Previous studies of ASFV isolation used primary pulmonary alveolar macrophage (PAM) cells because of their high adaptation [2, 6, 9, 20]; however, this cell line can only be utilized in research and is not a candidate to grow large amounts of ASF virus for vaccine production due to high costs. In addition, there is also an issue of animal ethics with the large-scale collection of primary cells; meanwhile, several other commercial cell lines exhibit no evidence of stable replication of the ASF virus. Recently, a commercial cell type (MA104) was determined to be highly stable when used for the isolation of clinical samples . Our results in this study appear to be in keeping with the findings of the study by Rai et al., 2020; however, the COVID-19 pandemic interrupted some of the final steps necessary to obtain sufficient scientific information to enable publication. However, it should be noted that the results obtained by our study and previous work  demonstrate that MA104 is not only a stable cell line for the isolation of ASFV obtained from field samples but also exhibits strong potential as a candidate cell line for commercial vaccine development.
A notable finding of this study was that in the same MA104 cells, ASFV from various sample sources (blood and tissue) exhibited significant differences in adaptability and replication. In addition, modification of the medium composition in the maintenance and growth medium for the same MA104 cells also affected viral growth over 15 passages. The viral load was calculated via the Ct value of qPCR, and the ICC staining technique used a p30 polyclonal antibody. Therefore, medium components may play essential roles in enhancing the reproduction of the ASF virus in culture cell lines. The results of this study showed that there was gradual adaptation and higher growth of ASFV in the serial subpassage cultures, particularly after the 10th to 15th passages. Interestingly, it was noted in this study that ASFV isolated from blood on MA104 cells exhibited considerably better adaptation up to the 10th passage; in contrast, ASFV isolated from tissue samples exhibited slow adaptation, and it was terminated after the 10th passage.
Other cell lines, such as PAM, MARC-145, Vero and PK15, have been described by previous studies on the adaptive capacity of ASFV [2, 16, 19]. However, according to the results of this study, MA104 cells exhibited the most robust ASFV replication, which was in keeping with the findings of a recent publication . Therefore, further research to determine the best biological properties, MOI, variability, and optimal medium composition for the development of ASFV in MA104 cells may facilitate the development of an effective ASF vaccine. This study developed a model to enhance the adaptation of ASFV to MA104 cells. The adapted virus showed high growth potential, but no mutations occurred in structural proteins (p30, p54, and p72), and no change was observed in the antigenic regions of proteins p30 and p54. This lack of change is important in increasing the probability of maintaining immunogenicity when developing a vaccine candidate.
The evidence of propagation of ASFV in the cells in this study was determined by two methods, quantitative PCR (qPCR) (according to the Ct value) and immunocytochemistry (ICC), and highly homologous results were obtained. Hemadsorption (HA) is often the criterion for determining the presence of ASF virus in assays, but good blood quality for testing is not always available in every laboratory; moreover, some strains of virulent ASFV may not possess erythrocytic adsorption properties [3, 17, 22], and the application of ICC in MA104 cells with clear backgrounds is more effective than employing this technique in primary macrophage cells with peroxidase activity .