Mason Jessica, Zurita Mariceny, Martignette Lauren, Simmons Janine, Neilan John G, Puckette Michael
Plum Island Animal Disease Center, SAIC, P.O. Box 848 11944, Greenport, New York, USA.
U.S. Department of Homeland Security Science and Technology Directorate, Plum Island Animal Disease Center, P.O. Box 848, Greenport 11944, New York, USA.
Transbound Emerg Dis. 2025 Jul 30;2025:1160908. doi: 10.1155/tbed/1160908. eCollection 2025.
African swine fever (ASF) has emerged as a preeminent threat to worldwide pork production. Research and diagnostics of ASF virus (ASFV) is dependent upon culturing virus in primary cells, such as peripheral blood macrophages (PBMC) derived from swine blood, or pulmonary alveolar macrophages (PAM) extracted from swine lungs. The methodologies for production of these cells can be laborious, requiring significant investment in vivarium, personnel, and technical resources. As an alternative, the buffy coat cell fraction from blood contains a mixture of cell types, including undifferentiated monocytes that can be easily isolated by centrifugation. Herein, we culture buffy coat cells in macrophage (M∅) base media, containing L929 conditioned media to induce monocyte differentiation and enhance sensitivity to ASFV. Culturing the buffy coat cell fraction in M∅ base media enhanced the abundance of rosettes and number of detectable ASFV genome copies relative to buffy coat cells grown without L929 conditioned media. Buffy coat fraction cells were used to propagate ASFV directly from blood of infected swine and subsequent sequencing of extracted viral DNA yielded full genome coverage and identification of point mutations. This work demonstrated that growing ASFV in cells of the buffy coat fraction for pig blood was an effective alternative to using the traditionally isolated primary cell types for ASFV propagation, isolation, and sequencing.
非洲猪瘟（ASF）已成为全球猪肉生产的主要威胁。非洲猪瘟病毒（ASFV）的研究和诊断依赖于在原代细胞中培养病毒，例如源自猪血的外周血巨噬细胞（PBMC）或从猪肺中提取的肺泡巨噬细胞（PAM）。生产这些细胞的方法可能很繁琐，需要在饲养室、人员和技术资源方面投入大量资金。作为一种替代方法，血液中的血沉棕黄层细胞部分包含多种细胞类型的混合物，包括可以通过离心轻松分离的未分化单核细胞。在此，我们在含有L929条件培养基的巨噬细胞（M∅）基础培养基中培养血沉棕黄层细胞，以诱导单核细胞分化并增强对ASFV的敏感性。相对于在没有L929条件培养基的情况下生长的血沉棕黄层细胞，在M∅基础培养基中培养血沉棕黄层细胞部分可提高玫瑰花结的丰度和可检测到的ASFV基因组拷贝数。血沉棕黄层细胞部分用于直接从受感染猪的血液中繁殖ASFV，随后对提取的病毒DNA进行测序可获得全基因组覆盖并鉴定点突变。这项工作表明，在猪血的血沉棕黄层细胞部分中培养ASFV是使用传统分离的原代细胞类型进行ASFV繁殖、分离和测序的有效替代方法。
