ANNOTATED SEQUENCE RECORD Molecular characterization of a novel amalgavirus from the entomopathogenic fungus Beauveria bassiana Igor Koloniuk 1 • Lenka Hraba´kova´ 1,2 • Karel Petrzik 1 Received: 3 November 2014 / Accepted: 30 March 2015 Ó Springer-Verlag Wien 2015 Abstract Beauveria bassiana is a ubiquitous ento- mopathogen infecting hundreds of insect species. We have determined the genomic organization and the complete nucleotide sequence of a novel virus isolated from the isolate A24 of B. bassiana. Phylogenetic analysis of the polymerase gene reveals that the virus, tentatively named Beauveria bassiana virus 1, belongs to the family Amal- gaviridae and represents a distinct lineage of amal- gaviruses infecting fungi. A growing number of viruses have been detected recently in ascomycetous and, less frequently, basidiomycetous (pathogenic as well as industrially cultivated micro- and macrofungi) fungal species. The taxonomic system for these viruses is continuously growing and being refined. Since 2009, a few new viruses with an undivided, relatively small dsRNA genome separately infecting fungal and plant hosts have been described. Due to similarities in their RdRp sequences with those of members of the families Totiviridae and Partitiviridae but significant differences in genome organization, a family named Amalgaviridae has been newly established [1]. Here, we describe a new RNA virus infecting the as- comycete fungus Beauveria bassiana (Balls.-Criv.) Vuill. The virus has been provisionally named Beauveria bassi- ana virus 1 (BbV1-A24). B. bassiana is a ubiquitous en- tomopathogen infecting hundreds of insect species. It is commercially used for microbial control as a mycoinsec- ticide, and its isolates are used for producing a large number of biologically active secondary metabolites [2]. To date, two viruses have been reported to infect B. bassiana cultures. Both of these are members of the family Totiviridae [3, 4]. A series of fungal isolates belonging to Beauveria bassiana sensu lato obtained from the collection of the Faculty of Agriculture at the University of South Bohemia were screened for the presence of dsRNA elements. Selective extraction of dsRNA was carried out as described by Castillo et al. [5]. The isolate A24 was found to harbor a dsRNA element with an approximate molecular size of 3 kbp and was further investigated. A random cDNA li- brary was prepared from the excised band using a Maxima First Strand cDNA Synthesis Kit for RT-qPCR (Fermentas, Lithuania) with 0.5 pmol of dN6 random primer 5 0 - CCTGAATTCGGATCCTCCNNNNNN-3 0 (synthesized by Generi Biotech, Czech Republic) according to a pro- tocol described by Darissa et al. [6]. PCR reactions were carried out using 2x PPP Master Mix (Top-Bio, Czech Republic) in total volume 30 lL with 0.5 lM of 5 0 - CCTGAATTCGGATCCTCC-3 0 SPA primer (Generi Bio- tech, Czech Republic). The products obtained were re- solved on a 2 % agarose gel and stained with SYBR Green I dye. The PCR products were visualized as a smear with sizes of 100 bp to *1 kbp. Fragments larger than 500 bp were excised from the gel, purified using a GeneJET Gel Electronic supplementary material The online version of this article (doi:10.1007/s00705-015-2416-0) contains supplementary material, which is available to authorized users. & Igor Koloniuk koloniuk@umbr.cas.cz 1 Department of Plant Virology, Institute of Plant Molecular Biology, Biology Centre of the Czech Academy of Sciences, v.v.i., Branisˇovska´ 31, 370 05 C ˇ eske´ Budeˇjovice, Czech Republic 2 Department of Genetics, Faculty of Science, University of South Bohemia in C ˇ eske´ Budeˇjovice, Branisˇovska´ 31a, 370 05 C ˇ eske´ Budeˇjovice, Czech Republic 123 Arch Virol DOI 10.1007/s00705-015-2416-0