## 4 Discussion
ASaV isolates displayed differences in stop codons, which were also detected in the ORF3 region of the ASMaV and RYRSaV isolates [21; 27]. The type of stop codon can affect non-canonical translation mechanisms in RNA viruses and play a regulatory role in modulating the amount of protein translation [38; 39]. However, the impact of stop codon variation on ASaV RNA3 remains unclear. There was an indel of 3 or 5 nt in ORF3-I isolates, which also occurred in ASaV RNA4 and RNA5, as the 3'-UTRs of these segments also consist of repetitive adenine and uracil sequences. These events are more likely to occur in homopolymeric regions where repetitive nucleotide sequences heighten the chances of replication machinery slippage [40]. A variation in the length of RNA3 has also been reported in other emaravirus species, such as European mountain ash ringspot-associated virus (EMARAv), Wheat mosaic virus (WMoV), and AsMaV [21; 41]. ASaV RNA2, RNA4, and RNA5 were not phylogenetically informative. Similar findings have been reported for other members of emaraviruses such as AsMaV, RRV, and PCLSaV [21; 31; 35]. The ASaV MP sequence was the most conserved gene among the ASaV genome segments studied. This is not the case in all emaraviruses; for example, the identity of the MP gene in the blackberry leaf mottle-associated virus (BLMaV) ranged from 86-99% at the nt level and 95-100% at the aa level [33]. Overall, a maximum divergence of 0.14 was observed among the ASaV isolates. However, most genomic regions of the ASaV genome exhibited more than 90% identity at the nucleotide level, with even higher identity observed in the coding regions and at the amino acid level. The nucleotide diversity ranged between 0.02700 \(\pm\) 0.00126 and 0.05957 \(\pm\) 0.00775, denoting that ASaV overall has a conserved genome. ASaV in comparison to other emaravirus members showed less nucleotide diversity, for example, the N terminal regions of RdRP (0.40), MP (0.49), and NP (0.21). The nucleotide diversity of different genomic regions ranged from 0.049 to 0.077 for AsMaV and from 0.022 to 0.043 for FMV NP [21; 26]. This range of nucleotide diversity was in a similar range for some other woody plant-invading viruses, such as citrus tristeza virus (0.038) and citrus leaf blotch virus (0.021) [24; 26; 41] but was very low in comparison to grapevine fanleaf virus (0.15) [42]. It has been reported that the nucleotide diversity of blackberry leaf mottle-associated virus (BLMaV) was rather low when compared to FMV and European mountain ash ringspot-associated virus (EMARaV) [30; 43], ranging from 0.034 to 0.057 for NP and MP genes, respectively. The AsaV NP and MP genes showed similar or even lower nucleotide diversity, 0.037 and 0.027, for the corresponding regions. This could be explained by the very strong purifying selection pressure on the ASaV genome (Table 2). Negative values of [PERSON]'s \(D\), [PERSON] and [PERSON]'s \(D^{*}\), and [PERSON] and [PERSON]'s \(F^{*}\) were obtained for RNA2-4, suggesting an excess of low-frequency polymorphisms. This pattern may be the result of demographic events, such as population expansion or background selection [44], where deleterious alleles are removed through purifying selection. In contrast, positive values of [PERSON]'s \(D\), [PERSON] and [PERSON]'s \(D^{*}\), and [PERSON] and Li's \(F^{*}\) in RNA1 (ORF1 C-proximal) and RNA5 suggest that variation in these genomic regions deviates from neutrality, with an excess of intermediate-frequency alleles. This may be driven by balancing selection or a population bottleneck [45]. In the viruses with segmented genomes, reassortment is also an important factor in the genetic diversity and evolution of these viruses. Placing different genomic RNAs of ASaV in different phylogenetic groups indicated the possibility of recombination or reassortment (Figure 2).