Detection and Molecular Characterization of Novel dsRNA Viruses Related to the Totiviridae Family in Umbelopsis ramanniana

Abstract

<i>Umbelopsis ramanniana</i> is an oleaginous fungus belonging to the Mucoromycotina subphylum. Our group had previously detected four double-stranded RNA (dsRNA) bands in the <i>U. ramanniana</i> NRRL 1296 strain by gel electrophoresis. Here we describe the molecular characterization of its dsRNA elements as well as the discovery of four novel dsRNA viruses: Umbelopsis ramanniana virus 1 (UrV1), Umbelopsis ramanniana virus 2 (UrV2), Umbelopsis ramanniana virus 3 (UrV3), and Umbelopsis ramanniana virus 4 (UrV4). Full genomes of UrV1, UrV3, and UrV4 were determined using the full-length amplification of cDNAs (FLAC) technique; they contain two open reading frames (ORF), which putatively encode the coat protein (CP) and the RNA dependent RNA polymerase (RdRp), respectively. In case of UrV2, a partial ORF encoding a partial RdRp gene could be determined. Based on the phylogeny inferred from the RdRp sequences, UrV1 and UrV4 belong to the genus <i>Totivirus</i>, while UrV2 may belong to the genus <i>Victorivirus</i>. UrV3 nested to a novel, unclassified group of <i>Totiviridae</i>, which is related to the genus <i>Totivirus</i>. Hybridization analysis revealed that the dsRNA molecules of UrV1 and UrV4 correspond to the same 5.0-kbp electrophoretic band, whilst the probe for the UrV3 hybridized to the largest, 5.3-kbp and the 3.0-kbp bands of the dsRNA pattern of <i>U. ramanniana</i>. Interestingly, the probe for the UrV2 sequence did not hybridized to any dsRNA bands, but it could be amplified from the isolated 3.0-kbp fragment. By transmission electron microscopy, two different isometric virus particles with about 50 and 35 nm in diameter were detected in <i>U. ramanniana</i> NRRL 1296 indicating that this strain harbor multiple viruses. Beside <i>U. ramanniana</i>, dsRNA elements were also detected in other <i>Umbelopsis</i> isolates with different patterns consisting of 2 to 4 discrete and different sized (0.7-5.3-kbp) dsRNA molecules. Based on a hybridization analysis with UrV1 CP and RdRp probes, the bands with the size of around 5.0-kbp, which were present in all tested <i>Umbelopsis</i> strains, are presumed as possible full mycovirus genomes.