Revisiting a pollen-transmitted ilarvirus previously associated with angular mosaic of grapevine.

We report the characterization of a novel tri-segmented RNA virus infecting Mercurialis annua, a common crop weed and model species in plant science. The virus, named "Mercurialis latent virus" (MeLaV) was first identified in a mixed infection with the recently described Mercurialis orthotospovirus 1 (MerV1) on symptomatic plants grown in glasshouses in Lausanne (Switzerland). Both viruses were found to be transmitted by Thrips tabaci, which presumably help the inoculation of infected pollen in the case of MeLaV. Complete genome sequencing of the latter revealed a typical ilarviral architecture and close phylogenetic relationship with members of the Ilarvirus subgroup 1. Surprisingly, a short portion of MeLaV replicase was found to be identical to the partial sequence of grapevine angular mosaic virus (GAMV) reported in Greece in the early 1990s. However, we have compiled data that challenge the involvement of GAMV in angular mosaic of grapevine, and we propose alternative causal agents for this disorder. In parallel, three highly-conserved MeLaV isolates were identified in symptomatic leaf samples in The Netherlands, including a herbarium sample collected in 1991. The virus was also traced in diverse RNA sequencing datasets from 2013 to 2020, corresponding to transcriptomic analyses of M. annua and other plant species from five European countries, as well as metaviromics analyses of bees in Belgium. Additional hosts are thus expected for MeLaV, yet we argue that infected pollen grains have likely contaminated several sequencing datasets and may have caused the initial characterization of MeLaV as GAMV.

Development of full-length infectious cDNA clones and host range identification of an echinacea strain of tobacco streak virus.

Tobacco streak virus induces severe diseases on a wide range of plants and becomes an emerging threat to crop yields. However, the infectious clones of TSV remain to be developed for reverse genetics studies. Here, we obtained the full genome sequence of a TSV-CNB isolate and analyzed the phylogenetic characteristics. Subsequently, we developed the full-length infectious cDNA clones of TSV-CNB driven by 35 S promoter using yeast homologous recombination. Furthermore, the host range of TSV-CNB isolate was determined by Agrobacterium infiltration and mechanical inoculation. The results reveal that TSV-CNB can infect 10 plant species in 5 families including Glycine max, Vigna radiate, Lactuca sativa var. Ramosa, Dahlia pinnate, E. purpurea, Calendula officinalis, Helianthus annuus, Nicotiana. Benthamiana, Nicotiana tabacum and Chenopodium quinoa. Taken together, the TSV infectious clones will be a useful tool for future studies on viral pathogenesis and host-virus interactions.

The Virome of Babaco (Vasconcellea × heilbornii) Expands to Include New Members of the Rhabdoviridae and Bromoviridae.

Babaco (Vasconcellea × heilbornii) is a subtropical species in the Caricaceae family. The plant is native to Ecuador and represents an important crop for hundreds of families. The objective of this study was to characterize, at the genomic level, two new babaco viruses identified by high-throughput sequencing. The viruses, an ilarvirus and a nucleorhabdovirus, were found in a symptomatic babaco plant from a commercial nursery in the Azuay province of Ecuador. The tripartite genome of the new ilarvirus, provisionally named babaco ilarvirus 1 (BabIV-1), is related to subgroup 3 ilarviruses, including apple mosaic virus, apple necrotic mosaic virus, and prunus necrotic ringspot virus as the closest relatives. The genome of the nucleorhabdovirus, provisionally named babaco nucleorhabdovirus 1 (BabRV-1), showed the closest relation with joa yellow blotch-associated virus and potato yellow dwarf nucleorhabdovirus. Molecular-based detection methods found BabIV-1 and BabRV-1 in 21% and 36%, respectively, of plants surveyed in a commercial babaco nursery, highlighting the importance of enforcing virus testing and nursery certification programs for babaco.

Development of one step colorimetric RT-LAMP assays for rapid detection of Apple mosaic virus and Prunus necrotic ringspot virus.

