Viroid-like RNA Research Articles

Novel Fig-Associated Viroid-Like RNAs Containing Hammerhead Ribozymes in Both Polarity Strands Identified by High-Throughput Sequencing.

Based on high-throughput sequencing (HTS) data, the existence of viroid-like RNAs (Vd-LRNAs) associated with fig trees grown in the Hawaiian Islands has been predicted. One of these RNAs has been characterized as a circular RNA ranging in size from 357 to 360 nucleotides. Structural and biochemical features of this RNA, tentatively named fig hammerhead viroid-like RNA (FHVd-LR), markedly resemble those previously reported for several viroids and viroid-like satellite RNAs (Vd-LsatRNAs), which are non-protein-coding RNAs infecting their hosts autonomously and in combination with a helper virus, respectively. The full-length sequence of FHVd-LR variants was determined by RT-PCR, cloning, and sequencing. Despite a low global sequence identity with known viroids and Vd-LsatRNAs, FHVd-LR contains a hammerhead ribozyme (HRz) in each polarity strand. Northern blot hybridization assays identified the circular and linear forms of both polarity strands of FHVd-LR and showed that one strand, assigned the (+) polarity, accumulates at higher levels than the (−) polarity strand in vivo. The (+) polarity RNA assumes a rod-like secondary structure of minimal free energy with the conserved domains of the HRzs located in opposition to each other, a feature typical of several viroids and Vd-LRNAs. The HRzs of both FHVd-LR polarity strands were shown to be active in vitro during transcription, self-cleaving the RNAs at the predicted sites. These data, together with the sequence variability observed in the cloned and sequenced full-length variants, indicate that FHVd-LR is a novel viroid or Vd-LsatRNA. According to HTS data, the coexistence of FHVd-LR of different sizes in the same host cannot be excluded. The relationships of FHVd-LR with previously reported viroids and Vd-LsatRNAs, and the need to perform bioassays to conclusively clarify the biological nature of this circular RNA, are discussed.

Viral Small RNA-Based Screening of Malus spp. Reveals Citrus Leaf Blotch Virus Infection of Kaido Crab Apple in China

Viral Small RNA-Based Screening of <i>Malus</i> spp. Reveals Citrus Leaf Blotch Virus Infection of Kaido Crab Apple in China

A Plum Marbling Conundrum: Identification of a New Viroid Associated with Marbling and Corky Flesh in Japanese Plums.

Over the past 2 decades, fruit symptoms resembling a marbling pattern on the fruit skin or corking of the fruit flesh were observed on Japanese plums in South Africa, resulting in unmarketable fruit. The ability of high-throughput sequencing (HTS) to detect known and unknown pathogens was exploited by assaying affected and unaffected fruit tree accessions to identify the potential aetiological agent of marbling and/or corky flesh disease. In this study, it is shown that the disease is associated with a previously undescribed small RNA with typical viroid structural features. The potential viroid was the only pathological agent consistently detected in all symptomatic trees by HTS, and the association with the symptoms was confirmed in field surveys over two seasons. To date, this RNA was not detectable by RT-PCR in seedlings raised from seeds collected from infected trees. Although the autonomous replication of this viroid-like RNA was not proven, it was shown to be transmissible by grafting and associated with a range of symptoms that include marbling on the fruit skin, corky flesh, reduced fruit size, irregular shape, and uneven fruit surface depending on the cultivar. Moreover, the circular RNA genome, consisting of 317 nucleotides, strongly supports that this viroid-like RNA is most likely a viroid for which the name plum viroid I (PVd-I) is proposed. The primary structure of this viroid showed a less than 90% nucleotide sequence identity to viroids of the genus Apscaviroid, with which it has close phylogenetic relationships and shares conserved structural motifs.

