Semi-artificial datasets as a resource for validation of bioinformatics pipelines for plant virus detection

The widespread use of High-Throughput Sequencing (HTS) for detection of plant viruses and sequencing of plant virus genomes has led to the generation of large amounts of data and of bioinformatics challenges to process them. Many bioinformatics pipelines for virus detection are available, making the choice of a suitable one difficult. A robust benchmarking is needed for the unbiased comparison of the pipelines, but there is currently a lack of reference datasets that could be used for this purpose. We present 7 semi-artificial datasets composed of real RNA-seq datasets from virus-infected plants spiked with artificial virus reads. Each dataset addresses challenges that could prevent virus detection. We also present 3 real datasets showing a challenging virus composition as well as 8 completely artificial datasets to test haplotype reconstruction software. With these datasets that address several diagnostic challenges, we hope to encourage virologists, diagnosticians and bioinformaticians to evaluate and benchmark their pipeline(s)

First Report of Little cherry virus 1 affecting European Plum (Prunus domestica) in Belgium

Little cherry disease (LChD), one of the major viral diseases of cherry worldwide, can be caused by two viruses (Little cherry virus 1 and 2), both Closteroviridae members. LChD has an important impact on both yield and fruit quality in commercial sweet and sour cherry (Prunus avium L. and P. cerasus L.) (Ruiz-Garcia et al. 2016). LChV-1 (genus Velarivirus) is known to be graft-transmissible and is spread via infected propagated plant material, but no vector has been identified. For LChV-2 (genus Ampelovirus), at least two species of mealybugs (Hemiptera, Pseudococcidae) are known to transmit the virus, namely the apple mealybug (Phenacoccus aceris Signoret) and grape mealybug (Pseudococcus maritimus Ehrhorn). During two growing seasons (2013–15), intensive surveys were conducted in Belgium to monitor the incidence of LChD in sweet and sour cherries and in ornamental Prunus spp., revealing widespread occurrence of both LChV-1 and 2 (De Jonghe et al. 2016). In the close vicinity of a sweet cherry (P. avium cv. Coralise) orchard with an infection rate of 30% with LChV-1, plum (P. domestica L. cv. Opal) trees growing at the edge of a plum orchard and showing sporadic undetermined leaf symptoms such as premature leaf reddening and chlorosis were observed and sampled. RNA of leaves and roots collected from 50 plum trees was extracted using the Spectrum Total Plant RNA kit (Sigma-Aldrich, Machelen, Belgium) and tested using RT-PCR with LChV-1 specific primers as follows: LCUW7090 (5′-GGTTGTCCTCGGTTGATTAC-3′)/LCUWc7389 (5′-GGCTTGGTTCCATACATCTC-3′) (Bajet et al. 2008), amplifying a 300-bp fragment spanning the ORF1b encoding the RNA dependent RNA-polymerase (RdRp) gene and 1LC_12776F (5′-TCAAGAAAAGTTCTGGTGTGC-3′)/1LC_13223R (5′-CGAGCTAGACGTATCAGTATC-3′) (Nagyova et al. 2015), targeting a 456-bp fragment of the coat protein (CP) gene. The presence of LChV-1 was confirmed in 12% of the samples. Bidirectional sequencing (Macrogen, Amsterdam) was done for each LChV-1 amplicon. BLAST searches of the assembled sequences revealed a distinct variability between the Belgian plum and cherry isolates (8% and 6% divergence in the amplified RdRp and CP sequences, respectively) from the respective adjacent orchards, suggesting separate introduction events. RdRp gene sequences of the Belgian plum isolates (GenBank accession nos. KY173002 and KY173004) shared 99% identity with the Greek cherry (HG792418) and peach isolates (HG792399), while the Belgian cherry isolate (KY173001) showed 99% homology with the deposited RdRp gene sequences of the Greek cherry (HG792420, HG792398). Partial CP gene sequence of the Belgian plum isolates (KY173006, KY173008) were the closest to Italian ITMAR (EU715989) and German V2356 (JX669615) cherry isolates, sharing 96% and 94% identity, respectively. Further investigation is in progress to assess the importance of LChV natural host shift among Prunus spp., its epidemiology in propagation material, and its association with potential vectors. To our knowledge, this description of LChV-1 associated with P. domestica constitutes the first report in Belgium

An Advanced One-Step RT-LAMP for Rapid Detection of Little cherry virus 2 Combined with High-Throughput Sequence-Based Phylogenomics Reveal Divergent Flowering Cherry Isolates.

peer reviewedLittle cherry virus 2 (LChV-2, genus Ampelovirus) is considered to be the main causal agent of the economically damaging little cherry disease, which can only be controlled by removal of infected trees. The widespread viral disease of sweet cherry (Prunus avium L.) is affecting the survival of long-standing orchards in North America and Europe, hence the dire need for an early and accurate diagnosis to establish a sound disease control strategy. The endemic presence of LChV-2 is mainly confirmed using laborious time-consuming reverse-transcription (RT-PCR). A rapid reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay targeting a conserved region of the coat protein was developed and compared with conventional RT-PCR for the specific detection of LChV-2. This affordable assay, combined with a simple RNA extraction, deploys desirable characteristics such as higher ability for faster (<15 min), more analytically sensitive (100-fold), and robust broad-range diagnosis of LChV-2 isolates from sweet cherry, ornamental flowering cherry displaying heterogenous viral etiology and, for the first time, newly identified potential insect vectors. Moreover, use of Sanger and total RNA high-throughput sequencing as complementary metaviromics approaches confirmed the LChV-2 RT-LAMP detection of divergent LChV-2 isolates in new hosts and the relationship of their whole-genome was exhaustively inferred using maximum-likelihood phylogenomics. This entails unprecedented critical understanding of a novel evolutionary clade further expanding LChV-2 viral diversity. In conclusion, this highly effective diagnostic platform facilitates strategical support for early in-field testing to reliably prevent dissemination of new LChV-2 outbreaks from propagative plant stocks or newly postulated insect vectors. Validated results and major advantages are herein thoroughly discussed, in light of the knowledge required to increase the potential accuracy of future diagnostics and the essential epidemiological considerations to proactively safeguard cherries and Prunus horticultural crop systems from little cherry disease

Occurrence and Variability of Little cherry virus 1 and 2.

