## 4 Discussion  
The expanding catalog of emaraviruses, including new members and sequenced isolates, has enriched our understanding of their genome structure, detection methods, diversity, evolution, and host adaptation. Advances such as high-throughput sequencing have facilitated the identification of conserved motifs (e.g., motifs A-E) in the RNA-dependent RNA polymerase (RdRP) of emaraviruses, enabling the design of genus-specific primers for virus detection [22]. Additionally, the pDAP13 primer, which targets a conserved 13-nucleotide sequence at both the 5\({}^{\prime}\) and 3\({}^{\prime}\) ends of all emaravirus genomic RNAs, has been widely used to amplify entire genomes [23]. However, its efficiency varies by genomic region, necessitating supplementary primers for precise amplification. In this study, we analyzed the genetic diversity of ASaV using two regions of RNA1 and near-complete sequences of RNA2-RNA5 from isolates collected across two _Faxenius_ species and diverse geographic locations. At the time of primer design in 2022, only one complete ASaV genome in GenBank (E55270, Basel, Switzerland, _F. excelsior_, acc. no. OU466880-84) covering RNA1-5 and one fully sequenced RNA1 genome (S10, Germany, Hamburg, _F. ornus_, acc. no. OU466875) were available from high-throughput sequencing. Screening with primers from previous studies [6] amplified short fragments (243-500 bp), which were not sufficient for the genetic diversity studies, but newly designed primers (Table 1) demonstrated robust amplification across all genomic regions, particularly RNA3. However, primers for the ORF1 N- and C-proximal showed lower efficiency, leading to a lower number of successfully amplified RNA1 fragments. This could be addressed by utilizing additional full-length RNA1 sequences in future studies. The pDAP13 primer \begin{table}
\begin{tabular}{c c c c c c c c c} \hline \hline
**Population I** & **Population II** & \(K_{\text{s}}\)** & \(p\)**-Value** & \(Z\)** & \(p\)**-Value** & **Snn** & \(p\)**-Value** & \(F_{ST}\) \\ \hline Clade ORF3-I & Clade ORF3-II & 2.17071 & 0.0070 *** & 2.31398 & 0.0000 *** & 1.0000 & 0.0013 ** & 0.76152 \\ Clade ORF3-I & Clade ORF3-III & 2.86276 & 0.0000 *** & 5.42576 & 0.0000 *** & 1.0000 & 0.0000 *** & 0.75412 \\ Clade ORF3-II & Clade ORF3-III & 2.98920 & 0.0000 *** & 5.51200 & 0.0000 *** & 1.0000 & 0.0010 ** & 0.61905 \\ \hline \hline \end{tabular}  
* 0.001 \(<p<\) 0.01, *** \(p<\) 0.001, indicating statistical significance  
\end{table} 
Table 3: Genetic differentiation among phylogroups of RNA3. was effective for RNA3-RNA5 but required downstream gel purification for separation of the RNA5 and cloning to distinguish RNA3 and RNA4 due to their similar lengths. This highlights the practical advantage of using region-specific primers like those developed here for genomic studies. While the majority of genetic diversity studies on emaraviruses rely on short fragments from different genomic regions [6; 18; 21; 24; 25; 26; 27; 28; 29; 30; 31], some studies have successfully utilized larger genomic segments and a greater number of isolates [21; 32; 33; 34; 35]. A significant focus has been placed on the core genomic RNA molecules, RNA1-RNA4, which are present in all emaraviruses. Among these, RNA3, encoding the nucleocapsid protein (NP), has been identified as a promising candidate for genetic diversity studies due to its critical role in the virus life cycle and its variability [21]. Other RNA molecules, such as RNA1, RNA2, and RNA4, have also demonstrated different levels of variability but are less explored in genetic diversity studies. It has been reported that the aspen mosaic-associated virus (AsMaV) RdRP N-terminus showed 85.2-99.7 identity, which was similar to the level of the AsMaV RNA3 identity (86.8-100%) at the nt level and the phylogenetic tree based on this region showed similar profiling [21]. The ASaV ORF1 N-proximal and C-proximal showed 92.88-100% and 87.62-99.91% identity in a pairwise comparison of the sequenced isolates while the same value for RNA3 was 91.97-100%.