Next-generation high throughput sequencing technologies became available at the onset of the 21st century. They provide a highly efficient, rapid, and low cost DNA sequencing platform beyond the reach of the standard and traditional DNA sequencing technologies developed in the late 1970s. They are continually improved to become faster, more efficient and cheaper. They have been used in many fields of biology since 2004. In 2009, next-generation sequencing (NGS) technologies began to be applied to several areas of plant virology including virus/viroid genome sequencing, discovery and detection, ecology and epidemiology, replication and transcription. Identification and characterization of known and unknown viruses and/or viroids in infected plants are currently among the most successful applications of these technologies. It is expected that NGS will play very significant roles in many research and non-research areas of plant virology.

Next-generation sequencing (NGS) has been applied to plant virology since 2009. NGS provides highly efficient, rapid, low cost DNA, or RNA high-throughput sequencing of the genomes of plant viruses and viroids and of the specific small RNAs generated during the infection process. These small RNAs, which cover frequently the whole genome of the infectious agent, are 21–24 nt long and are known as vsRNAs for viruses and vd-sRNAs for viroids. NGS has been used in a number of studies in plant virology including, but not limited to, discovery of novel viruses and viroids as well as detection and identification of those pathogens already known, analysis of genome diversity and evolution, and study of pathogen epidemiology. The genome engineering editing method, clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has been successfully used recently to engineer resistance to DNA geminiviruses (family, Geminiviridae) by targeting different viral genome sequences in infected Nicotiana benthamiana or Arabidopsis plants. The DNA viruses targeted include tomato yellow leaf curl virus and merremia mosaic virus (begomovirus); beet curly top virus and beet severe curly top virus (curtovirus); and bean yellow dwarf virus (mastrevirus). The technique has also been used against the RNA viruses zucchini yellow mosaic virus, papaya ringspot virus and turnip mosaic virus (potyvirus) and cucumber vein yellowing virus (ipomovirus, family, Potyviridae) by targeting the translation initiation genes eIF4E in cucumber or Arabidopsis plants. From these recent advances of major importance, it is expected that NGS and CRISPR-Cas technologies will play a significant role in the very near future in advancing the field of plant virology and connecting it with other related fields of biology.

This comprehensive volume presents indispensable and up-to-date information on viroids and viroid diseases. It provides a single source of information on the properties of viroids, the economic impact of viroid diseases, and methods for their detection and control. It examines the diseases associated with different plant species, the geographic distribution and epidemiology of viroids, diseases of possible viroid etiology, and the future applications of viroids. Viroids examines the biology of viroids, molecular characteristics, localization and movement, replication, pathogenesis, viroids and gene silencing, classification, viroid-like satellite RNAs, detection of viroids using bioamplification hosts, biological indexing, polyacrylamide gel electrophoresis, molecular hybridisation and polymerase chain reaction. The book looks at the geographical distribution and epidemiology of viroids in North America, Australasia, China, Japan, Europe, the Middle East, Africa, South America, and at the global level. It covers the control of viroids including quarantine of imported germplasm, availability of viroid-tested propagation materials, thermotherapy, tissue culture, and other conventional strategies as well as biotechnological control approaches. Special topics such as ribozyme reaction of viroids and economic advantages of viroid infection are also included. Other chapters summarise the current state of knowledge concerning viroid diseases of the crop in question and aspects of the natural history of viroids in horticulture. Among the crops covered are potato, tomato, tobacco, cucumber, pome fruits, stone fruits, avocado, citrus, grapevines, hop, chrysanthemum, coleus, columnea, and coconut palm. The four eminent editors of this watershed volume have assembled an international group of more than 70 scientists who have substantial experience with viroids and viroid diseases. They have produced a cohesive and comprehensive work that can be used by students, researchers, extension agents, and regulators. It may also be of a great value to science managers, policy makers, and industries in formulating policies and products to obtain viroid-free plants and control viroid diseases. The information on plant quarantine and certification programs will help anyone concerned with the safe movement of plant material across international boundaries or within a single country.

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Peach latent mosaic viroid (PLMVd) is widely distributed (approximately 55%) in peach germplasm from Europe, Asia, North America, and South America. PLMVd, or a closely related viroid, was occasionally detected in cherry, plum, and apricot germplasm from countries in Europe or Asia. The cherry isolate of PLMVd is 337 nucleotides in length and is 91 to 92% homologous to PLMVd isolates from peach. Molecular hybridization experiments demonstrated that PLMVd is not related to the agent of peach mosaic disease. PLMVd was readily transmitted (50 to 70%) by contaminated blades to green shoots and lignified stems of peach GF-305 plants. These results indicate that the viroid may be transmitted in orchards with contaminated pruning equipment.

Hepatitis C virus (HCV) is a major cause of acute and chronic hepatitis with over 180 million cases worldwide. Vaccine development for HCV has been difficult. Presently, the virus cannot be grown in tissue culture and there is no vaccine or effective therapy against this virus. In this research, we describe the development of an experimental plant-derived subunit vaccine against HCV. A tobamoviral vector was engineered to encode a consensus sequence of hypervariable region 1 (HVR1), a potential neutralizing epitope of HCV, genetically fused to the C-terminal of the B subunit of cholera toxin (CTB). This epitope was selected from among the amino acid sequences of HVR1 "mimotopes" previously derived by phage display technology. The nucleotide sequence encoding this epitope was designed utilizing optimal plant codons. This mimotope is capable of inducing cross-neutralizing antibodies against different variants of the virus. Plants infected with recombinant tobacco mosaic virus (TMV) engineered to express the HVR1/CTB chimeric protein, contained intact TMV particles and produced the HVR1 consensus peptide fused to the functionally active, pentameric B subunit of cholera toxin. Plant-derived HVR1/CTB reacted with HVR1-specific monoclonal antibodies and immune sera from individuals infected with virus from four of the major genotypes of HCV. Intranasal immunization of mice with a crude plant extract containing the recombinant HVR1/CTB protein elicited both anti-CTB serum antibody and anti-HVR1 serum antibody which specifically bound to HCV virus-like particles. Using plant-virus transient expression to produce this unique chimeric antigen will facilitate the development and production of an experimental HCV vaccine. A plant-derived recombinant HCV vaccine can potentially reduce expenses normally associated with production and delivery of conventional vaccines.

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