4. Discussion
The primary goals of viral genome characterization during the 2021 ASFV outbreak in the Dominican Republic were to describe geographical and local transmission and to track virus variation over time to inform disease control and future surveillance efforts. We successfully sequenced 73 whole ASFV genomes and characterized their genetic variations within 24-72 hours of receipt using Oxford Nanopore and Illumina sequencing technologies. We anticipate that the genomic data derived from this study, in conjunction with other epidemiological data, including cases reported to the World Organization for Animal Health (OIE), will provide a clearer picture of ASFV transmission and geographical spread during the 2021 DR outbreak.
In addition to resolving patterns of local viral transmission, whole genome sequencing has been used to place an outbreak in the context of global viral isolates for virus tracing. The DR sequences shared 4 ancestral SNPs with European and Asiatic isolates but lacked a SNP present in the Asiatic isolates, suggesting that the DR sequences were most closely related to historically characterized European isolates (Supplementary File 2). These 4 ancestral SNPs appeared to have been acquired sometime between the Georgia 2007 outbreak and the subsequent outbreaks described in Europe and Asia from 2016-2018, according to when they were first described in public ASFV genomes. This, together with the fact that sequences from the DR did not contain a particular SNP that was characteristic of the Asiatic genomes, suggest that the DR isolates were more closely related to Eastern European ASFV sequences from 2016-2018 than those from the 2018 Asia outbreak, which was reflected in the phylogenetic tree (Figure 2).
However, the DR sequences contained at least 8 SNPs that had not been publicly described in other ASFV genomes; this suggests that the DR sequences were divergent by some time from the most recently characterized European isolates. Since no genomes were available with intermediate variations to link the public isolates to the DR sequences, it was difficult to accurately determine a divergence time or rate. It may be that the 8 or more SNPs present in the DR genomes are also present in ASFV genomes circulating elsewhere and have yet to be described in the public databases. Determining where the most recent common ancestor of the DR sequences was circulating prior to its introduction into the DR will require increased ASFV surveillance and whole or targeted genome characterization efforts.
Furthermore, our analysis revealed some insertions and deletions in the genome of the DR ASFV isolates (Table 1). It was previously established that some insertions and deletions have been associated with attenuation of virulence in ASFV Georgia 2007 strains (Li et al., 2021). Such insertions and deletions may provide a plausible explanation for the reduced virulence and subacute clinical manifestations of ASF observed among domestic pig populations during the DR outbreak. However, there was insufficient information obtained from this outbreak to correlate specific insertions and deletions to ASFV virulence, and further studies will be required to establish such a correlation.
Considering the field observations of reduced virulence and subacute clinical manifestation of ASF in the DR outbreak, it is possible that ASFV was not detected by initial surveillance efforts and has been circulating in this region for a longer time than anticipated. If so, this demonstrates that reliance on detection of increased swine mortality alone may not be a sufficient indicator of an ASFV outbreak or introduction. Serology and molecular detection may be valuable surveillance tools to use in conjunction with epidemiological measures for continued surveillance of ASFV in the DR and neighboring regions.
Within the ASFV genomes from the DR, we described two distinct genetic clusters that were divergent from one another by at least 5 SNPs (Supplementary File 3). Genetic cluster 1 contained the majority of ASFV genomes sequenced from the DR (68/73 genomes). Based on phylogenetic results, we hypothesized that the prototypical sequence from genetic cluster 1 was the strain that initially spread across the DR provinces. This “prototypical” sequence contained only the 11 SNP backbone characteristic of all DR sequences plus the single SNP defining genetic cluster 1 at position NC_044959.2:90280 G->A (e.g. strain DR/Duarte/2021/9682773, Supplementary File 3). Of the 73 genomes sequenced, 35 had this prototypical genetic profile from cluster 1 and were identified across 12 of the 18 provinces with dates ranging from July 5 to September 30, 2021.
The remainder of the genetic cluster 1 genomes (33/73 sequences) contained SNPs in addition to the prototypical cluster 1 sequence, and the additional SNPs groupings were more often found within the same province. This accumulation of mutations by geographic region was suggestive of localized transmission and evolutionary adaptation, at least within the provinces from which they were sequenced. It is possible that reduced animal movement following the initial ASFV detection resulted in more localized transmission patterns and therefore SNP profiles clustered by geographic region.
Additional data are needed to determine the origin and emergence of these distinct ASFV genetic clusters in the DR; however, sufficient time must have passed for these genomes to diverge from those characterized in public genome databases. Whether this variation occurred within the DR or elsewhere over a longer timeframe with subsequent introduction into the DR is currently unclear given the genomic data available. Sequencing of additional samples from the DR and a thorough epidemiological investigation could help address this question.