This study was conducted as part of the national African swine fever surveillance program in Indonesia and involved the collection of EDTA blood and serum samples from pigs presented for slaughter and from farms under routine animal health investigation. The sampling activities did not involve experimental infection, treatment trials, or any procedures beyond standard veterinary diagnostic and surveillance practices. Therefore, separate approval from a formal Institutional Animal Care and Use Committee was not required. All field procedures were performed by trained veterinarians and authorized animal health officers in accordance with institutional surveillance protocols, national animal welfare principles, and applicable biosafety regulations for handling suspected transboundary animal disease materials. Sample collection was carried out with minimal distress to animals and only under routine field or slaughterhouse conditions. In addition, all laboratory procedures involving potentially ASFV-positive materials were conducted under biosafety level 2 conditions following national biosafety guidance for veterinary diagnostic laboratories.

This study was conducted from 2021 to 2025 in several provinces of Indonesia, including North Sumatra, East Nusa Tenggara, and DKI Jakarta. Samples were collected from slaughterhouses and pig-producing areas as part of routine molecular surveillance activities.

This study was designed as a cross-sectional molecular surveillance of ASFV conducted from 2021 to 2025. Sampling used a combination of convenience and risk-based methods, targeting both slaughterhouses and pig-producing areas to monitor ASFV circulation along the production and marketing chain. Provinces were chosen based on previous ASF reports, pig population density, and logistical feasibility for routine surveillance activities.

Whole blood (EDTA) and serum samples were gathered by trained veterinarians and animal health officers in accordance with standard biosafety precautions for ASF-suspected materials. Blood and serum were not always collected from the same animals because samples were obtained opportunistically from slaughterhouses and farms based on field availability. After collection, samples were stored at 4°C in the field and transported to the laboratory within 24–48 h, then kept at −20°C until DNA extraction.

EDTA blood and serum samples were collected using a cross-sectional molecular surveillance design that combined convenience and risk-based sampling from slaughterhouses and pig-producing areas. Samples were obtained from apparently healthy pigs presented for slaughter as well as pigs from farms with recent or suspected ASF history, depending on field access and availability. Metadata recorded for each sample included the sampling site, year of collection, sample code, and specimen type. Sampling procedures followed institutional surveillance guidelines for animal health monitoring, and all activities were performed in accordance with national biosafety and ethical regulations.

Total DNA was extracted from 200 µL of EDTA-treated blood or serum using the High Pure PCR Template Preparation Kit (Roche Molecular Biochemicals) following the manufacturer’s instructions. The final elution volume for each sample was 50 µL. Extracted DNA was stored at −20°C until further analysis. A negative extraction control (nuclease-free water) was included in each batch to monitor potential cross-contamination. DNA concentration and purity were not routinely measured because samples were directly subjected to PCR as part of standard ASFV surveillance procedures.

Conventional PCR assays targeting the B646L (p72) gene were performed using two primer sets with different analytical purposes. For diagnostic screening, the PPA1/PPA2 primers were used to amplify a 257-bp fragment (PPA1: 5′-AGTTATGGGAAACCCGACCC-3′; PPA2: 5′-CCCTGAATCGGAGCATCCT-3′) [18]. For genotyping and sequencing, the P72-U/P72-D primer pair was used to amplify a 478-bp region (P72-U: 5′-GGCACAAGTTCGGACA TGT-3′; P72-D: 5′’-GTACTGTAACGCAGCACAG-3′) [19].

Each PCR reaction consisted of 1× PCR buffer (50 mM KCl, 10 mM Tris-HCl), 2 mM MgCl₂, 0.2 mM dNTPs, 0.2 µM of each primer, 0.625 U Taq DNA polymerase, and 2 µL DNA template in a final volume of 25 µL.

Thermal cycling conditions for the PPA1/PPA2 assay were as follows: initial denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, 62°C for 30 s, and 72°C for 30 s, with a final extension at 72°C for 7 min. For amplification with the P72-U/P72-D primers, cycling conditions consisted of an initial denaturation at 96°C for 20 s, followed by 35 cycles of 96°C for 12 s, 50°C for 20 s, and 72°C for 25 s, with a final extension at 72°C for 5 min.

A previously confirmed ASFV-positive field sample from the 2019 outbreak in East Nusa Tenggara was used as a positive control in all PCR runs, whereas nuclease-free water served as a no-template negative control. PCR assays were performed in single reactions per sample in accordance with routine surveillance protocols. PCR products were separated on 2% agarose gels stained with ethidium bromide and visualized under ultraviolet illumination to confirm the expected amplicon sizes before sequencing.

Amplicons produced using the P72-U/P72-D primers (478 bp) underwent bidirectional Sanger sequencing, which was outsourced to Macrogen (PT. Indolab Utama, Indonesia). Raw chromatogram files were reviewed and trimmed with Chromas software to eliminate low-quality bases, and forward and reverse reads were assembled into consensus sequences. Only sequences with clear and unambiguous peaks throughout most of the target region were kept for further analysis.

