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Characterization of New Isolates of Apricot vein clearing-associated virus and of a New Prunus-Infecting Virus: Evidence for Recombination as a Driving Force in Betaflexiviridae Evolution.

Marais A, Faure C, Mustafayev E, Candresse T - PLoS ONE (2015)

Bottom Line: Phylogenetic relationships and nucleotide comparisons suggested that together with AVCaV, CPrV could define a new genus (proposed name: Prunevirus) in the family Betaflexiviridae.A molecular test targeting both members of the new genus was developed, allowing the detection of additional AVCaV isolates, and therefore extending the known geographical distribution and the host range of AVCaV.The sequences reported in the present manuscript have been deposited in the GenBank database under accession numbers KM507061-KM504070.

View Article: PubMed Central - PubMed

Affiliation: INRA, UMR 1332 BFP, Villenave d'Ornon, France; Université de Bordeaux, UMR 1332 BFP, Villenave d'Ornon, France.

ABSTRACT
Double stranded RNAs from Prunus samples gathered from various surveys were analyzed by a deep-sequencing approach. Contig annotations revealed the presence of a potential new viral species in an Azerbaijani almond tree (Prunus amygdalus) and its genome sequence was completed. Its genomic organization is similar to that of the recently described Apricot vein clearing associated virus (AVCaV) for which two new isolates were also characterized, in a similar fashion, from two Japanese plums (Prunus salicina) from a French germplasm collection. The amino acid identity values between the four proteins encoded by the genome of the new virus have identity levels with those of AVCaV which fall clearly outside the species demarcation criteria. The new virus should therefore be considered as a new species for which the name of Caucasus prunus virus (CPrV) has been proposed. Phylogenetic relationships and nucleotide comparisons suggested that together with AVCaV, CPrV could define a new genus (proposed name: Prunevirus) in the family Betaflexiviridae. A molecular test targeting both members of the new genus was developed, allowing the detection of additional AVCaV isolates, and therefore extending the known geographical distribution and the host range of AVCaV. Moreover, the phylogenetic trees reconstructed with the amino acid sequences of replicase, movement and coat proteins of representative Betaflexiviridae members suggest that Citrus leaf blotch virus (CLBV, type member of the genus Citrivirus) may have evolved from a recombination event involving a Prunevirus, further highlighting the importance of recombination as a driving force in Betaflexiviridae evolution. The sequences reported in the present manuscript have been deposited in the GenBank database under accession numbers KM507061-KM504070.

No MeSH data available.


Related in: MedlinePlus

Schematic representation of the genomes of Apricot vein clearing associated virus (AVCaV, reference isolate and Pair isolate), Caucasus prunus virus (Aze204 agent) and Citrus leaf blotch virus (CLBV).The size in amino acids of the encoded proteins is indicated between brackets. The four genomes are aligned on the basis of the C-ter of the polymerase. The hatched boxes represent parts of the Pol and MP genes which are either absent or not expressed, respectively, in the AVCaV reference isolate but present and expressed in the Iran1, Pair and 13025 isolates. The black arrows represent the amplified fragments using the two primer pairs Pol-F1/R1 and NB-F1/R1. MP, Movement protein; CP, Coat protein; NB, Nucleic acid binding protein.
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pone.0129469.g001: Schematic representation of the genomes of Apricot vein clearing associated virus (AVCaV, reference isolate and Pair isolate), Caucasus prunus virus (Aze204 agent) and Citrus leaf blotch virus (CLBV).The size in amino acids of the encoded proteins is indicated between brackets. The four genomes are aligned on the basis of the C-ter of the polymerase. The hatched boxes represent parts of the Pol and MP genes which are either absent or not expressed, respectively, in the AVCaV reference isolate but present and expressed in the Iran1, Pair and 13025 isolates. The black arrows represent the amplified fragments using the two primer pairs Pol-F1/R1 and NB-F1/R1. MP, Movement protein; CP, Coat protein; NB, Nucleic acid binding protein.

