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.

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.

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.

Rapid detection of Tobacco streak virus (TSV) in cotton (Gossypium hirsutum) based on Reverse Transcription Loop Mediated Isothermal Amplification (RT-LAMP).

Tobacco Streak Virus (TSV) belongs to the genus Ilarvirus of the family Bromoviridae an emerging pathogen posing threat to the crop species worldwide. Identification of symptoms due to TSV infection by visual observation of plants often results in misdiagnosis as symptoms produced by this virus can match with those reflecting physiological and nutritional disorders affecting cotton. Development of diagnostic tools with rapidity will have immense role to play in detection and management of the emerging virus. The protocol for rapid diagnosis of TSV infected samples by using Reverse Transcription-Loop Mediated Isothermal Amplification (RT-LAMP) was optimised and this is the first report of its use for diagnosis of TSV on cotton and Soybean. The colorimetric detection for diagnostic simplicity of amplified RT-LAMP product by using different dyes lead to enhanced applicability of this technique. The RT-LAMP diagnostic tool can be utilized not only for laboratory research but also for quarantine and field diagnosis of this important emerging pathogen affecting cotton.

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.

Characterization of the genome of a novel ilarvirus naturally infecting Cape gooseberry (Physalis peruviana).

As part of an initiative to characterize viruses infecting Cape gooseberry in the province of Antioquia (Colombia), we report the genome sequence of a new member of the genus Ilarvirus (family Bromoviridae). This virus was identified in a Cape gooseberry plot in the municipality of Marinilla in a mixed infection with potato virus Y (PVY) as part of high-throughput sequencing initiative. Results were confirmed by nested RT-PCR and DAS-ELISA. Phylogenetic analysis suggested that the Cape gooseberry ilarvirus is a new member of subgroup 1 and it is most closely related to ageratum latent virus (AgLV). The name "Cape gooseberry ilarvirus 1" (CGIV-1) is proposed for this new ilarvirus.

Rapid Detection of Prunus Necrotic Ringspot Virus by Reverse Transcription-cross-priming Amplification Coupled with Nucleic Acid Test Strip Cassette.

Prunus necrotic ringspot virus (PNRSV) is one of the most devastating viruses to Prunus spp. In this study, we developed a diagnostic system RT-CPA-NATSC, wherein reverse transcription-cross-priming amplification (RT-CPA) is coupled with nucleic acid test strip cassette (NATSC), a vertical flow (VF) visualization, for PNRSV detection. The RT-CPA-NATSC assay targets the encoding gene of the PNRSV coat protein with a limit of detection of 72 copies per reaction and no cross-reaction with the known Prunus pathogenic viruses and viroids, demonstrating high sensitivity and specificity. The reaction is performed on 60 °C and can be completed less than 90 min with the prepared template RNA. Field sample test confirmed the reliability of RT-CPA-NATSC, indicating the potential application of this simple and rapid detection method in routine test of PNRSV.

Rapid detection of fifteen known soybean viruses by dot-immunobinding assay.

A dot-immunobinding assay (DIBA) was optimized and used successfully for the rapid detection of 15 known viruses [Alfalfa mosaic virus (AMV), Bean pod mottle virus (BPMV), Bean yellow mosaic virus (BYMV), Cowpea mild mottle virus (CPMMV), Cowpea severe mosaic virus (CPSMV), Cucumber mosaic virus (CMV), Peanut mottle virus (PeMoV), Peanut stunt virus (PSV), Southern bean mosaic virus (SBMV), Soybean dwarf virus (SbDV), Soybean mosaic virus (SMV), Soybean vein necrosis virus (SVNV), Tobacco ringspot virus (TRSV), Tomato ringspot virus (ToRSV), and Tobacco streak virus (TSV)] infecting soybean plants in Oklahoma. More than 1000 leaf samples were collected in approximately 100 commercial soybean fields in 24 counties of Oklahoma, during the 2012-2013 growing seasons. All samples were tested by DIBA using polyclonal antibodies of the above 15 plant viruses. Thirteen viruses were detected, and 8 of them were reported for the first time in soybean crops of Oklahoma. The highest average incidence was recorded for PeMoV (13.5%) followed by SVNV (6.9%), TSV (6.4%), BYMV, (4.5%), and TRSV (3.9%), while the remaining seven viruses were detected in less than 2% of the samples tested. The DIBA was quick, and economical to screen more than 1000 samples against 15 known plant viruses in a very short time.

Genome Sequences of Spinach Deltapartitivirus 1, Spinach Amalgavirus 1, and Spinach Latent Virus Identified in Spinach Transcriptome.

