# 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 RNA4  
The length of RNA4 in sequenced isolates was 1557 nucleotides in size and there were no indel events. The length of 5\({}^{\prime}\) UTR, ORF4, and 3\({}^{\prime}\) UTR of RNA 4 were 86, 1086 (361 aa), and 385 nucleotides, respectively. The level of 95.1-99.9% and 95.8-99.4% identity on nucleotide level was estimated in pairwise comparisons of the isolates from Swedish and Finnish locations, respectively. Samples E58227 and E58209 from Sweden and the isolates E58421 and E58491 from Finland were the most divergent sequences of each geographical region. Like RNA3, the 3\({}^{\prime}\) UTR was the most divergent region of the genomic RNA4 (Table 2).  
The ORF4 in the sequenced isolates of AsMaV initiates with an AUG at position 87-90 and terminates with a UGA stop codon at position 1170-1172 (numbers based on the GenBank Acc. No. acc. no. LR742464). Interestingly, there were one or two UAA stop codons (in frame) in positions 1176-1181. This might denote that the size of MP plays an important role in the function of this protein. Alignment of the deduced amino acid sequences from published emaraviruses identified the following conserved amino acids or motifs: K\({}_{58}\), Y\({}_{72}\), Rss\({}_{19}\)XXXXXX(W/Y)\({}_{97}\)XP\({}_{98}\), D\({}_{113}\)XR\({}_{115}\), V\({}_{146}\), D\({}_{168}\)I\({}_{169}\)X(K/Y)\({}_{171}\)(I/V)\({}_{172}\), M\({}_{183}\), W\({}_{199}\)XT\({}_{199}\), (F/Y)\({}_{215}\), E\({}_{224}\), and (I/L)\({}_{326}\). Similar motifs have been previously reported for PPSMV-1 and PPSMV-2, RLBV and RRV movement proteins and it has been suggested that some of these motifs showed a similarity to membrane-located protein and they might be involved in virus cell-to-cell movement [59; 60].  
In the CMP tree, constructed based on the AsMaV-RNA4, the 25 isolates sequenced in this study (18 from Finland and 7 from Sweden) and one previously reported isolate E55089 with 1000 bootstrap replicates at the nt level were placed in two main groups (R4-G1 and R4-G2). Isolate E58213 (Sweden) was placed in a separate clade close to the outgroups (FMV, PiVB, and PPSMV-2) (Figure 3). The R4-G1-A includes E58227, E58299, and E55089 from Sweden and an isolate from Finland, E58413. R4-G1-B includes three isolates from Finland, i.e., E58405, E58421, and E58434. The rest of the isolates from Sweden were placed as a clade in R4-G2 and the remaining Finnish isolates were placed in three subclades or remained ungrouped.  
Most of the AsMaV isolates remained ungrouped in the CMP tree constructed based on the ORF4 at the amino acid level (data not shown). It might be that these sequences were not parsimoniously or phylogenetically informative. According to the CMD tree drawn based on the nucleotide sequence of ORF4, the Swedish isolates were grouped in two clades, ORF4-G2 and ORF4-G7, and isolate E58213 remained ungrouped. Five Finnish isolates were not placed in any group and isolate E58413 was placed between Swedish isolates in ORF4-G7. The rest of the Finnish isolates were grouped into five groups.