Apple mosaic virus (ApMV) and Prunus necrotic ringspot virus (PNRSV), belonging to genus Ilarvirus, cause significant losses to rose and other plants of the family Rosaceae. They are easily transmitted through mechanical or vegetative means. In our previous study, the occurrence of ApMV and PNRSV in rose plants was reported. In this study, as a first step towards the development of a colorimetric Reverse Transcriptase - Loop Mediated Isothermal Amplification (RT-LAMP) assay, two primer sets were designed, each containing six primers (F3, B3, FIP, BIP, LF and LB) targeting the coat protein genes of ApMV and PNRSV. After incubation of RT-LAMP reaction mix at an isothermal temperature (65 °C/30 min), the amplified products were visually confirmed with the nucleic acid intercalation dye SYBR Green I and the indicator dye Hydroxy-Naphthol Blue. The developed assays were virus specific and showed no cross amplification. Their sensitivity was 103 times higher than that of the corresponding RT-PCRs. The LAMP assays developed in this study are inexpensive, rapid and reliable for the early detection of ApMV and PNRSV, and could therefore be used in plant quarantine to control the risk of their spread.

The coat protein of the ilarvirus prunus necrotic ringspot virus mediates long-distance movement.

The coat protein (CP) of plant viruses generally has multiple functions involving infection, replication, movement and pathogenicity. Functions of the CP of prunus necrotic ringspot virus (PNRSV), the causal agent of several threatening diseases of Prunus fruit trees, are poorly studied. Previously, we identified a novel virus in apple, apple necrotic mosaic virus (ApNMV), which is phylogenetically related to PNRSV and probably associated with apple mosaic disease in China. Full-length cDNA clones of PNRSV and ApNMV were constructed, and both are infectious in cucumber (Cucumis sativus L.), an experimental host. PNRSV exhibited higher systemic infection efficiency with more severe symptoms than ApNMV. Reassortment analysis of genomic RNA segments 1-3 found that RNA3 of PNRSV could enhance the long-distance movement of an ApNMV chimaera in cucumber, indicating the association of RNA3 of PNRSV with viral long-distance movement. Deletion mutagenesis of the PNRSV CP showed that the basic motif from amino acids 38 to 47 was crucial for the CP to maintain the systemic movement of PNRSV. Moreover, we found that arginine residues 41, 43 and 47 codetermine viral long-distance movement. The findings demonstrate that the CP of PNRSV is required for long-distance movement in cucumber, which expands the functions of ilarvirus CPs in systemic infection. For the first time, we identified involvement of Ilarvirus CP protein during long-distance movement.

Rose Virome Analysis and Identification of a Novel Ilarvirus in Taiwan.

Rose (Rosa spp.), especially R. hybrida, is one of the most popular ornamental plants in the world and the third largest cut flower crop in Taiwan. Rose mosaic disease (RMD), showing mosaic, line patterns and ringspots on leaves, is a common rose disease caused by the complex infection of various viruses. Due to pests and diseases, the rose planting area in Taiwan has been decreasing since 2008; however, no rose virus disease has been reported in the past five decades. In the spring of 2020, rose samples showing RMD-like symptoms were observed at an organic farm in Chiayi, central Taiwan. The virome in the farm was analyzed by RNA-seq. Rose genomic sequences were filtered from the obtained reads. The remaining reads were de novo assembled to generate 294 contigs, 50 of which were annotated as viral sequences corresponding to 10 viruses. Through reverse transcription-polymerase chain reaction validation, a total of seven viruses were detected, including six known rose viruses, namely apple mosaic virus, prunus necrotic ringspot virus, rose partitivirus, apple stem grooving virus, rose spring dwarf-associated virus and rose cryptic virus 1, and a novel ilarvirus. After completing the whole genome sequencing and sequence analysis, the unknown ilarvirus was demonstrated as a putative new species, tentatively named rose ilarvirus 2. This is the first report of the rose virus disease in Taiwan.

Selection and Validation of Reference Genes for Gene Expression Studies Using Quantitative Real-Time PCR in Prunus Necrotic Ringspot Virus-Infected Cucumis sativus.

