Viroids are the smallest infectious pathogens known, consisting solely of short strands of circular, single-stranded RNA without protein coats. They are mostly plant pathogens, some of which are of economical importance. Viroid genomes are extremely small in size, ranging from 246 to 467 nucleobases.  In comparison, the genome of the smallest known viruses capable of causing an infection by themselves are around 2,000 nucleobases in size. The human pathogen hepatitis D virus is a defective RNA virus similar to viroids.
Viroids, the first known representatives of a new domain of "sub-viral pathogens," were discovered, initially characterized, and named by Theodor Otto Diener, plant pathologist at the U.S Department of Agriculture's Research Center in Beltsville, Maryland, in 1971. The first viroid to be identified was Potato spindle tuber viroid (PSTVd). Some 33 species have been identified.
Viroids do not code for any protein. Viroid's replication mechanism uses RNA polymerase II, a host cell enzyme normally associated with synthesis of messenger RNA from DNA, which instead catalyzes "rolling circle" synthesis of new RNA using the viroid's RNA as template. Some viroids are ribozymes, having catalytic properties which allow self-cleavage and ligation of unit-size genomes from larger replication intermediates.
With Diener’s 1989 hypothesis that viroids may represent “living relics” from a hypothetical, ancient, and non-cellular RNA world before the evolution of DNA or protein, viroids have attained significance beyond plant virology to evolutionary biology by representing the most plausible molecules capable of explaining crucial intermediate steps in the evolution of life from inanimate matter. Although Diener’s hypothesis lay dormant for 25 years, in 2014 it was resurrected, and its plausibility further enhanced by additional characteristics of viroids and viroid-like satellite viruses.
- Family Pospiviroidae
- Genus Pospiviroid; type species: Potato spindle tuber viroid ; 356–361 nucleotides(nt) 
- Genus Pospiviroid; type species: Citrus exocortis ; 368–467 nt 
- Genus Hostuviroid; type species: Hop stunt viroid ; 294–303 nt 
- Genus Cocadviroid; type species: Coconut cadang-cadang viroid; 246–247 nt
- Genus Apscaviroid; type species: Apple scar skin viroid ; 329–334 nt 
- Genus Coleviroid; type species: Coleus blumei viroid 1 ; 248–251 nt 
- Family Avsunviroidae
Viroid infections are transmitted by cross contamination following mechanical damage to plants as a result of horticultural or agricultural practices. Some are transmitted by aphids and they can also be transferred from plant to plant by leaf contact.
Viroids replicate in the nucleus (Pospiviroidae) or chloroplasts (Avsunviroidae) of plant cells in three steps through an RNA-based mechanism. They require RNA polymerase II, a host cell enzyme normally associated with synthesis of messenger RNA from DNA, which instead catalyzes "rolling circle" synthesis of new RNA using the viroid as template. Some viroids are ribozymes, having catalytic properties which allow self-cleavage and ligation of unit-size genomes from larger replication intermediates.
Viroids and RNA silencing
There has long been uncertainty over how viroids are able to induce symptoms in plants without encoding any protein products within their sequences. Evidence now suggests that RNA silencing is involved in the process. First, changes to the viroid genome can dramatically alter its virulence.This reflects the fact that any siRNAs produced would have less complementary base pairing with target messenger RNA. Secondly, siRNAs corresponding to sequences from viroid genomes have been isolated from infected plants. Finally, transgenic expression of the noninfectious hpRNA of potato spindle tuber viroid develops all the corresponding viroid-like symptoms. This evidence indicates that when viroids replicate via a double stranded intermediate RNA, they are targeted by a dicer enzyme and cleaved into siRNAs that are then loaded onto the RNA-induced silencing complex. The viroid siRNAs contain sequences capable of complementary base pairing with the plant's own messenger RNAs, and induction of degradation or inhibition of translation causes the classic viroid symptoms.
Living relics of the RNA World?
