Characterization of badnaviruses infecting Dioscorea spp. in the Pacific reveals two putative novel species and the first report of dioscorea bacilliform RT virus 2

Abstract

The complete genome sequences of three new badnaviruses associated with yam (Dioscorea spp.) originating from Fiji, Papua New Guinea and Samoa were determined following rolling circle amplification of the virus genomes. The full-length genomes consisted of a single molecule of circular double-stranded DNA of 8106bp for isolate FJ14, 7871bp for isolate PNG10 and 7426bp for isolate SAM01. FJ14 and PNG10 contained three open reading frames while SAM01 had an additional open reading frame which partially overlapped the 3' end of ORF 3. Amino acid sequence analysis of ORF 3 from the three isolates confirmed the presence of conserved motifs typical of other badnaviruses. Phylogenetic analysis revealed the sequences to be closely related to other Dioscorea-infecting badnaviruses. FJ14 and PNG10 appear to be new species, which we have tentatively named dioscorea bacilliform ES virus (DBESV) and dioscorea bacilliform AL virus 2 (DBALV2), respectively, while SAM01 represents a Pacific isolate of the recently published dioscorea bacilliform RT virus 2 and is described as dioscorea bacilliform RT virus 2-[4RT] (DBRTV2-[4RT]).

Rolling Circle Amplification to Screen Yam Germplasm for Badnavirus Infections and to Amplify and Characterise Novel Badnavirus Genomes

“…germplasm in the 1970s (Harrison and Roberts, 1973), several hundred partial badnavirus reverse transcriptase (RT)-ribonuclease H (RNaseH) sequences have been characterised (Kenyon et al, 2008;Bousalem et al, 2009), but only a few complete Dioscorea bacilliform virus (DBV) genome sequences have been reported (Phillips et al, 1999;Seal and Muller, 2007;Bömer et al, 2016 andSukal et al, 2017;Umber et al, 2017). We have optimised a workflow involving total nucleic acid extractions and rolling circle amplification (RCA) combined with restriction enzyme analysis for the detection and amplification of DBVs present in yam germplasm.…”

Characterization and genetic diversity of Dioscorea bacilliform viruses present in a Pacific yam germplasm collection

“…esculenta accession from Fiji (DE/FJ14; Sukal et al . , ), this virus was not detected in any additional D. esculenta accessions, or accessions of other yam species from either Fiji or any other countries included in the present study. Previously, Kenyon et al .…”

“…Digest products were electrophoresed through 1.5% agarose gels, stained with SYBR Safe (Thermo Fisher Scientific) and fragments of interest were excised, purified and cloned as described in Sukal et al . (). Sequencing was carried out using BadnaFP/RP primers (Yang et al .…”

“…At present, seven distinct DBV species have been taxonomically accepted by the ICTV, namely Dioscorea bacilliform AL virus (DBALV), Dioscorea bacilliform AL virus 2 (DBALV2), Dioscorea bacilliform ES virus (DBESV), Dioscorea bacilliform RT virus 1 (DBRTV1), Dioscorea bacilliform RT virus 2 (DBRTV2), Dioscorea bacilliform SN virus and Dioscorea bacilliform TR virus (Adams and Carstens, ; Sukal et al . , ; Adams et al . , ).…”

“…Sukal et al . () identified three DBVs, namely DBALV2, DBESV, and DBRTV2, in the Pacific germplasm collection at CePaCT using a rolling circle amplification (RCA)‐based strategy. In this study, RCA was used to identify and further characterize the molecular diversity of badnaviruses infecting the Pacific yam germplasm collection.…”