## 3 Results
### Indel Events and Motifs in Different Genomic RNAs of ASaV  
The alignment of RNA2 from 21 ASaV isolates revealed that all sequences terminate with a UAA stop codon, differing from the GenBank-retrieved E55270 isolate (OU466881), which terminates with a UAG stop codon. A putative cleavage site at KA \(\downarrow\) DD [17], located between A\({}_{182}\) and D\({}_{183}\), was identified in all isolates analyzed. This site is predicted to generate a smaller 21 kDa glycoprotein (Gn) and a larger 51 kDa glycoprotein (Gc). The Pi\({}_{a}\)/Pi\({}_{s}\) values were calculated as 0.063 for Gn and 0.034 for Gc region of ASaV GPP. The GCYDCQSG motif [17] was conserved across all ASaV isolates at positions 475-482. The predicted N-glycosylation sites at four positions (N\({}_{62}\), N\({}_{382}\), N\({}_{390}\), and N\({}_{623}\)) with high confidence were also conserved across all isolates. SignalP-5.0 predicts a signal peptide with the cleavage site occurring at positions 22-23 in the VYN-HF context with a 55.07% probability. Four transmembrane helix (TMH) domains were identified between residues 2-24, 112-134, 170-187, and 581-603. The conserved sequence motifs NXXSXNNXXXA, NRLA, and GYFE, believed to be involved in RNA binding within the NP of emaraviruses [18], were consistently present across all studied isolates. These motifs correspond to the sequences NIVSFNKACA at positions 130-139, NRLA at 178-181, and GYFE at 192-202 nt. The isolates belonging to clade ORF3-I exhibit distinguishing features that differentiate them from those in clades ORF3-II and ORF3-III. Protein synthesis in these isolates terminates at an opal (UGA) stop codon, and clade ORF3-II and ORF3-III isolates terminate at an ochre (UAA) stop codon. Two PaEV isolates retrieved from GenBank with two different stop codons (PaEV-17 acc no. MN399721 with UAA and PaEV-18 acc. no. MN399722 with UGA) were classified as clades ORF3-III and ORF3-I. Interestingly, shorter RNA3 lengths, unique amino acid substitutions, and different stop codons were observed in the ASaV RNA3 clade ORF3-I isolates. Alignment of the RNA4 sequences confirmed that the signal-peptide sequence at both the P4 N-terminus and the C-terminus, along with motifs critical for cell-to-cell movement (K\({}_{55}\), Y\({}_{72}\), R\({}_{88}\)XA\({}_{90}\)XXXXXXW\({}_{96}\)XP\({}_{98}\), D\({}_{113}\)XR\({}_{115}\), V\({}_{146}\), D\({}_{168}\)I\({}_{169}\)X\({}_{171}\)I\({}_{172}\), M\({}_{183}\), W\({}_{197}\)XT\({}_{199}\), F\({}_{215}\), E\({}_{224}\), and I\({}_{326}\)[19; 20; 21]), were conserved in all studied isolates. All studied isolates terminated with a UAG stop codon (antigenome position 1169-1172 nt). Within this region, the ORF4 gene consists of 1086 nucleotides and encodes a protein of 362 amino acids, corresponding to antigenome positions 83 to 1168 in the reference sequence OU466883. An indel event was identified at antigenome position 55 (Adenine) of the 5\({}^{\prime}\)-UTR, resulting in a variable length of 49-50 nucleotides (antigenome positions 34-84; accession no. OU466883). The alignment of the 3\({}^{\prime}\)-UTR, encompassing 330 out of 350 nucleotides (antigenome positions 1168-1518; accession no. OU466883), revealed that indels occur in the AT-rich region, resulting in varying lengths of 324 to 330 nucleotides. Due to indels in this region, E62907 was the longest, while E62037, E62053, and E62902 (Melzower forest, Germany) were the shortest. Interestingly, this deletion occurred in the isolates, including five out of eight from Melzower forest and one isolate (E62017) out of four from Berlin. As with RNA3 and RNA4, the isolates studied for RNA5 exhibited variations in sequence lengths due to indels in the 3\({}^{\prime}\)-UTR. The RNA5 segment length of the iso lates ranges between 1277 and 1288 nucleotides, compared to the full RNA5 length of 1333 nucleotides (antigenome positions 28-1302, acc. no. OU466884). The ORF5 consists of 693 nucleotides (antigenome positions 70-762, acc. no. OU466884), encoding a protein of 231 amino acids.