A Highly Permissive K-C4 Cell Clone for Genotype II African Swine Fever Virus Reveals Passage-Dependent Hemadsorption Changes Associated with EP402R Deletion

African swine fever virus (ASFV) research commonly relies on primary porcine alveolar macrophages (PAMs), whose limited availability and variability hinder standardized virus isolation and propagation. This study aimed to establish a continuous cell line highly susceptible to genotype II ASFV and characterize phenotypic and genomic changes during serial passage. Thirty COS-1-derived clones were screened after infection with Korea/Pig/PAJU1/2019 using quantitative real-time polymerase chain reaction, immunofluorescence, and hemadsorption assays. Selected clones underwent serial passage, and viruses from passages 5 and 10 in the most susceptible clone were analyzed by whole-genome sequencing. K-C4 supported stable ASFV replication, with a mean viral genome level of 8.80 ± 0.55 log10 copies/mL, compared with 7.38 ± 0.90 log10 copies/mL in PAMs. Productive infection was confirmed by p72-specific immunofluorescence. The parental and passage 1 viruses retained hemadsorption, whereas the passage 5 virus lacked detectable hemadsorption despite continued replication, and hemadsorption reappeared by passage 10. Whole-genome analysis identified passage-associated deletions of differing extent within EP402R, encoding the hemadsorption-associated CD2-like viral protein (CD2v), which corresponded with the hemadsorption phenotype at each passage. These findings establish K-C4 as a highly permissive platform for genotype II ASFV propagation and characterization while emphasizing the need to monitor phenotypic and genomic stability during serial passage.