# Characterization of a Novel Emaravirus Affecting Ash Species (_Fraximus_ spp.) in Europe
## 4 Discussion
The function and importance of these analogous proteins in the lifecycle of these emaraviruses, however, remains elusive. A specific interaction with host or vector proteins, or a role in virus pathogenicity has previously been speculated for such unknown proteins [33; 41]. Viral suppressors of host RNA silencing mechanisms, for example, are well documented in other plant viruses [42; 43], including the emaravirus High Plains wheat mosaic virus (HPWMoV), in which the P8 was reported to have suppressor activity [44; 45]. However, no significant homology of the ASaV P26 was found to this protein. Phylogenetic analyses based on amino acid alignments of the RdRp and N proteins of ASaV and selected emaraviruses revealed cladograms with four distinct subgroups (A, B, C and D), whereby AsaV consistently clustered in subgroup A. This not only corroborates the cumulative findings of previous emaravirus studies [21; 22], but also confirms AsaV to be a new distinct emaravirus species, being to the best of our knowledge the first to have been detected in _Frazinus_ spp. Surprisingly, the N protein (RNA3) of AsaV was found to be nearly identical (99%) to that of the recently discovered pea-associated emaravirus (PaEV) which infects green peas (_Pisum sativum_ L.) [39]. This raises the question as to whether both viruses are in fact the same species. With the exception of mechanical transmission of emaraviruses occurring under experimental conditions [46; 47; 48], emaraviruses have largely been found to infect a single host or relatively few, closely related host species. Although a natural infection of both ash and peas would therefore seem unlikely, it would raise new epidemiological questions, if this were the case. However, as only the RNA3 of PaEV is currently available in the databases for analysis, the characterization of further PaEV genome segments is necessary to determine the phylogenetic relationship of both viruses. 3\({}^{\prime}\) terminal ends of the RNA1 and RNA2 were confirmed by tailing PCR as being the same as those generated by the genus specific PDAP213 primer [33]. The remaining terminal ends of ASaV are still to be confirmed. Possible explanations for the difficulties experienced in amplifying particular termini by this method may include an unequal distribution of virus particles in the host plant, or low concentrations of RNA complementary to the genomic viral strands. [PERSON] et al. [49] found that the concentrations of negative sense RNA were up to 20 times higher than those of sense viral RNA in HPWMoV infected samples. Rapid amplification of cDNA ends (RACE) [50; 51] or inverse PCR methods [52] could be used as alternative methods in future studies to confirm remaining terminal sequences. During this study, a successful RT-PCR-based method was established for the detection of ASaV in _Frazimus_ spp., applying virus-specific primer sets targeting the five RNA segments. All primer pairs are located within the coding regions of each genome segment and comparable detection results for ASaV were achieved for all sets. The existence of more divergent ASaV variants, which may not be detected by these primer sets, cannot, however, be excluded due to the limited sequence information currently available. [PERSON] et al. (2014) [49] demonstrated the existence of HPWMoV variants differing significantly in the identity of only a single genome segment. To avoid the possibility of false negative results, samples should therefore be tested by applying primer sets for at least two different genome segments, as suggested by [PERSON] et al. [47]. However, as a 5% maximum nucleotide diversity was found between two ASaV variants, each identified in a different _Frazimus_ species, this suggests that the overall sequence variability of ASaV is not expected to be high. A close association between the presence of AsaV and the described host symptoms could be demonstrated in _F. excelsior_ and _F. ornus_, indicating the virus to be the probable causal agent.