Dendrobium viroid, a new monocot-infecting apscaviroid

Viroids are small circular RNA molecules which have been found to infect many dicot species. Only coconut cadang-cadang viroid and coconut tinangaja viroid have been reported so far to infect a monocot (coconut). Data mining in silico has proven an efficient approach to identify new viruses/viroids, and a systematic screen of public transcriptomic data revealed a 648 nucleotides (nt) sequence potentially representing a novel viroid-like RNA in a transcriptome shotgun assembly from Dendrobium officinale. This sequence contained two central conserved regions (CCRs) characteristic of members of the genus Apscaviroid, indicating that the viroid-like RNA is 324 nt in length. The infectivity of dimeric RNA transcripts generated by in vitro transcription of a synthetic cDNA, was demonstrated by directly injecting into the stems of young Dendrobium officinale plants. The presence of this novel viroid, tentatively designated as Dendrobium viroid (DVd), in the inoculated plants was confirmed by 2D-PAGE together with northern hybridization. DVd is predicted to have a rod-like secondary structure containing a CCR and a terminal conserved region (TCR), and phylogenetic analysis shows that it groups with the known members of the genus Apscaviroid. It is most closely related to citrus viroid V (56% nt identity). A field survey revealed a low DVd incidence (0.96%) in Dendrobium species in China. To our best knowledge, DVd is the only viroid known to infect orchids and the third one from monocotyledonous plants.

Analysis of the genetic diversity of genome sequences of variants of apple hammerhead viroid

A dataset of approximately 85 apple hammerhead viroid (AHVd) genome sequences was analysed to determine genetic diversity. Sequences analysed included 18 new AHVd variants detected in a 2017/2018 survey, as well as suitable sequences deposited in GenBank. Two phylogenetically distinct clades were observed. A range of indel events was identified with most AHVd sequences ranging in size from 433 to 435 nt. None of these events appear to affect the predicted secondary structure of AHVd, which was well supported by a prevalence of unambiguous covariation events and or substitutions resulting in the conversion of canonical to wobble sequences, or vice versa. All AHVd variants analysed possess the 13 nt that are conserved in most hammerheads of viroids and viroid-like satellite RNA, except at positions 146 and 149. No significant recombination event was identified.

First Report of Citrus viroid V Naturally Infecting Grapefruit and Calamondin Trees in California.

The Citrus Clonal Protection Program–National Clean Plant Network, University of California, Riverside (UCR), received citrus budwood samples in June 2016 from the California Department of Food and Agriculture (CDFA) for virus and viroid testing under the mandatory California (CA) §3701 Citrus Nursery Stock Pest Cleanliness Program. Samples originated from nonsymptomatic budwood sources, two redblush grapefruit (RG) (Citrus paradisi Macfadyen) and four variegated calamondin (VC) (C. madurensis Lour.), and were found in a nursery in Tulare County, CA. TRIzol-extracted RNA was tested with a SYBR green I reverse transcription quantitative polymerase chain reaction (RT-qPCR) for the universal detection of citrus viroids (Osman et al. 2017). Amplicons with melting temperatures similar to the Citrus viroid V (CVd-V) control (Vidalakis and Wang 2013) were produced in 50% of the RG and 25% of the VC samples. Citron (C. medica L.) Arizona 861-S-1 indicators were graft-inoculated with RG and VC blind buds (two citrons each) and maintained under warm greenhouse conditions (27 to 41°C). By 8 months, all citrons had developed mild stunting and leaf bending coupled with midvein necrosis, indicative of viroid infection. Sequential polyacrylamide gel electrophoresis of the symptomatic citrons identified circular and linear viroid-like RNAs with electrophoretic mobility similar to CVd-V. RT-PCR with primers specific for the full CVd-V genome (Wang et al. 2013) produced amplicons of the expected size of 294 bp from both the RG and VC samples and from the four indicator citrons. Cloning and sequencing of the RT-PCR CVd-V amplicons produced complete viroid genome sequences (GenBank MF477857 to MF477876). The RG isolates (MF477857 to MF477866) had 99% similarity with the KM isolate (JQ348924, Pakistan), with one nucleotide change (A₆₅ > U). The VC isolates (MF477867 to MF477876) had 99% similarity with the MO isolate (JQ348925, Pakistan), with two nucleotide changes (G₆₉ > A and A₂₅₁ > U). To further test the infectivity of the identified viroid, RNA extracts from the RG and VC graft-inoculated citrons were slash-inoculated onto four new citrons budded on rough lemon (C. jambhiri Lush.) rootstock. All citrons developed symptoms, and CVd-V was identified with RT-PCR, cloning, and sequencing. The CVd-V identification results were independently verified by CDFA’s Plant Pest Diagnostic Center, and the original source trees were destroyed. CVd-V was first reported in Spain (Serra et al. 2008a) and has been detected in various samples including a source from UCR, but the source of the UCR field material was not recorded (Serra et al. 2008b). This note reports the natural occurrence of CVd-V in CA and corroborates the CVd-VCᴬ variant report from the UCR source (Serra et al. 2008b). This note also demonstrates the importance of regulatory testing of propagative materials and elimination of infected citrus germplasm source trees to prevent the spread of viroids into commercial citrus.

