Comparison of low-density arrays, RT-PCR and real-time TaqMan RT-PCR in detection of grapevine viruses

Low-density arrays (LDA) have been designed based on the real-time RT-PCR (TaqMan) assays for the specific detection of 13 viruses that infect Grapevines in addition to the housekeeping gene 18S rRNA. The viruses included in the study are Grapevine leafroll associated viruses 1, 2, 3, 4, 5, and 9, Grapevine leafroll associated virus-2 Redglobe (GLRaV-2RG) strain, Ruspestris stem pitting associated virus, Grapevine vitivirus A, Grapevine vitivirus B, Grapevine fanleaf virus, Tomato ringspot virus (ToRSV), and Grapevine fleck virus (GFkV). This study includes three new TaqMan RT-PCR assays that have been developed for GLRaV-2RG, GFkV and ToRSV and have been included in the TaqMan RT-PCR and LDA detection. The LDAs were evaluated against a wide range of isolates distributed geographically. Geographical locations included Africa, Europe, Australia, Asia, Latin America and the United States. High-throughput detection of these viruses using LDAs was compared to RT-PCR and real-time TaqMan RT-PCR. The efficiency of different RNA extraction methodologies and buffers were compared for use in low-density array detection. In addition improving the RNA extraction technique and testing the quality of the RNA using the 18S ribosomal RNA TaqMan assay as an RNA specific internal control proved to generate better diagnostic assays. This is the first report on the use of LDA for the detection of plant viruses.     

A sensitive one-step real-time RT-PCR method for detecting Grapevine leafroll-associated virus 2 variants in grapevine

Grapevine leafroll syndrome is caused by a complex of up to nine different Grapevine leafroll-associated viruses (GLRaV-1-9) with GLRaV-2 being reported as one of the most variable species of this group. Many methods, including indexing, serological and molecular procedures, have been developed for the detection of GLRaV-2. However, due to the low concentration of the virus in plants and the high variability of GLRaV-2, a method with improved sensitivity and with the capacity to detect of all known variants is required. Such improvement is essential for grapevine rootstocks, as these are suspected to harbour frequent GLRaV-2 infections difficult to detect, thus contributing to the spread of the leafroll disease. The development of new universal primers is described using a target sequence located in the 3' end of the virus genome. These primers were combined with a one-step SYBR Green real-time RT-PCR assay to achieve quantitative detection. All 43 GLRaV-2 isolates tested in this study were identified readily and reproducibly, regardless of their geographical origin or variety of grapevine. Using the procedure developed in this study, the sensitivity was increased 125 times compared to a conventional single-tube RT-PCR. This real-time method opens new perspectives for the sanitary selection of grapevine and in leafroll 2 disease monitoring.     

Comparison of three techniques for detection of grapevine leafroll-associated virus 1

Thirty seven plants of grapevine from the Research Station of Viticulture, Karlstejn was examined for the presence of leafroll viruses. Grapevine leafroll-associated virus 1 (GLRaV-1) was detected in the grapevines plants tested using double-antibody sandwich ELISA (DAS-ELISA), RT-PCR and molecular hybridization with non-radioactive RNA probes. Both molecular methods were based on a detection of the GLRaV-1 heat-shock protein 70 (HSP70) gene and showed a higher sensitivity in the detection of GLRaV-1 compared to DAS-ELISA. RNA probes are considered more suitable for the GLRaV-1 detection, as their application can overcome potential minor sequence variability, which may cause the detection by RT-PCR less reliable, especially when the variability occurs in the genome region targeted by RT-PCR primers. Based on additional DAS-ELISA, a mixed infection of GLRaV-1 and Grapevine leafroll-associated virus 3 (GLRaV-3) occurred frequently, while a mixed infection of GLRaV-1 and Grapevine virus A (GVA) or Grapevine fleck virus (GFkV) or a multiple infection of GLRaV-1, GLRaV-3 and GFkV occurred rarely in the tested plants. A mixed infection of all the four viruses mentioned above was not observed.     

