Results and Discussion
All partial B646L and EP402R sequences of the ASFV isolates were identical to those of the original Korean isolate, Korea/19S804/wb/2019 (GenBank accession nos. MN817977 and MN817978). These sequences were categorised into the genotype II (Fig. 2A) and serogroup 8 (Fig. 2B) groups, respectively. Most IGR fragments also showed 100% sequence identity to the corresponding region of the Korea/19S804/wb/2019 isolate (GenBank accession no. MN817979), except for those of two isolates collected in Paju (Korea/19S3965/wb/2019 on December 3 and Korea/19S5464/wb/2019 on December 30). The IGR fragment contained an STR (5′-GGAATATATA-3′), with a repeat time varying in or among ASFV populations (Goller et al., 2015; Nix et al., 2006). The 10-bp STR was inserted three times in the corresponding regions of the major Korean ASFV isolates from the wild boar (IGR variant II), similar to those in the Russia/Volgograd/wb/2014 (GenBank accession no. KP137637), Belgium/Etalle/wb/2018 (GenBank accession no. MH998359.1), and China/2018/Domestic pig (GenBank accession no. MH 735144) strains. The genome of another Korean isolate from a domestic pig (Korea/2019/Domestic pig, GenBank accession no. MN603969 [Kim H. J. et al., 2020]) also belonged to the variant II group. On the other hand, the nucleotide stretch was repeated two and four times in the genomes of the Korea/19S3965/wb/2019 (IGR variant I) and Korea/19S5464/wb/2019 (IGR variant III) isolates, respectively (Table 2).
Because the genomes of DNA viruses, including ASFV, show relatively low evolutionary divergence, few informative molecular markers have been detected within the genic regions of the viral genomes, as observed for the by B646L and EP402R sequences in this study. However, the lengths of genomic STR are readily expanded or contracted during DNA replication largely by slipped strand mispairing (slippage mechanism). If the affected STR is in an intergenic noncoding region, the allelic variant can be fixed in the population because of the low levels of purifying selection (Gemayel et al., 2010). ASFV strains with variant IGR genotypes have been discovered in Russia (Goller et al., 2015) and China (Ge et al., 2019; Li et al., 2019). However, unlike the IGR variants in these countries, those identified in this study were sympatrically distributed with the pre-existing type in a small county (Fig. 1B). The time intervals for their emergence were also very short (approximately two and three months after the first outbreak in wild boars). Taken together, these facts may suggest that the Korean IGR variants I and III were sporadically generated rather than being independently transported from other countries through replication error in the pre-existing IGR variant II population.
In this study, we identified ASFV variants with different genotypes collected during the comprehensive survey of wild boars in small counties of South Korea surrounding the original ASF outbreak point. Considering the short emergence periods of less than three months and sympatric distributions within a narrow geographical region, these variant strains are likely to have spontaneously emerged in the local viral population through a molecular mechanism(s) such as replication slippage.
The probable transmission routes of ASF can be predicted by analysing the spatiotemporal distributions of ASFV with distinct IGR genotypes (Goller et al., 2015). Therefore, the polymorphic STR was suggested as an informative marker to discriminate closely related ASFV strains (Ge et al., 2019). Currently, we have no evidence supporting clonal expansion of these variants in the relevant region, which may be because of their recent emergence. Surveillance of wild boars will be continued until the viral disease is eliminated. If simultaneous propagation of these IGR variants is observed in the near future, our data will provide a highly informative genetic marker for molecular epidemiological approaches to trace both local and global transmission of ASFV.