# Characterization of a Novel Emaravirus Affecting Ash Species (_Fraximus_ spp.) in Europe
## 3 Results
Protein sequences encoded by ORFs of RNA1-RNA4 showed sequence identities and contained conserved domains characteristic for the viral replicase (RdRp, RNA1), a glycoprotein precursor (GPP, RNA2), the nucleocapsid protein (N, RNA3), and the movement protein (MP, RNA4) to certain members of the genus _Emaravirus_ (Table 3 and Table S2, Supplementary Material). The RNA1 was found to be 7031 nucleotides (nt) long, with a single ORF (6891 nucleotides, 2297 aa) between 45 and 6935 nt in the positive sense (mRNA) encoding the viral Rdkp. The 268 kDa protein exhibited a highest sequence identity of 77.9% to the Rdkp \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Virus** & \begin{tabular}{c} **RNA1** \\ **(RdRp)** \\ \end{tabular} & \begin{tabular}{c} **RNA2** \\ **(GPP)** \\ \end{tabular} & \begin{tabular}{c} **RNA3** \\ **(N)** \\ \end{tabular} & \
\begin{tabular}{c} **RNA4** \\ **(MP)** \\ \end{tabular} &
\begin{tabular}{c} **RNA5** \\ **(P 26–28 kDa)** \\ \end{tabular} \\ \hline AcCRaV & 48.7 & 39.5 & 35.6 & 29.0 & 10.8 (P5, 27 kDa) \\ AcV-2 & 77.9 & 54.7 & 73.1 & 73.6 & 42.4 (P6, 27 kDa) \\ AsMaV & 71.6 & 53.8 & 57.2 & 65.0 & 34.4 (P28, 28 kDa) \\ BLMaV & 68.4 & 50.6 & 59.1 & 56.1 & 28.7 (P5, 26 kDa) \\ CjaV-1 & 25.5 & 19.1 & 16.4 & 10.2 & - \\ CjaV-2 & 26.2 & 18.3 & 13.1 & 9.4 & - \\ CORAv & 31.1 & 21.7 & 17.6 & 16.4 & - \\ EMARaV & 49.1 & 36.5 & 34.8 & 28.2\({}^{\dagger}\) & 10.6 (P4, 27 kDa) \({}^{2}\) \\ FMV & 73.8 & 55.2 & 69.2 & 76.1 & 31.0 (P6, 26 kDa) \\ HPWMoV & 30.3 & 21.6 & 15.1 & 16.5 & - \\ JYMaV & 31.5 & 21.8 & 20.5 & 15.8 & - \\ LiCRaV & 48.6 & 38.2 & 34.3 & 30.7 & 13.0 (P5, 26 kDa) \\ MaMaV & 67.5 & 50.1 & 47.6 & 62.6 & 32.7 (P6, 27 kDa) \\ PCLSaV & 28.0 & 14.2 & 14.8 & 9.2 & - \\ PerMV & 25.3 & 18.4 & 16.3 & 10.2 & 7.5 (P, 27 kDa) \\ PaEV & - & - & 99.0 & - & - \\ PiVB & 71.0 & 53.6 & 57.7 & 69.8 & 30.7 (P6, 28 kDa) \\ PPSMV-1 & 53.2 & 44.0 & 40.6 & 38.5 & 32.7 (P6, 27 kDa) \\ PPSMV-2 & 73.4 & 58.2 & 71.1 & 73.4 & 32.3 (P6, 27 kDa) \\ PVBV & 31.4 & 19.3 & 15.2 & 16.1 & - \\ RLBV & 31.3 & 21.4 & 18.8 & 17.3 & - \\ RRV & 68.3 & 52.7 & 59.8 & 59.8 & 28.9 (P6b, 27 kDa) \\ KYRSaV & 48.2 & 40.5 & 35.8 & 30.2 & 11.6 (P5, 26 kDa) \\ TiRSaV & 31.0 & 20.8 & 18.5 & 14.0 & - \\ \hline \hline \end{tabular} \({}^{\dagger}\)EMARav RNA5 [28], \({}^{\ddagger}\)EMARav RNA4 [26].  
\end{table} 
Table 3: Amino acid sequence identities (%) of Rdkp, GPP, N, MP and P5, encoded by the RNA1-5 of the novel virus identified in a diseased _Frazinus excelsior_ (E55270, accession numbers: RNA1 OU46680, RNA2 OU466881, RNA3 OU466882, RNA4 OU466883, RNA5 OU466884), with corresponding proteins of classified and selected putative emaraviruses. Note: Accession numbers of putative and classified emaraviruses listed in supplementary information, Table S2. of AcV-2. The RNA2 is 2243 nt in length, with a single ORF (1926 nt, 642 nt) between 57 and 1982 nt, encoding a 73 kDa glycoprotein precursor (GPP). The GPP was found to have the highest similarity to the pigeon pea sterility mosaic virus -2 (PPSMV-2) GPP (58.2%). The RNA3 is 1483 nt long, with a single ORF (948 nt, 316 aa) between 104 nt and 1051 nt, encoding a 35 kDa nucleocapsid protein (N). The N protein is 99% identical to the complementary P3 protein of the recently identified pea-associated emaravirus (PaEV) [39]. The RNA4 is 1518 nt in length with a single ORF (1086 nt, 362 aa) between 83 and 1168 nt, thought to encode the movement protein (MP). The 41 kDa protein was found to be 76.1% identical to the MP of FMV. The RNA5 is 1333 nt in length, with a single ORF (696 nt, 232 aa) between 70 and 765 nt. The function of the 26 kDa protein remains unknown. However, blastx revealed similarities to other undetermined proteins, of classified emaraviruses, including AcV-2 P6 (42.4%) and AsMaV (34.4%). The 5\({}^{\prime}\) and 3\({}^{\prime}\) termini of all five identified genome segments are conserved and show high complementary to each other.