M. oryzae strain NJ39 was isolated from a lesion of a rice neck-panicle sample that was collected in the city of Nanjing, Jiangsu province, China, and it showed abnormal biological phenotype such as less pigment, less aerial hyphae, and no production of conidia. High-throughout RNA sequencing revealed that strain NJ39 was infected only a new virus, which was tentatively named “Magnaporthe oryzae mymonavirus 1” (MoMNV1).
M. oryzae strains were stored on rice stem nodes at -20 °C and cultured on potato dextrose agar at 28 °C. Design specific primers based on the high-throughput analysis results of MoMNV1 to determine its entire genome sequence and used for RT-PCR to confrm the accuracy of the original sequence by resequencing. Using a ligase mediated end terminal amplification method to amplify cDNA at the 5 'and 3' ends in order to determine the terminal sequences of the dsRNA[6][14][23]. In both orientations, every base was determined by sequencing at three or more independent overlapping clones. The complete nucleotide sequence of MoMNV1 has been deposited in the GenBank database （the accession number OL415836.1）. The amino acid (aa) sequence of the putative RdRp of MoMNV1 was aligned with other virus RdRp sequences using the Clustal Omega program (https://www.ebi.ac.uk/Tools/msa/clustalo/). RNA secondary structures of the termini of MoMNV1 were predicted using the UNAFold web server [27](http://www.unafold.org/Dinamelt/applications/quickfold.php). Conserved domains were identifed using the NCBI Conserved Domain Database (https://www.ncbi.nlm.nih.gov/cdd). On the basis of the aligned sequences, a phylogenetic tree was constructed by the neighbor-joining method using MEGA version 6.0 [12].
Sequence properties
The full-length nucleotide sequence of MoMNV1 was determined and found to be 10,515 nucleotides (nt) long with a GC content of 48.2%. It was used the standard genetic to conclude that contains 6 ORFs (ORF Ⅰ- ORF Ⅵ)， with ORF VI being the largest, encoding RNA-dependent RNA polymerase (RDRP), containing 5837 nt, starting at nucleotide position 4542 and ending at position 10379. And the ORF V being the smallest, containing 512 nt, starting at nucleotide position 3715 and ending at position 4227. The MoMNV1 genome has a 5’- untranslated region (UTR) of 130 nt and a 3’-UTR of 136 nt, and between them are no significant complementarity (Fig. 1).
The ORFⅠ， ORFⅡ， ORFⅢ， ORFⅣ and ORFⅤ encoded five proteins of 704 aa， 1196 aa, 581 aa, 632 aa and 512 aa in length, respectively. In addition, The Nucleic Acid Base consistency rate of five UTRs detected among six ORFs reached 58.6% (Fig 2). The 5’-terminal sequence (nt positions 1–60) and the 3’-terminal sequence (nt positions 10470–10515) of MoMNV1 were predicted to be folded into potentially stable stem-loop structures with ΔG values of -3.03 and -2.11 A, respectively (Fig. 3).
A homology search of the GenBank database using BLASTp showed that MoMNV1 RdRp was most closely related to the RdRps of some members of the genus Penicillimonavirus in the family Mymonaviridae, including Plasmopara viticola lesion associated mononegaambi virus 7 (YP_010798439.1; identity, 59%; query coverage, 99%; e-value, 0), Magnaporthe oryzae mononegaambi virus 1 (NC_077110.1; identity, 90%; query coverage, 99%; e-value, 0), and Erysiphe necator associated negative-stranded RNA virus 6 (NC_077042.1; identity, 52%; query coverage 99%; e-value, 0). Furthermore, a conserved domain database search and a multiple amino acid sequence alignment confrmed that the protein encoded by MoMNV1 contains four typical conserved motifs that are characteristic of the RdRps of (-) ssRNA viruses[26] (Fig. 4). To analyze the taxonomic position of MoMNV1, , a molecular phylogenetic tree was constructed using aa sequences of the RdRp regions of MoMNV1 and 83 other selected viruses of the families Mymonaviridae, Artovuridae and Nyamiviridae. NIne viruses of the family Artovuridae and Nyamiviridae were used as an outgroup. As shown in Fig. 4, the neighbor-joining tree strongly suggested that MoMNV1 is a new member of the family Mymonaviridae (Fig. 5).