Jeremy R. Thompson

Complete Genome Sequence of a New Circular DNA Virus from Grapevine

ABSTRACT A novel circular DNA virus sequence is reported from grapevine. The corresponding genomic organization, coding potential, and conserved origin of replication are similar to those of members of the family Geminiviridae, but the genome of 3,206 nucleotides is 4% larger than the largest reported geminiviral genome and shares only 50% overall sequence identity.

A survey of geminiviruses and associated satellite DNAs in the cotton-growing areas of northwestern India

Severe symptoms of cotton leaf curl disease (CLCuD) are caused by the association of a single-stranded circular DNA satellite (betasatellite) with a helper begomovirus. In this study, we analyzed 40 leaf samples (primarily cotton with CLCuD symptoms and other plants growing close by) from four sites between New Delhi and the Pakistan/India border, using rolling-circle amplification (RCA) and PCR. In total, the complete sequences of 12 different helper viruses, eight alphasatellites, and one betasatellite from five different plant species were obtained. A recombinant helper virus molecule found in okra and a novel alphasatellite-related DNA from croton are also described. This is the first report of the presence of both DNA components (helper virus and betasatellite) associated with resistance-breaking CLCuD in India, and it highlights the need for further work to combat its damage and spread.

Twenty Years of Transgenic Plants Resistant to Cucumber mosaic virus

Plant genetic engineering has promised researchers improved speed and flexibility with regard to the introduction of new traits into cultivated crops. A variety of approaches have been applied to produce virus-resistant transgenic plants, some of which have proven to be remarkably successful. Studies on transgenic resistance to Cucumber mosaic virus probably have been the most intense of any plant virus. Several effective strategies based on pathogen-derived resistance have been identified; namely, resistance mediated by the viral coat protein, the viral replicase, and post-transcriptional gene silencing. Techniques using non-pathogen-derived resistance strategies, some of which could offer broader resistance, generally have proven to be much less effective. Not only do the results obtained so far provide a useful guide to help focus on future strategies, but they also suggest that there are a number of possible mechanisms involved in conferring these resistances. Further detailed studies on the interplay between viral transgene-derived molecules and their host are needed in order to elucidate the mechanisms of resistance and pathogenicity.

A new potato virus in a new lineage of picorna-like viruses

Summary.On constructing a cDNA library for potato, ‘contaminating’ sequences with a significant identity to Apple latent spherical virus (ALSV) were found. Determination of the remaining genome sequence indicated the presence of a bipartite virus with an RNA1 and 2 of 7034 and 3315 nucleotides, respectively, excluding a poly(A)tail. RNA1 encodes a single polyprotein (233 kDa) and shares highest amino acid identity with ALSV at 65%. Conserved amino acid motifs typical for helicase, protease and RNA-dependent polymerase (RdRp) functions are present. RNA2 encodes a single polyprotein (106 kDa) with amino acid identities to the flat apple isolate of Cherry rasp leaf virus (CRLV-FA) (97%) and ALSV (70%), suggesting this is a potato strain of CRLV (CRLV-pot). Phylogenetic analysis using the RdRp region shows that this virus falls within a group separate from the Comoviridae that includes members of the Sequiviridae and the taxonomically unassigned viruses ALSV, Strawberry mottle virus, Satsuma dwarf virus and Navel orange infectious mottling virus. Other regions of the genome have highest identities with both plant and animal infecting members of the picorna-like virus superfamily. The evolutionary context of CRLV-pot and related viruses is discussed. Similar viral sequences from an EST library of peppermint are also analysed.

Grapevine red blotch-associated virus Is Widespread in the United States.

Grapevine red blotch disease has been recognized since 2008 as affecting North American grape production. The presence of the newly described Grapevine red blotch-associated virus (GRBaV) is highly correlated with the disease. To more effectively detect and monitor the presence of the virus, a sample processing strategy and multiplex polymerase chain reaction assay were developed. A total of 42 of 113 vine samples collected in or received from seven of the United States were shown to harbor the virus, demonstrating the virus is widely distributed across North America. Phylogenetic analyses of a viral replication-associated protein (Rep) gene fragment from the 42 isolates of GRBaV demonstrated distinct clades of the virus (1 and 2), with clade 1 showing the greatest variability. The full-length genome of six virus isolates was sequenced, and phylogenetic analyses of 14 whole genomes recapitulated results seen for the Rep gene. A comparison of GRBaV genomes revealed evidence of recombination underlying some of the variation seen among GRBaV genomes within clade 1. Phylogenetic analyses of coat and replicase-associated protein sequences among single-stranded DNA viruses showed GRBaV to group within the family Geminiviridae. This grouping is distinct from members of the families Nanoviridae and Circoviridae, with limited significant affinities to both recognized genera and novel plant-infecting, gemini-like viruses.

Encapsidation of DNA, a protein and a fluorophore into virus-like particles by the capsid protein of cucumber mosaic virus.

