A Polyvalent Tool for Detecting Coguviruses in Multiple Hosts Allowed the Identification of a Novel Seed-Transmitted Coguvirus Infecting Brassicaceae.

The genus Coguvirus, a recently established genus in the family Phenuiviridae, includes several species whose members infect both woody and herbaceous hosts, suggesting a broader host range and wider distribution than previously. To gain insights into the epidemiology and biology of coguviruses, a polyvalent reverse transcription-PCR assay using degenerate primers was developed. The specificity of the assay for coguviruses was confirmed by testing citrus and apple plants infected by previously reported coguviruses and/or several unrelated viruses. The expected 236-bp amplicon was obtained from citrus, apple, pear, watermelon, and several species of the family Brassicaceae. Sequencing of the PCR amplicons allowed the identification, for the first time in Italy and/or Europe, of several coguviruses in multiple hosts, confirming the effectiveness of the assay. Moreover, a new virus, tentatively named Brassica oleracea Torzella virus 1 (BoTV1), was detected in several plants of Torzella cabbage. The complete +genome of BoTV1, determined by high-throughput sequencing and 5' rapid amplification of cDNA ends, revealed that it has the typical molecular features of coguviruses and fulfils the current criteria to be classified as a member of a new species, for which the tentative name Coguvirus torzellae is proposed. The same polyvalent assay was also used to investigate and confirm that BoTV1 is transmitted through seeds in black cabbage, thus providing the first evidence on the relevance of this natural transmission mode in the epidemiology of coguviruses. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

On the early identification and characterization of pear blister canker viroid, apple dimple fruit viroid, peach latent mosaic viroid and chrysanthemum chlorotic mottle viroid.

In the 90's, pear blister canker viroid (PBCVd), apple dimple fruit viroid (ADFVd), peach latent mosaic viroid (PLMVd) and chrysanthemum chlorotic mottle viroid (CChMVd) were identified and characterized in the Ricardo Flores' laboratory. In these studies, the autonomous replication of these infectious RNAs and their involvement in the elicitation of diseases in their natural hosts were also shown. Their discovery was achieved by classical approaches based on the physical purification of the viroid RNAs from polyacrylamide gels followed by the sequencing of their genomic RNAs and by bioassays to assess their autonomous replication and the fulfillment of Koch's postulates. The molecular characterization of these four viroids, including the study of their sequence variability, contributed to the establishment of the concept of quasispecies for viroids and to the development of reliable molecular diagnostic methods that have facilitated the control of the diseases they caused. Most importantly, some of these viroids became valuable experimental model systems that are still used nowadays to study structural-functional relationships in RNAs and to dissect evolutionary and pathogenic pathways underlying plant-viroid interaction. The differences between early viroid discovery strategies, relying on biological and pathogenic issues, and the current high-throughput sequencing-based approaches, that frequently allow the discovery of new viroids and viroid-like RNAs in symptomless hosts, is also discussed, clarifying why the traditional molecular and biological studies mentioned above are still required to conclusively define the nature of any novel viroid-like RNA.

Actinidia chlorotic ringspot-associated virus: a novel emaravirus infecting kiwifruit plants.

By integrating next-generation sequencing (NGS), bioinformatics, electron microscopy and conventional molecular biology tools, a new virus infecting kiwifruit vines has been identified and characterized. Being associated with double-membrane-bound bodies in infected tissues and having a genome composed of RNA segments, each one containing a single open reading frame in negative polarity, this virus shows the typical features of members of the genus Emaravirus. Five genomic RNA segments were identified. Additional molecular signatures in the viral RNAs and in the proteins they encode, together with data from phylogenetic analyses, support the proposal of creating a new species in the genus Emaravirus to classify the novel virus, which is tentatively named Actinidia chlorotic ringspot-associated virus (AcCRaV). Bioassays showed that AcCRaV is mechanically transmissible to Nicotiana benthamiana plants which, in turn, may develop chlorotic spots and ringspots. Field surveys disclosed the presence of AcCRaV in four different species of kiwifruit vines in five different provinces of central and western China, and support the association of the novel virus with symptoms of leaf chlorotic ringspots in Actinidia. Data on the molecular features of small RNAs of 21-24 nucleotides, derived from AcCRaV RNAs targeted by host RNA silencing mechanisms, are also reported, and possible molecular pathways involved in their biogenesis are discussed.

Variants of Peach latent mosaic viroid inducing peach calico: uneven distribution in infected plants and requirements of the insertion containing the pathogenicity determinant.