Several members of the genus Ilarvirus infect fruit trees and are distributed worldwide. Prunus necrotic ringspot virus (PNRSV) is one of the most prevalent viruses, causing significant losses. Cucumissativus can be infected by several ilarviruses, leading to obvious symptoms, including PNRSV, which suggests that cucumbers could be good hosts for the study of the pathogenesis of ilarviruses. Real-time quantitative PCR is an optimal choice for studying gene expression because of its simplicity and its fast and high sensitivity, while its accuracy is highly dependent on the stability of the reference genes. In this study, we assessed the stability of eleven reference genes with geNorm, NormFinder, ΔCt method, BestKeeper, and the ranking software, RefFinder. The results indicated that the combined use of EF1α and F-BOX was the most accurate normalization method. In addition, the host genes AGO1, AGO4, and RDR6 were selected to test the reliability of the reference genes. This study provides useful information for gene expression analysis during PNRSV infection and will facilitate gene expression studies associated with ilarvirus infection.

Prunus necrotic ringspot virus in Turkey: an immigrant population.

An outbreak in northwestern Turkey of prunus necrotic ringspot virus (PNRSV, genus Ilarvirus, family Bromoviridae) was sampled in 2016-2018. Gene sequences from these isolates, together with all of the gene sequence data for this virus in the GenBank database (>300 non-recombinant coat protein (CP) genes and 20 complete genomic sequences) were analysed to determine the relationship of the Turkish PNRSV isolates to those from other parts of the world. Phylogenetic and population genetic methods independently showed that the most recent common ancestor of the world PNRSV population was probably American, not Eurasian. PNRSV has spread to Turkey on several occasions, as its CP sequences are among the terminal branches of three of the most sampled CP phylogroups. The complete PNRSV genome consists of three segments (RNA1, RNA2, and RNA3), with the larger two encoding replicases and the smallest encoding the movement protein and the CP. One quarter of the RNA1 and RNA2 genes were recombinants. The phylogenies of the CP and MP genes (i.e., different regions of RNA3) were closely correlated but did not correlate with those of RNA1 and RNA2, indicating that some of the isolates were reassortants. However, the non-reassortant ancestor could not be identified, probably because none of the complete genome sequences were from isolates from the basal CP phylogroups. Our results emphasize the importance of strict quarantine, both international and local, for the world's stone fruit crops.

A novel high-throughput sequencing approach reveals the presence of a new virus infecting Rosa: rosa ilarvirus-1 (RIV-1).

Roses are one of the most valuable ornamental flowering shrubs grown worldwide. Despite the widespread of rose viruses and their impact on cultivation, they have not been studied in detail in the United Kingdom (UK) since the 1980's. As part of a survey of rose viruses entering the UK, 35 samples were collected at Heathrow Airport (London, UK) and were tested by RT-qPCR for different common rose viruses. Of the 35 samples tested using RT-qPCR for prunus necrotic ringspot virus (PNRSV; genus Ilarvirus), 10 were positive. Confirmatory testing was performed using RT-PCR with both PNRSV-specific and ilarvirus-generic primers, and diverse results were obtained: One sample was exclusively positive when using the ilarvirus-generic primers, and subsequent sequencing of the RT-PCR product revealed homology to other ilarviruses but not PNRSV. Further work to characterise the virus was performed using high throughput sequencing, both the MinION Flongle and Illumina MiSeq. The sequencing confirmed the presence of a new virus within group 2 of the genus Ilarvirus and we propose the name "rosa ilarvirus-1″ (RIV-1). Here, we describe the identification of a novel virus using the low-cost Flongle flow cell and discuss its potential as a front-line diagnostic tool.

Complete genome sequence of a putative novel ilarvirus isolated from Eleocharis dulcis.

The complete genomic sequence of a novel ilarvirus from Eleocharis dulcis, tentatively named "water chestnut virus A" (WCVA), was determined using next-generation sequencing (NGS) combined with reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) PCR. The three genomic RNA components of WCVA were 3578 (RNA1), 2873 (RNA2), and 2073 (RNA3) nucleotides long, with four predicted open reading frames containing conserved domains and motifs typical of ilarviruses. Phylogenetic analysis of each predicted protein consistently placed WCVA in subgroup 4 of the genus Ilarvirus, together with prune dwarf virus, viola white distortion associated virus, Fragaria chiloensis latent virus, and potato yellowing virus. The genetic distances and lack of serological reaction to antisera against other ilarviruses suggest that WCVA is a novel member of the genus.

Molecular Identification of Prune Dwarf Virus (PDV) Infecting Sweet Cherry in Canada and Development of a PDV Full-Length Infectious cDNA Clone.