Diener’s 1989 hypothesis proposed that unique properties of viroids make viroids more plausible candidates than introns, or other RNAs considered in the past, as possible “living relics” of a hypothetical, pre-cellular RNA world. If so, viroids have attained potential significance beyond plant pathology to evolutionary biology, by representing the most plausible macromolecules known capable of explaining crucial intermediate steps in the evolution of life from inanimate matter. Diener's hypothesis lay dormant until 2014 when it was resurrected in a review article by Flores et al. which summarized Diener’s arguments supporting his hypothesis and conflated them with two additional ones (numbers 2 and 4 below). These properties are:
- viroid’s small size, imposed by error-prone replication;
- their high guanine and cytosine content, which increases stability and replication fidelity;
- their circular structure, which assures complete replication without genomic tags;
- existence of structural periodicity, which permits modular assembly into enlarged genomes;
- their lack of protein-coding ability, consistent with a ribosome-free habitat; and
- replication mediated in some by ribozymes—the fingerprint of the RNA world.
In the 1920s, symptoms of a previously unknown potato disease were noticed in farmers’ New York and New Jersey fields. Because tubers on affected plants become elongated and misshaped, they named it the potato spindle tuber disease.
The symptoms appeared on plants onto which pieces from affected plants had been budded—indicating that the disease was caused by a transmissible pathogenic agent. However, a fungus or bacterium could not be found consistently associated with symptom-bearing plants, and therefore, it was assumed the disease was caused by a virus. Despite numerous attempts over the years to isolate and purify the assumed virus, using increasingly sophisticated methods, these were unsuccessful when applied to extracts from potato spindle tuber disease-afflicted plants.
In 1971 Theodor O. Diener showed that the agent was not a virus, but a totally unexpected novel type of pathogen, one-80th the size of typical viruses, for which he proposed the term “viroid.” Parallel to agriculture-directed studies, more basic scientific research elucidated many of viroids’ physical, chemical, and macromolecular properties. Viroids were shown to consist of short stretches (a few hundred nucleobases) of single-stranded RNA and, unlike viruses, not to have a protein coat. Compared with other infectious plant pathogens, viroids are extremely small in size, ranging from 246 to 467 nucleobases; they thus consist of fewer than 10,000 atoms. In comparison, the genomes of the smallest known viruses capable of causing an infection by themselves are around 2,000 nucleobases long.
In 1976, Sänger et al. presented evidence that potato spindle tuber viroid is a “single-stranded, covalently closed, circular RNA molecule, existing as a highly base-paired rod-like structure"—believed to be the first such molecule described. Circular RNA, unlike linear RNA, forms a covalently closed continuous loop, in which the 3' and 5' ends present in linear RNA molecules have been joined together. Sänger et al. also provided evidence for the true circularity of viroids by finding that the RNA could not be phosphorylated at the 5’ terminus. Then, in other tests, they failed to find even one free 3’ end, which ruled out the possibility of the molecule having two 3’ ends. Viroids thus are true circular RNAs.
The single-strandedness and circularity of viroids was confirmed by electron microscopy and the complete nucleotide sequence of potato spindle tuber viroid was determined in 1978 by Gross et al. PSTV was the first pathogen of a eukaryotic organism for which the complete molecular structure has been established. Over thirty plant diseases have since been identified as viroid-, not virus-caused, as had been assumed.
- Lewin, Benjamin.; Krebs, Jocelyn E.; Kilpatrick, Stephen T.; Goldstein, Elliott S.; Lewin, Benjamin. Genes IX. (2011). Lewin's genes. Sudbury, Mass.: Jones and Bartlett. p. 23. ISBN 9780763766320.
- Alves, C; Branco, C; Cunha, C (2013). "Hepatitis delta virus: A peculiar virus". Advances in Virology 2013: 560105. doi:10.1155/2013/560105. PMC 3807834. PMID 24198831.
- Diener TO (August 1971). "Potato spindle tuber "virus". IV. A replicating, low molecular weight RNA". Virology 45 (2): 411–28. doi:10.1016/0042-6822(71)90342-4. PMID 5095900.
- "ARS Research Timeline – Tracking the Elusive Viroid". 2006-03-02. Retrieved 2007-07-18.
- Tsagris EM, de Alba AE, Gozmanova M, Kalantidis K; Martínez De Alba; Gozmanova; Kalantidis (September 2008). "Viroids". Cell. Microbiol. 10 (11): 2168–79. doi:10.1111/j.1462-5822.2008.01231.x. PMID 18764915.
