Adrian Fox

Next Generation Sequencing for Detection and Discovery of Plant Viruses and Viroids: Comparison of Two Approaches

Next generation sequencing (NGS) technologies are becoming routinely employed in different fields of virus research. Different sequencing platforms and sample preparation approaches, in the laboratories worldwide, contributed to a revolution in detection and discovery of plant viruses and viroids. In this work, we are presenting the comparison of two RNA sequence inputs (small RNAs vs. ribosomal RNA depleted total RNA) for the detection of plant viruses by Illumina sequencing. This comparison includes several viruses, which differ in genome organization and viroids from both known families. The results demonstrate the ability for detection and identification of a wide array of known plant viruses/viroids in the tested samples by both approaches. In general, yield of viral sequences was dependent on viral genome organization and the amount of viral reads in the data. A putative novel Cytorhabdovirus, discovered in this study, was only detected by analysing the data generated from ribosomal RNA depleted total RNA and not from the small RNA dataset, due to the low number of short reads in the latter. On the other hand, for the viruses/viroids under study, the results showed higher yields of viral sequences in small RNA pool for viroids and viruses with no RNA replicative intermediates (single stranded DNA viruses).

New aphid vectors and efficiency of transmission of Potato virus A and strains of Potato virus Y in the UK

The aphid-transmitted viruses Potato virus Y (PVY) and Potato virus A (PVA) commonly affect seed potatoes in the UK. The transmission efficiency for aphid species is used to calculate a potential transmission risk and is expressed as a relative efficiency factor (REF). These REFs have not previously been calculated for UK strains of viruses or aphid clones. Using a previously published method, REFs have been calculated for the aphid species and viruses commonly occurring in UK potatoes. The efficiency of transmission of Myzus persicae is nominally set to a REF of 1 and REFs for other species are calculated relative to this. These data represent the first set of REFs calculated for PVA transmission. Macrosiphum euphorbiae (REF 0.91) was almost as efficient as M. persicae at PVA transmission. The data were further analysed to compare transmission rates of PVY and PVA using a binomial (logit) generalized mixed model to take into account the potential influence of variation in virus titre between leaves. This approach found that there is little variation between the efficiency of transmission between clones of each aphid species or between strains within a virus species. This is a first report that Aphis fabae, Metopolophium dirhodum, Sitobion avenae, Acyrthosiphon pisum and Cavariella aegopodii have the ability to vector PVA. This study also represents a first report that C. aegopodii has the ability to vector PVY and confirms the potential of S. avenae, A. fabae, M. euphorbiae and Rhopalosiphum padi as important PVY vectors.

Diagnosis of Plant Viruses Using Next-Generation Sequencing and Metagenomic Analysis

Next generation sequencing (NGS) is revolutionising the diagnosis of plant viral disease. This chapter describes the sequencing platforms (Illumina, 454, Pacific Biosciences, IonTorrent, Nanopore) and techniques used to produce and analyse NGS virus data. At present NGS has been used for plant viral disease diagnosis in a number of exemplar cases but, as yet, it has still to be routinely adopted for frontline diagnostic applications. The barriers to this uptake including access, cost, analysis, validation and interpretation are discussed.

Detection and transmission of Carrot torrado virus, a novel putative member of the Torradovirus genus.

A new Torradovirus tentatively named Carrot torrado virus (CaTV) was an incidental finding following a next generation sequencing study investigating internal vascular necrosis in carrot. The closest related viruses are Lettuce necrotic leaf curl virus (LNLCV) found in the Netherlands in 2011 and Motherwort yellow mottle virus (MYMoV) found in Korea in 2014. Primers for reverse transcriptase-PCR (RT-PCR) and RT-qPCR were designed with the aim of testing for the presence of virus in plant samples collected from the field. Both methods successfully amplified the target from infected samples but not from healthy control samples. The specificity of the CaTV assay was also checked against other known carrot viruses and no cross-reaction was seen. A comparative study between methods showed RT-qPCR was the most reliable method, giving positive results in samples where RT-PCR fails. Evaluation of the Ct values following RT-qPCR and a direct comparison demonstrated this was due to improved sensitivity. The previous published Torradovirus genus specific RT-PCR primers were tested and shown to detect CaTV. Also, virus transmission experiments carried out suggest that unlike other species of the same genus, Carrot torrado virus could be aphid-transmitted.