Prune dwarf virus (PDV) is a member of ilarviruses that infects stone fruit species such as cherry, plum and peach, and ornamentally grown trees worldwide. The virus lacks an RNA silencing suppressor. Infection by PDV either alone, or its mixed infection with other viruses causes deteriorated fruit marketability and reduced fruit yields. Here, we report the molecular identification of PDV from sweet cherry in the prominent fruit growing region of Ontario, Canada known as the Niagara fruit belt using next generation sequencing of small interfering RNAs (siRNAs). We assessed its incidence in an experimental farm and determined the full genome sequence of this PDV isolate. We further constructed an infectious cDNA clone. Inoculation of the natural host cherry with this clone induced a dwarfing phenotype. We also examined its infectivity on several common experimental hosts. We found that it was infectious on cucurbits (cucumber and squash) with clear symptoms and Nicotiana benthamiana without causing noticeable symptoms, and it was unable to infect Arabidopsis thaliana. As generating infectious clones for woody plants is very challenging with limited success, the PDV infectious clone developed from this study will be a useful tool to facilitate molecular studies on PDV and related Prunus-infecting viruses.

Molecular characterization and pathogenicity analysis of prunus necrotic ringspot virus isolates from China rose (Rosa chinensis Jacq.).

Prunus necrotic ringspot virus (PNRSV) is a viral pathogen with worldwide distribution, infecting many commercial fruit trees and ornamental plants. So far, the correlation between PNRSV infection and China rose mosaic disease has not been studied. Rose mosaic disease is characterized by severe symptoms, including mosaic, line pattern, and ringspot. Six viruses that were potentially associated with mosaic disease, including PNRSV, were tested in China roses. Only PNRSV was detected in China roses showing mosaic disease, and asymptomatic samples tested negative for this virus. This result was confirmed by small RNA sequencing, but rose leaf rosette-associated virus and rose spring dwarf-associated virus were also identified in both samples with mosaic disease and asymptomatic samples. This implied that PNRSV might be associated with China rose mosaic disease. Full genome sequences of two PNRSV isolates were determined, and the RNA1, 2 and 3 segments were found to be 3,332, 2,594 and 1,951 nucleotides (nt) in length, respectively. The three RNA segments shared 88.7-89.1% nt sequence identity in the 3'UTR, while RNA2 and RNA3 shared 98.2-99.4% identity. The higher variability in RNA1 suggests that it might have been under greater selection pressure. Phylogenetic analysis showed that the two PNRSV isolates clustered in group PV-32. Full-length infectious cDNA clones of PNRSV from China rose were constructed and used to agroinfiltrate cucumber seedlings. The inoculated cucumber leaves showed yellowing, chlorotic spots, necrosis, dwarfing, and decline at 23 to 39 days post-inoculation, demonstrating the virulence of the PNRSV isolate from China rose. These data lay a foundation for determining the molecular mechanism of rose mosaic disease caused by PNRSV.

A Prunus necrotic ringspot virus (PNRSV)-Based Viral Vector for Characterization of Gene Functions in Prunus Fruit Trees.

Virus-induced gene silencing (VIGS) is a gene silencing mechanism by which an invading virus targets and silences the endogenous genes that have significant sequence similarity with the virus. It opens the door for us to develop viruses as powerful viral vectors and modify them for molecular characterization of gene functions in plants. In the past two decades, VIGS has been studied extensively in plants, and various VIGS vectors have been developed. Despite the fact that VIGS is in particular practical for functional genomic study of perennial woody vines and trees with a long life cycle and recalcitrant to genetic transformation, not many studies have been reported in this area. Here, we describe a protocol for the use of a Prunus necrotic ringspot virus (PNRSV)-based VIGS vector we have recently developed for functional genomic studies in Prunus fruit trees.

Role of Thrips palmi and Parthenium hysterophorus pollen in active spread of tobacco streak virus in the cotton ecosystem.