Apple hammerhead viroid-like RNA is a bona fide viroid: Autonomous replication and structural features support its inclusion as a new member in the genus Pelamoviroid

Apple hammerhead viroid-like RNA (AHVd RNA) has been reported in different apple cultivars and geographic regions and, considering the presence of hammerhead ribozymes in both polarity strands, suspected to be either a viroid of the family Avsunviroidae or a viroid-like satellite RNA. Here we report that dimeric head-to-tail in vitro transcripts of a 433-nt reference variant of AHVd RNA from cultivar “Pacific Gala” are infectious when mechanically inoculated to apple, thus showing that this RNA is a bona fide viroid for which we have kept the name apple hammerhead viroid (AHVd) until its pathogenicity, if any, is better assessed. By combining thermodynamics-based predictions with co-variation analyses of the natural genetic diversity found in AHVd we have inferred the most likely conformations for both AHVd polarity strands in vivo, with that of the (+) polarity strand being stabilized by a kissing loop-interaction similar to those reported in peach latent mosaic viroid and chrysathemum chlorotic mottle viroid, the two known members of the genus Pelamoviroid (family Avsunviroidae). Therefore, AHVd RNA fulfills the biological and molecular criteria to be allocated to this genus, the members of which, intriguingly, display low global sequence identity but high structural conservation.

First Report of Grapevine hammerhead viroid-like RNA Infecting Grapevine (Vitis vinifera) in France

Grapevine hammerhead viroid-like RNA (GHVd) is a poorly characterized circular, single-stranded RNA molecule discovered through the bioinformatic analysis of small interfering RNAs of Italian grapevines of the Pinot noir variety (Wu et al., 2012). It is 375 nucleotides long and shares many structural features of viroids or of some small circular satellite RNA molecules. In particular, it has a predicted secondary structure similar to those of members of the Pelamoviroid genus with a branched region stabilized by a kissing-loop pseudoknot, and an extensively base-paired arm that encodes in its plus and minus strands self-cleaving hammerhead ribozymes (Wu et al., 2012; Giguere et al., 2014). Outside of conserved nucleotides in these hammerhead ribozymes, the primary sequence of GHVd has no significant similarities to any entries in Genbank. Besides the original Italian Pinot noir grapes, GHVd has been detected in Pinot noir plants of the 115 clone grown in California (Wu et al., 2012). Given that infectivit...

