High intragenotypic diversity, serogroup heterogeneity, and recombination of African swine fever virus strains circulating in sylvatic and domestic hosts

Although the sylvatic cycle is central to African swine fever virus (ASFV) maintenance in sub-Saharan Africa, its contribution to viral genetic diversification, antigenic heterogeneity, and recombination remains poorly characterized. We investigated ASFV diversity in Zambia by analyzing soft ticks collected from Mosi-oa-Tunya, South Luangwa and Kafue National Parks, together with tissues and blood from warthogs and archived domestic pig isolates. ASFV detection was performed by PCR, warthog sera were screened by ELISA, and ticks were identified by mitochondrial gene sequencing. ASFV-positive samples were further characterized by p72 , p54 , B602L /CVR, and EP402R / CD2v sequencing. Compared with the previously reported ASFV detection rate of 17.7% in tested Ornithodoros moubata ticks from Mosi-oa-Tunya, where genotype I viruses were detected, lower pool-level detection rates were observed in Ornithodoros porcinus ticks from South Luangwa (1/29; 3.4%) and Kafue (1/25; 4.0%), which carried p72 genotypes XII and XIV, respectively. Warthogs showed high ASFV seroprevalence but no detectable viral DNA. Genotype I viruses exhibited marked intragenotypic and serogroup-associated heterogeneity, including multiple p54/CVR variants and seven CD2v lineages. Recombination analyses suggested genotype I-related contributions to genotype II, XII, and XIV viruses, highlighting the need for broadly protective vaccines for endemic regions that account for intragenotypic diversity, CD2v-associated antigenic heterogeneity, and recombination-driven diversification.