Virus Research 140 (2009) 179–187 Contents lists available at ScienceDirect Virus Research journal homepage: www.elsevier.com/locate/virusres A novel mycovirus associated with four double-stranded RNAs affects host fungal growth in Alternaria alternata  Nanako Aoki a , Hiromitsu Moriyama a,∗ , Motoichiro Kodama c , Tsutomu Arie b , Tohru Teraoka b , Toshiyuki Fukuhara a a Laboratories of Molecular and Cellular Biology, Department of Applied Biological Sciences, Tokyo University of Agriculture and Technology, 3-5-8 Saiwaicho, Fuchu, Tokyo 183-8509, Japan b Laboratory of Plant Pathology, Department of Applied Biological Sciences, Tokyo University of Agriculture and Technology, 3-5-8 Saiwaicho, Fuchu, Tokyo 183-8509, Japan c Laboratory of Plant Pathology, Faculty of Agriculture, Tottori University, 4-101 Koyama-Minami, Tottori 680-8553, Japan article info Article history: Received 13 July 2008 Received in revised form 30 November 2008 Accepted 4 December 2008 Available online 15 January 2009 Keywords: Mycovirus dsRNA elements Alternaria alternata RNA-dependent RNA polymerase Virus particles abstract Four double-stranded RNAs (dsRNAs), referred to as dsRNA 1 (3617bp), dsRNA 2 (2794bp), dsRNA 3 (2576bp) and dsRNA 4 (1420bp), were detected in the EGS 35-193 strain of Alternaria alternata at high concentration (∼3 g/g dried mycelium). This strain had an impaired growth phenotype. By exposing the strain to cycloheximide during hyphal tip isolation, we isolated strains which had normal mycelial growth and pigmentation, in which decreased levels of the dsRNAs were observed (∼0.3 g/g dried mycelium). These results indicate that this dsRNA mycovirus might be involved in modulating traits of its fungal host, A. alternata. The buoyant density of isometric virus particles (about 33 nm in diameter) containing these dsRNAs in CsCl was 1.35–1.40g/cm 3 depending on the size of the packaged dsRNAs. The dsRNA 1 encodes a single open reading frame (3447nt) containing the conserved motifs of viral RNA-dependent RNA polymerase (RdRp), which is related to the ORF encoded by dsRNA 1 of Aspergillus mycovirus 341. It is noteworthy that all of the coding strands of the four dsRNA genomes have 3 ′ -poly (A) tails ranging from 33 to 50nt in length. We named this novel dsRNA mycovirus in the EGS 35-193 strain A. alternata virus-1 (AaV-1). © 2008 Elsevier B.V. All rights reserved. 1. Introduction In simple eukaryotic cells, such as protozoa and fungi, dsRNA viruses are common infectious agents. The dsRNA components that are found in filamentous fungi are known as mycoviruses (Ghabrial, 1998). Although mycoviruses are usually latent in nature, and their biological manifestations can be thought of as cyto- plasmically inherited determinants rather than as viruses, several mycoviruses can affect the phenotype of host growth. In the yeast Saccharomyces cerevisiae, a lethal phenotype is caused by infection with a cytoplasmic-persisting dsRNA virus in the Totiviridae family, called Saccharomyces cerevisiae virus L-A (Wickner et al., 2005). In plant pathogenic fungi, several mycoviruses reduce host vir- ulence, causing distinct morphological and physiological changes, including toxin production (Magliani et al., 1997), cytologi- cal alteration of cellular organelles (Newhouse et al., 1983), and virulence-associated traits such as altered growth rate  The GenBank/ENBL/DDBJ accession numbers of the sequences reported in this paper are AB368492 and AB438027 through AB438029. ∗ Corresponding author. Tel.: +81 42 367 5622; fax: +81 42 360 8830. E-mail address: hmori714@cc.tuat.ac.jp (H. Moriyama). (Boland, 1992), sporulation (Bottacin et al., 1994), pigmentation (Anagnostakis and Day, 1979), and enzymatic activities (Rigling and Van Alfen, 1993). A cytoplasmically controlled degenerative disease of Rhizoctonia solani has been reported (Castanho and Butler, 1978), in which particular dsRNA elements might up- or down-regulate virulence in R. solani (Tavantzis et al., 2002). A well-known example of virus-mediated modulation of fungal virulence is the chest- nut blight fungus Cryphonectria parastica infected by the dsRNA mycovirus CHV1-EP713, which is classified as a member of the Hypoviridae family (Nuss, 2005). Another group of mycoviruses in the Mycoreovirus genus of the Reoviridae family has been detected in C. parastica, and also affects the host phenotype (Hillman et al., 2004). In the white root rot fungus, Rosellinia necatrix, two types of mycoviruses, which are classified in the Mycoreovirus genus and the Partitivirus genus, have been detected (Kanematsu et al., 2004; Sasaki et al., 2006). Both of these mycoviruses are associated with the hypovirulence phenomenon. A large dsRNA in Helicobasidium monpa strain V670 of the violet root rot fungus has been identified as a hypovirulence factor and assigned to the genus Endornavirus (Osaki et al., 2006). In black Aspergillus, 1 out of 64 mycovirus infected strains showed an abnormal phenotype (Diepeningen et al., 2006). Penicillium chrysogenum viruses used to be classified with the family Partitiviridae; however, these viruses are now defined 0168-1702/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2008.12.003