Identification of a viroid-like RNA in a lychee Transcriptome Shotgun Assembly

In the course of a systematic screen of plant transcriptomic data, the sequence of a novel viroid-like RNA was identified in four contigs in a recent Transcriptome Shotgun Assembly (TSA) from lychee in China. Portions of this sequence are closely related to the central conserved region (CCR) and terminal conserved region (TCR) present in members of the genus Apscaviroid, sequences that are important criteria for viroid classification. RT-PCR with two divergent adjacent primers amplified the full 304 nucleotide sequence of this viroid-like RNA which can be folded into the rod-like secondary structure that is a typical feature of viroids in the family Pospiviroidae. Northern-blot hybridization following denaturing PAGE and sequential polyacrylamide gel electrophoresis (sPAGE) revealed the presence of both circular and linear forms of the viroid-like RNA in lychee leaf tissue. Phylogenetic analysis indicates that this RNA groups with known members of the genus Apscaviroid with a maximum pairwise sequence identity of <62%. Field surveys revealed the presence of this viroid-like RNA in most lychee samples collected in China; however, it was not found in the accessions in a germplasm collection of lychees that originated from a wide range of other countries. None of the four herbaceous hosts and one woody host tested were susceptible to infection with the lychee viroid-like RNA via agroinoculation and RNA inoculation. The provisional name lychee viroid-like RNA (LVd-like RNA) is proposed for this tentative new species in the genus Apscaviroid.

Hepatitis Delta Virus: Replication Strategy and Upcoming Therapeutic Options for a Neglected Human Pathogen.

The human Hepatitis Delta Virus (HDV) is unique among all viral pathogens. Encoding only one protein (Hepatitis Delta Antigen; HDAg) within its viroid-like self-complementary RNA, HDV constitutes the smallest known virus in the animal kingdom. To disseminate in its host, HDV depends on a helper virus, the human Hepatitis B virus (HBV), which provides the envelope proteins required for HDV assembly. HDV affects an estimated 15–20 million out of the 240 million chronic HBV-carriers and disperses unequally in disparate geographical regions of the world. The disease it causes (chronic Hepatitis D) presents as the most severe form of viral hepatitis, leading to accelerated progression of liver dysfunction including cirrhosis and hepatocellular carcinoma and a high mortality rate. The lack of approved drugs interfering with specific steps of HDV replication poses a high burden for gaining insights into the molecular biology of the virus and, consequently, the development of specific novel medications that resiliently control HDV replication or, in the best case, functionally cure HDV infection or HBV/HDV co-infection. This review summarizes our current knowledge of HBV molecular biology, presents an update on novel cell culture and animal models to study the virus and provides updates on the clinical development of the three developmental drugs Lonafarnib, REP2139-Ca and Myrcludex B.

Molecular and phylogenetic identification of unique isolates of hammerhead viroid-like RNA from ‘Pacific Gala’ apple (Malus domestica) in Canada

Ten isolates of apple hammerhead viroid-like RNA (AHVd RNA) were detected by next-generation sequencing (NGS) and/or by RT-PCR analysis of apple (Malus domestica) ‘Pacific Gala’ (PG) from Canada. All consisted of 433 nucleotides in size, and their circular structure and sequence were supported by reverse transcription amplification with two separate pairs of abutted primers, cloning and sequencing. Two regions were defined for the computer-predicted secondary structure of AHVd RNA-PG isolates – a hammerhead region (HHR) and a loop-rich region (LRR). The HHR contained the core and flanking nucleotides typical of natural hammerhead ribozymes. The secondary structure associated with the LRR was distinct from that predicted for the corresponding region of AHVd RNA isolates previously described in ‘Fuji’ apple in China. However, both structures share conserved stem-loops in the LRR, identified as S1, S2, S3 and S4.

Hepatitis delta virus: insights into a peculiar pathogen and novel treatment options.

