The pulmonary alveolar macrophages (PAMs) used in this study were prepared from 2- to 3-month-old piglets. Briefly, the intact lung was filled with sterilized phosphate-buffered saline (PBS), and we then collected the bronchoalveolar lavage fluid. Cells were collected via centrifugation at 560 × g for 10 min and resuspended using PBS. After removing the erythrocytes and rinsing with PBS, the cell pellets were resuspended, and the cells were grown in RPMI 1640 (Gibco) supplemented with 10% fetal bovine serum (Gibco). Cells were cultured in an incubator at 37°C under 5% CO2. We performed nucleic acid testing according to China national standards to ensure that there was no contamination with viruses infecting swine, which include ASFV, classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies virus (PRV), porcine parvovirus (PPV), and porcine circovirus 1/2 (PCV1/2).
The ASFV SY18 strain (GenBank accession no.MH766894 ), a field ASFV isolate, was isolated from pig specimens after the first outbreak of ASF in China in August 2018 by our laboratory, which caused acute infection in pigs and resulted in up to 100% fatality in the farm. The virus was passaged in primary PAMs and stored at −80°C. The SY18ΔMGF/CD2v strain, an attenuated strain of ASFV bearing deletions of the MGF505-1R, MGF505-2R, MGF505-3R, MGF360-12L, MGF505-13L, MGF505-14L, and EP402R ORFs through homologous recombination, was also used in this study for comparison in vaccination and challenge trials. It has been proven to protect vaccinated pigs from virulent isolate SY18 infection when challenged by the oral administration of 103.0 TCID50 (18 ).
ASFV strain SY18 was titrated using 96-well plates and observed by an immunofluorescence assay, with staining with a fluorescein isothiocyanate (FITC)-labeled monoclonal antibody against the ASFV protein p30. Briefly, virus suspensions were 10-fold diluted and inoculated onto the cell monolayer. After incubation for 5 days at 37°C, the cultures were fixed using 80% cold acetone and incubated with the FITC-labeled p30 monoclonal antibody (prepared in our laboratory) for 1 h at 37°C, followed by observation under a fluorescence microscope. For the gene-deleted strains, the fluorescence emitted directly from eGFP was observed. The Reed-Muench method was used to calculate the virus titer (43 (link)).
The ASFV SY18 strain (GenBank accession no.
ASFV strain SY18 was titrated using 96-well plates and observed by an immunofluorescence assay, with staining with a fluorescein isothiocyanate (FITC)-labeled monoclonal antibody against the ASFV protein p30. Briefly, virus suspensions were 10-fold diluted and inoculated onto the cell monolayer. After incubation for 5 days at 37°C, the cultures were fixed using 80% cold acetone and incubated with the FITC-labeled p30 monoclonal antibody (prepared in our laboratory) for 1 h at 37°C, followed by observation under a fluorescence microscope. For the gene-deleted strains, the fluorescence emitted directly from eGFP was observed. The Reed-Muench method was used to calculate the virus titer (43 (link)).
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