Explore the vast range of titles available.

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.

An interactive relationship between vitiviruses and grapevine leafroll viruses was characterized in grapevine. Grapevine viruses A and B (GVA and GVB) were found more frequently in the presence of co-infecting Grapevine leafroll associated viruses (GLRaV-1, −2 or −3) than in their absence. The titers of the vitiviruses in co-infection with leafroll viruses were found to be higher than were their titers in the absence of leafroll virus infection. The occurrence of vitivirus-associated stem-pitting symptoms was correlated with leafroll virus co-infection. Specific pairing associations on the species level were found between different viti- and leafroll virus species: GVB was associated preferentially with GLRaV-2; GVA was associated preferentially with GLRaV-1 and GLRaV-3. In contrast to the increase in vitivirus titer seen with leafroll virus co-infection, the incidence and titer of grapevine leafroll virus appeared to be unaltered by vitivirus co-infection. The potential for a synergistic enhancement of grapevine disease in co-infected vines is discussed.

We have characterized the virome in single grapevines by 454 high-throughput sequencing of double-stranded RNA recovered from the vine stem. The analysis revealed a substantial set of sequences similar to those of fungal viruses. Twenty-six putative fungal virus groups were identified from a single plant source. These represented half of all known mycoviral families including the Chrysoviridae, Hypoviridae, Narnaviridae, Partitiviridae, and Totiviridae. Three of the mycoviruses were associated with Botrytis cinerea, a common fungal pathogen of grapes. Most of the rest appeared to be undescribed. The presence of viral sequences identified by BLAST analysis was confirmed by sequencing PCR products generated from the starting material using primers designed from the genomic sequences of putative mycoviruses. To further characterize these sequences as fungal viruses, fungi from the grapevine tissue were cultured and screened with the same PCR probes. Five of the mycoviruses identified in the total grapevine extract were identified again in extracts of the fungal cultures.

Real-time quantitative RT-PCR (RT-qPCR) was developed for the detection of Cherry rasp leaf virus (CRLV), Cherry green ring mottle virus (CGRMV), Cherry necrotic rusty mottle virus (CNRMV), Cherry virus A (CVA) and Apple chlorotic leaf spot virus (ACLSV) in stone fruits, including apricot, cherry, peach, plum and almond. The RT-qPCR assays were designed after aligning nucleotide sequences derived from the most conserved regions of the coat protein gene of geographically diverse isolates and isolates for which sequence information is available in Gen-Bank. RT-qPCR assays were validated and compared to conventional RT-PCR using 221 isolates. Our data showed that more isolates were detected by RT-qPCR than by RTPCR; 0.9%, 6.3%, 1.29%, 5% and 8.14% more for CRLV, CGRMV, CNRMV, CVA and ACLSV, respectively.

Reverse transcription quantitative PCR (RT-qPCR) assays were developed for the detection of the ilarviruses Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), Apple mosaic virus (ApMV), and American plum line pattern virus (APLPV), and the nepoviruses Tomato ringspot virus (ToRSV) and Cherry leafroll virus (CLRV). These viruses affect various stone fruits such as apricots, cherries, peaches, plums, and almonds. The goal of this work was to improve the RT-qPCR detection of PNRSV, PDV, and ApMV in addition to developing three new RT-qPCR assays for the detection of APLPV, ToRSV and CLRV. Primers for conventional RT-PCR as well as primers and probes for RT-qPCR assays were designed after aligning coat protein (CP) gene sequences of geographically diverse isolates with the corresponding CP gene sequences from the GenBank, targeting regions with 100% sequence identity. The efficiency of each RT-qPCR assay, as well as the intra- and inter-assay variability were determined. These conventional RT-PCR and RT-qPCR assays were validated using purified total RNAs from 221 trees from the USDA Clonal Germplasm Repository orchards. The data showed that more isolates were detected by RTqPCR than by RT-PCR.

A virus species provisionally named Grapevine virus F (GVF) was found in grapevine by deep sequencing. GVF is a vitivirus distinctly different from the other four members of this genus, as shown by nucleotide sequence and phylogenetic analysis. An RT-PCR test was developed for the detection of this virus. In a collection of 454 grapevine accessions from worldwide sources, an infection rate of 7% was found.