Chronic hepatitis D is the most severe form of viral hepatitis, affecting ∼20 million HBV-infected people worldwide. The causative agent, hepatitis delta virus (HDV), is a unique human pathogen: it is the smallest known virus; it depends on HBV to disseminate its viroid-like RNA; it encodes only one protein (HDAg), which has both structural and regulatory functions; and it replicates using predominantly host proteins. The failure of HBV-specific nucleoside analogues to suppress the HBV helper function, and the limitations of experimental systems to study the HDV life cycle, have impeded the development of HDV-specific drugs. Thus, the only clinical regimen for HDV is IFNα, which shows some efficacy but long-term virological responses are rare. Insights into the receptor-mediated entry of HDV, and the observation that HDV assembly requires farnesyltransferase, have enabled novel therapeutic strategies to be developed. Interference with entry, for example through blockade of the HBV-HDV-specific receptor sodium/taurocholate cotransporting polypeptide NTCP by Myrcludex B, and inhibition of assembly by blockade of farnesyltransferase using lonafarnib or nucleic acid polymers such as REP 2139-Ca, have shown promising results in phase II studies. In this Review, we summarize our knowledge of HDV epidemiology, pathogenesis and molecular biology, with a particular emphasis on possible future developments.

Engineering Periodic shRNA for Enhanced Silencing Efficacy.

RNA interference (RNAi) provides a versatile therapeutic approach via silencing of specific genes, particularly undruggable targets in cancer and other diseases. However, challenges in the delivery of small interfering RNA (siRNA) have hampered clinical translation. Polymeric or periodic short hairpin RNAs (p-shRNAs)-synthesized by enzymatic amplification of circular DNA-are a recent development that can potentially address these delivery barriers by showing improved stability and complexation to enable nanoparticle packaging. Here, we modify these biomacromolecules via structural and sequence engineering coupled with selective enzymatic digestion to generate an open-ended p-shRNA (op-shRNA) that is cleaved over ten times more efficiently to yield siRNA. The op-shRNA induces considerably greater gene silencing than p-shRNA in multiple cancer cell lines up to 9 days. Furthermore, its high valency and flexibility dramatically improve complexation with a low molecular weight polycation compared to monomeric siRNA. Thus, op-shRNA provides an RNAi platform that can potentially be packaged and efficiently delivered to disease sites with higher therapeutic efficacy.

In memory of Professor Biao Ding (1960-2015).

It is with profound sadness that we mourn the loss of Dr.Biao Ding,a full-professor in the Department of Molecular Genetics,Ohio State University(OSU),and a world-renowned plant biologist with leading authority in viroid research.He died suddenly on June 26,2015 in Prague,Czech Republic,while attending the International Conference on Viroids and Viroid-Like RNAs as a Keynote Speaker.Dr.Ding was born in 1960,in Kunming,Yunnan Province,China.He received his B.S.from Beijing Forestry University in1982,and was then selected as one of the first group of

First report of Dahlia latent viroid and Potato spindle tuber viroid mixed‐infection in commercial ornamental dahlia in Japan

