Samples and virus:
African swine fever viruses isolated from diagnostic samples received at
ICAR-National Institute of High Security animal diseases, Bhopal during
the first outbreaks of ASF were used in this study (Rajukumar et al.,
2021). Two ASFV isolates (second passage), from Arunachal
Pradesh(IND/AR/SD-61/2020) and Assam(IND/AS/SD-02/2020), were further
propagated for one more passage in porcine monocyte culture as described
previously by Borca et al., 2020. Briefly, PBMCs of healthy pig were
cultured in 10% fetal bovine serum containing RPMI-1640 medium (growth
medium). After 48 hours of incubation at 37°C under 5%
CO2 concentration, monocytes wereharvestedin Dulbecco’s
PBSwith 1mM EDTA after removing unattached cells by thorough washing
using PBS. Harvested monocytes were cultured in 24 well culture dishes
at the concentration of 2x106 cells per mL in growth
medium. The monocytes were infected with supernatants obtained in the
second passage at 1/10 dilution.Two wells were maintained as uninfected
controls. One hundred µl of freshly prepared 1% swine RBCs in growth
medium were added to each well and observed for Haemadsorption for up to
5 days post infection.