Previous characterization of Peach latent mosaic viroid (PLMVd) variants from a single peach calico (PC) isolate showed that PC symptoms are induced by variants with a 12-13 nt insertion at a specific position and folding into a hairpin with a U-rich loop. Here, this study was extended to two other PC isolates. PLMVd variants with insertions similar to those reported previously (type 1), predominated in one isolate (PC-P2). The second (PC-P1), in addition to these variants, contained others with insertions in the same position and of the same size, but with the hairpin capped by a GA-rich loop (type 2). When symptomatic and non-symptomatic tissues from both isolates were used to inoculate GF-305 peach seedlings, they reproduced the phenotype of the inoculum source, indicating that variants differing in pathogenicity are unevenly distributed within single plants. Moreover, characterization of the progeny from inoculations with the PC-P1 source showed that variants with insertions of type 1 and 2 were predominant in the symptomatic and non-symptomatic seedlings, respectively, confirming the association between PC and variants with type 1 but not type 2 insertions. Inoculations with dimeric in vitro transcripts from PLMVd variants with type 1, type 2 and with a chimeric insertion showed that the variant with type 2 insertion was latent and established that the U-rich capping loop has a major role in PC, although the adjacent stem may also have some influence. Insertions can be acquired and lost during infection, suggesting that latent variants can evolve into pathogenic variants and vice versa.

Cherry chlorotic rusty spot and Amasya cherry diseases are associated with a complex pattern of mycoviral-like double-stranded RNAs. I. Characterization of a new species in the genus Chrysovirus.

Cherry chlorotic rusty spot (CCRS) and Amasya cherry disease (ACD) display similar symptoms and are associated with a series of dsRNAs. However, a direct comparison has been lacking. Here, a side-by-side analysis confirmed that both diseases were symptomatologically very similar, as were the number (10-12) and size of their associated dsRNAs. Sequence determination of four of these dsRNAs revealed that they were essentially identical for CCRS and ACD. The largest (3399 bp), which potentially encoded a protein of 1087 aa with the eight motifs conserved in RNA-dependent RNA polymerases of dsRNA mycoviruses, had the highest similarity to those coded by dsRNA 1 of viruses belonging to the genus Chrysovirus and was termed CCRS or ACD chrys-dsRNA 1. The three closely migrating dsRNAs had the properties of the other components of a chrysovirus and in CCRS and ACD versions, respectively, were chrys-dsRNA 2 (3125 and 3128 bp), chrys-dsRNA 3 (2833 bp) and chrys-dsRNA 4 (2499 and 2498 bp), potentially encoding the major capsid protein (993 and 994 aa) and two proteins (884 and 677 aa, respectively) of unknown function. The four 5'- and 3'-UTRs shared internal similarities and had conserved GAAAAUUAUGG and AUAUGC termini, respectively. The 5'-UTRs contained the 'Box 1' motif followed by a stretch rich in CAA, CAAA and CAAAA repeats, characteristic of chrysovirus dsRNAs. Because species of the genus Chrysovirus have only been described as infecting fungi, this suggests a fungal aetiology for CCRS and ACD, a proposal supported by the properties of two other CCRS- and ACD-associated dsRNAs (see accompanying paper by Coutts et al., 2004, in this issue).

Cherry chlorotic rusty spot and Amasya cherry diseases are associated with a complex pattern of mycoviral-like double-stranded RNAs. II. Characterization of a new species in the genus Partitivirus.

Two dsRNAs from cherry trees affected with cherry chlorotic rusty spot (CCRS) in Italy and Amasya cherry disease (ACD) in Turkey were sequenced and found to be essentially identical. The larger dsRNA 1 (2021 or 2006 bp, respectively) potentially encoded a protein of 621 aa containing the conserved motifs of the RNA-dependent RNA polymerases (RdRp) of dsRNA mycoviruses, having highest similarity with those in the genus Partitivirus. The smaller dsRNA 2 (1841 or 1839 bp, respectively) had properties consistent with the second genomic component of a partitivirus and potentially encoded the coat protein (CP) of 504 aa. Phylogenetic analysis based on the RdRp and CP was coincidental and indicated that species in the genus Partitivirus could be separated into two subgroups. Because species of this genus only infect fungi, these observations suggest a fungal aetiology for CCRS and ACD, further substantiating a previous proposal (see accompanying paper by Covelli et al., 2004, in this issue).