Endornaviruses are viruses with linear single-stranded positive-sense RNA genomes ranging from approximately 9.7 to17.6 kb reported to infect plants, fungi, and oomycetes [22, 23]. Their genome codes for a single polyprotein which contains conserved domains. Although, true virions have not been reported, cytoplasmic vesicles have been associated with some endornaviruses [8, 17]. Endornaviruses are classified in the family Endornaviridae which contains two genera, Alphaendornavirus and Betaendornavirus . In plants, endornaviruses do not cause apparent effects on the phenotype and are transmitted only vertically [2, 23]. Endornaviruses have been reported in many cultivated and wild plant species [2, 3, 5, 23].
Common buckwheat (Fagopyrum esculentum Moench) and Tartary buckwheat (F. tataricum Gaertner) are members of the family Polygonaceae. They are cultivated worldwide for their nutritional and medicinal values, which includes gluten-free and a broad spectrum of flavonoids with antioxidant effects [7, 18].
Symptomless common buckwheat plants cv. Kitawasesoba growing in the field of Ibaraki Agricultural Center in Kasama, Japan were tested for the presence of large dsRNAs by the spin column method  and found to contain an approximately 16 kbp dsRNA. Because this dsRNA was similar in size to dsRNAs found associated with many plant endornaviruses, we cloned and Sanger-sequenced random-primed cDNA and RACE products as described previously [11, 15]. Amino acid sequences were assembled and analyzed using Genetyx Ver9 (GENETYX corporation, Japan). Multiple sequence alignments of deduced amino acid sequences were conducted using Clustal X . A Maximum Likelihood-based phylogenic tree was constructed using MEGA ver. 6.0 . Bootstrap tests were performed with 1,000 resamplings. Preliminary sequence analyses yielded conserved domains typical of plant endornaviruses. Further sequence analyses suggested that a novel endornavirus was infecting Kitawasesoba common buckwheat. The complete sequence was submitted to the GenBank (Accession No. LC500285) and the virus was provisionally named Fagopyrum esculentum endornavirus 1 (FeEV1).
The nucleotide sequence of FeEV1 was determined to be 15,677 bp in length containing a single open reading frame (ORF) in one strand (designated as the plus strand), starting at nt 73 (the second AUG codon) and ending at nt 15,645. The ORF potentially encoded a polyprotein of 5,190 aa with an estimated molecular mass of 589 kDa (Fig.1). The first AUG codon was found at nt 22, however, it was not considered to be the favorable initiation codon. The 5'-UTR and 3'-UTR of FeEV1 consisted of 72 and 32 nt long, respectively. The 3'-UTR ended with 12 cytocines. A BLAST search using the aa sequence detected conserved domains of a putative viral methyltransferase (MT) (aa 311-454), viral RNA helicase 1 (HEL) (aa 1467-1712), UDP-glycosyltransferase (UGT) (aa 3262-3608), and viral RNA dependent RNA polymerase 2 (aa 4884-5116) (Fig.1). These four domains are also reported in some plant endornaviruses [10, 13, 15, 16]. In addition, a unique conserved domain, MSCRAMM family adhesion SdrC, was found in between the HEL and UGT domains (aa 1,945-2077). Features of this protein family include a YSIRK-type signal peptide at the N-terminus and a variable-length C-terminal region of Ser-Asp (SD) repeats followed by an LPXTG motif for surface immobilization by sortase . FeEV1 also included the YSIRK motif at the N-terminus of the polyprotein (aa 28-32) and five SD motifs followed by the motif LPSTG (aa 1,950-1,954) in the MSCRAMM family domain. The aa sequence LPSTG may be the protease recognition site for the cleavage of the polyprotein. In silico analysis showed that a cysteine-rich region (CRR), identified in other endornaviruses and suggested as a candidate for a viral protease that can process the polyprotein into functional units [4, 15, 21], was also found in FeEV1. The polyprotein of FeEV1 had three CXCC signatures between MT and HEL domains (aa 773-776, 778-791, and 820-823). Like in the case of other plant endornaviruses [14, 15], a site-specific nick (nt 891) near the 5' region of the plus strand was detected in the FeEV1 dsRNA by 5' RACE experiments (Fig.1). The role of the nick is unknown, but it is thought to be involved in regulation of virus replication .
Seeds of 34 plant introductions of F. esculentum and two of F. tataricum were obtained from the USDA-ARS, Plant Genetic Resources Unit, Geneva, New York, USA and planted in the laboratory under fluorescent lights. Visual inspections of the plants did not show any virus-like symptoms. Foliar samples were collected from individual plants at maturity (flowering stage) and dsRNA extracted and analyzed by electrophoresis as described by Khankhum et al. . Analyses of dsRNA extractions yielded dsRNAs of approximately 16 kb in 16 Fagopyrum plant introductions (from nine countries) but not in 20 others (Supplementary Table 1). DsRNA-positive plants were tested by RT-PCR using general endornavirus primers  and the endornavirus nature of the dsRNAs was confirmed. These results suggest that FeEV1 or perhaps other endornaviruses are present in the F. esculentum and F. tataricum germplasm.
BLASTP analysis showed that the polyprotein of FeEV1 shared the highest identity (45.84%) with that of winged bean endornavirus 1 (91% query cover). The genome organization of FeEV1 is similar to that of endornaviruses of winged bean (winged bean endornavirus 1), pepper (bell pepper endornavirus and hot pepper endornavirus), and common bean (Phaseolus vulgaris endornavirus 2). In addition, the phylogenetic tree of the RdRp places FeEV1 in a group including these four endornaviruses (Fig. 2). In conclusion, the data provided here support placing the virus isolated from common buckwheat as a novel species in the genus Alphaendornavirus in the family Endornaviridae.