Mentions: Complete genome sequences of three AVCaV isolates and one partial (3,037 nt 3' terminal) are now available. Both complete AVCaV sequences determined here are 8,358 nt long, significantly longer than the reference isolate (7,315 nt). Their overall genomic organization and gene order are conserved as all encode four proteins from 5' to 3' (Fig 1): a replication-associated protein (Pol), a movement protein (MP), a coat protein (CP) and a nucleic acid binding protein (NB). The CP is the only protein conserved in size (221 amino acids [aa]) between the four AVCaV isolates (S2 Table). The Pol of the 13025 and Pair isolates contains an additional 342 aa inserted at aa position 405 of the Pol reference isolate, so that the size of their protein is 2,021 aa instead of 1,679 for the previously sequenced isolate [21]. The five domains methyltransferase (MeT), AlkB, endopeptidase, helicase and RNA-dependent RNA polymerase are conserved, with the large indel positioned between the MeT and AlkB domains, in the hypervariable part of the Betaflexiviridae Pol protein. Interestingly, in the 13025 and Pair isolates, the indel is bordered by the duplicated heptanucleotide sequence GCAACTT. The MP of the reference isolate is also significantly shorter (292 aa) due to a frameshift mutation leading to premature MP ORF termination as compared to the other three isolates, which have a MP of 460 aa. Three of the four AVCaV isolates encode a NB of 139 aa, but that of the Iran1 isolate is 23 aa longer due to a point mutation (T to C) at nucleotide 419 in the NB gene, which suppresses the first stop codon at the end of the ORF. The large indel in the Pol gene and the mutations affecting the length of the various ORFs were all confirmed by targeted sequencing of the relevant isolates. The 5' non coding region (NCR) of the Pair and 13025 isolates is 78 nt long, five nt longer than that of the reference isolate, due to indels. The 3' NCR is also variable is size, being 152 nt long in the Pair and 13025 isolates (13 nt longer at the 3' end than in the reference isolate) but only 84 nt long in the Iran1 isolate, due to the extension of the NB gene to the second in frame stop codon. Overall, and when not taking into account the large indel in the Pol gene, the three complete genomes show a very high level of nucleotide identity (96%-96.8%). As shown in S2 Table, the same applies when considering the different genome regions (and the fourth, partially sequenced isolate), with the exception of the 5' NCR, which is highly divergent in the reference isolate and shows only about 58% identity with that of the other two isolates.


Characterization of New Isolates of Apricot vein clearing-associated virus and of a New Prunus-Infecting Virus: Evidence for Recombination as a Driving Force in Betaflexiviridae Evolution.

Marais A, Faure C, Mustafayev E, Candresse T - PLoS ONE (2015)

Schematic representation of the genomes of Apricot vein clearing associated virus (AVCaV, reference isolate and Pair isolate), Caucasus prunus virus (Aze204 agent) and Citrus leaf blotch virus (CLBV).The size in amino acids of the encoded proteins is indicated between brackets. The four genomes are aligned on the basis of the C-ter of the polymerase. The hatched boxes represent parts of the Pol and MP genes which are either absent or not expressed, respectively, in the AVCaV reference isolate but present and expressed in the Iran1, Pair and 13025 isolates. The black arrows represent the amplified fragments using the two primer pairs Pol-F1/R1 and NB-F1/R1. MP, Movement protein; CP, Coat protein; NB, Nucleic acid binding protein.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4472227&req=5