The family Closteroviridae revised

Summary.  Recently obtained molecular and biological information has prompted the revision of the taxonomic structure of the family Closteroviridae. In particular, mealybug-transmitted species have been separated from the genus Closterovirus and accommodated in a new genus named Ampelovirus (from ampelos, Greek for grapevine). Thus, the family now comprises three genera. Their major properties are (i) Closterovirus: type species Beet yellows virus, genome monopartite, 15.5–19.3 kb in size, a 22–25 kDa major coat protein (CP), the gene encoding the divergent CP analogue (CPd) upstream of the CP cistron, transmission by aphids, a membership of 8 definitive and 4 tentative species; (ii) Ampelo-virus: type species Grapevine leafroll virus 3, genome monopartite 16.9–19.5 kb in size, a 35–37 kDa major CP, a CPd cistron generally located downstream of the CP gene, transmission by pseudococcid and coccid mealybugs, a membership of 6 definitive and 5 tentative species; (iii) Crinivirus: type species Lettuce infectious yellows virus, genome essentially bipartite 15.3–19 kb in size, a 28–33 kDa CP, a CPd cistron downstream of the CP gene, transmission by whiteflies (Bemisia, Trialeurodes), a membership of 7 definitive and 3 tentative species. There are five unassigned species in the family.     

Genome Sequences and Structures of Two Biologically Distinct Strains of <Emphasis Type="Italic">Grapevine leafroll</Emphasis>-<Emphasis Type="Italic">associated virus</Emphasis> 2 and Sequence Analysis

Grapevine leafroll-associated virus 2 (GLRaV-2), a member of the genus Closterovirus within Closteroviridae, is implicated in several important diseases of grapevines including “leafroll”, “graft-incompatibility”, and “quick decline” worldwide. Several GLRaV-2 isolates have been detected from different grapevine genotypes. However, the genomes of these isolates were not sequenced or only partially sequenced. Consequently, the relationship of these viral isolates at the molecular level has not been determined. Here, we group the various GLRaV-2 isolates into four strains based on their coat protein gene sequences. We show that isolates “PN” (originated from Vitis vinifera cv. “Pinot noir”), “Sem” (from V. vinifera cv. “Semillon”) and “94/970” (from V. vinifera cv. “Muscat of Alexandria”) belong to the same strain, “93/955” (from hybrid “LN-33”) and “H4” (from V. rupestris “St. George”) each represents a distinct strain, while Grapevine rootstock stem lesion-associated virus.     

Genomic and biological analysis of Grapevine leafroll-associated virus 7 reveals a possible new genus within the family Closteroviridae

Deep sequencing analysis of an asymptomatic grapevine revealed a virome containing five RNA viruses and a viroid. Of these, Grapevine leafroll-associated virus 7 (GLRaV-7), an unassigned closterovirus, was by far the most prominently represented sequence in the analysis. Graft-inoculation of the infection to another grape variety confirmed the lack of the leafroll disease symptoms, even though GLRaV-7 could be detected in the inoculated indicator plants. A 16,496 nucleotide-long genomic sequence of this virus was determined from the deep sequencing data. Its genome architecture and the sequences encoding its nine predicted proteins were compared with those of other closteroviruses. The comparison revealed that two other viruses, Little cherry virus-1 and Cordyline virus-1 formed a well supported phylogenetic cluster with GLRaV-7.     

The coat protein gene of grapevine leafroll associated closterovirus-3: cloning, nucleotide sequencing and expression in transgenic plants

Summary.  A lambda ZAP II cDNA library was constructed by cloning cDNA prepared from a high molecular weight double-stranded RNA (dsRNA, ca. 18 kb) isolated from grapevine leafroll associated closterovirus-3 (GLRaV-3) infected tissues. This cDNA library was immuno-screened with GLRaV-3 coat protein specific polyclonal and monoclonal antibodies and three immuno-positive clones were identified. Analysis of nucleotide sequences from these clones revealed an open reading frame (ORF) which was truncated at the end; the remainder of this ORF was obtained by sequencing a fourth clone that overlapped with one of the immunopositive clones. A total of 2028 bp was sequenced. The putative GLRaV-3 coat protein ORF, 939 bp, encodes a protein (referred to as p35) with a calculated of 34 866. Multiple alignment of the p35 amino acid sequence with coat protein sequences from other clostero-viruses revealed that the consensus amino acid residues (R and D) of filamentous plant viruses are preserved in the expected locations. The GLRaV-3 coat protein gene was then engineered for sense and antisense expression in transgenic plants. Transgenic Nicotiana benthamiana plants that contain the sense GLRaV-3 coat protein gene produced a 35 kDa protein that reacted with GLRaV-3 antibody in Western blot. Accepted January 10, 1997; Received November 12, 1996     

Rugose wood-associated viruses do not appear to be involved in Shiraz (Syrah) decline in South Africa

