Molecular epidemiology of African swine fever genotype I and II viruses: insights from genome sequencing

African swine fever (ASF), a haemorrhagic disease of domestic pigs caused by ASF virus (ASFV) was confined to Africa until 1957. Since then, two other excursions have occurred, in 1960 and in 2007. The first was a genotype I virus introduction to the Iberian Peninsula in 1957 and the second a genotype II virus introduction to Georgia in 2007. Both excursions were associated with a range expansion within and outside Africa, which in the absence of vaccination and treatments options had significant impact to the pork industry globally. Eradication of genotype I from mainland Europe took almost four decades to achieve, and the genotype II pandemic is ongoing. The origin of the latter virus introduction could not be determined despite the availability of gene sequence data for viruses causing outbreaks in Mozambique and two Indian Ocean islands (Mauritius and Madagascar) in the decade prior to the 2007 introduction to the Caucasus. In this study, next generation sequencing (NGS) technologies were used to generate complete genome sequences for eight genotype II viruses from eight Southern African Development Community (SADC) countries spanning a 26-year period (1993-2019). In addition to identifying the Mozambique/2005 as the most likely origin of Georgia 2007/1, the data permitted assessment of the diversity and phylogenomic relatedness of genotype II ASFVs from Africa. The identification of an atypical pre-2007 genotype II virus from Zambia (1993) warranted comprehensive analysis of this strain, which, based on genome sequencing, was shown to contain features of both genotypes (I and II) under study. In addition, eight genotype I strains, representative of three discrete outbreak clusters (2016-2019) were selected for NGS. The generation of sixteen genome sequences, with equal representation for each of the ASFV genotypes (I and II), allowed for identification of single nucleotide polymorphisms (SNPs), deletions in multigene family (MGF) genes and variation on tandem repeat sequences that are useful for distinguishing between genotype II viruses grouping within the six clades (I-IV) identified in this study, underscoring the value of a whole genome sequencing approach. In contrast, the genome data generated for virus’s representative of three genotype I sub-clusters, recovered the same clusters that were discernible based on partial p72 gene sequences and the central variable region (CVR). The high levels of variation and recovery of three sub-clusters (Ia, Ib and Ic) despite a short field presence (2016-2019) suggests independent transboundary introductions. In conclusion, the study has provided insight in the most likely origin of Georgia 2007/1 virus and confirmed high levels of intra-genotypic diversity for genotype II viruses of African origin. The findings also highlighted that the genotype II virus from Zambia may represent a naturally occurring recombinant based on the observed similarities between the 1993 Lusaka isolate and genotype I viruses. By generating reference genome sequences for genotype I and II viruses from the SADC region, this study contributes to global efforts to expand ASF genome databases to include genotypes and geographical regions that are under-represented.