Emergence of Non-Hemadsorbing African Swine Fever Virus Genotype II Variants and the Evolution of a Vaccine-Derived Strain in Vietnam

Figure 1. Geographic locations of the sampled provinces in Vietnam. Colored provinces (Vinh Phuc, Hung Yen, and Thai Binh) indicate the sampled locations. White areas represent other provinces in northern Vietnam, and grey areas represent provinces in central and southern Vietnam.

Figure 2. Hemadsorption (HAD) assay results for the three isolates obtained from whole blood samples collected from pigs showing prolonged clinical signs. (A) Uninoculated porcine alveolar macrophages (PAMs) without added red blood cells. (B) Uninoculated PAMs with added red blood cells. (C) Positive control: PAMs infected with VNUA/HY-ASF1 [46], demonstrating characteristic hemadsorption (HAD) rosette formation. (D–F) PAMs infected with VNUA/ASFV/VP2023-isolate1, VNUA/ASFV/TB2024-isolate2, and VNUA/ASFV/HY2024-isolate3, respectively, showing cytopathic effects (CPE) in the absence of HAD. Images were captured at 40× magnification.

Figure 3. Maximum likelihood phylogenetic tree based on whole-genome sequences of ASFV strains. The tree was generated using IQ-TREE (1000 bootstrap replicates) and visualized with iTOL Version 7.4.1 (Interactive Tree of Life, https://itol.embl.de (accessed on 26 January 2026). The three isolates from this study are shown in bold red.

Figure 4. Multiple sequence alignment of the I73R–I329L intergenic region of the three isolates and reference strains belongs to IGR I to IV variants. Dashed vertical lines indicate distinct repeat blocks.

Figure 5. Circular plot comparing the genomes of ASFV Georgia 2007/1 and the three isolates from this study based on ORF-level similarity. Each concentric ring represents a viral genome, with ORFs displayed according to their genomic positions. Gray-labeled ORFs indicate coding regions that are expected to be located within the missing terminal regions at the 5′ and/or 3′ ends of the genomes belonging to the three isolates. Red-labeled ORFs denote genes harboring major mutations, including large deletions, frameshift mutations, or premature stop codons, relative to the reference genome. The height of each ORF segment is proportional to its nucleotide identity percentage compared with ASFV Georgia 2007/1.

Figure 6. Disruption of the EP402R (CD2v) coding sequence by premature stop codons. Black boxes with asterisks indicate positions containing premature stop codons.

Figure 7. Sequence alignment of the modified I177L locus in VNUA/ASFV/VP2023-isolate1 against a reference ASFV Georgia 2007/1. Grey regions represent aligned sequence regions shared between the compared ASFV strains, black regions indicate unmatched sequence regions.