An important property of some spherical plant viruses is their ability to reassemble in vitro from native capsid protein (CP) and RNA into infectious virus-like particles (VLPs). Virions of cucumber mosaic virus (CMV) are stabilized by protein-RNA interactions and the nucleic acid is essential for assembly. This study demonstrated that VLPs will form in the presence of both ssDNA and dsDNA oligonucleotides, and with a lower size limit of 20 nt. Based on urea disruption assays, assembled VLPs from CMV CP and RNA (termed ReCMV) exhibited a level of stability similar to that of virions purified from plants, whilst VLPs from CMV CP and a 20mer exhibited comparable or greater stability. Fluorescent labelling of VLPs was achieved by the encapsidation of an Alexa Fluor 488-labelled 45mer oligonucleotide (ReCMV-Alexa488-45) and confirmed by transmission electron and confocal microscopy. Using ssDNA as a nucleating factor, encapsidation of fluorescently labelled streptavidin (53 kDa) conjugated to a biotinylated oligonucleotide was observed. The biological activity and stability of ReCMV and ReCMV-Alexa488-45 was confirmed in infectivity assays and insect vector feeding assays. This work demonstrates the utility of CMV CP as a protein cage for use in the growing repertoire of nanotechnological applications.

Assessment of the benefits and risks for engineered virus resistance.

Viral diseases of cultivated crops are responsible for the worldwide loss of billions of US dollars in agricultural productivity every year. Historically, this loss has been reduced or minimized principally by the implementation of specific agricultural/phytosanitary measures, and by the introduction of naturally occurring virus-resistance genes into appropriate cultivars by plant breeding. Since the first report of virus-resistant transgenic plants (VRTPs) in 1986, a remarkable diversity of virus-resistance transgenes has been developed. Despite this, to a large part due to controversy surrounding the use of genetically modified organisms, the number of commercially available VRTPs remains small. However, since the potential risks associated with VRTPs were first formulated in the early 1990s, fundamental research on plant-virus interactions and also research specifically aimed at resolving biosafety issues have greatly circumscribed the potential impact of the risks envisaged. Yet, in spite of the advances, both in strategies for creating VRTPs and in the assessment of potential risks, it remains remarkably difficult to weigh the risks/costs and benefits of different means to manage plant viral diseases, and even to make scientifically well-founded choices of the most appropriate strategy for creating VRTPs. Many of the outstanding issues concern the lack of sufficient knowledge of the breadth and durability of the resistance of VRTPs under field conditions. VRTPs will only take their appropriate place in modern agriculture when their potential users will be able to base their choices on realistic assessments of their efficacy, durability, and safety.

Profiling viral infections in grapevine using a randomly primed reverse transcription-polymerase chain reaction/macroarray multiplex platform.

Crop-specific diagnostics to simultaneously detect a large number of pathogens provides an invaluable platform for the screening of vegetative material prior to its propagation. Here we report the use of what is to-date the largest published example of a crop-specific macroarray for the detection of 38 of the most prevalent or emergent viruses to infect grapevine. The reusable array consists of 1,578 virus-specific 60 to 70mer oligonucleotide probes and 19 plant and internal control probes spotted onto an 18 × 7 cm nylon membrane. In a survey of 99 grapevines from the United States and Europe, virus infections were detected in 46 selections of Vitis vinifera, V. labrusca, and interspecific hybrids. The majority of infected vines (30) was singly infected, while 16 were mixed-infected with viruses from two or more families. Representatives of the four main virus families Betaflexiviridae, Closteroviridae, Secoviridae, and Tymoviridae present in grapevines were found alone and in combination, with a notable bias in representation by members of the family Tymoviridae. This work demonstrates the utility of the macroarray platform for the multiplex detection of viruses in a single crop, its potential for characterizing grapevine virus associations, and usefulness for rapid diagnostics of introduced material in quarantine centers or in certification programs.

Macroarray detection of grapevine leafroll-associated viruses

Grapevine leafroll-associated viruses (GLRaVs) are an emerging group of viruses that represent a significant threat to the global productivity and sustainability of the grapevine industry. Their control is achieved through the identification and elimination of infected vines, and the use of planting material derived from virus-tested, certified stocks. As such, much effort has been invested in developing reliable molecular diagnostic techniques. In this work, we report the development of a macroarray assay for the detection of the principal GLRaVs. In total 314 70-mer oligonucleotides specific to GLRaV-1, -2, -3, -4, -7, and GLRaV-4 strains 5, 6, 9 and Pr were spotted onto a 11×7cm nylon membrane. Thirty-four grapevine samples from various origins were tested by the macroarray, RT-PCR and ELISA. Thirty were positive for virus infection using RT-PCR, 28 by ELISA and 25 by the macroarray. Mixed infections were identified by macroarray in two samples and confirmed by RT-PCR or ELISA. There were a few discrepancies between methods that were most likely due to differences in the sensitivity of detection, and in the case of the macroarray, limitations in the sequence data available for certain virus species in the design of the oligonucleotides. This work demonstrates the successful application of macroarray methodology using randomly primed and sequence-nonspecific amplified cDNAs derived from grapevine total RNA extracts, and provides a proof-of-principal for unbiased multiplex detection using a single robust platform.

Compensatory Capsid Protein Mutations in Cucumber Mosaic Virus Confer Systemic Infectivity in Squash (Cucurbita pepo)

ABSTRACT Cucumber mosaic virus (CMV) systemically infects both tobacco and zucchini squash. CMV capsid protein loop mutants with single-amino-acid substitutions are unable to systemically infect squash, but they revert to a wild-type phenotype in the presence of an additional, specific single-site substitution. The D118A, T120A, D192A, and D197A loop mutants reverted to a wild-type phenotype but did so in combination with P56S, P77L, A162V, and I53F or T124I mutations, respectively. The possible effect of these compensatory mutations on other, nonsystemically infecting loop mutants was tested with the F117A mutant and found to be neutral, thus indicating a specificity to the observed changes.