Project description:A detection system based on a multiplex reverse transcription (RT) polymerase chain reaction (PCR) was developed to simultaneously identify multiple viruses in the lily plant. The most common viruses infecting lily plants are the cucumber mosaic virus (CMV), lily mottle virus (LMoV), lily symptomless virus (LSV). Leaf samples were collected at lily-cultivation facilities located in the Kangwon province of Korea and used to evaluate the detection system. Simplex and multiplex RT-PCR were performed using virus-specific primers to detect single-or mixed viral infections in lily plants. Our results demonstrate the selective detection of 3 different viruses (CMV, LMoV and LSV) by using specific primers as well as the potential of simultaneously detecting 2 or 3 different viruses in lily plants with mixed infections. Three sets of primers for each target virus, and one set of internal control primers were used to evaluate the detection system for efficiency, reliability, and reproducibility.
Project description:<h4>Background</h4>The lily is a perennial flowering plant belonging to the genus Lilium in the family Liliaceae. Most cultivated lily plants are propagated by bulbs. Therefore, numerous lily bulbs are frequently infected by diverse viruses causing viral diseases. To date, no study has examined the viromes of plants of one type with identical genetic backgrounds collected from different geographical regions.<h4>Results</h4>Here, we examined different viromes of the lily cultivar "Sorbonne" using 172 gigabytes of transcriptome data composed of 23 libraries from four different projects for the cultivar "Sorbonne." We identified 396 virus-associated contigs from all but one library. We identified six different viruses, including Plantago asiatica mosaic virus (PlAMV), Cucumber mosaic virus (CMV), Lily symptomless virus (LSV), Tulip virus X (TVX), Lily mottle virus (LMoV), and Tobacco rattle virus (TRV). Of them, PlAMV was the most common virus infecting the lily. Scale and flower samples possessed a high number of virus-associated reads. We assembled 32 nearly complete genomes for the six identified viruses possessing the polyadenylate tails. Genomes of all six viruses were highly conserved in the lily cultivar "Sorbonne" based on mutation analysis. We identified defective RNAs from LSV, TVX, and PlAMV localized in the triple gene block region. Phylogenetic analyses showed that virus genomes are highly correlated with geographical regions and host plants.<h4>Conclusions</h4>We conducted comprehensive virome analyses of a single lily cultivar, "Sorbonne," using transcriptome data. Our results shed light on an array of lily virome-associated topics, including virus identification, the dominant virus, virus accumulation in different plant tissues, virus genome assembly, virus mutation, identification of defective RNAs, and phylogenetic relationships of identified viruses. Taken together, we provide very useful methods and valuable results that can be applied in other virome-associated studies.
Project description:Background:Plums are a kind of stone fruit, a category that includes peaches, cherries, apricots, and almonds. In Korea, Japanese plum trees are usually cultivated as they best suit the climate. To date, there have been few studies in Korea on viruses infecting plum trees compared to those infecting peach trees. Methods:To identify viruses and viroids infecting plum trees, we collected leaf samples from six different plum cultivars and subjected them to RNA-sequencing (RNA-seq). Six different plum transcriptomes were de novo assembled using the Trinity assembler followed by BLAST searching against a viral reference database. Results:We identified hop stunt viroid (HSVd) and six viruses, including apple chlorotic leaf spot virus (ACLSV), little cherry virus-1 (LChV-1), peach virus D (PeVD), peach leaf pitting-associated virus (PLPaV), plum bark necrosis stem pitting-associated virus (PBNSPaV), and prunus necrotic ringspot virus (PNRSV), from six plum cultivars by RNA-seq. RT-PCR confirmed the infection of HSVd and three viruses-ACLSV, PBNSPaV, and PNRSV-in plum trees. However, RT-PCR demonstrated that plum trees in this study were not infected by LChV-1, PeVD, or PLPaV. It is likely that the three viruses LChV-1, PeVD, and PLPaV as identified by RNA-seq were contaminants from other peach libraries caused by index misassignment, which suggests that careful confirmation by other methods should be carried out in next-generation sequencing (NGS)-based virus identification. Taken together, we identified a viroid and three viruses infecting plum trees in Korea.