Consensus sequences were aligned with a curated global ASFV reference dataset representing genotypes I–XXIV, in which each genotype was represented by one or two well-characterized reference strains. Multiple sequence alignment was performed using the ClustalW algorithm implemented in MEGA X [20]. Phylogenetic trees were constructed using the Neighbor-Joining method with the Kimura 2-parameter model with gamma distribution (K2+G), selected because of its suitability for relatively conserved DNA sequences and its common use in ASFV p72-based genotyping studies. Node reliability was evaluated using 1,000 bootstrap replicates.

The nucleotide sequences generated in this study have been submitted to GenBank and are currently under processing. Accession numbers can be obtained either from GenBank or the corresponding author.

PCR and sequencing results were summarized descriptively based on sampling location and specimen type. Positivity rates were calculated by dividing the number of PCR-positive samples by the total samples tested for each site and matrix. All sequence-confirmed ASFV cases were recorded along with sample identifiers, collection sites, specimen types, and related amplicon information. These data were combined with phylogenetic findings to describe the distribution and lineage features of ASFV circulating in Indonesia.

A total of 272 clinical specimens, including EDTA blood and serum samples collected from seven provinces between 2021 and 2025, were screened for ASFV using conventional PCR targeting the B646L (p72) gene. Overall, seven samples (2.6%) showed positive amplification signals (Table 1). No ASFV DNA was found in samples from Singkawang, West Kalimantan. In contrast, low but consistent positivity was observed in several other regions. One sample from East Nusa Tenggara tested positive (1/18; 5.6%). In North Sumatra, ASFV was detected in both blood and serum samples, with one positive sample each from Deli Serdang blood (1/27; 3.7%), Deli Serdang serum (1/17; 5.9%), and Tana Karo serum (1/25; 4.0%). Additional positive cases were identified at the Medan City abattoir (2/70; 2.9%) and the Kapuk abattoir in Jakarta (1/125; 0.8%). All ASFV-positive samples were identified during the 2023 and 2025 sampling periods, while no positive samples were found in 2021, indicating sustained but low-level circulation of the virus several years after the initial outbreak.

Both PCR assays produced clear and distinct amplicons of the expected sizes. The diagnostic PPA1/PPA2 primer set consistently generated a 257-bp fragment confirming ASFV detection (Figure 1), while the P72-U/P72-D primers produced the 478-bp fragment used for sequencing and genotyping (Figure 2). Band clarity and uniformity across both EDTA blood and serum samples indicate that these specimen types are suitable for conventional PCR-based ASFV screening. No samples were positive by PPA1/PPA2 but failed amplification with the P72-U/P72-D primers, and no ambiguous or mixed infection bands were observed.

All seven PCR-positive samples amplified with the P72-U/P72-D primer pair were successfully sequenced, resulting in high-quality chromatograms suitable for further analysis. Partial B646L (p72) gene sequences were aligned and compared with a curated global reference panel that includes ASFV genotypes I–XXIV. Phylogenetic reconstruction using the Neighbor-Joining method with the Kimura 2-parameter + Gamma model and 1,000 bootstrap replicates revealed a consistent and well-supported clustering pattern.

Pairwise nucleotide comparisons showed that the Indonesian ASFV sequences shared over 99% identity with Georgia 2007–derived genotype II reference strains from China, Vietnam, the Philippines, and previously reported Indonesian isolates. Only a few synonymous nucleotide substitutions were observed, and no amino acid–changing mutations were found in the analyzed p72 region. All ASFV sequences obtained in this study clustered clearly within the genotype II lineage, with bootstrap values of 80 or higher. These sequences closely grouped with representative isolates of the Georgia 2007–derived Eurasian lineage, including reference strains from Vietnam (2019), China (2019), the Philippines (2022), India (2023), and previously reported Indonesian isolates such as the Bali 2023 strain. No evidence of divergent clustering, unique sub-lineages, or additional genotype introductions was observed (Figure 3). These findings confirm the ongoing circulation of the pandemic Eurasian genotype II strain in the sampled regions of Indonesia.

This study offers one of the first multi-year molecular surveillance datasets of ASFV in Indonesia since its initial introduction in 2019. Unlike early outbreak-focused investigations, the results show low-level yet ongoing circulation of ASFV across geographically distant areas, including production zones and slaughterhouses, suggesting an endemic transmission pattern rather than a single epidemic wave.