First Report of Carrot torradovirus 1 (CaTV1), a Member of the Torradovirus Genus, Infecting Carrots in France

First Report of [i]Carrot torradovirus 1[/i] (CaTV1), a Member of the [i]Torradovirus[/i] Genus, Infecting Carrots in France

Transmission and Epidemiology of Potato virus Y

As obligate parasites, plant viruses, require in order to survive, to be transmitted to another plant. Experimentally, viruses such as Potato virus Y (PVY) can be transmitted by mechanical means such as wounding and grafting. In its natural environment, PVY transmission is mediated by sap-feeding aphid vector, or vegetatively through propagated organs such as potato tubers. A vast number of aphid species have been reported to transmit PVY in a non-persistent manner with variable efficiency to a large number of solanaceous and non-solanaceous plant species including weeds and ornamentals. Several sensory stimuli will influence host selection and feeding behaviour of the aphid and will strongly influence virus epidemiology. The interactions between the virus, its vector, and the environment are complex and are the focus of many studies aiming to understand the molecular basis of these interactions and their impact on disease development. This chapter will present the current knowledge of PVY transmission, epidemiology, and its management in different countries.

First report of Prunus necrotic ringspot virus and Mulberry cryptic virus 1 in mulberry ( Morus alba ) in the United Kingdom

In September 2017, a sample of mulberry leaf (Morus alba cv. Capsrum) was submitted from a nursery in Worcestershire, to Fera Science Ltd. Over 100 trees were affected with a chlorotic oak leaf line pattern on the fully developed leaves …

Application of HTS for Routine Plant Virus Diagnostics: State of the Art and Challenges

Department of Genetics, Stellenbosch University, Stellenbosch, South Africa, 2 Agricultural Research Council, Infruitec-Nietvoorbij, The Fruit, Vine and Wine Institute, Stellenbosch, South Africa, Department of Plant Protection, Fera Science Ltd., York, United Kingdom, Department of Plant Pathology, University of California, Davis, Davis, CA, United States, 5 School of Natural and Environmental Sciences, University of Newcastle, Newcastle Upon Tyne, United Kingdom, UMR 1332 Biologie du Fruit et Pathologie, INRA, University of Bordeaux, Bordeaux, France

The impact of high throughput sequencing on plant health diagnostics

High throughput sequencing informed diagnostics is revolutionising plant pathology. The application of this technology is most advanced in plant virology, where it is already becoming a front-line diagnostic tool and it is envisaged that for other types of pathogen and pests this will be the case in the near future. However, there are implications to deploying this technology due to a number of technical and scientific challenges. Firstly, interpretation of data and the assessment of plant health risk against a limited baseline of existing knowledge of the presence of pathogens in a given geographic region. Secondly, evidence of causality and the separation of pathogenic from commensal organisms in the sequence data, thirdly, the tension between the generation of a rapid sequence result with the necessary but laborious epidemiological characterisation in support of plant health risk assessment. Finally, the validation and accreditation of methods based on this rapidly evolving technology. These in turn present challenges for plant health policy and regulation. This review discusses the development of this technology, its application in plant health diagnostics, and explores the implications of applying this technology in the plant health setting.

Complete sequence and genomic annotation of carrot torradovirus 1

Carrot torradovirus 1 (CaTV1) is a new member of the genus Torradovirus within the family Secoviridae. CaTV1 genome sequences were obtained from a previous next-generation sequencing (NGS) study and were compared to other members and tentative new members of the genus. The virus has a bipartite genome, and RACE was used to amplify and sequence each end of RNA1 and RNA2. As a result, RNA1 and RNA2 are estimated to contain 6944 and 4995 nucleotides, respectively, with RNA1 encoding the proteins involved in virus replication, and RNA2 encoding the encapsidation and movement proteins. Sequence comparisons showed that CaTV1 clustered within the non-tomato-infecting torradoviruses and is most similar to motherwort yellow mottle virus (MYMoV). The nucleotide sequence identities of the Pro-Pol and coat protein regions were below the criteria established by the ICTV for demarcating species, confirming that CaTV1 should be classified as a member of a new species within the genus Torradovirus.