- Daròs JA, Elena SF, Flores R; Elena; Flores (2006). "Viroids: an Ariadne's thread into the RNA labyrinth". EMBO Rep. 7 (6): 593–8. doi:10.1038/sj.embor.7400706. PMC 1479586. PMID 16741503.
- Diener, T.O. (1989). "Circular RNAs: Relics of precellular evolution?". PNAS 86 (23): 9370–9374. Bibcode:1989PNAS...86.9370D. doi:10.1073/pnas.86.23.9370. PMC 298497. PMID 2480600. Retrieved November 1, 2014.
- Flores, R.; Gago-Zachert, S.; Serra, P.; Sanjuan, R.; Elena, S.F. (June 18, 2014). "Viroids: Survivors from the RNA World?". Annual Review of Microbiology 68: 395–414. doi:10.1146/annurev-micro-091313-103416. PMID 25002087. Retrieved December 7, 2014.
- Brian W. J. Mahy, Marc H. V. Van Regenmortel (ed.). Desk Encyclopedia of Plant and Fungal Virology. Academic Press. pp. 71–81. ISBN 978-0123751485.
- De Bokx, J. A. and P. G. M. Piron (1981). "Transmission of potato spindle tuber viroid by aphids." Netherlands Journal of Plant Pathology 87(2): 31-34. ISSN: 0929-1873
- Flores R, Serra P, Minoia S, Di Serio F, Navarro B. "Viroids: from genotype to phenotype just relying on RNA sequence and structural motifs." Front Microbiol. 18, 217 2012. doi: 10.3389/fmicb.2012.00217. eCollection 2012.
- Daros JA, Elena SF, Flores R "Viroids: an Ariadne's thread into the RNA labyrinth.Review. EMBO Rep.7(6):593-8. 2006. PMID:16741503
- Hammond RW. "Analysis of the virulence modulating region of potato spindle tuber viroid (PSTVd) by site-directed mutagenesis." Virology.187,654-62 (1992)PMID:1546460
- Wang MB, Bian XY, Wu LM et al. (2004). "On the role of RNA silencing in the pathogenicity and evolution of viroids and viral satellites". Proc. Natl. Acad. Sci. U.S.A. 101 (9): 3275–80. Bibcode:2004PNAS..101.3275W. doi:10.1073/pnas.0400104101. PMC 365780. PMID 14978267.
- Pallas, V; Martinez, G; Gomez, G (2012). "Antiviral Resistance in Plants:The Interaction Between Plant Viroid-Induced Symptoms and RNA Silencing". Methods in molecular biology (Clifton, N.J.). Methods in Molecular Biology 894: 323–43. doi:10.1007/978-1-61779-882-5_22. ISBN 978-1-61779-881-8. PMID 22678590.
- Zimmer, C (September 25, 2014). "A Tiny Emissary From the Ancient Past". New York Times. Retrieved November 22, 2014.
- Owens, R.A. and J.Th.J. Verhoeven. 2009. Potato spindle tuber. The Plant Health Instructor. DOI: 10.1094/PHI-I-2009-0804-01
- "ARS Research Timeline – Tracking the Elusive Viroid". 2006-03-02. Retrieved 2014-11-22.
- Pommerville, Jeffrey C (2014). Fundamentals of Microbiology. Burlington, MA: Jones and Bartlett Learning. p. 482. ISBN 978-1-284-03968-9.
- Sänger, HL; Klotz, G; Riesner, D; Gross, HJ; Kleinschmidt, AK (1976) . "Single-stranded covalently closed circular RNA molecules, existing as highly base-paired rod-like structures". Proc.Natl.Acad.Sci.USA 73 (11): 3852–56. PMID 1069269
- Sogo, JM, Koller T, Diener TO. (1973) “Potato spindle tuber viroid. X. Visualization and size determination by electron microscopy.” Virology.(1973) 55(1):70–80.PMID: 4728831
- Gross, H. J.; Domdey, H; Lossow, C; Jank, P; Raba, M; Alberty, H; Sänger, H. L. (1978). "Nucleotide sequence and secondary structure of potato spindle tuber viroid". Nature 273 (5659): 203–8. PMID 643081.
- Hammond, R. W. and Owens, R. A. 2006. Viroids: New and Continuing Risks for Horticultural and Agricultural Crops. Online. APSnet Features. doi: 10.1094/APSnetFeature-2006-1106