Beauveria bassiana Vuillemin is currently one of the most widely commercialized and applied biological insecticides in the world [1]. Previous studies have shown that B. bassiana could be infected by not only a separate [2,3] but also multiple mycoviruses [1,4]. However, the viruses reported in B. bassiana are mainly double stranded RNA (dsRNA) viruses [5-7], and there are few reports of single stranded RNA (ssRNA) viruses infected with B. bassiana, although the only positive single stranded RNA virus Beauveria bassiana small Narna-like virus (BbSNLV) has been found in B. bassiana [4], there is no record of negative single stranded RNA (-ssRNA) virus infection on B. bassiana.
Negative stranded viruses are recently discovered in fungi, and only a few of them have been so far characterized through virus purification and whole genome characterization [8]. The order Mononegavirales includes 11 families and some unclassified viruses, in which the family Mymonaviridae is the only classified viral family related to mycoviruses [9,10]. Viruses in the order Mononegavirales are mostly monopartite with multiple open reading frames (ORFs) [11]. The most surprising result in the Mononegavirales was that its characterization of the genome organization of some complete and putatively incomplete genomes coding for two ORFs [12].
The virus-infected B. bassiana strain GusB2 of was isolated from the diseased Ostrinia furnacalis larva in a corn field located in Jingyu County, Jilin Province, China (42°06′N, 126°30′E) in 2022, and identified following the methodology described by Morris et al [13]. To obtain the viral genomic sequence, we conducted a series of experimental operations according to the protocol outlined by Kang et al [3], including the total fungal RNA and dsRNA extraction, the dsRNA purification, and the cDNA library construction utilizing the Illumina HiSeq platform from Novogene Bioinformation Technology Co., Ltd. (Beijing, China) to obtain the raw data (approximately 3.8 G), which was then analyzed with SPAdes (V3.13.1). Finally, the terminal sequences of the three RNA segments were obtained according to a classical RACE protocol in two separate experiments[14].
To determine the taxonomical status of the viruses, we predicted the open reading frames (ORFs), analyzed the amino acid (aa) sequences and conserved domains in the viral genomes, and conducted the phylogenetic trees to perform a multiple aa sequence alignment. All of these were performed according to the description of Kang et al [3].The B. bassiana strain GusB2 experienced a co-infection by two distinct types of nucleic acid viruses (Fig. 1A), namely -ssRNA (Fig. 1B) and dsRNA (Fig. 1C). The complete genetic sequences of both viruses were deposited in GenBank under the accession number PRJNA1027002. The -ssRNA virus is a single segment, which genome length is 6169 bp (Genbank no. OR737625) with a GC content of 49%, the sequence of which 5,-UTR and 3,-UTR were 165 and 154 bp in length, respectively. It encodes a single large ORF that produces a protein consisting of 1949 aa and weighing approximately 220.1 kDa (Fig. 1D).
Phylogenetic analysis based on a BLASTx analysis searching showed that the ORF of the -ssRNA segment exhibited a 59.79% homology to the RNA dependent RNA polymerase (RdRp) of Plasmopara viticola lesion associated mononegaambi virus 2 (PvLamonoambiV2) (GenBank No. QHD64771.1, Query coverage = 99%, E-value=0), and 59.42% homology to that of PvLamonoambiV4 (GenBank No. QHD64774.1, Query coverage = 99%, E-value=0) [11 ] (Fig. 2A). The identified virus was designated as BbNSRVs1. A search conducted in the CDD database indicated that the protein encoded by this ORF contained a conserved Mononeg-RNA-polsuper family domain with an E value of 2.74e−87 and a Mononeg-mRNAcap superfamily domain with an E value of 2.28e−11 (Fig. 2D). Consequently, BbNSRVs1 can be considered as a new member of Mononegavirales.
The dsRNA virus genome is composed of two segments, namely dsRNA1 and dsRNA2, respectively (Fig 1C). The length of dsRNA1 is 2164 bp (Genbank no. OR737623) with a GC content of 55%. It contains an ORF that codes for a protein consisting of 664 aa (76.06 kDa). Both the 5'-UTR and 3'-UTR regions have lengths of 83 bp and 86 bp, respectively (Fig 1E). The dsRNA2 has a length of 1765 bp (Genbank no. OR737624) with a GC content of 58%. It carries an ORF encoding a protein comprising of 317 aa (35.28 kDa). The lengths of its respective 5'-UTR and -3' UTR regions are approximately 102 bp and 709 bp (Fig 1E). Importantly, it should be noted that both the 5′- and 3′terminals of the two segments exhibit high conservation (Fig 1F).
According to the BLASTx analysis, the two ORFs encoded by two dsRNA segments exhibited a high degree of similarity, reaching up to 98.95% and 99.05%, respectively, with the RdRp (GenBank No. WEI52980.1, Query coverage=100%, E-value=0) and coat protein (CP) (GenBank No. WEI52981.1, Query coverage=100 %, E-value=0) of Beauveria bassiana dsRNA mycovirus 1 reported in Genbank from B. bassiana samples, thus it was determined that both viruses are identical. These segments have been designated as BbdsRNAMV1 for identification purposes.
Phylogenetic analysis was conducted using the complete aa sequence of motifs RdRp and CP. The ORF1 of BbdsRNAMV1 shows a homology of 70.75% with the RdRp of Fusarium graminearum dsRNA mycovirus 5 (FgdsRNAMV5) (GenBank No. APP91269.1, Query coverage = 92%, E-value=0), as determined by a BLASTx analysis (Fig. 2B). A conserved RT-like superfamily domain (E value = 1.64e−11) was identified in the protein encoded by ORF1 through a search in CDD (Fig. 2E). The ORF2 of BbdsRNAMV1 shares a homology of 64.26% with the CP of FgdsRNAMV5 (GenBank No. APP91270.1, Query coverage=99%, E-value=9e-141) according to a BLASTx analysis (Fig. 2C). Notably, the RdRp region of this virus displayed a conserved GDD motif (Fig. 2E). Therefore, similar to FgdsRNAMV5, the taxonomic classification for virus BbdsRNAMV1 remains undetermined at present.