Propagation and Sequencing of African Swine Fever Virus on Porcine-Derived Buffy Coat Fraction Cells

Abstract

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.

Keywords: African swine fever virus; buffy coat; hemadsorption; primary cells; sequencing; swine; virus isolation.

Figure 1

(A) Inclusion of L929 cell culture media in M∅ base media increased the abundance of rosettes compared to those observed in M∅ mimic media. (B) A statistically significant decrease, p ≤ 0.001 as determined by a Student's T-test, in average C_т values was observed at 7-, 10-, and 14-days postinfection in M∅ base media (orange triangles) when compared with M∅ mimic media (blue circles) demonstrating greater viral genome numbers in M∅ base media samples. (C) Rosette formation on buffy coat cells for six ASFV isolates: Brazil '78, Haiti '79, Killean III, Lisbon '60, and Tengani. (D) Buffy coat cells cryopreserved and stored at −150°C for 12 days retained viability and (E) the ability to produce rosettes upon infection.

Figure 2

(A) Cells extracted per 1 mL of blood using 4 mL of EDTA-treated blood or 5, 15, or 50 mL of heparin-treated blood. Blood was either processed upon receipt (green) or stored at 4°C overnight (blue). (B) Buffy coat cells retained the ability to produce rosettes upon ASFV infection regardless of anticoagulant used or overnight storage at 4°C.