Dahlia (Asteraceae) has been cultivated since 1841 in Japan. Two viroids belonging to the family Pospiviroidae, Potato spindle tuber viroid (PSTVd) and Dahlia latent viroid (DLVd), were identified separately from dahlia plants in Japan and in the Netherlands, respectively (Tsushima et al., 2011; Verhoeven et al., 3). Because DLVd was characterised as a novel species, the findings drew attention towards the study of DLVd infection in dahlia and its potential risk in domestic dahlia cultivation. A total of four dahlia samples infected with PSTVd-dahlia (Tsushima et al., 2) were collected, and total RNA was extracted from approximately 200 mg of leaf tissue using TRIzol® reagent (Life Technologies, Carlsbad, USA) according to manufacturer's instructions. RNA was separated in 2-dimensional polyacrylamide gel (2D-PAGE) (Schumacher et al., 1) and two specific bands appeared, one corresponding to PSTVd infection and the other being unknown (Fig. 1). The unknown circular RNA samples were gel-excised and first tested with PSTVd-specific primers using a two-step reverse-transcription-polymerase chain reaction (RT-PCR) (Tsushima et al., 2), with, however, negative results. Therefore, a DLVd-specific primer set, DLVd-P1 (5'-GGGGCTCCTCAGAGAGTCTC-3') and DLVd-P2 (5'-GGGGCAACTCCGAGAGTGCTG-3') (Verhoeven et al., 3), was then used in two-step RT-PCR for the unknown viroid-like circular RNAs. The amplified DNA bands of the expected size (~340 bp) from these positive samples were purified, ligated to pGEM®-T easy vector (Promega, Madison, USA) and sequenced. Identical 342 bp sequences were obtained from all four dahlia isolates (GenBank Accession No. LC036322), corresponding with DLVd reported from the Netherlands (JX263426). Furthermore, a total of 78 dahlia leaf samples were collected from fields around Japan, irrespective of disease symptoms, and verified by two-step RT-PCR using the same DLVd-specific and pospiviroid-specific primers (Tsushima et al., 2). Of these samples, DLVd infection was detected in 38, suggesting that DLVd is widespread in Japan. New PSTVd infection was not found. To evaluate the pathogenicity of DLVd and PSTVd, these viroids were co-inoculated into wild hop (Humulus lupulus var. cordifolius) and dahlia seedlings. DLVd alone was inoculated into members of the Asteraceae family, Chrysanthemum pacificum, Tithonia rotundifolia, Helianthus annuus and Glebionis coronaria. Inoculated dahlia seedlings were positive for both DLVd and PSTVd, but wild hop and the remaining members of Asteraceae were negative. Dahlia seedlings that were positive for DLVd and PSTVd showed mild leaf curling in the early stage of growth (Fig. 2). To the best of our knowledge, this is the first report of DLVd infecting commercial dahlia in Japan and also of the mixed-infection of DLVd and PSTVd in commercial dahlia plants. A possible negative synergistic interaction of DLVd and PSTVd during dahlia cultivation must be considered. This work was supported in part by Japan Society for the Promotion of Science KAKENHI grant no. 24380026. TT received Research Fellowship of the Japan Society for the Promotion of Science.

Discovery of replicating circular RNAs by RNA-seq and computational algorithms.

Replicating circular RNAs are independent plant pathogens known as viroids, or act to modulate the pathogenesis of plant and animal viruses as their satellite RNAs. The rate of discovery of these subviral pathogens was low over the past 40 years because the classical approaches are technical demanding and time-consuming. We previously described an approach for homology-independent discovery of replicating circular RNAs by analysing the total small RNA populations from samples of diseased tissues with a computational program known as progressive filtering of overlapping small RNAs (PFOR). However, PFOR written in PERL language is extremely slow and is unable to discover those subviral pathogens that do not trigger in vivo accumulation of extensively overlapping small RNAs. Moreover, PFOR is yet to identify a new viroid capable of initiating independent infection. Here we report the development of PFOR2 that adopted parallel programming in the C++ language and was 3 to 8 times faster than PFOR. A new computational program was further developed and incorporated into PFOR2 to allow the identification of circular RNAs by deep sequencing of long RNAs instead of small RNAs. PFOR2 analysis of the small RNA libraries from grapevine and apple plants led to the discovery of Grapevine latent viroid (GLVd) and Apple hammerhead viroid-like RNA (AHVd-like RNA), respectively. GLVd was proposed as a new species in the genus Apscaviroid, because it contained the typical structural elements found in this group of viroids and initiated independent infection in grapevine seedlings. AHVd-like RNA encoded a biologically active hammerhead ribozyme in both polarities, and was not specifically associated with any of the viruses found in apple plants. We propose that these computational algorithms have the potential to discover novel circular RNAs in plants, invertebrates and vertebrates regardless of whether they replicate and/or induce the in vivo accumulation of small RNAs.

Elucidation of the structures of all members of the Avsunviroidae family.

