Complete genome sequence of a new quadrivirus infecting a member of the genus Thelonectria

A novel dsRNA virus named “Thelonectria quadrivirus 1” (TQV1) was found in a member of the genus Thelonectria (Ascomycota), isolated from a root associated with stem collar necrosis of Fraxinus excelsior L. The complete genome of TQV1 is composed of four segments, each containing a single ORF on the positive sense RNA. The sequence of the 5´ (5´-(C/T)ACGAAAAA-3´) and 3´termini (5´AT(T/G)AGCAATG(T/C)GC(G/A)CG-3’) of dsRNA 1 (4876 bp), dsRNA 2 (4312 bp), dsRNA 3 (4158 bp), and dsRNA 4 (3933 bp) are conserved. Based on its genome organization and phylogenetic position, TQV1 is suggested to be a new member of the family Quadriviridae. This is the first report of a mycovirus infecting a member of the genus Thelonectria.

The most widespread mycoviruses are viruses with a dsRNA genome. According to Kotta-Loizou et al. [1], dsRNA mycoviruses are arranged in seven families (Totiviridae, Partitiviridae, Megabirnaviridae, Chrysoviridae, Quadriviridae, Endornaviridae and Reoviridae) and one genus (Botybirnavirus). At present, the family Quadriviridae consists of the single genus Quadrivirus. Within this genus, only one member, Rosellinia necatrix quadrivirus 1-W1075, has been confirmed so far [2,3], but several other viruses have been proposed to belong to the family Quadriviridae.
The quadrivirus genome consists of four dsRNA segments, which are packed in non-enveloped spherical particles, 45 nm in diameter. The particles are composed of the structural proteins P2 and P4 and enclose the RdRP, which is encoded on segment 3. Segment 1 encodes a hypothetical protein with unknown function. The dsRNA segments range in size from 3.5 to 5.0 kbp, comprising 16.8-17.1 kbp in total [3].
Members of the genus Thelonectria P. Chaverri & Salgado are widespread fungi that belong to the family Nectriaceae Tul. & C. Tul., phylum Ascomycota [4], that can exist in a cylindrocarpon-like asexual state. Typically, they maintain a saprophytic lifestyle; however, they can cause small cankers or root rot on their hosts. Fruiting bodies are mostly spread on the bark of diseased, dying or recently dead broadleaf host trees [5,6]. To date, no virus has been described infecting a fungus of this genus.

Provenance of the virus material
The Thelonectria strain NW-FVA-1901 (GenBank accession ID: OK161009) was isolated from a necrotic root associated with stem collar necrosis of Fraxinus excelsior L. It was collected in the Waldgehege Fahrenstedthof, mark 24860, Böklund, Abt. 3410a in Schleswig-Holstein, Germany. Isolation and identification as a member of the species Thelonectria was performed as described by Langer [7]. Mycelium was cultivated on potato-dextrose agar (PDA), from which virus-like particles were purified as described by Lutz et al. [8]. Nucleic acids were extracted from particles using a Double-RNA -Viral dsRNA Extraction Kit (iNtRON Biotechnology, Seongnam-Si, South Korea). Isolated dsRNA was subjected to next-generation sequencing. Libraries were prepared using a Nextera XT DNA Library Preparation Kit (Illumina Inc., San Diego, CA, USA) and sequenced on a NextSeq 2000 (Illumina Inc., San Diego,

Sequence properties
The complete genome sequence of TQV1 has been deposited in the GenBank database (accession ID: OK077750-OK077753). As it is typical for members of the family Quadriviridae [2,12,13], each of the four dsRNA segments contains a single ORF on the positive-sense RNA strand ( Fig. 2A). The GC content of each segment ranges from 49 to 53%. The sequenced segments corresponded in number and size to the bands detected by agarose gel electrophoresis (Fig. 1), which showed bands ranging from 4.9 to 3.9 kbp. Segment 1 is 4876 bp in length. Its ORF spans from nucleotide position 46 to 4821 and encodes a protein with 1591 aa and a calculated molecular weight of 176.21 kDa.
In total, the genome of TQV1 consists of 17.279 bp. To determine the taxonomic position of TQV1, a maximumlikelihood tree was constructed based on a BLASTp search of the sequence of the putative RdRP (P3) of TQV1. It was aligned with P3 sequences of confirmed and tentative members of the families Quadriviridae and Chrysoviridae (Fig. 2B). The phylogenetic analysis showed that TQV1 may represent a new clade between the clade including RnQV1 and the other viruses tentatively assigned to the family Quadriviridae. However, there is currently little sequence information about quadriviruses, and this lack of additional sequences correlates directly with the low bootstrap values observed in the tree. Therefore, the taxonomic position of TQV1 can only be assumed based on this phylogenetic analysis. Nonetheless, based on its genome organization and its phylogenetic position, TQV1 is suggested to be a new member of the family Quadriviridae.
Funding Open Access funding enabled and organized by Projekt DEAL. This project was financed by the Agency for Renewable Resources (FNR) in the program "Waldklimafonds" (Forest and Climate Fund) (2219WK22A4 and 2219WK22G4) funded by the German Federal Ministry of Food and Agriculture and the German Federal Ministry for Environment, Nature Conservation and Nuclear Safety.
Availability of data and material The datasets generated and/or analyzed in the current study are available in the GenBank database (accession ID: OK077750-OK077753 and OK161009).

Declarations
Conflicts of interest All authors declare that they have no conflict of interest.
Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors.
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