# Molecular Population Genetics of Aspen Mosaic-Associated Virus in Finland and Sweden
## 3 Results and Discussion
### Complete Sequence of AsMaV-RNA1, RNA2 and RNA 5 of Distant Isolates  
Based on the information provided, it appears that the isolates Kuninkaansaari, Finland), and E58632 (Vallisaari, Finland) were more divergent from compared isolates of AsMaV. This divergence is observed at both the nucleotide and amino acid levels in the pairwise alignment and their position in the constructed phylogenetic trees based on the RNA3, RNA4, and partial RNA1. To investigate this further, the complete RNA1, RNA2, and RNA5 of these isolates were amplified by RT-PCR, cloned, and sequenced. The size of RNA1 was 7103 in E58627 and 7106 nucleotides in E58632, respectively. An indel event of three nucleotides was observed at positions 4033-4036, resulting in a shorter RdRP protein in E57627. However, the RdRP protein in these isolates still showed 98-99% similarity with the published isolate E55089 from Sweden at the amino acid level. E58632 and E55809 showed 97% identity at the nucleotide level, while E58627 showed 88% identity with E55809 and E58632. The ORF1 in the sequenced isolates of AsMaV initiates with an AUG at position 46-48 and terminates with an UGA stop codon at position 6955-6957 or 6952-6954 (numbers based on the GenBank Acc. No. LR742464). The RdRP was 2302 aa in isolate E58627 and 2303 in isolate 2303, respectively. Three different domains, N-terminus SRD-1, central region SRD-2, and C-terminus SRD-3 have been reported for RdRP in emaraviruses. The SRD-1 harbor endonuclease sequences like _Peribunyaviridae_ polymerase and probably is involved in \"cap-snatching\". It contains four motifs located between amino acids 66 and 242. The motifs RHD\({}_{98-100}\) and TPD\({}_{136-138}\) are universally conserved in different genera of _Peribunyaviridae_[61]. AsMaV RdRP sequences obtained in this study revealed that these motifs were in the same position and there were no changes in these motifs. But motifs EVK\({}_{152-154}\) and KTDL\({}_{158-161}\) vary across different genera [61]. It should be mentioned that the emaraviruses have been grouped based on their phylogenetic relations in four clades (A to D), in which AsMaV is placed in the clade A [6]. In clade A species of emaraviruses, the motif EVK\({}_{152-154}\) has been reported for PPSMAV-1 and FMV and instead ELV\({}_{152-154}\) was the dominant motif in most of the species including AsMaV (Figure 4). The KTDL\({}_{158-161}\) motif was replaced by RKTDL\({}_{158-161}\) in the RdRP of AsMaV isolates and could be R/H/N/Q/K\({}_{158}\)T\({}_{159}\)D/N\({}_{160}\)L\({}_{161}\) in different species of clade A species but its function is not yet determined. The presence of six polymerase motifs (designated preA, A-E) characteristic of negative-strand RNA viruses [62; 6] have been identified in the RdRP of AsMaV RNA1. This region was conserved in the sequenced isolates. A similar domain-like reported motif of DPLTYNYWVMPTN\({}_{1947-1959}\) in the C-terminus of PPSMV-1 RdRP in the SRD-3 domain was identified as DSRVYNHWLVPTN in the AsMaV isolates sequenced in this study. Probably, this motif is a cap-binding site (CBS). The RNA2 was 2287 nucleotides in length and ORF2 spanned on the nucleotide positions 58-1983 encodes for GPP (641 aa). There were no indel events. The RNA2 and GPP in isolates E58627 and E58632 shared 91% and 95% identity with E55089 at the nucleotide and amino acid levels, respectively. The two sequenced isolates shared 98% and 99% identity, respectively. It has been reported that the GPP of emaraviruses contains a N-terminal signal sequence, transmembrane domains, potential glycosylation sites, and a motif also found in the glycoprotein precursors of phleboviruses (family _Peribunaviridae_) [37,63] which is processed by a protease into two glycoproteins, a smaller N-terminal one (Gn), and a larger C-terminal one (Gc) (Figure 1).