The Isolation and Replication of African Swine Fever Virus in Primary Renal-Derived Swine Macrophages

Abstract

African swine fever virus (ASFV) causes hemorrhagic disease in domestic pigs by replicating mainly in monocyte/macrophage lineages. Various primary cells including pulmonary alveolar macrophages have been used for the propagation of ASFV on this account. However, ethical constraints and consistency problems exist as it is necessary to harvest same phenotype of primary cells in order to continue a study. We suggested renal-derived swine macrophages as a novel primary cell candidate to address these issues. These primary cells proved to be permissive to both cell adapted ASFV and a wild-type ASFV. Compared to the commercial cell line MA-104, the renal-derived macrophages were more suitable to isolate the field virus. The consistent molecular characteristics of the renal-derived macrophages were demonstrated by immunocytochemistry with antibodies against macrophage cell surface markers including CD163, CD172a, and Iba-1. Viral protein p30 and p72 expression in ASFV infected macrophages was confirmed by immunocytochemistry by use of specific monoclonal antibodies. We observed increase of cell-free viral DNA and infectious virus titer in infected cell supernatant in successive days-post-infection. These results demonstrated that primary renal-derived swine macrophages are useful for ASFV isolation and propagation in terms of cell phenotypes, susceptibility to the virus, and virus production.

Cell Lines for the Development of African Swine Fever Virus Vaccine Candidates: An Update

“…Despite the many advantages of PAMs, working with these primary cells includes some disadvantages, such as high time consumption, expensive cell extraction, and animal welfare considerations [ 18 ]. In addition, other issues are the high variation hindering consistent macrophage phenotypes among different pigs and the low reproducibility from day to day [ 49 , 50 ].…”

“…Macrophages can be efficiently isolated from primary porcine kidney cell cultures [ 51 ], and Oh and colleagues recently characterized these cells as renal-derived macrophages presenting the molecular and morphological characteristics of monocyte/macrophage lineage, with high continuity and reproducibility. In detail, immunocytochemistry analyses revealed that these cells expressed macrophage markers CD163, CD172a, and Iba1 [ 50 ], indicating that they were a mature macrophage population, and most importantly, that they were susceptible to infection with both wild-type (MW039157) and cell-adapted (MW287337) ASFV genotype II.…”

“…The phenotype of these cells remained consistent for at least two months in different batches, and overall data suggested that these primary renal-derived macrophages would be a suitable alternative to PAM or moMФ [ 50 ]. In addition, researchers observed that both strains were efficiently replicated in these cells (using an MOI of 0.3).…”

“…In addition, researchers observed that both strains were efficiently replicated in these cells (using an MOI of 0.3). These cells were also more permissive to the wild-type virus than the continuous cell line MA-104 (see Section 4.3 ), suggesting that they can be used for ASFV isolation, propagation, and production [ 50 ]. Although more studies are needed to evaluate whether the genome and immunogenicity of the virus isolates change after several passages in renal-derived macrophages, these cells might be an alternative cell line for the large-scale production of modified live ASF vaccine [ 50 ].…”

“…MA-104 is a commercial cell line that originated from kidney epithelial cells derived from the African green monkey Cercopithecus aethiops [ 97 ]. Its susceptibility to ASFV and its ability to support viral growth was recently tested by several groups [ 50 , 97 , 98 ].…”