Project description:Whole-genome sequencing of infectious agents enables the identification and characterization of emerging viruses. The MinION device is a portable sequencer that allows real-time sequencing in fields or hospitals. <i>Hantaan orthohantavirus</i> (Hantaan virus, HTNV), harbored by <i>Apodemus agrarius</i>, causes hemorrhagic fever with renal syndrome (HFRS) and poses a critical public health threat worldwide. In this study, we aimed to evaluate the feasibility of using nanopore sequencing for whole-genome sequencing of HTNV from samples having different viral copy numbers. Amplicon-based next-generation sequencing was performed in <i>A. agrarius</i> lung tissues collected from the Republic of Korea. Genomic sequences of HTNV were analyzed based on the viral RNA copy numbers. Amplicon-based nanopore sequencing provided nearly full-length genomic sequences of HTNV and showed sufficient read depth for phylogenetic analysis after 8 h of sequencing. The average identity of the HTNV genome sequences for the nanopore sequencer compared to those of generated from Illumina MiSeq revealed 99.8% (L and M segments) and 99.7% (S segment) identities, respectively. This study highlights the potential of the portable nanopore sequencer for rapid generation of accurate genomic sequences of HTNV for quicker decision making in point-of-care testing of HFRS patients during a hantavirus outbreak.
Project description:The sweet potato in the family Convolvulaceae is a dicotyledonous perennial plant. Here, we conducted a comprehensive sweet potato virome study using 10 different libraries from eight regions in Korea and two different sweet potato cultivars by RNA-Sequencing. Comprehensive bioinformatics analyses revealed 10 different virus species infecting sweet potato. Moreover, we identified two novel viruses infecting sweet potato referred to as Sweet potato virus E (SPVE) in the genus Potyvirus and Sweet potato virus F (SPVF) in the genus Carlavirus. Of the identified viruses, Sweet potato feathery mottle virus (SPFMV) was the dominant virus followed by Sweet potato virus C (SPVC) and SPVE in Korea. We obtained a total of 30 viral genomes for eight viruses. Our phylogenetic analyses showed many potyvirus isolates are highly correlated with geographical regions. However, two isolates of SPFMV and a single isolate of Sweet potato virus G (SPVG) were genetically distant from other known isolates. The mutation rate was the highest in SPFMV followed by SPVC and SPVG. Two different sweet potato cultivars, Beni Haruka and Hogammi, were infected by seven and five viruses, respectively. Taken together, we provide a complete list of viruses infecting sweet potato in Korea and diagnostic methods.
Project description:<i>Lilium tsingtauense,</i> known as 'Korean wheel lily' and 'Twilight lily', is a lily species naturally distributed in Korea. The complete chloroplast genome sequences of <i>L. tsingtauense</i> was obtained by <i>de novo</i> assembly using whole-genome next-generation sequencing data. The chloroplast genome of <i>L. tsingtauense</i> was 152,710?bp in length and consisted of four distinct regions, such as large single-copy region (82,059?bp), small single-copy region (17,619?bp) and a pair of inverted repeat regions (26,516?bp). The genome contained a total of 113 genes, including 79 protein-coding genes, 30 tRNA genes and 4 rRNA genes. Phylogenetic analysis with the reported chloroplast genomes revealed that <i>L. tsingtauense</i> is most closely related to <i>Lilium hansonii</i>, a lily species native to Korea.
Project description:Potato virus Y (PVY) is the most economically important virus infecting cultivated potato (Solanum tuberosum L.). Accurate diagnosis is crucial to regulate the trade of tubers and for the sanitary selection of plant material for propagation. However, high genetic diversity of PVY represents a challenge for the detection and classification of isolates. Here, the diversity of Irish PVY isolates from a germplasm collection and commercial sites was investigated using conventional molecular and serological techniques. Recombinant PVY isolates were prevalent, with PVYNTNa being the predominant genotype. In addition, we evaluated Nanopore sequencing to detect and reconstruct the whole genome sequence of four viruses (PVY, PVX, PVS, PLRV) and five PVY genotypes in a subset of eight potato plants. De novo assembly of Nanopore sequencing reads produced single contigs covering greater than 90% of the viral genome and sharing greater than 99.5% identity to the consensus sequences obtained with Illumina sequencing. Interestingly, single near full genome contigs were obtained for different isolates of PVY co-infecting the same plant. Mapping reads to available reference viral genomes enabled us to generate near complete genome sequences sharing greater than 99.90% identity to the Illumina-derived consensus. This is the first report describing the use of Oxford Nanopore's MinION to detect and genotype potato viruses. We reconstructed the genome of PVY and other RNA viruses; indicating the technologies potential for virus detection in potato production systems, and for the study of genetic diversity of highly heterogeneous viruses such as PVY.