The presence of rugose-wood-associated viruses of the genera Foveavirus and Vitivirus in the family Betaflexiviridae was investigated in various clones of own-rooted and grafted Vitis vinifera cv. Shiraz that were affected, or not, by Shiraz decline, and in rootstocks. RT nested-PCR amplification of double-stranded RNA using degenerate primers for the simultaneous detection of foveaviruses and vitiviruses (Dovas CI, Katis NI in J Virol Meth 170:99–106, 2003), cloning of DNA amplicons, SSCP analysis of clones, sequencing and computer-assisted analysis of sequences was used to characterize viral genetic variability. A total of 1,137 clones were analysed by SSCP, and, of those, 371 clones were sequenced. The results revealed that variants of five molecular groups belonging to the species Grapevine rupestris stem pitting-associated virus (GRSPaV), including highly divergent variants related to strain SY (Lima MF et al. in Arch Virol 151:1889–1894, 2006) were present in plants of various clones of Shiraz regardless of their Shiraz decline status, and in rootstocks. Grapevine virus A (GVA) and grapevine virus B (GVB) were detected in a relatively small number of plants. This study suggested no involvement of GRSPaV, GVA or GVB in Shiraz decline.     

Biological properties and molecular characterization of beet chlorosis virus (BChV)

Summary.  Two distinct viruses belonging to the Polerovirus genus, in the family Luteoviridae, have been described as being able to induce mild yellowing on sugar beet: Beet mild yellowing virus (BMYV) and more recently, beet chlorosis virus (BChV). We have analysed biological properties and molecular organisation of two strains of BChV, one collected in England and the second from California. The biological data suggested that BChV displayed a narrower host range compared to BMYV and Beet western yellows virus lettuce isolate (BWYV). The complete genomic RNA sequence of the American isolate BChV-California and the European isolate BChV-2a showed a genetic organisation and expression typical of other Polerovirus members including 6 open reading frames (ORFs). Interspecific and intraspecific phylogenetic studies suggested that BChV arose by recombination events between a Polerovirus-like ancestor donating P0 and the replicase complex and either a BMYV or a BWYV progenitor providing the 3′ ORFs [3, 4 and 5]. The 5′- and 3′-parts of the BChV genome have evolved differently in the two continents, possibly due to different selection pressures to allow adaptation to the various environments, hosts and vectors. BChV is a distinct species of the Polerovirus genus. Received February 27, 2001 Accepted November 15, 2001     

Complete nucleotide sequence and genome organization of Beet soilborne mosaic virus, a proposed member of the genus Benyvirus

Summary.  The complete nucleotide sequences of RNAs 1 to 4 of Beet soilborne mosaic virus (BSBMV) were determined. The genomic organization of BSBMV is identical to Beet necrotic yellow vein virus (BNYVV), the type species of the genus Benyvirus. BSBMV RNA1 encodes a single large open reading frame (ORF) with similar replicase-associated motifs identified for BNYVV. BSBMV RNA2 has six potential ORFs with an organization resembling BNYVV RNA2. RNA3 and RNA4 resemble the analogous BNYVV RNAs, which encode proteins associated with symptom development and fungal transmission, respectively. The predicted ORFs on BNYVV and BSBMV reveal 23% to 83% amino acid identity and the overall nucleotide sequences are 35% to 77% identical. Based on sequence analyses, BSBMV is a new benyvirus that can be distinguished from BNYVV. Received February 20, 2001 Accepted August 2, 2001     

Genome organization, serology and phylogeny of Grapevine leafroll-associated viruses 4 and 6: taxonomic implications

Complete nucleotide sequences of the type isolate of Grapevine leafroll-associated virus 4 (GLRaV-4) and of an isolate of GLRaV-6 from cv ‘Estellat’ (GLRaV-6Est) were generated and compared mutually and with related viruses. The genome organization of both viruses resembled that of members of Subgroup I in the genus Ampelovirus (fam. Closteroviridae). The availability of these sequences, along with previously existing data on related GLRaVs, allowed critical review of the taxonomy and nomenclature of these viruses. In phylogenetic analyses, GLRaV-4 and -6Est consistently grouped with GLRaV-5, -9, and -Pr forming a poorly resolved sub-cluster (“GLRaV-4 group”) within the genus Ampelovirus. In-depth study showed that genetic distances between these viruses do not exceed the intra-species diversity observed in other closteroviruses. In Western blots, partially purified preparations of GLRaVs -4, -5, -6 and -9 reacted only with homologous monoclonal antibodies, but were all recognized by polyclonal antisera to GLRaV-5 and GLRaV-9. Serological relatedness among these viruses was further confirmed in DAS-ELISA. In immuno-electron microscopy, GLRaV-6 particles appeared uniformly decorated with homologous monoclonal antibodies, whereas GLRaV-2, used as a control, showed “bipolar” morphology of the virion. Results of this study challenge taxonomy and nomenclature of several GLRaVs suggesting that they are divergent isolates of Grapevine leafroll-associated virus 4 and not, as has been assumed, distinct species (definitive and/or putative) in the genus Ampelovirus.     

