African swine fever (ASF) is a highly lethal disease caused by the ASF virus (ASFV) and poses a significant threat to the swine industry worldwide. This study investigated the pathogenicity and pathological characteristics of VNUA/rASFV/HD1/23, a recently identified recombinant ASFV genotype I/II in northern Vietnam. Sixteen healthy, seven-week-old pigs divided into four groups were inoculated intramuscularly (IM) with different virus concentrations (10², 10³, and 10⁴ HAD₅₀/mL), and their clinical signs, survival times, and pathological alterations were evaluated. All experimentally infected pigs exhibited acute clinical signs characterized by fever, anorexia, depression, diarrhea, and death (4–10 days after injection). The pathological findings included splenomegaly with infarcts, hemorrhagic lymph nodes, and severe pulmonary congestion. The pigs that received the highest dose (10⁴ HAD₅₀/mL) IM showed the earliest onset of clinical signs and the shortest survival time. This study provides important insights into the virulence and the pathological lesions induced by the recombinant genotype I/II ASFV strains that emerged in Vietnam.

Highly virulent African swine fever virus (ASFV) genotype II strains have been responsible for the global epidemic in domestic pigs and are typically characterized by a hemadsorption (HAD)-positive phenotype mediated by the CD2v protein encoded by the EP402R gene. Here, we report the detection and genetic characterization of three non-HAD genotype II ASFV isolates (VNUA/ASFV/VP2023-isolate1, VNUA/ASFV/TB2024-isolate2, and VNUA/ASFV/HY2024-isolate3) recovered from whole-blood samples collected from pigs exhibiting prolonged clinical signs in northern Vietnam. Whole-genome analysis revealed nonsense mutations in the EP402R gene (G57A in isolates VNUA/ASFV/TB2024-isolate2 and VNUA/ASFV/HY2024-isolate3, and G132A in VNUA/ASFV/VP2023-isolate1), resulting in premature stop codons and a HAD-negative phenotype. Furthermore, additional genetic alterations, including deletions and frameshift mutations, were identified within multigene families (MGF110, MGF360, and MGF505), which are known to play critical roles in virulence, host range, and immune evasion. Notably, VNUA/ASFV/VP2023-isolate1 harbored a partial deletion of the I177L gene along with the insertion of an mCherry marker gene, suggesting possible evolution of the modified live ASFV-G-ΔI177L vaccine strain under field conditions. Collectively, these findings underscore the ongoing evolution and genomic plasticity of ASFV strains circulating in Vietnam.