamese strains. Initial in silico analysis indicated that this novel p54 protein contained a longer α-helix structure, which could influence viral movement and interactions with host cells. Overall, these subtle yet significant genetic shifts imply that ASFV might be rapidly evolving intra-host under selective pressure from widespread immunization and/or diverse introduction sources during repopulation, potentially enabling it to evade the host immune system. This highlights the essential need for robust biosecurity protocols, stringent regulatory oversight of LAV deployment, and the required genetic surveillance. For diagnosis and management, the urgent development of reliable DIVA (Differentiating Infected and Vaccinated Animals) tools and real-time, adaptable molecular identification methods, coupled with a vaccination-tracking system, is necessary for accurate and sustained ASF control.