BACKGROUND and OBJECTIVES Little Cherry disease (LChD) can be caused by two distinct viruses (Little cherry virus 1 and 2), and is having a large impact on yield and quality in commercial sweet (Prunus avium L.) and sour cherry (Prunus cerasus L.) production (Rott and Jelkmann, 2001). Several other Prunus spp. can be infected - yet often latently - by both viruses, including the popular oriental flowering cherry (P. serrulata L.). For LChV-1, no vector has been identified so far, but at least two distinct species of mealybugs (Hemiptera, Coccoidea, Pseudococcidae) are known to transmit LChV-2, namely the apple mealybug (Phenacoccus aceris Signoret) and grape mealybug (Pseudococcus maritimus Ehrhorn) (Slykhuis et al., 1980; Mekuria et al. 2013). MATERIALS and METHODS During 2014 and 2015, an intensive survey has been conducted to monitor the incidence and spread of LChV- 1 and -2 in symptomatic and non-symptomatic host trees, eventually leading to re-evaluate the status of LChV infections in Belgium. Leaf symptoms such as premature reddening or bronzing, as well as the development of small fruits, uneven ripening and an insipid taste were observed in many orchards. A total of 306 trees were sampled and tested by RT-PCR for the 2 viruses. LChV-1 or -2 -specific PCR products spanning different open reading frames, were subsequently sequenced and used as markers for characterization. RESULTS Both viruses were widely detected in individual or mixed infections, with a slightly higher incidence for LChV- 2 in samples from sweet and sour cherries. The disease was found to be prevalent in many cherry production areas in nearly all places where cherries are grown. Additionally, both viruses were also found in ornamental Prunus spp. in private gardens and in lane trees. Along with published homologous genomic data from other isolates, the genetic diversity of Belgian Little cherry virus (LChV-1 & 2) isolates originating from different hosts and geographic locations was assessed, based on sequences corresponding to the partial RNA-dependent RNA polymerase (RdRp), Heat-Shock Protein homologue (HSP70h) and Coat Protein (CP) genes. CONCLUSIONS Preliminary phylogenetic analysis revealed a low genetic variability for the Belgian LChV-1 and LChV-2 isolates, yet suggests a long-term establishment for both viruses in our region

Detection of single nucleotide polymorphisms in virus genomes assembled from high-throughput sequencing data: large-scale performance testing of sequence analysis strategies

Recent developments in high-throughput sequencing (HTS) technologies and bioinformatics have drastically changed research in virology, especially for virus discovery. Indeed, proper monitoring of the viral population requires information on the different isolates circulating in the studied area. For this purpose, HTS has greatly facilitated the sequencing of new genomes of detected viruses and their comparison. However, bioinformatics analyses allowing reconstruction of genome sequences and detection of single nucleotide polymorphisms (SNPs) can potentially create bias and has not been widely addressed so far. Therefore, more knowledge is required on the limitations of predicting SNPs based on HTS-generated sequence samples. To address this issue, we compared the ability of 14 plant virology laboratories, each employing a different bioinformatics pipeline, to detect 21 variants of pepino mosaic virus (PepMV) in three samples through large-scale performance testing (PT) using three artificially designed datasets. To evaluate the impact of bioinformatics analyses, they were divided into three key steps: reads pre-processing, virus-isolate identification, and variant calling. Each step was evaluated independently through an original, PT design including discussion and validation between participants at each step. Overall, this work underlines key parameters influencing SNPs detection and proposes recommendations for reliable variant calling for plant viruses. The identification of the closest reference, mapping parameters and manual validation of the detection were recognized as the most impactful analysis steps for the success of the SNPs detections. Strategies to improve the prediction of SNPs are also discussed

Biological and Genetic Characterization of Physostegia Chlorotic Mottle Virus in Europe Based on Host Range, Location, and Time.

peer reviewedApplication of high throughput sequencing (HTS) technologies enabled the first identification of Physostegia chlorotic mottle virus (PhCMoV) in 2018 in Austria. Subsequently, PhCMoV was detected in Germany and Serbia on tomatoes showing severe fruit mottling and ripening anomalies. We report here how prepublication data-sharing resulted in an international collaboration across eight laboratories in five countries, enabling an in-depth characterization of PhCMoV. The independent studies converged toward its recent identification in eight additional European countries and confirmed its presence in samples collected 20 years ago (2002). The natural plant host range was expanded from two to nine species across seven families, and we confirmed the association of PhCMoV presence with severe fruit symptoms on economically important crops such as tomato, eggplant, and cucumber. Mechanical inoculations of selected isolates in the greenhouse established the causality of the symptoms on a new indexing host range. In addition, phylogenetic analysis showed a low genomic variation across the 29 near-complete genome sequences available. Furthermore, a strong selection pressure within a specific ecosystem was suggested by nearly identical sequences recovered from different host plants through time. Overall, this study describes the European distribution of PhCMoV on multiple plant hosts, including economically important crops on which the virus can cause severe fruit symptoms. This work demonstrates how to efficiently improve knowledge on an emergent pathogen by sharing HTS data and provides a solid knowledge foundation for further studies on plant rhabdoviruses.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.European Union’s Horizon 2020 Research and Innovation Progra