Tobacco streak virus incidence in the cotton field, cv.CO14 at Department of Cotton, Tamil Nadu Agricultural University (TNAU), Coimbatore, India was nearly 36.50 %. Cotton plants infected with TSV exhibits different types of symptoms, including necrotic spots, lesions, mosaic, purplish necrotic rings, square drying, veinal necrosis and drying of terminal shoots. The highly prevalent thrips species in this cotton ecosystem was established as Thrips palmi (60.00 %) by morphological (ESEM) and molecular methods (RT-PCR using mtCOI primers). The density of the alternate weed host, Parthenium hysterophorus, was 15.05 plants per m2 in these fields. Association of Thrips palmi with Parthenium was confirmed, when observed under environmental scanning electron microscope (ESEM), Parthenium pollen grains (i.e., average size @ 15000X =12.94 µm) were found adhering to its body. Molecular studies through RT-PCR confirmed the presence of TSV in the leaves and pollen grains of symptomatic and symptom-free Parthenium plants collected from the cotton field (cv. CO14). Therefore, the combined role of Thrips palmi and the Parthenium pollen grains in the transmission of TSV was examined; acquiring of TSV and its presence in the body of Thrips palmi instars and adults after 72 h of AAP was convincingly demonstrated using RT-PCR, NASH and qPCR. However virus acquired thrips could not transmit the virus. Pollen from TSV infected Parthenium plants when dusted on cotton (ANKUR 2110) seedlings along with virus acquired or non-acquired thrips led to symptom development 22 days after sowing. From the study it is evident that thrips only facilitate the movement of TSV borne pollen grains, and thereby contributing to active spread of the virus.

Characterization of distinct strains of an aphid-transmitted ilarvirus (Fam. Bromoviridae) infecting different hosts from South America.

Potato yellowing virus (PYV, original code SB-22), an unassigned member of the Genus Ilarvirus Family Bromoviridae, has been reported infecting potatoes in Peru, Ecuador and Chile. It is associated with symptomless infections, however yellowing of young leaves has been observed in some potato cultivars. Thirteen potato and yacon isolates were selected after routine screening of CIP-germplasm and twenty-four were identified from 994 potato plants collected in Peru whereas one was intercepted from yacon in the UK. These isolates were identified using high throughput sequencing, ELISA, host range and RT-PCR. Here we report the sequence characterization of the complete genomes of nine PYV isolates found infecting Solanum tuberosum, four complete genome isolates infecting Smallanthus sonchifolius (yacon), and in addition 15 complete RNA3 sequences from potato and partial sequences of RNA1, 2 and 3 of isolates infecting potato and yacon from Ecuador, Peru and Bolivia. Results of phylogenetic and recombination analysis showed RNA3 to be the most variable among the virus isolates and suggest potato infecting isolates have resulted through acquisition of a movement protein variant through recombination with an unknown but related ilarvirus, whereas one yacon isolate from Bolivia also had resulted from a recombination event with another related viruses in the same region. Yacon isolates could be distinguished from potato isolates by their inability to infect Physalis floridana, and potato isolates from Ecuador and Peru could be distinguished by their symptomatology in this host as well as phylogenetically. The non-recombinant yacon isolates were closely related to a recently described isolate from Solanum muricatum (pepino dulce), and all isolates were related to Fragaria chiloensis latent virus (FCiLV) reported in strawberry from Chile, and probably should be considered the same species. Although PYV is not serologically related to Alfalfa mosaic virus (AMV), they are both transmitted by aphids and share several other characteristics that support the previous suggestion to reclassify AMV as a member in the genus Ilarvirus.

Development of RT-qPCR assays for the detection of three latent viruses of pome.

Latent fruit tree viruses present economic threat to the industry and nurseries as diseases they cause not only reduce fruit quality and production yield, but can also be spread inadvertently through propagation due to the lack of viral symptoms on an infected mother plant. As a result, these viruses require appropriate detection tools for effective management. In this study we developed RT-qPCR assays for the detection of three latent viruses of pome, apple chlorotic leaf spot virus (ACLSV), apple stem pitting virus (ASPV), and apple mosaic virus (ApMV), using the alignment of representative sequences from the NCBI database. The optimized assays were shown to be specific by successfully amplifying the target from positive controls without showing any detectable amplification in negative and non-target controls, and revealed high sensitivity by reliably detecting as low as 101 copies per reaction. The results also demonstrated that both the choice of extraction method and the reagents used for RT-qPCRcould play a critical role in virus detection outcome. These assays were both reliable and robust compared to the extant RT-PCR methods, and they could be a viable tool for making informed management decisions.

Detection and quantification of four viruses in Prunus pollen: Implications for biosecurity.