Viroids are small single-stranded RNA pathogens which cause significant damage to plants. As their nucleic acids do not encode for any proteins, they are dependant solely on their structure for their propagation. The elucidation of the secondary structures of viroids has been limited because of the exhaustive and time consuming nature of classic approaches. Here, the method of high-throughput selective 2'-hydroxyl acylation analysed by primer extension (hSHAPE) has been adapted to probe the viroid structure. The data obtained using this method were then used as input for computer-assisted structure prediction using RNA structure software in order to determine the secondary structures of the RNA strands of both (+) and (–) polarities of all Avsunviroidae members, one of the two families of viroids. The resolution of the structures of all of the members of the family provides a global view of the complexity of these RNAs. The structural differences between the two polarities, and any plausible tertiary interactions, were also analysed. Interestingly, the structures of the (+) and (–) strands were found to be different for each viroid species. The structures of the recently isolated grapevine hammerhead viroid-like RNA strands were also solved. This species shares several structural features with the Avsunviroidae family, although its infectious potential remains to be determined.To our knowledge, this article represents the first report of the structural elucidation of a complete family of viroids.

Viroid-like RNAs from cherry trees affected by leaf scorch disease: further data supporting their association with mycoviral double-stranded RNAs

Cherry trees from Spain affected by cherry leaf scorch (CLS), a fungal disease proposed to be caused by Apiognomonia erythrostoma, show symptoms (translucent-chlorotic leaf spots evolving into rusty areas) very similar to those of cherry chlorotic rusty spot disease (CCRS) and Amasya cherry disease, reported in Italy and Turkey, respectively. The three maladies are closely associated with 10-12 double-stranded viral RNAs, and CCRS is additionally associated with two cherry small circular RNAs (cscRNA1 and cscRNA2). Here, we report that a small viroid-like RNA similar to the CCRS-associated cscRNA1 is also present in CLS-affected trees, thus extending the link between the two diseases. Both CLS and CCRS cscRNA1 elements have common features, including sequence identity (88%), a predicted quasi rod-like conformation with short bifurcations at both termini, and the presence of hammerhead ribozymes in the strands of both polarities. However, cscRNA2, apparently derived from cscRNA1 by deletion of a short hairpin, was not detected in CLS-affected material. Although the biological nature of cscRNAs is unknown, the identification of at least cscRNA1 in different cherry cultivars and in two distinct geographic areas (Spain and Italy), always in close association with the same mycoviral dsRNAs, supports that these viroid-like RNAs could be satellite RNAs.

Dahlia latent viroid: a recombinant new species of the family Pospiviroidae posing intriguing questions about its origin and classification

A viroid-like RNA has been detected in two asymptomatic dahlia accessions by return and double PAGE. It appeared smaller than Chrysanthemum stunt viroid and Potato spindle tuber viroid, the two members of the genus Pospiviroid, family Pospiviroidae, reported in this ornamental previously. RT-PCR with primers designed for amplifying all pospiviroids produced no amplicons, but RT-PCR with random primers revealed a 342 nt RNA. The sequence of this RNA was confirmed with specific primers, which additionally revealed its presence in many dahlia cultivars. The RNA was named Dahlia latent viroid (DLVd) because it replicates autonomously, but symptomlessly, in dahlia and shares maximum sequence identity with other viroids of less than 56 %. Furthermore, DLVd displays characteristic features of the family Pospiviroidae: a predicted rod-like secondary structure of minimum free energy with a central conserved region (CCR), and the ability to form the metastable structures hairpins I and II. Its CCR is identical to that of Hop stunt viroid (HSVd, genus Hostuviroid). However, DLVd: (i) has the terminal conserved region present in members of the genus Pospiviroid, but absent in HSVd, and (ii) lacks the terminal conserved hairpin present in HSVd. Phylogenetic reconstructions indicate that HSVd and Pepper chat fruit viroid (genus Pospiviroid) are the closest relatives of DLVd, but DLVd differs from these viroids in its host range, restricted to dahlia so far. Therefore, while DLVd fulfils the criteria to be a novel species of the family Pospiviroidae, its recombinant origin makes assignment to the genera Pospiviroid or Hostuviroid problematic.

