1 Department of Biological Sciences, Imperial College London, Imperial College Road, London SW7 2AZ, UK
2 Instituto de Biología Molecular y Celular de Plantas (UPV-CSIC), Universidad Politécnica de Valencia, Avenida de los Naranjos s/n, 46022 Valencia, Spain
3 Dipartimento di Arboricoltura, Botanica e Patologia Vegetale, Università di Napoli, 80055 Portici, Italy
4 Dipartimento di Protezione delle Piante e Microbiologia Applicata, Università degli Studi and Istituto di Virologia Vegetale del CNR, Sezione di Bari, 70126 Bari, Italy
5 Tekirdag Ziraat Fakültesi, Trakya Universitesi, 59030 Tekirdag, Turkey
6 Adnan Menderes University, Agricultural Faculty, Plant Pathology Department, 09100 Aydin, Turkey
Correspondence
Ricardo Flores
rflores{at}ibmcp.upv.es
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers of the sequences reported in this paper are AJ781168 and AJ781167 for ACD-associated dsRNAs 1 and 2 and AJ781401 and AJ781402 for CCRS-associated dsRNAs 1 and 2.
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MAIN TEXT |
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Members of the family Partitiviridae (Ghabrial et al., 2004), which together with the Chrysoviridae, Hypoviridae and Totiviridae constitute the four fungal dsRNA virus families (Ghabrial, 2001
; Ghabrial & Castón, 2004
), are isometric, non-enveloped viruses, 3040 nm in diameter, with a genome composed of two linear dsRNAs of approximately 14003000 bp (Ghabrial et al., 2004
). The smaller dsRNA encodes the coat protein (CP) and the larger the virion-associated RNA-dependent RNA polymerase (RdRp) (Ghabrial et al., 2004
). Additional satellite or defective RNAs may also be present. The family Partitiviridae has been divided into four genera, Partitivirus and Chrysovirus for viruses that infect fungi, and Alphacryptovirus and Betacryptovirus for viruses that infect plants (Ghabrial, 2001
). Recently, the genus Chrysovirus was removed from the family Partitiviridae and placed into the new family Chrysoviridae (Ghabrial & Castón, 2004
), which, in addition to Penicillium chrysogenum virus (PcV) (Jiang & Ghabrial, 2004
), the type species, and Helminthosporium victoriae 145S virus (Hv145SV) (Ghabrial & Castón, 2004
), now includes a tentative new species present in dsRNA extracts produced from ACD- and CCRS-affected cherry trees (Covelli et al., 2004
). Most of these viruses cause latent infections and may have originated from totiviruses (Ghabrial, 1998
). We previously suggested that two of the 1012 dsRNAs (of approx. 1800 and 2000 bp) associated with both of the above diseases probably comprised the genome of a partitivirus (see accompanying paper by Covelli et al., 2004
, in this issue) and here we report its molecular characterization.
The dsRNAs were isolated from infected sweet cherry leaves collected in Turkey and Italy exhibiting typical symptoms of ACD and CCRS, respectively, and after separation by electrophoresis on polyacrylamide or agarose gels they were either collectively or individually used for reverse transcription, PCR amplification, cloning and sequencing as described previously (Covelli et al., 2004). cDNA clones of the presumed ACD-associated partitivirus dsRNAs 1 and 2 were obtained by random priming of methyl mercuric hydroxide-denatured dsRNA, with further DNA manipulations being performed as previously described (Sambrook et al., 1989
). For the synthesis of additional cDNAs covering their complete sequence, purified dsRNAs 1 and 2 of the presumed ACD-associated partitivirus were denatured with methyl mercuric hydroxide and subjected to a single-primer, genome-walking RT-PCR protocol developed previously to sequence plant virus RNAs (Livieratos et al., 1999
). An RNA ligase-mediated (RLM)-RACE PCR procedure was used to determine the 5'- and 3'-terminal sequences of the dsRNAs (Coutts & Livieratos, 2003
). The sequences of the equivalent CCRS-associated dsRNAs were obtained by the single-primer method as described previously (Covelli et al., 2004
). Nucleotide and amino acid sequences were manipulated (Deveraux et al., 1984
), aligned using CLUSTAL X (Thompson et al., 1997
) and drawn using the MEGA 2 program (Kumar et al., 2001
).
The complete dsRNA 1 sequences of the presumed ACD- and CCRS-associated partitiviruses were 2006 and 2021 bp, respectively, and each included one ORF potentially encoding a putative protein of 621 aa with a molecular mass of approximately 73 kDa. The two predicted proteins were 98 % identical to one another and contained the conserved motifs characteristic of the RdRps of partitiviruses and dsRNA viruses infecting simple eukaryotes (Bruenn, 1993) (Fig. 1a
). A phylogenetic reconstruction using the fragments containing these conserved motifs from some representative toti-, chryso- and partitiviruses provided corroboration that the presumed ACD- and CCRS-associated partitiviruses clustered together with members of this group (Covelli et al., 2004
). To delimit more precisely the relationships within the genus Partitivirus, a phylogenetic tree was constructed based on multiple alignment of the fragments containing the RdRp conserved motifs from all species of this genus deposited in databases using Beet cryptic virus (BCV-3) as an outgroup. The tree showed that ACD- and CCRS-associated partitiviruses formed a subgroup with the Helicobasidium mompa virus strain V70 (HmV-V70) (Osaki et al., 2002
), the Heterobasidion annosum virus P-type (HaV-Pt) (Ihrmark, 2001
), the Atkinsonella hypoxylon virus isolate 2H (AhV-2H) (Oh & Hillman, 1995
), the Fusarium poae virus 1 (FpV-1) (Compel et al., 1999
) and the Rhizoctonia solani virus 717 (RhsV-717) (Strauss et al., 2000
), with the remaining five members of the genus forming a second subgroup (Fig. 1b
).
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In summary, we have characterized the two dsRNA components of the ACD- and CCRS-associated partitiviruses. The phylogenetic analysis derived from the RdRp potentially encoded by dsRNA 1 of the known partitiviruses was essentially coincidental with the current taxonomy of the genus (Ghabrial et al., 2004) and located the ACD- and CCRS-associated partitiviruses in one specific subgroup. Interestingly, a parallel analysis based on the more limited data available for the putative CP potentially encoded by dsRNA 2 also led to the same subgroups, thus providing additional support for the present classification scheme. Within this scheme, ACD- and CCRS-associated partitiviruses clustered in the same subgroup with a predominance of dsRNA viruses that infect basidiomycetous fungi (HmV-V70, HaV-Pt and RhsV-717). What significance in evolutionary terms this has necessitates the isolation and molecular characterization of further partitiviruses from a variety of fungal species in the future.
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ACKNOWLEDGEMENTS |
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Received 8 April 2004;
accepted 19 July 2004.
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