## 2 Materials and Methods
### Sequence Data Analysis, Recombination, Phylogeny, and Population Parameters  
The obtained sequence data were assembled using the BioEdit Sequence Alignment Editor version 7.2.5 [13] and deposited in GenBank with acc. no. PQ438405 to PQ438536. The assembled sequences were aligned using the MUSCLE algorithm, implemented in MEGA11 [14].  
Seven recombination detection methods (RDP, BOOTSCAN, GENECONV, MAXCHI SISCAN, 3 SEQ, and LARD) implemented in the RDP4 software were applied to detect recombination events in the aligned sequences [15]. Recombination events identified by less than four independent methods were considered unreliable. Phylogenetic trees were constructed using the neighbor-joining (NJ) and maximum likelihood (ML) methods with bootstrap analysis in MEGA11 software to investigate the evolutionary relationships among the sequence isolates of the virus. It should be mentioned that the branching patterns of the NJ and ML trees were essentially similar; however, the NJ trees are shown in this  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline \multicolumn{1}{c}{**Primer Name**} & \multicolumn{1}{c}{**Primer Sequence (\(5^{\prime}\to 5^{\prime}\))**} & \multicolumn{1}{c}{**Genome**} & \multicolumn{1}{c}{**Primer Position**} & \multicolumn{1}{c}{**Ued For**} & \multicolumn{1}{c}{**PCR Product**} & \multicolumn{1}{c}{**Size (bp)**} & \multicolumn{1}{c}{**Reference**} \\ \hline AsaV-R1-840-s & ACAGACTACTACTACTGCGACA & RN1A1 & 840-859 & cloning & 1035 & this study \\ AsaV-R1-875-as & GTCTGAGATACTACTTAATGACG & RN1A1 & 4361–4380 & & 1113 & this study \\ AsaV-R1-4501-s & GCTCTGAGATTAGAGATAGGC & RN1A1 & 4361–4380 & cloning & 1113 & this study \\ AsaV-R1-547-as & CATCATCATCATCATCATCATCATCATCATCAT & RN2A & 164–41 & cloning & 2225 & this study \\ AsaV-R2-16-s & ATTAG  
paper. Branches with bootstrap values lower than 90% were collapsed. Corresponding regions of _Emaravirus kiwi_ (acc. no. NC_055646, NC_055647, NC_055648, NC_055649, and NC_055651), _Emaravirus fici_ (acc. no. PP196567, PP196569, PP228020, PP228021, and PP228022), and _Emarovirus toordali_ (acc. no. HF912243, HF912244, HF912245, HF912246, and HG939490) were used as outgroups for phylogenetic trees. For the phylogenetic analyses of ASaV RNA5, the RNA6 of _Emaravirus fici_ (PP196569), _Emaravirus toordali_ (HG939490), and _Emaravirus kiwi_ (NC_055651), were used as an outgroup as the P6 of these emaraviruses share more homologous motifs with ASaV P5 [6]. SignalP 6.0 ([[https://services.healthtech.dtu.dk/services/SignalP-6.0/](https://services.healthtech.dtu.dk/services/SignalP-6.0/)]([https://services.healthtech.dtu.dk/services/SignalP-6.0/](https://services.healthtech.dtu.dk/services/SignalP-6.0/)) (accessed on 10 July 2024)) and TMHMM v2.0 ([[https://services.healthtech.dtu.dk/services/TMHMM-2.0/](https://services.healthtech.dtu.dk/services/TMHMM-2.0/)]([https://services.healthtech.dtu.dk/services/TMHMM-2.0/](https://services.healthtech.dtu.dk/services/TMHMM-2.0/)) (accessed on 10 July 2024)) were used to predict the presence and characteristics of signal peptides and transmembrane helices. NetNGlyc-1.0 ([[https://services.healthtech.dtu.dk/services/NetNGlyc-1.0/](https://services.healthtech.dtu.dk/services/NetNGlyc-1.0/)]([https://services.healthtech.dtu.dk/services/NetNGlyc-1.0/](https://services.healthtech.dtu.dk/services/NetNGlyc-1.0/)) (accessed on 10 July 2024)) was used to predict N-glycosylation sites in the ASaV isolates' GPP sequences. DnaSP version 6.12.03 software [16] was used to assess different population parameters.