Despite the relatively low PCR positivity rate (2.6%) from samples collected between 2021 and 2025, the detection of ASFV DNA across several geographically distant regions, including North Sumatra, East Nusa Tenggara, and Jakarta, indicates ongoing viral circulation within different components of the swine value chain [21]. The detection of ASFV in both EDTA blood and serum further supports the usefulness of these matrices for routine molecular surveillance, consistent with earlier reports showing that ASFV can be reliably detected in various clinical specimens during acute and subacute infections [22].

The relatively low ASFV detection rate (2.6%) aligns with a post-epidemic endemic phase, during which widespread mortality has already occurred, and surviving pig populations, market flows, and improved biosecurity measures lower the likelihood of detecting actively infected animals. Additionally, slaughterhouse-based sampling often includes apparently healthy animals that may carry ASFV at low viral loads or during subclinical infection, further reducing PCR positivity compared to outbreak-driven farm investigations. The detection of ASFV in slaughterhouse samples from Medan and Jakarta emphasizes the epidemiological significance of abattoir-based surveillance as an early warning system. Slaughterhouses process pigs from multiple sources and trading networks, making them key locations for identifying silent ASFV circulation that might not be detected through farm-based surveillance alone.

The molecular detection pattern observed in this study aligns with the epidemiological history of ASF outbreaks in Indonesia since its first confirmation in 2019 [23]. The sporadic yet persistent detection in slaughterhouses and pig-producing areas indicates possible underreporting of cases, the presence of subclinical infections, and the movement of infected but clinically normal pigs within local trade systems [24]. Similar patterns have been reported in other Southeast Asian countries, where ASFV continues to circulate endemically several years after its initial introduction due to incomplete depopulation strategies, inadequate biosecurity, and challenges in controlling intra-island and inter-province pig movement [25].

The results of this study align with previous molecular research from Indonesia and nearby Southeast Asian countries, which consistently identified genotype II as the main circulating lineage. However, most earlier studies in Indonesia focused on outbreak investigations or covered limited geographic areas, whereas this study shows ongoing genotype II circulation across multiple provinces over several years under routine surveillance conditions.

Phylogenetic analysis of the partial p72 gene revealed that all Indonesian ASFV sequences obtained in this study grouped within genotype II, consistent with previous research conducted by national institutions [26]. Strong bootstrap support and close genetic similarity to reference strains from Georgia (2007), China (2019), Vietnam (2019), and the Philippines (2022) further confirm that the Indonesian outbreaks are part of the broader Eurasian genotype II epidemic that has dominated global ASFV spread over the past decade [3]. No genetic divergence, distinct branching, or emerging sub-lineages were observed among the Indonesian isolates, indicating that genotype II circulating in Indonesia remains genetically conserved [27].

This pattern is typical for ASFV, which generally exhibits low mutation rates in the p72 region, especially over limited geographic and temporal scales [28]. The lack of divergent sub-lineages or unique amino acid substitutions in the p72 region suggests that the ASFV genotype II circulating in Indonesia has remained genetically stable despite extended circulation. This finding contrasts with reports of emerging microvariants in other regions and implies that virus evolution in Indonesia may be limited by minimal selective pressure or the absence of new variant introductions.

The phylogenetic clustering also supports the hypothesis that ASFV introduction into Indonesia was closely linked to regional dissemination routes from Asia, consistent with patterns reported in neighboring countries [26]. The absence of additional genotypes suggests that Indonesia probably experienced a single primary introduction event followed by local spread rather than multiple independent ones [21]. These findings have significant implications for national surveillance programs. Ongoing genomic monitoring is necessary to identify potential recombination events or new strain introductions, especially given the extensive pig transport networks connecting farms, traders, and slaughterhouses [29].

Although the B646L (p72) gene remains the global standard for ASFV genotyping, its limited ability to resolve intra-genotype variation presents an inherent limitation [30]. Future studies that combine p72 sequencing with more variable genomic regions, such as the central variable region (CVR), intergenic regions (IGRs), or whole-genome sequencing, could enable more precise tracing of transmission chains and viral evolution within Indonesia [31]. Expanding sampling coverage to backyard farms, trader networks, and wild boar populations could further enhance understanding of ASFV circulation [32].

A major limitation of this study is the use of partial sequencing of the B646L (p72) gene, which provides reliable genotype classification but limited resolution for identifying detailed transmission routes or recent evolutionary changes. Including more variable genomic regions or performing whole-genome sequencing would allow for more accurate reconstruction of transmission pathways and viral adaptation. Nevertheless, p72-based genotyping remains the international standard for ASFV classification and enables direct comparison with historical and global datasets, making it suitable for long-term lineage tracking and surveillance across countries.

The findings of this study show that Eurasian genotype II ASFV still exists in Indonesia and highlight the need for ongoing molecular surveillance for early detection and outbreak control. The repeated detection of genotype II across different provinces underscores the importance of strengthening biosecurity, improving movement controls, and incorporating genomic data into epidemiological decisions to enable more effective ASF management [33].