Pollen transmitted viruses require accurate detection and identification to minimize the risk of spread through the global import and export of pollen. Therefore in this study we developed RT-qPCR assays for the detection of Cherry leaf roll virus (CLRV), Prune dwarf virus (PDV), Prunus necrotic ringspot virus (PNRSV), and Cherry virus A (CVA), four viruses that infect pollen of Prunus species. Assays were designed against alignments of extant sequences, optimized, and specificity was tested against known positive, negative, and non-target controls. An examination of assay sensitivity showed that detection of virus at concentrations as low as 101 copies was possible, although 102 copies was more consistent. Furthermore, comparison against extant assays showed that in both pollen and plant samples, the newly developed RT-qPCR assays were more sensitive and could detect a greater range of isolates than extant endpoint RT-PCR and ELISA assays. Use of updated assays will improve biosecurity protocols as well as the study of viruses infecting pollen.

Characterization of Tomato Necrotic Spot Virus, a Subgroup 1 Ilarvirus Causing Necrotic Foliar, Stem, and Fruit Symptoms in Tomatoes in the United States.

The genomic, biological, and serological characterization of tomato necrotic spot virus (ToNSV), a virus first described infecting tomato in California, was completed. The complete genomic sequence identified ToNSV as a new subgroup 1 ilarvirus distinct from the previously described tomato-infecting ilarviruses. We identified ToNSV in Indiana in 2017 and 2018 and in Ohio in 2018. The coat protein coding region of the isolates from California, Indiana, and Ohio have 94 to 98% identity, while the same isolates had 99% amino acid identity. ToNSV is serologically related to TSV, a subgroup 1 ilarvirus, and shows no serological relationship to ilarviruses in the other subgroups. In tomato, ToNSV caused symptoms of necrotic spots and flecks on leaves, necrotic streaking on stems, and necrotic spots and circular patterns on fruit resulting in a yield loss of 1 to 13%. These results indicate that ToNSV is a proposed new subgroup 1 ilarvirus causing a necrotic spotting disease of tomato observed in California, Indiana, and Ohio.

Genomic Analysis, Sequence Diversity, and Occurrence of Apple necrotic mosaic virus, a Novel Ilarvirus Associated with Mosaic Disease of Apple Trees in China.

China accounts for over 50% of apple production worldwide. Very recently, a novel ilarvirus, Apple necrotic mosaic virus (ApNMV), was isolated from apple trees showing mosaic symptoms in Japan. This study compared different types of mosaic symptoms observed in apple trees in China under field conditions. Complete nucleotide sequences were obtained for six isolates of ApNMV. The genomic components varied in size from 3,378 to 3,380 nt (RNA1), 2,778 to 2,786 nt (RNA2), and 1,909 to 1,955 nt (RNA3), respectively. Although nucleotide sequence similarities with subgroup 3 ilarviruses were low (49.2 to 64.3%), results of phylogenetic analysis indicated that Chinese ApNMV isolates were clustered in subgroup 3 together with Prunus necrotic ring spot virus (PNRSV) and Apple mosaic virus (ApMV). Apple mosaic disease occurred widely in apple producing areas of China with a very high percentage (92.1%, 268 out of 291) of symptomatic trees being infected with ApNMV but not with ApMV. The data suggested that ApNMV might be the main pathogen causing apple mosaic disease in China. The genomes of the six studied Chinese ApNMV isolates demonstrated substantial sequence diversity. Here, we demonstrated a strong association of ApNMV with the mosaic disease of apple trees in China.

Complete genome sequence of peanut virus C, a putative novel ilarvirus.

We determined the complete genome sequence of a putative novel ilarvirus, tentatively named "peanut virus C" (PVC), identified in peanut (Arachis hypogaea). The three segmented genomic RNA molecules of PVC were 3474 (RNA1), 2925 (RNA2), and 2160 (RNA3) nucleotides in length, with five predicted open reading frames containing conserved domains and motifs that are typical features of ilarviruses. The three genomic RNAs shared nucleotide sequence similarity (74% identity and 93% query coverage for RNA1, 75% identity and 85% query coverage for RNA2, and 72% identity and 70% query coverage for RNA3) with the most closely related ilarvirus, parietaria mottle virus. These results suggest that PVC is a novel member of the genus Ilarvirus in the family Bromoviridae.