The genus Metarhizium encompass cosmopolitan entomopathogenic fungi widely used to control pests of agricultural importance [1]. Besides the entomopathogenic life cycle, these species can also be found associated with plants, as endophytes; or in soil and litter, as saprophytes [2]. The biological control potential of the genus Metarhizium has been explored and has great relevance in countries where the agricultural economy is predominant, such as Brazil [1]. Notably, several isolates of Metarhizium harbor potential mycoviruses [3–5]. However, the function and importance of these viral segments in fungal biology are still unexplored.
Mostly of the knowledge about mycoviruses is derived from phytopathogenic-infecting viral sequences, due to the agricultural importance of the hosts. Moreover, several mycoviruses have been linked to hypovirulent-phenotypes [6, 7]. However, recently, virome approaches have also been applied to understand the diversity of viral sequences in entomopathogens, such as Beauveria bassiana [8, 9]. The potential interference of viruses on fungal development, sporulation, and virulence against arthropod-hosts are the main factors explored in infected strains [10, 11]. Although a high prevalence of viral sequences has been found in Metarhizium species, only three viral genomes are available to date. Notably, all available genomes belong to Partiviridae (dsRNA) family [12–14].
The family Totiviridae comprises five genera: Totivirus, Victorivirus, Leishmaniavirus, Trichomonasvirus, and Giardiavirus. The Victorivirus genus, which currently comprises 14 species, harbors filamentous-fungi-infecting viruses (International Committee on Taxonomy of Viruses - ICTV: https://ictv.global/taxonomy/ release 2021). The genome of Victorivirus members is characterized by one linear dsRNA segment (4 to 6 kbp), encoding a single protein RNA-dependent RNA polymerase (RdRp) and a coat protein (CP). Here we describe the genome of a new member of the family Totiviridae, from Victorivirus genus, found in M. anisopliae strain M5. This new victorivirus has been named Metarhizium anisopliae victorivirus 1 (MaVV1).
Metarhizium anisopliae strain M5 was isolated from an insect cadaver (Deois sp. - Homoptera, Cercopidae) in Pernambuco, Brazil and belongs to the Escola Superior de Agricultura Luiz de Queiroz (ESALQ-USP/Brazil) collection of entomopathogenic fungi [15]. The fungus was maintained in Cove’s Complete Medium (MCc) agar plates for routine uses [16]. For dsRNA extraction, mycelium was cultivated in 50 mL liquid MCc (180 rpm; 28 °C; 72 hours) and cells were grinded to powder in mortar and pestle with liquid nitrogen. Nucleic acid extraction was performed employing the phenol/chloroform method [17] and dsRNA segments were precipitated with LiCl [18]. The samples were treated with DNAse and S1 Nuclease (Promega) for 1 hour at 37 ºC. To check dsRNA integrity, electrophoresis was performed in a 1.2% TAE agarose gel containing 0.5 µg/mL ethidium bromide (Figure 1A). The dsRNA samples were lyophilized and then sequenced on an Illumina NovaSeq Platform (Macrogen, Republic of Korea). Contigs were obtained by assembling using Q30-filtered raw data and SPAdes and were subsequently subjected to BLASTx platform against the non-redundant protein database at NCBI GenBank. A potential virus genome was identified as belonging to the Totiviridae family and considered for full description.
To determine both 5’ and 3’ untranslated regions (UTR), the RNA Ligase Mediated – Rapid Amplification of cDNA Ends (RLM-RACE) protocol was performed. Total dsRNAs were extracted from the mycelia as described above. Approximately 250 nanograms of dsRNA were used as template for synthesis of first-strand cDNA using an adapter and a complementary primer as described by Xie et al. with minor modifications [19]. DNA amplicons corresponding to the 5’ and 3’ terminus sequences were cloned into the pUC18 plasmid, transformed into 10β Escherichia coli cells, and sequenced using M13 primers, regions which are designed to flank the cDNA insert.
The full MaVV1 genome is 5,353 bp in length with 58.45% GC content. A motif search on the NCBI Conserved Domain Database (CDD) revealed that MaVV1 has a coat domain as well as a RdRp domain in the same single dsRNA segment (cl25797 – Totivirus Coat protein, and pfam02123 – viral RNA-directed RNA-polymerase) (Figure 1B). The ORFs were identified using the NCBI ORFfinder web software and were subjected to BLASTx (NCBI) to detect sequence similarities. ORF1, which encodes a coat protein (2,274 bp) has 68,23% identity to a coat protein from Ustilaginoidea virens RNA virus L. ORF2, which encodes a RdRp (2,520 bp) has 58.11% identity to a RdRp protein from Ustilaginoidea virens RNA virus 1. Overlapping the two ORFs an unusual octanucleotide (AUGAGUAA) containing both the ORF2 start codon (AUG) and ORF1 stop codon (UAA) was observed (Figure 1C). The genome is completed by the 5' and 3' UTR regions corresponding to 507 and 60 bp, respectively. MaVV1 complete genome sequence was deposited in the GenBank database (accession number OP959068 - temporary).
Phylogenetic analyses were performed to clarify the taxonomic placement of MaVV1. Sequences employed by Khalifa and MacDiarmid as well as sequences from the GenBank/NCBI database were included [20]. Multiple sequence alignments of RdRp amino acid sequences were generated using THE GUIDANCE2 SERVER employing MAFFT algorithm [21]. The phylogenetic tree (Figure 2) was constructed using the PhyML (Maximum likelihood) platform [22] and employing the best substitution model (Q.pfam +R+F) predicted by SMS [23]. Branches lower than 0.8 SH-like branch support were collapsed using TreeGraph 2 software.
These results support previous work in which the taxonomy of viral particles present in the Metarhizium anisopliae M5 strain was inferred from protein and structural characteristics [3]. Therefore, we propose MaVV1 to be a new member of the genus Victorivirus (Gharbivirales: Totiviridae) based on phylogenetic analysis and RdRp sequence comparisons. This Victorivirus was designated as "Metarhizium anisopliae victorivirus 1".