Deep sequencing analysis of viral short RNAs from an infected Pinot Noir grapevine

Virus-derived short interfering RNAs (vsiRNAs) isolated from grapevine V. vinifera Pinot Noir clone ENTAV 115 were analyzed by high-throughput sequencing using the Illumina Solexa platform. We identified and characterized vsiRNAs derived from grapevine field plants naturally infected with different viruses belonging to the genera Foveavirus, Maculavirus, Marafivirus and Nepovirus. These vsiRNAs were mainly of 21 and 22 nucleotides (nt) in size and were discontinuously distributed throughout Grapevine rupestris stem-pitting associated virus (GRSPaV) and Grapevine fleck virus (GFkV) genomic RNAs. Among the studied viruses, GRSPaV and GFkV vsiRNAs had a 5′ terminal nucleotide bias, which differed from that described for experimental viral infections in Arabidopsis thaliana. VsiRNAs were found to originate from both genomic and antigenomic GRSPaV RNA strands, whereas with the grapevine tymoviruses GFkV and Grapevine Red Globe associated virus (GRGV), the large majority derived from the antigenomic viral strand, a feature never observed in other plant-virus interactions.     

Grapevine fleck virus-like viruses in Vitis

Summary.  Two sets of degenerate primers for the specific amplification of 572–575 nt and 386 nt segments of the methyltransferase and RNA-dependent RNA polymerase cistrons of members of the genera Tymovirus and Marafivirus and of the unassigned virus Grapevine fleck virus (GFkV) were designed on the basis of available sequences. These primers were used for amplifying and subsequent cloning and sequencing part of the open reading frame 1 of the genome of GFkV, Grapevine asteroid mosaic-associated virus (GAMaV) and of another previously unreported virus, for which the name Grapevine red globe virus (GRGV) is proposed. Computer-assisted analysis of the amplified genome portions showed that the three grapevine viruses are phylogenetically related with one another and with sequenced tymoviruses and marafiviruses. The relationships with tymoviruses was confirmed by the type of ultrastructural modifications induced in the host cells. RdRp-specific degenerate primers were successfully used for the aspecific detection of the three viruses in crude grapevine sap extracts. Specific virus identification was obtained with RT-PCR using antisense virus-specific primers. Accepted September 27, 1999     

Genomic analysis of grapevine leafroll associated virus-5 and related viruses

The grapevine leafroll-associated viruses (GLRaVs) (Closteroviridae) represent an emerging threat to world grape production. One group of GLRaVs within the genus Ampelovirus, the GLRaV-4-like viruses (GLRaV-4LVs), contains a fragmented collection of seven viruses only two of which (GLRaV-Pr and GLRaCV) are fully sequenced. Here in reporting the sequence of GLRaV-5, a member of GLRaV-4LVs, we identify genomic elements common to the GLRaV-4LV group. Exclusive properties include a highly conserved p5 gene product and phylogenies for complete genes that, except for the p23 gene, are reliably monophyletic. In comparison with other members of the genus Ampelovirus, GLRaV-4LVs form a tight cluster for all genes analyzed. In addition, they all possess a conserved AlkB domain which is most similar to the more distantly related GLRaV-3, suggesting recombination. In silico RNA structural analyses revealed a conserved five stem-loop structure at the 3′ untranslated region that extends to all GLRaV-4LVs, and the ampeloviruses Pineapple mealybug wilt-associated virus 1 and Pineapple mealybug wilt-associated virus 3. A conserved G-U rich stem loop was also found upstream of the ORF1a stop and 1b start codons. Taken together, this work allows for a more thorough contextualization of GLRaV-5 and the GLRaV-4LVs as a group within the genus Ampelovirus.     

Nucleotide sequence and genome organisation of cherry mottle leaf virus and its relationship to members of the Trichovirus genus

Summary.  The nucleotide sequence of cherry mottle leaf virus (CMLV) was determined and compared to sequences of a number of plant viruses including the type member of the Trichovirus genus (apple chlorotic leafspot virus, ACLSV), and members of the Vitivirus genus including grapevine virus B, (GVB). The CMLV genome was determined to consist of 8003 nt excluding the poly(A) tail at the 3′ end of the genome. The overall A+U content of CMLV genomic RNA was 59. 1%, which is similar to ACLSV, but significantly different from GVB. Four putative open reading frames were identified (ORFs 1, 2, 3, and 4) encoding proteins of M_r 215. 8 kDa, 47 kDa, 21.6 kDa, and 15. 3 kDa, respectively. This differs from ACLSV which has 3 ORFS, and GVB which has 5 ORFs. Protein database searches showed no matches of CMLV ORF4 with ACLSV sequences, but found similarities between ORF4 of CMLV and ORF5 of GVB, suggesting that this may be a nucleic acid-binding protein. CMLV and ACLSV formed a common virus clade in phylogenetic analysis of the coat protein amino acid sequence and except for CMLV’s ORF4, these viruses show high levels of similarity throughout the genome. CMLV appears to be a member of the Trichovirus genus. Accepted November 19, 1999/Received August 12, 1999     

