# Molecular Population Genetics of Aspen Mosaic-Associated Virus in Finland and Sweden
## 3 Results and Discussion
### Genetic Diversity and Phylogeny of AsMaV Isolates Based on RNA3  
The length of AsMaV-RNA3 in sequenced isolates was 1588-1589 nucleotides in size, with 86.8-100% sequence identity among the isolates. Isolates E58213 (Sweden), E58627, and E57260 (Finland) were the most divergent isolates. The 3\({}^{\prime}\)UTR region exhibited the highest level of sequence diversity (Table 2). The length of AsMaV RNA3 was previously reported to be 1587 nucleotides (nts) for isolate E55089 (GenBank Accession number LR742463). In 80% of the sequenced isolates, RNA3 was 1588 nts long, while in the remaining isolates, it was 1589 nucleotides in size. The ORF3 started at nt 101 and ended at nt 1057 with the exception of isolates E58627, E58213, and E57260 which ended at nt 1060 (Figure 1). Consequently, the length of the P3 gene or NP gene was 957 or 960 nucleotides encoding for 318-319 amino acids (aa), respectively. As previously published, RNA3 has a very long 3\({}^{\prime}\) UTR, i.e., 530 nts [5]. Three different stop codons were detected in AsMaV ORF3, with the amber stop codon (UGA) being the most prevalent in the majority of isolates (18 out of 27). The ochre (UAA) and opal (UGA) stop codons were detected in six and three isolates, respectively (Figure 1). Stop codons have varying termination efficiencies (UAA > UAG > UGA). In plant viruses, either amber (UAG) or opal (UGA) stop codons are read through; there are no examples of read-through of the natural ochre (UAA) stop codon [56]. The presence of several stop codons in different frame shifts downstream of the ORF3 might be denoting the importance of the NP size. The three conserved motifs previously reported for the NP of emaraviruses (NXXS-XNNXXA, NXLA, and GYEF) [57; 58] were also found in AsMaV-NP. The first motif identified in AsMaV-NP was NIVSFNKACA\({}_{131-140}\) which was present in all sequenced isolates, except for E58446 and E58434 where the last amino acid was P instead of A. The motif NRLA and GYEF were found in positions 179-182 and 200-203, respectively. The first motif has not been reported for (PerMV) and motif GYEF was replaced with GVEF\({}_{159-162}\). The aa \(X_{139}\) was also R in the motif NXLA\({}_{138-141}\) of PerMV. In the AsMaV and PerMV, there is a 21 aa distance between the second and third motif, which appears to be conserved among the emaraviruses and essential for the folding or functioning of the protein. However, the distance between the first and second motif (35-39 amino acids) was not conserved among the emaraviruses. Sequence data confirmed the presence of AanI site in RNA3 at the positions 627-632, 1069-1074, 1142-1147, 1406-1411, and 1473-1478 based on the nt sequence of the isolate E55089 (GenBank Acc. No. LR742463). Among these restriction sites, only one of them was placed in the ORF3 (nts 627-632), and the rest of the AanI restriction sites were detected in the 3\({}^{\prime}\) UTR. The first nt of AanI restriction site (T\({}_{628}\)) was placed in the position of nt 627-632 (5\({}^{\prime}\)-CITATAATC-3\({}^{\prime}\)) and is the second codon position of aa leucine. Therefore, the second nucleotide position of the AanI site is the third-codon position of leucine in which any changes in that position do not result in non-synonymous substitutions. Interestingly, the nt 627-632 AanI site is adjacent to the emaravirus motif NxLA (Table 1). The rest of the AanI sites are spanned in 3\({}^{\prime}\) UTR which was the largest untranslated region of AsMaV and the most divergent part of RNA3. Searching for the secondary structure of RNA