# Characterization of a Novel Emaravirus Affecting Ash Species (_Fraximus_ spp.) in Europe
## 4 Discussion  
We identified a novel plant virus infecting European ash (_Fraximus excelsior_) and manna ash (_F. ornus_) across Europe. Infected trees displayed symptoms including chlorotic ringspots, mottle, line pattern and leaf deformations of curling and shoestring. The virus is proposed as being a new member of the plant virus genus _Emanavirus_ (Order: _Bunyavirales_; Family: _Fimoviridae_) [24], fulfilling established criteria for classification to the genus [23]: (i) A multipartite genome of at least four negative-sense single-stranded RNA segments each with a single open reading frame (ORF) encoding one protein. (ii) A significant homology of these proteins to the viral replicase (RdRP, RNA1), a glycoprotein precursor (GPP, RNA2), a nucleocapsid protein (N, RNA3) and a putative movement protein (MP, RNA4) of other recognized emaraviruses. (iii) Genome segments possessing conserved, complementary terminal nucleotide sequences; and (iv) the presence of spherical double-membrane bodies (DMBs) with an approximate diameter of 70-80 nm. The tentative name ash shoestring-associated virus (ASaV) is proposed for the novel emaravirus, after the most prominent symptom observed in diseased ash. We identified five RNA genome segments (RNA1-5) of the novel virus in both affected ash species by HTS in combination with full-length PCR and Sanger sequencing and could attribute them to the putative emaravirus ASaV. The sequence divergence of the five ASaV encoded proteins to the analogous proteins of other emaraviruses known to date clearly demonstrates the novel virus ASaV as being a separate species. Despite the fact that the divergence of ASaV encoded proteins is less than 25%, when compared to the divergence of respective proteins of different members of the genus, this applies only to  
Figure 6: Thin section of mesophyll cells of a Fraxinus excelsior leaf exhibiting chlorotic ringspots and chloroses infected with the novel emaravirus as confirmed by virus-specific RT-PCR. White arrows indicate spherical particles (double-membrane bodies, DMB) of different sizes (70–80 nm in diameter) typically associated with emaravirus-diseased leaf tissues. Squares indicate sections enlarged in the respective picture to the right. one of the respective protein sequences (RdRp, GPP, N), which are considered as relevant for species demarcation on a molecular level [23]. When comparing the ASaV encoded protein sequences to AcV2, only the RdRp shows identity above 75%, while the other four encoded proteins (GPP, N, MP, P26) exhibit diversities above 25% to the respective homologs. In the case of the putative N protein of AsaV, the aa sequence is nearly identical with the P3 of PaEV, suggesting that the emaravirus identified in pea may be the same as the novel virus we identified in ash. However, as RdRp, GPP, and MP sequences of PaEV are not available in the databases to date, this cannot be ascertained. In any case, the putative ASaV GPP and P26 demonstrate a diversity greater than 25%, when compared to the analogous protein of any other emaravirus. Therefore, the species demarcation criteria established for the genus _Emaravirus_[23] are fulfilled. Whether these five RNA segments represent the complete genome of ASaV remains to be clarified. Although all emaraviruses comprise of at least four core RNA segments, the number of additional genome segments is known to vary [23; 33] with the 10 of perilla mosaic-associated virus (PerMV) being the highest number known to date [40]. However, as the five RNA segments attributed to AsaV were identified in two HTS datasets from pooled _F. excelsior_ and _F. ornus_ and also additionally by full-length RT-PCR, our results strongly suggest that we determined the complete genome of AsaV. The fifth RNA5 encoded protein of AsaV was found to be homologous to a group of similar-sized, non-structural emaravirus proteins, designated P26-P28 according to their size (26-28 kDa), as described by [PERSON] et al. [22].