Whole-Genome Sequencing of African Swine Fever Virus in Serbian Samples 2019–2023

African swine fever (ASF) is a highly fatal viral hemorrhagic disease with devastating economic impacts on global swine production. In Serbia, ASF was first detected in domestic pigs in 2019, followed by its reemergence in wild boar in 2020, where it has persisted. This study aimed to assess genomic changes in the ASF virus by performing shotgun metagenomic sequencing on wild boar and domestic pigs samples. Spleen samples from hunted or found dead wild boar and dead domestic pigs were processed. Five samples were selected based on nucleic acid concentration and purity and sequenced using Illumina technology at Novogene Ltd. (UK). Sequenced reads were trimmed with BBduk v.38.84 and de novo assembled using metaSPAdes v.3.13.1 to generate contigs, which were mapped to the ASF reference genome Georgia 2007/1 using Minimap2 v.2.0. as plugins in Geneious Prime software suite v. 2022.1.1. Genome alignments were performed with MAFFT v.1.5 and phylogenetic analysis was conducted using MEGA X. Five high-quality genomes were obtained and deposited in NCBI GenBank (accession numbers OR660695–OR660699). Phylogenetic analysis, incorporating seven additional genomes, revealed three subgroups: strains NC044959, ON108571, and LR722599 formed the first subgroup; MT847622, MT847623, and LR899193 formed the second; while sequences from this study clustered with MK543947 in the third subgroup. Mutational analysis revealed both major and minor mutations. Major mutations contributed to enhanced strain classification and potential impacts on protein functionality. In contrast, minor mutations included extended poly-C and poly-G regions in MGF 110 and 360 gene families and ACD genes, as well as single deletions in intergenic regions. Silent mutations and poly-C/G insertions, although functionally unclear, were also observed. This study provides critical insights into ASF virus genetic diversity and evolutionary dynamics in Serbia, contributing to the global understanding of its transmission and persistence. These findings underscore the importance of genomic surveillance in controlling this devastating disease.