Dominique Colinet

Identification of a sweet potato feathery mottle virus isolate from China (SPFMV-CH) by the polymerase chain reaction with degenerate primers

Four degenerate oligonucleotide primers derived from conserved regions of the genome of potyviruses have been designed. A combined assay of reverse transcription and the polymerase chain reaction utilizing these primers on total RNA extracted from Ipomoea purpurea infected with a sweet potato feathery mottle virus isolate from China (SPFMV-CH), amplified a 1.35 kb and a 830 bp fragment. These amplified fragments were cloned into the Bluescript plasmid and sequenced. The comparison of the sequence of the N-terminal part of the coat protein of SPFMV-CH with those published for two other strains of SPFMV, showed a strong relationship between SPMV-CH and -RC.

Molecular Evidence That The Whitefly-Transmitted Sweetpotato Mild Mottle Virus Belongs To A Distinct Genus Of The Potyviridae

SummaryComplementary DNA representing 2 108 nucleotides at the 3′ end of the genomic RNA of the whitefly-transmitted sweetpotato mild mottle virus (SPMMV) was cloned after PCR. Sequence analysis revealed an open reading frame of 1 797 nucleotides which codes for a protein of 599 amino acids, followed by a 3′ non-coding region of 311 nucleotides. Alignment of the deduced amino acid sequence with corresponding sequences of other members of thePotyviridae demonstrated that part of the presumptive RNA-dependent RNA polymerase and the coat protein coding regions of SPMMV are found at the 3′ end of its genome, in that order. Alignment of the amino acid sequence of the core of SPMMV coat protein with those of selected members of thePotyviridae showed limited identity, thus demonstrating — with phylogenetic analysis — that SPMMV belongs to a distinct genus of the familyPotyviridae.

The complete nucleotide sequences of the coat protein cistron and the 3' non-coding region of a newly-identified potyvirus infecting sweetpotato, as compared to those of sweetpotato feathery mottle virus.

SummaryComplementary DNA representing 728 nucleotides of the 3′ end of the genomic RNA of sweetpotato virus G (SPV-G), a newly-identified potyvirus infecting sweetpotato, was cloned and sequenced. This sequence was combined with that previously determined for the 5′ terminal part of the coat protein cistron of the virus. The whole sequence contained a single open reading frame (ORF) of 1065 nucleotides, with the capacity to encode a coat protein of 355 amino acids, significantly larger than that of other potyviruses. The ORF was followed by an untranslated region of 222 nucleotides and a poly (A) tail. The coat protein of SPV-G was only distantly related to that of known potyviruses, with the exception of sweetpotato feathery mottle virus (SPFMV). Indeed, sequence identity in the C-terminal three quarters of the coat protein (more than 80%) and in the 3′ untranslated region (more than 70%) indicate that SPV-G should be considered as closely related to, though distinct from SPFMV. This subset relationship is similar to that previously reported for members of the bean yellow mosaic virus subgroup or the bean common mosaic virus subgroup.

Detection of Apple Stem Grooving Virus in Dormant Apple Trees with Crude Extracts as Templates for One-Step RT-PCR

Partial nucleotide sequences of amplification products obtained from four European apple stem grooving virus (ASGV) isolates using degenerate primers showed 80 to 85% similarity with the published ASGV sequence of a Japanese strain but 98 to 100% identities among themselves. Based on these sequences, two ASGV-specific primers (ASGV4F-ASGV4R) were designed to amplify a 574-bp fragment located in the putative viral RNA polymerase. With these primers, six European and five American ASGV isolates, maintained in herbaceous hosts (Chenopodium quinoa, Nicotiana glutinosa, and N. occidentalis) or in apple trees, were readily detected by reverse transcription-polymerase chain reaction (RT-PCR). Using these specific ASGV primers, dsRNA preparations have been shown to constitute good templates for reliable amplification of ASGV sequences from leaves and bark tissues of apple trees, both in a two-step RT-PCR protocol and in the one-step Titan One-Tube RT-PCR. System. Furthermore, the one-step RT-PCR system allowed a specific amplification of ASGV sequences directly from clarified crude extracts of leaves and bark tissues of apple trees during both active growth and the dormant season.

Determination of the taxonomic position and characterization of yam mosaic virus isolates based on sequence data of the 5′-terminal part of the coat protein cistron

SummaryThe sequences of the N-terminal part of the coat protein cistron from six isolates of yam mosaic virus (YMV-TOG, YMV-COT, YMV-12, YMV-CAR, YMV-BU1 and YMV-BU2) were determined. The analysis of the deduced amino acid sequences revealed the presence of consensus motifs characteristic of the potyvirus genus supporting the classification of YMV as a potyvirus member. The alignment of the N-terminal part of the coat protein of YMV-TOG, YMV-COT, YMV-12 and YMV-CAR showed that they were identical in size (152 aa) while YMV-BU1 and YMV-BU2 were shorter (140 aa) due to a deletion of 12 aa. These amino acid sequences exhibit an overall sequence identity ranging from 70.4% to 97.4% while the identity level with the other potyviruses sequenced in the considered region is below 50%, confirming that YMV is a distinct member of the potyvirus genus. The detailed analysis of the amino acid sequence alignment and of the identity levels observed between the N-terminal part of the coat protein of the six YMV isolates lead us to suggest that they have to be considered as distantly related strains of YMV rather than closely related but distinct viruses.