pone.0129469.g001: Schematic representation of the genomes of Apricot vein clearing associated virus (AVCaV, reference isolate and Pair isolate), Caucasus prunus virus (Aze204 agent) and Citrus leaf blotch virus (CLBV).The size in amino acids of the encoded proteins is indicated between brackets. The four genomes are aligned on the basis of the C-ter of the polymerase. The hatched boxes represent parts of the Pol and MP genes which are either absent or not expressed, respectively, in the AVCaV reference isolate but present and expressed in the Iran1, Pair and 13025 isolates. The black arrows represent the amplified fragments using the two primer pairs Pol-F1/R1 and NB-F1/R1. MP, Movement protein; CP, Coat protein; NB, Nucleic acid binding protein.
Mentions: Complete genome sequences of three AVCaV isolates and one partial (3,037 nt 3' terminal) are now available. Both complete AVCaV sequences determined here are 8,358 nt long, significantly longer than the reference isolate (7,315 nt). Their overall genomic organization and gene order are conserved as all encode four proteins from 5' to 3' (Fig 1): a replication-associated protein (Pol), a movement protein (MP), a coat protein (CP) and a nucleic acid binding protein (NB). The CP is the only protein conserved in size (221 amino acids [aa]) between the four AVCaV isolates (S2 Table). The Pol of the 13025 and Pair isolates contains an additional 342 aa inserted at aa position 405 of the Pol reference isolate, so that the size of their protein is 2,021 aa instead of 1,679 for the previously sequenced isolate [21]. The five domains methyltransferase (MeT), AlkB, endopeptidase, helicase and RNA-dependent RNA polymerase are conserved, with the large indel positioned between the MeT and AlkB domains, in the hypervariable part of the Betaflexiviridae Pol protein. Interestingly, in the 13025 and Pair isolates, the indel is bordered by the duplicated heptanucleotide sequence GCAACTT. The MP of the reference isolate is also significantly shorter (292 aa) due to a frameshift mutation leading to premature MP ORF termination as compared to the other three isolates, which have a MP of 460 aa. Three of the four AVCaV isolates encode a NB of 139 aa, but that of the Iran1 isolate is 23 aa longer due to a point mutation (T to C) at nucleotide 419 in the NB gene, which suppresses the first stop codon at the end of the ORF. The large indel in the Pol gene and the mutations affecting the length of the various ORFs were all confirmed by targeted sequencing of the relevant isolates. The 5' non coding region (NCR) of the Pair and 13025 isolates is 78 nt long, five nt longer than that of the reference isolate, due to indels. The 3' NCR is also variable is size, being 152 nt long in the Pair and 13025 isolates (13 nt longer at the 3' end than in the reference isolate) but only 84 nt long in the Iran1 isolate, due to the extension of the NB gene to the second in frame stop codon. Overall, and when not taking into account the large indel in the Pol gene, the three complete genomes show a very high level of nucleotide identity (96%-96.8%). As shown in S2 Table, the same applies when considering the different genome regions (and the fourth, partially sequenced isolate), with the exception of the 5' NCR, which is highly divergent in the reference isolate and shows only about 58% identity with that of the other two isolates.

Bottom Line: Phylogenetic relationships and nucleotide comparisons suggested that together with AVCaV, CPrV could define a new genus (proposed name: Prunevirus) in the family Betaflexiviridae.A molecular test targeting both members of the new genus was developed, allowing the detection of additional AVCaV isolates, and therefore extending the known geographical distribution and the host range of AVCaV.The sequences reported in the present manuscript have been deposited in the GenBank database under accession numbers KM507061-KM504070.

View Article: PubMed Central - PubMed

Affiliation: INRA, UMR 1332 BFP, Villenave d'Ornon, France; Université de Bordeaux, UMR 1332 BFP, Villenave d'Ornon, France.

ABSTRACT
Double stranded RNAs from Prunus samples gathered from various surveys were analyzed by a deep-sequencing approach. Contig annotations revealed the presence of a potential new viral species in an Azerbaijani almond tree (Prunus amygdalus) and its genome sequence was completed. Its genomic organization is similar to that of the recently described Apricot vein clearing associated virus (AVCaV) for which two new isolates were also characterized, in a similar fashion, from two Japanese plums (Prunus salicina) from a French germplasm collection. The amino acid identity values between the four proteins encoded by the genome of the new virus have identity levels with those of AVCaV which fall clearly outside the species demarcation criteria. The new virus should therefore be considered as a new species for which the name of Caucasus prunus virus (CPrV) has been proposed. Phylogenetic relationships and nucleotide comparisons suggested that together with AVCaV, CPrV could define a new genus (proposed name: Prunevirus) in the family Betaflexiviridae. A molecular test targeting both members of the new genus was developed, allowing the detection of additional AVCaV isolates, and therefore extending the known geographical distribution and the host range of AVCaV. Moreover, the phylogenetic trees reconstructed with the amino acid sequences of replicase, movement and coat proteins of representative Betaflexiviridae members suggest that Citrus leaf blotch virus (CLBV, type member of the genus Citrivirus) may have evolved from a recombination event involving a Prunevirus, further highlighting the importance of recombination as a driving force in Betaflexiviridae evolution. The sequences reported in the present manuscript have been deposited in the GenBank database under accession numbers KM507061-KM504070.

No MeSH data available.


Related in: MedlinePlus