Project description:Metagenomic next-generation sequencing (mNGS), the shotgun sequencing of RNA and DNA from clinical samples, has proved useful for broad-spectrum pathogen detection and the genomic surveillance of viral outbreaks. An additional target enrichment step is generally needed for high-sensitivity pathogen identification in low-titre infections, yet available methods using PCR or capture probes can be limited by high cost, narrow scope of detection, lengthy protocols and/or cross-contamination. Here, we developed metagenomic sequencing with spiked primer enrichment (MSSPE), a method for enriching targeted RNA viral sequences while simultaneously retaining metagenomic sensitivity for other pathogens. We evaluated MSSPE for 14 different viruses, yielding a median tenfold enrichment and mean 47% (±16%) increase in the breadth of genome coverage over mNGS alone. Virus detection using MSSPE arboviral or haemorrhagic fever viral panels was comparable in sensitivity to specific PCR, demonstrating 95% accuracy for the detection of Zika, Ebola, dengue, chikungunya and yellow fever viruses in plasma samples from infected patients. Notably, sequences from re-emerging and/or co-infecting viruses that have not been specifically targeted a priori, including Powassan and Usutu, were successfully enriched using MSSPE. MSSPE is simple, low cost, fast and deployable on either benchtop or portable nanopore sequencers, making this method directly applicable for diagnostic laboratory and field use.
Project description:Currently, next generation sequencing (NGS) is the mainly used approach for identification and monitorization of viruses with a potential public health threat in clinical and environmental samples. To facilitate detection in NGS, the sequence-independent, single-primer-amplification (SISPA) is an effective tool for enriching virus sequences. We performed a preliminary assessment of SISPA-nanopore sequencing as a potential approach for screening tick-borne viruses in six specimens with detectable Crimean-Congo hemorrhagic fever virus (CCHFV) and Jingmen tick virus (JMTV) sequences. A comparison of unbiased NGS and SISPA followed by nanopore sequencing was carried out in 4 specimens with single and pooled ticks. The approach was further used for genome sequencing in culture-grown viruses. Overall, total/virus-specific read counts were significantly elevated in cell culture supernatants in comparison to single or pooled ticks. Virus genomes could be successfully characterized by SISPA with identities over 99%. Genome coverage varied according to the segment and total read count. Base calling errors were mainly observed in tick specimens and more frequent in lower viral loads. Culture-grown viruses were phylogenetically-related to previously-reported local viruses. In conclusion, the SISPA + nanopore sequencing was successful in generating data comparable to NGS and will provide an effective tool for broad-range virus detection in ticks.
Project description:Asian pear (Pyrus pyrifolia) is a widely cultivated and commercially important fruit crop, which is occasionally subject to severe economic losses due to latent viral infections. Thus, the aim of the present study was to examine and provide a comprehensive overview of virus populations infecting a major pear cultivar ('Singo') in Korea. From June 2017 to October 2019, leaf samples (n = 110) of pear trees from 35 orchards in five major pear-producing regions were collected and subjected to RNA sequencing. Most virus-associated contigs matched the sequences of known viruses, including apple stem grooving virus (ASGV) and apple stem pitting virus (ASPV). However, some contigs matched the sequences of apple green crinkle-associated virus and cucumber mosaic virus. In addition, three complete or nearly complete genomes were constructed based on transcriptome data and subjected to phylogenetic analyses. Based on the number of virus-associated reads, ASGV and ASPV were identified as the dominant viruses of 'Singo.' The present study describes the virome of a major pear cultivar in Korea, and looks into the diversity of viral communities in this cultivar. This study can provide valuable information on the complexity of genetic variability of viruses infecting pear trees.