Complete genome sequence of ornithogalum virus 3.

Ornithogalum thyrsoides, a widely cultivated bulbous ornamental plant endemic to South Africa, has significant commercial value as a pot plant and for the production of cut flowers. However, infection by viruses threatens the success of commercial cultivation, as symptoms negatively affect the appearance of the plant and flowers. To date, four Ornithogalum-infecting viruses have been reported. Complete genome sequence data are available for three of these viruses, but the genome of the potyvirus ornithogalum virus 3 (OV3) has not been fully sequenced. In this study, the complete sequence of OV3 was determined by high-throughput sequencing (HTS) and validated by Sanger sequencing. Based on recognition of protease cleavage patterns and multiple sequence alignments with closely related viruses, the polyprotein of OV3 was predicted to be proteolytically cleaved to produce 10 mature peptides containing domains conserved in members of the genus Potyvirus. Phylogenetic analysis and species demarcation criteria confirm the previous classification of OV3 as a member of a separate species in this genus. This is the first report of a complete genome sequence of OV3.

Complete genome sequence of a novel polerovirus in Ornithogalum thyrsoides from South Africa.

Ornithogalum thyrsoides, commonly known as chincherinchee, is an indigenous ornamental plant widely cultivated in South Africa. It is commercially valued as a flowering pot plant and for the production of cut flowers. Virus infections resulting in the development of severe necrotic mosaic symptoms threaten the success of commercial cultivation. The virome of an O. thyrsoides plant displaying necrotic mosaic symptoms was determined using high-throughput sequencing (HTS). In this plant, ornithogalum mosaic virus and ornithogalum virus 3 were identified, as well as a previously unknown virus. The full genome sequence of this virus was confirmed by Sanger sequencing using overlapping amplicons combined with rapid amplification of cDNA ends (RACE). Based on genome organisation and phylogenetic analysis, this novel virus can be classified as a polerovirus.

Status of the current vitivirus taxonomy.

Since the establishment of the genus Vitivirus, several additional viruses have been sequenced and proposed to represent new species of this genus. Currently, the International Committee on Taxonomy of Viruses recognizes 15 vitivirus species. The report of new vitiviruses that fail to completely adhere to the species demarcation criteria, the incorporation of non-vitivirus grapevine viruses in the unofficial "naming system", and the existence of non-grapevine vitiviruses lead to inconsistencies in classification. In this report, we give a brief overview of vitiviruses and use currently available information to clarify the present status of the vitivirus taxonomy.

In Silico Probe-Based Detection of Citrus Viruses in NGS Data.

The conservation of plant biosecurity relies on the rapid identification of pathogenic organisms, including viruses. With next-generation sequencing (NGS), it is possible to identify multiple viruses within a metagenomic sample. In this study, we explored the use of electronic probes (e-probes) for the simultaneous detection of 11 recognized citrus viruses. E-probes were designed and screened against raw sequencing data to minimize the bioinformatic processing time required. The e-probes were able to accurately detect their cognate viruses in simulated datasets, without any false negatives or positives. The efficiency of the e-probe-based approach was validated with NGS datasets generated from different RNA preparations: double-stranded RNA (dsRNA) from 'Mexican' lime infected with different Citrus tristeza virus (CTV) genotypes, dsRNA from field samples, and small RNA and total RNA from grapefruit infected with the CTV T3 genotype. A set of probes was made available that is able to accurately detect CTV in sequence data regardless of the input dataset or the genotype that plants are infected with.

Relative quantitation goes viral: An RT-qPCR assay for a grapevine virus.

Accurate detection and quantitation of viruses can be beneficial to plant-virus interaction studies. In this study, three SYBR green real-time RT-PCR assays were developed to quantitate grapevine leafroll-associated virus 3 (GLRaV-3) in infected vines. Three genomic regions (ORF1a, coat protein and 3'UTR) were targeted to quantitate GLRaV-3 relative to three stably expressed reference genes (actin, GAPDH and α-tubulin). These assays were able to detect all known variant groups of GLRaV-3, including the divergent group VI, with equal efficiency. No link could be established between the concentration ratios of the different genomic regions and subgenomic RNA (sgRNA) expression. However, a significant lower virus concentration ratio for plants infected with variant group VI compared to variant group II was observed for the ORF1a, coat protein and the 3'UTR. Significant higher accumulation of the virus in the growth tip was also detected for both variant groups. The quantitation of viral genomic regions under different conditions can contribute to elucidating disease aetiology and enhance knowledge about virus ecology.

Complete nucleotide sequence of a new strain of grapevine leafroll-associated virus 3 in South Africa.

The complete genome sequences of two newly identified genetic variants of grapevine leafroll-associated virus 3 (GLRaV-3) are described. Two isolates, GH11 (18671 nt) and GH30 (18576 nt) were sequenced and found to be less than 70 % similar to other South African GLRaV-3 variants. The genome organization of GH11 and GH30 is similar to that of previously described GLRaV-3 isolates. However, no corresponding open reading frame 2 (ORF2) could be identified. Phylogenetic analysis indicates that GH11 and GH30 cluster in a subgroup (group VI) that is separate from the previously described GLRaV-3 isolates and should be regarded as a different strain of GLRaV-3.

Three genetic grapevine leafroll-associated virus 3 variants identified from South African vineyards show high variability in their 5'UTR.

Three genetic variants of grapevine leafroll-associated virus 3 (GLRaV-3) were identified in vineyards of the Western Cape, South Africa. The GLRaV-3 variants were identified by single-strand conformation polymorphism (SSCP) profiles generated from a region amplified in ORF5. ORF5 sequence data confirmed the three genetic variant groups, and a specific SSCP profile was assigned to each variant group. The results of SSCP analysis of this region in ORF5 showed that this method gives a fast and reliable indication of the GLRaV-3 variant status of a plant, which in many instances showed mixed infections. The full genome sequence of one representative of each variant group i.e. isolates 621 (group I), 623 (group II) and PL-20 (group III), was determined by sequencing overlapping cloned fragments of these isolates. The sequences of genomic 5' ends of these isolates were determined by RLM-RACE. Sequence alignment of the 5'UTRs indicated significant sequence and length variation in this region between the three South African variant groups. Alignment of the Hsp70h and CP gene regions of these isolates with those of isolates from elsewhere in the world, followed by phylogenetic analysis, further supported the presence of three variants of GLRaV-3 in South Africa and the presence of two or three additional variant groups elsewhere in the world.