New insights into the genetic structure and variability of grapevine leafroll-associated viruses (GLRaVs) gained through worldwide efforts in the last decade or so, and the production and use of new sets of serological reagents, have provided the solid foundation on which the present revision of the taxonomic structure of the family Closteroviridae, and the genus Ampelovirus in particular, is based. A comparative examination of the amino acid sequence divergence of three taxonomically relevant genes [RNA-dependent RNA polymerase (polymerase), heat shock protein 70 homologue (HSP70h) and coat protein (CP)] disclosed a difference among Grapevine leafroll-associated virus 4 (GLRaV-4), -5, -6 and -9 and a group of more recently described viruses (GLRaV-Pr, GLRaV-De and GLRaV-Car) below the 25% limit recently set by the International Committee on Taxonomy of Viruses (ICTV) as a discriminating criterion for the identification of species in the family Closteroviridae. This, plus the recognition that GLRaV4, -5, -6 and -9 are serologically related, have similar biological and epidemiological traits, and that these viruses and GLRaV-Pr, GLRaV-De, GLRaV-Car have a genome with the same structure and size, supports the notion that they are all genetically divergent variants of a single species, GLRaV-4. The genus Ampelovirus is split into two subgroups designated I and II in recognition of the wide difference in the size and structure of the genome of the present members. Finally, the establishment of a fourth genus within the family Closteroviridae, comprising the unassigned putative species Grapevine leafroll-associated virus 7 (GLRaV-7), Little cherry virus 1 (LChV-1) and Cordyline virus 1 (CoV-1), is justified based on their molecular and biological characteristics that differ from those of members of the other three genera of the family.

Tissue-imprint hybridization (TIH) assay was validated for large-scale detection of Peach latent mosaic viroid (PLMVd) and Hop stunt viroid (HSVd). All 72 collected leaves (100%) from 2 PLMVd- and 2 HSVd-infected trees were positive in TIH, regardless of the geographic orientation of the scaffold, level of the canopy and position of the leaf in the shoot. In a large-scale survey in Serbia, we tested by TIH 871 trees of stone fruits, representing 602 cultivars from fruit collections in Belgrade, Čačak and Novi Sad. PLMVd was detected in 185 (50%) peach trees or 95 (54%) cultivars and HSVd in 2 apricot trees and cultivars (2%). The occurrence of HSVd is a new report for Serbia. No viroid infection was found in European plums, sweet cherries, sour cherries and wild Prunus spp. PLMVd-infected peach cultivars originated from the world's main breeding centres of this crop. Western European and Asian cultivars were the most infected (58%) followed by those originating from North America (50%). Nine PLMVd and two HSVd isolates were sequenced and analyzed. All showed PMLVd sequences clustered together in the previously reported phylogenetic group III. Both HSVd isolates were found to be derived from recombinant events, but that of the cv. Saturn represented a putative new phylogenetic group of HSVd.

The oil-and table olive industry in California is growing rapidly. Propagation material is being requested in ever-larger quantities from the USDA National Clonal Germplasm Repository at the University of California, Davis. This collection is recognized as one of the richest sources of olive material in the U.S. The repository maintains 107 different olive varieties imported from 19 different countries. However, the collection's viral infection status has never been systematically analyzed. We have now completed the first comprehensive virus testing of the collection using molecular diagnostic tools. A total of 49 trees from 36 different cultivars were sampled. Though these trees were asymptomatic, the samples from 97.9% of them showed dsRNA profiles indicating viral infection. The 93.8% of these trees tested positive for Olive leaf yellowing-associated virus (OLYaV; an unassigned virus in the Closteroviridae) by RT-PCR analysis, while 34.7% were positive for Cucumber mosaic virus (CMV). PCR amplicons of the OLYaV heat shock 70 homologue (HSP70h) gene were cloned and sequenced to analyze diversity of isolates from trees originating from different geographical regions. Sequence analysis showed a maximum of 32% divergence between amplicons obtained from these selections.

One hundred twenty-five trees (each of a different cultivar) of sour and sweet cherry from two large varietal collections in Serbia were visually inspected for virus symptoms and tested for the presence of cherry viruses by ELISA, herbaceous host assays, graft-indexing on P. serrulata cv. Kwanzan, and RT-PCR. All samples were tested by ELISA for Prunus necrotic ring spot virus (PNRSV), Prune dwarf virus (PDV), Apple chlorotic leaf spot virus (ACLSV), Apple mosaic virus (ApMV), and Plum pox virus (PPV). The overall detection of PDV, PNRSV, and ACLSV was 63%. Additional ELISA tests were done on 80 trees for Arabis mosaic virus (ArMV), Cherry leaf roll virus (CLRV), Strawberry latent ring spot virus (SLRSV), Petunia asteroid mosaic virus (PetAMV), Raspberry ringspot virus (RpRSV), Tomato black ring virus (TBRV), Tobacco mosaic virus (TMV), and Tomato ringspot virus (ToRSV). In these tests, one tree tested positive for PetAMV RT-PCR testing of 44 trees detected another five viruses: Cherry green ring mottle virus (CGRMV), Cherry necrotic rusty mottle virus (CNRMV), Cherry virus A (CVA), European rusty mottle associated virus (ERMaV) and Plum bark necrosis and stem pitting-associated virus (PBNSPaV), but not Cherry mottle leaf virus (CMLV). In graft-indexing tests on Kwanzan with all 125 trees, samples from 38 trees induced symptoms of necrotic crook disease (causal agent unknown). Viruses reported for the first time in Serbia were CGRMV, CNRMV, CVA, ERMaV, PBNSPaV, and PetAMV.