High ex vivo recombination rate of African swine fever virus in porcine alveolar macrophages.

Using fluorescently labeled African Swine Fever Virus (ASFV) strains, a high (~0.2) quasi-recombinant frequency (qRF3-4) was detected ex vivo during 3-4 replication cycles in naïve porcine alveolar macrophages (PAM). While co-infection and recombination frequencies remained low in PAM, they increased sharply between the second and fourth viral cycles. Infection multiplicity and synchronicity significantly influenced ASFV recombination frequency, which correlated positively with genetic distance. Crossing the ASFV/Lv17/WB/Rie1 mutants yielded a saturation curve resembling the Haldane mapping function, plateauing at ~20% recombination frequency at 160 kbp. Crossing less homologous strains (ASFV/Lv17/WB/Rie1 × ASFV/NHV/P68) reduced the recombination frequency; however, at large marker distances (140 kbp), it approached values (0.18) observed in homologous strains. A novel mathematical model estimated the average co-infection rate to be 0.47 after three viral cycles. Empirical application of the model revealed that 1 centimorgan translates to ~550-1650 bp, meaning offspring virions from a single cell undergo an average of 1.15-3.45 recombination events. This high recombination rate is consistent with in vivo observations and has critical implications for ASFV prevention and vaccination strategies. Such genomic plasticity can facilitate rapid viral evolution, potentially undermining the efficacy of live-attenuated vaccines globally.