Complete nucleotide sequences of two isolates of Sunflower chlorotic mottle virus (SuCMoV) obtained from different hosts

BEJERMAN, N.; GIOLITTI, F.; DE BREUIL, S.; LENARDON, S.

Mar del Plata

Conferencia; 18th International Sunflower Conference; 2012

ASAGIR

Sunflower chlorotic mottle virus (SuCMoV), which belongs to the genus Potyvirus, is the most prevalent sunflower virus present in Argentina. This virus has a widespread occurrence in Argentina, where it infects naturally sunflower (Helianthus annuus), Dipsacus fullonum and Ibicella lutea. Two biologically different strains of SuCMoV have been reported on sunflower: the common strain (C) and the chlorotic ringspot strain (CRS) and theirs genomes have been completely sequenced. The objective of this work was to sequence and analyze the complete genomes of two SuCMoV isolates, SuCMoV-dip, obtained from Dipsacus fullonum and SuCMoV-ibi obtained from Ibicella lutea to further characterize SuCMoV´s molecular variability and the genetic structure of its population. Total RNA was extracted from D. fullonum, and I. lutea systematically infected tissues and used as template to amplify the SuCMoV full-length genome. Three SuCMoV-specific pair of primers were designed to generate three overlapping fragments cDNA fragments to cover the full-length genome of SuCMoV-dip and -ibi. Three independent clones were sequenced for each of the three fragments amplified. The full-length genomic sequences of -dip and -ibi was assembled from three overlapping fragments using CAP3 and pairwise nucleotide (nt) and amino acid (aa) comparison were made with the EMBOSS Needle Pairwise Alignment Algorithms with default parameters. Trees were constructed using the maximum likelihood method. Bootstrap analyses with 1000 replicates were performed to evaluate the significance of the interior branches.  The sequence of SuCMoV-dip and SuCMoV-ibi comprise 9953 nucleotides (nt), including both non-translated regions (5´-NCR with 135 nt and 3´-NCR with 257 nt, respectively). Both sequences include a large open reading frame (ORF) starting at position 136. The deduced amino acid (aa) sequences show a polyprotein with 3187 aa. The recently identified small ORF PIPO and the conserved G(1-2)A(6-7) motif associated with its putative frameshift translation were identified in the sequences of both isolates. The genomes of -dip and -ibi isolates are 1 aa longer than the SuCMoV-CRS genome, but 4 aa shorter than SuCMoV-C genome. Indels located in the N-terminal region of P1 determinate the difference in length within SuCMoV´s genome. A total of nine cleavage sites were identified in the polyproteins of both isolates. Those nine sites are completely conserved among the two isolates and the two strains of SuCMoV. The previously identified and highly conserved aa sequence motifs described in potyviruses, were identified in both SuCMoV isolates. Sequence comparison of the deduced aa sequences of isolates dip and ibi with those of SuCMoV´s C and CRS strains, confirmed the identity of the two mentioned isolates as SuCMoV: they display more than 90% sequence identity with SuCMoV-C and CRS for the complete polyprotein. Most of the nt changes occurred at the 3rd codon position and most of them were silent. The most conserved region was the NIb coding region. In contrast, the P1 coding region was the most variable regions. Phylogenetic analysis based on the complete polyprotein aa sequences supported the results from sequence comparison. SuCMoV-dip and -ibi isolates formed a monophyletic cluster with SuCMoV-C and CRS strains. We have determined the complete sequences of the genomes of two SuCMoV isolates from Dipsacus fullonum and Ibicella lutea. Our results demonstrate that there is a significant variation at the 5´-end of the SuCMoV´s genome. The results of this work deepen our understanding of SuCMoV´s variability and evolution, which will be useful to design stables and effective strategies to control its virus.