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.