Deep sequencing analysis of viruses infecting grapevines: Virome of a vineyard

Double stranded RNA, isolated from 44 pooled randomly selected vines from a diseased South African vineyard, has been used in a deep sequencing analysis to build a census of the viral population. The dsRNA was sequenced in an unbiased manner using the sequencing-by-synthesis technology offered by the Illumina Genome Analyzer II and yielded 837 megabases of metagenomic sequence data. Four known viral pathogens were identified. It was found that Grapevine leafroll-associated virus 3 (GLRaV-3) is the most prevalent species, constituting 59% of the total reads, followed by Grapevine rupestris stem pitting-associated virus and Grapevine virus A. Grapevine virus E, a virus not previously reported in South African vineyards, was identified in the census. Viruses not previously identified in grapevine were also detected. The second most prevalent virus detected was a member of the Chrysoviridae family similar to Penicillium chrysogenum virus. Sequences aligning to two other mycoviruses were also detected.     

The nucleotide sequence of Indian peanut clump virus RNA 2: sequence comparisons among pecluviruses

Summary.  The RNA-2 molecule of an isolate of the L serotype of Indian peanut clump virus (IPCV) was shown to consist of 4290 nucleotides with five open reading frames (ORF). The arrangement of the ORFs resembled that in RNA-2 of Peanut clump virus (PCV) from West Africa. The proteins encoded by the ORFs in IPCV-L RNA are between 32% and 93% identical to those encoded by PCV RNA. Partial sequence data for the RNA-2 of isolates of the H and T serotypes of IPCV show that the coat and P40 proteins encoded by the 5′-most ORFs of RNA-2 of IPCV-L, IPCV-H and IPCV-T are as similar to each other as any is to the corresponding proteins of PCV. A conserved motif ‘F-E-x₆-W’ is present near the C-termini of the coat proteins of all three IPCV serotypes and of PCV, as it is in the coat proteins of other viruses that have rod-shaped particles, such as Tobacco mosaic virus and Tobacco rattle virus. The results support the distinction of IPCV and PCV as separate virus species, but also raise the question of how the serotypes of IPCV should be classified. Received December 17, 1999/Accepted March 15, 2000     

A novel strain of <Emphasis Type="Italic">Beet western yellows virus</Emphasis> infecting sugar beet with two distinct genotypes differing in the 5′-terminal half of genome

The complete genomic sequences of two distinct Beet western yellows virus (BWYV) genotypes infecting sugar beet in Beijing, named as BWYV-BJ^A and BWYV-BJ^B (GenBank accession number HM804471, HM804472, respectively), were determined by RT-PCR sub-cloning approach. BWYV-BJ^A and BWYV-BJ^B were 5674 and 5626nt in length, respectively. BWYV-BJ^B was 48nt shorter than BWYV-BJ^A in the regions 1589–1615 and 1629–1649nt. Sequence alignment analysis showed that the full length of BWYV-BJ^A and BWYV-BJ^B shared 93% nucleotide sequence identity, with relatively high variability within ORFs 0, 1, 2 (at the nucleotide level was 86.3–88.8%) and high conservation within ORFs 3, 4, 5 (at the nucleotide level was 99.3–99.5%). The complete nucleotide sequences of BWYV-BJ^A and BWYV-BJ^B were most related to BWYV-US (80.6 and 79.0%, respectively). ORFs 1, 2 of BWYV-BJ^A and BWYV-BJ^B shared the highest homology with BWYV-US (nucleotide identity 91.2–93.3, 86.7–89.5%, respectively) and their ORFs 3, 4 were more closely related to BWYV-IM. However, their ORF5 were more closely related to that of Cucurbit aphid-borne yellows virus China strain (CABYV-CHN), with 68.1 and 68.5% nucleotide identity, respectively. Based on the sequence and phylogenetic analysis, we proposed that BWYV-BJ was at least a novel strain of BWYV, and BWYV-BJ^A, BWYV-BJ^B were two distinct genotypes of BWYV-BJ. In addition, phylogenetic analysis and recombination analysis suggested that BWYV-BJ^A and BWYV-BJ^B might be recombinant viruses.