Growth Kinetics of African Swine Fever Virus (Genotype II) Isolated from a Domestic Pig in Punjab, India

African swine fever (ASF), a deadly disease caused by African swine fever virus (ASFV), emerged in north-eastern India in 2020 and subsequently spread to several states, devastating swine herds due to the lack of commercial vaccines. We report the isolation, genetic characterization and growth kinetics of ASFV isolated from Punjab, India in primary porcine pulmonary alveolar macrophages (PAM). Partial sequencing of the B646L gene categorized the virus as genotype II, showing complete nucleotide identity with previously reported Indian genotype II isolates. Phylogenetic analysis placed the isolate amongst genotype II ASFVs circulating in Europe and the Asia-Pacific region since 2007. Multistep growth curves were constructed based on HAD50 titres and viral DNA copy numbers after infection of PAM cultures at 1.0 and 0.1 multiplicity of infection (MOI) observed up to 168 hrs post-infection (hpi). At 1.0 MOI, viral titres and genome copy numbers in the cell associated (CA) fraction peaked earlier (120-144 hpi) before declining, whereas the culture supernatant (CS) fraction showed a delayed but sustained increase, reaching maximum levels at 168 hpi. At 0.1 MOI, both HAD50 titres and viral genome copies increased progressively in CA and CS fractions, with higher peak levels observed in the CS fraction at later time points. By observing up to 168 hpi, this study reveals higher extracellular virus production with lower DNA copy to HAD ratio in PAM cultures infected at 0.1 MOI, demonstrating optimal duration for higher virus yield in cell culture supernatants, which would support efficient ASFV propagation, pathogenesis and vaccine development studies.