First Report of Coleus blumei viroid Infecting Coleus in India.

Members of the genus Coleviroid (Coleus blumei viroid [CbVd]), family Pospiviroidae have been reported to infect Coleus (Solenostemon sp.). CbVd-1 was first reported from Brazil, CbVd-2, -3, and -4 were first reported from Germany, whereas CbVd-5 and -6 were recently identified in China (2). In India, Coleus is extensively cultivated as an ornamental plant in home gardens. In March to June 2012, Coleus leaf samples with irregular chlorotic spots/patches were collected from home gardens of two different districts of Karnataka (Kodagu and Mysore districts), India, suspecting the presence of Coleus blumei viroids (CbVd 1 to 6). Low molecular weight RNAs were extracted using 2% CTAB buffer containing 1.4 M NaCl, 20 mM EDTA, pH 8.0 and 100 mM Tris-Cl, pH 8.0 (1). Viroid-like RNA was enriched by fractionation 2M LiCl soluble nucleic acids (4). A DNA fragment with the expected size of CbVd-1 was detected in 10 (including both districts) of 14 analyzed (incident rate of 71%) from reverse transcription (RT)-PCR assay using Coleviroid specific primers (forward 5'-TGGATCCAGCGCTGCAACGGAATCCA-3' and reverse 5'-TTGGATCCGCCAGGGAACCCAGGTAAG-3'). RT was performed at 37°C for 60 min in 25 μl reaction mix containing 5 μl RNA extracts, 1 μl reverse primer, 1× first strand buffer, 10 mM dNTPs, and 200U M-MuLV-RT (Invitrogen, USA). For PCR, 2 μl RT was mixed in 25 μl PCR mix containing 0.2 μM each forward and reverse primers and 2U LA Taq (Takara-Bio, Japan) according to the manufacturer's instruction. PCR parameter was one cycle at 94°C for 2 min, 35 cycles at 94°C for 45 s, 53°C for 30 s, and 72°C for 60 s, followed by final extension at 72°C for 10 min (4). Sequence analysis of cloned amplicons detected CbVd-1 in India. To confirm the sequence of the primer regions, an additional set of tail-tail primers were designed, CbVd1-136F (5'-CTTCGTGGAACGGCTCCGCG-3') and CbVd1-136Rev (5'-GAAGAAGCCGAAGCAACTCTC-3') and were used for RT-PCR. Amplified products were cloned, sequenced and compared with previously obtained data. Further, the presences of CbVd-1 in Coleus samples was confirmed by a RNA gel blot assay using digoxigenin-labeled CbVd-1 cRNA probe (3). Alignment of 19 sequences obtained from four representative Coleus samples found the presence of two sequence variants of CbVd-1, namely Ind-1 (GenBank Accession No. AB740017) and Ind-2 (AB740018). Ind-1 was found to differ from Ind-2 by two nucleotide substitutions at position 40 (C to T) and 211 (T to C). BLAST analysis of Ind-1 showed 100% sequence similarity with CbVd-1 isolates from China (DQ178399) and South Korea (EU 410620), whereas Ind-2 was 99% identical to these two Chinese and Koreans isolates. Furthermore, 97% and 96% sequence identity with CbVd 1-RL RNA (Accession no. X95366) was observed for Ind-1 and Ind-2, respectively. Isolates from India were 88% similar with Coleus blumei viroid 1-RG (X95291). To the best of our knowledge, this is the first molecular evidence for the presence of CbVd-1 infecting Coleus in India. Coleus harbors various viroid species and CbVd-1, reported widely, can transmit efficiently through seed and also could infect the other herbaceous plants (3). This report from India will contribute further understanding of a potential risk of Coleus viroids in ornamental species.