Background and Aim: African swine fever (ASF) is a highly contagious and often fatal hemorrhagic viral disease affecting domestic and wild pigs. Since its emergence in Indonesia in 2019, ASF virus (ASFV) has spread across multiple islands and become endemic in numerous provinces. Previous molecular investigations have consistently identified genotype II as the predominant circulating genotype; however, these studies offered limited information on intra-genotypic diversity, geographic distribution, and temporal genetic stability across distant regions. The present study aimed to determine the genotype and evaluate genetic variations of representative ASFV strains responsible for outbreaks in East Kalimantan, North Sumatra, and East Nusa Tenggara provinces during 2021–2023. Molecular characterization was performed using the partial B646L (p72) gene, the complete E183L (p54) gene, and the tandem repeat sequences within the intergenic region between I73R and I329L genes. To the best of our knowledge, this represents the first multi-province and multi-year analysis of ASFV in Indonesia employing this combined molecular approach. Materials and Methods: A total of 33 clinical specimens (organs and swabs) collected from affected backyard pigs and farm environments in Berau City (East Kalimantan), Pematang Siantar (North Sumatra) and Kupang (East Nusa Tenggara) were screened for ASFV by quantitative polymerase chain reaction (PCR) targeting the B646L (p72) gene. Five representative samples, selected based on cycle threshold values and geographic representation, were subjected to conventional PCR amplification and Sanger sequencing of the three target genomic regions. Nucleotide sequences were assembled, aligned, and analyzed phylogenetically using neighbor-joining and minimum evolution methods with 1 000 bootstrap replicates. Amino acid translations and tandem repeat motif comparisons were conducted to detect insertions, deletions, or sequence polymorphisms relative to reference strains. Results: ASFV DNA was detected in all 33 specimens. Phylogenetic analyses of partial B646L (p72) and full-length E183L (p54) sequences unambiguously classified the five Indonesian isolates within genotype II, clustering closely with contemporary Asian genotype II strains. Sequence analysis of the E183L (p54) gene revealed a previously unreported insertion of five amino acids (PVTDN motif) at positions 121–125 in the two isolates originating from North Sumatra; this duplication was absent from all other Indonesian and Southeast Asian genotype II sequences examined. Intergenic region analysis demonstrated that all isolates belonged to the IGR II variant, characterized by two identical 10-nucleotide GGAATATATA tandem repeat units. This motif showed 100% identity across the 2021–2023 samples and matched the configuration reported from the initial 2019 outbreak in North Sumatra, indicating sustained genetic homogeneity. Conclusion: The ASFV strains responsible for outbreaks in three geographically distant Indonesian provinces from 2021 to 2023 belonged exclusively to genotype II and exhibited low genetic diversity. The novel PVTDN insertion identified in the E183L (p54) gene of North Sumatra isolates constitutes a unique molecular marker with potential utility for lineage tracing. The identical IGR II profile across provinces and years confirms spatial and temporal genetic stability of the circulating virus. These findings underscore the value of integrated analyses of B646L (p72), E183L (p54), and the I73R/I329L intergenic region for refining the molecular epidemiology of ASFV and provide critical baseline data to support enhanced surveillance, diagnostic development, and control strategies in Indonesia. Keywords: African swine fever, ASFV genotype II, ASFV Indonesia, E183L gene, genetic characterization, I73R I329L intergenic region, molecular epidemiology, p72 gene.
Ratnawati et al. (Sun,) studied this question.