Complete genome sequences of three new plant RNA viruses, Spinach deltapartitivirus 1 (SpDPV1), Spinach amalgavirus 1 (SpAV1), and Spinach latent virus (SpLV), were identified from a spinach (Spinacia oleracea) transcriptome dataset. The RNA-dependent RNA polymerases (RdRps) of SpDPV1, SpAV1, and SpLV showed 72%, 53%, and 93% amino acid sequence identities with the homologous RdRp of the most closely related virus, respectively, suggesting that SpDPV1 and SpAV1 were novel viruses. Sequence similarity and phylogenetic analyses revealed that SpDPV1 belonged to the genus Deltapartitivirus of the family Partitiviridae, SpAV1 to the genus Amalgavirus of the family Amalgaviridae, and SpLV to the genus Ilarvirus of the family Bromoviridae. Based on the demarcation criteria, SpDPV1 and SpAV1 are considered as novel species of the genera Deltapartitivirus and Amalgavirus, respectively. This is the first report of these two viruses from spinach.

Detection of Strawberry necrotic shock virus using conventional and TaqMan(®) quantitative RT-PCR.

Graft-indexing of an advanced selection from the University of Florida strawberry breeding program produced virus-like symptoms on Fragaria vesca. However; RT-PCR testing of the material did not detect the presence of any of 16 strawberry virus species or members of virus groups for which strawberries are routinely indexed. Large scale sequencing of the material revealed the presence of an isolate of Strawberry necrotic shock virus. The nucleotide sequence of this isolate from Florida shows a significant number of base changes in the annealing sites of the primers compared to the primers currently in use for the detection of SNSV thereby explaining the most probable reason for the inability to detect the virus in the original screening. RT-PCR and Taqman(®) qPCR assays were developed based on conserved virus sequences identified in this isolate from Florida and other sequences for SNSV currently present in GenBank. The two assays were applied successfully on multiple samples collected from several areas across the United States as well as isolates from around the world. Comparison between the RT-PCR and the qPCR assays revealed that the qPCR assay is at least 100 times more sensitive than conventional PCR.

A Novel Ilarvirus Is Associated with Privet Necrotic Ringspot Disease in the Southern United States.

Necrotic ringspot disease (NRSD) is a graft-transmissible disorder of privet (synonym ligustrum), originally reported from Florida and Louisiana more than 50 years ago. In this communication we report an isometric virus isolated from Japanese privet (Ligustrum japonicum) collected in the southern United States displaying symptoms resembling those of NRSD. In mechanical transmission tests, the virus induced systemic infections in several herbaceous hosts. Double-stranded RNA analysis showed a pattern resembling replicative forms of members of the family Bromoviridae. The genome organization along with phylogenetic analyses and serological tests revealed that the virus belongs to subgroup 1 of the genus Ilarvirus. Pairwise comparisons with recognized ilarviruses indicated that the virus is a distinct, and as yet, undescribed member in the taxon, for which we propose the name Privet ringspot virus (PrRSV). Furthermore, the near-perfect association of PrRSV infections with symptoms, and apparent absence of any other virus(es) in studied samples, strongly suggest an important role of this virus in the etiology of NRSD of privet in the southeastern United States.

Genetic variability and evolutionary analysis of parietaria mottle virus: role of selection and genetic exchange.

The genetic variability and evolution of parietaria mottle virus (PMoV) of the genus Ilarvirus was studied by analyzing nucleotide sequences of 2b and CP genes from isolates collected in different countries. Phylogenetic analysis showed that PMoV isolates clustered in different clades: one (clade I) composed of only Italian isolates and three clades (clades II-IV) including the Spanish isolates. The Greek isolate GrT-1 used in this study was in clade IV for the CP phylogenetic tree whereas it formed a separate branch in the 2b phylogenetic tree. The nucleotide sequence diversity of both the 2b and CP genes was low (0.062 ± 0.006 and 0.063 ± 0.006 for 2b and CP, respectively) but higher than those of other ilarviruses. Distribution of synonymous and nonsynonymous substitutions revealed that 2b and CP proteins are under purifying selection, with some positions under diversifying selection. Genetic exchange among Spanish isolates was also detected.

Detection, discrimination and discovery of a new Tobacco streak virus strain.

Soybean plants that exhibited symptoms of virus infection were sampled from different counties of Oklahoma. These plants were tested serologically for 15 viruses known to infect soybean plants. Fifty-seven samples that exhibited typical virus-like symptoms did not test positive for any of the 15 viruses used in a dot-immunobinding assay (DIBA). Four samples were pooled and used for next generation sequencing using the 454-Roche protocol. Sequence and phylogenetic analysis of the sequences obtained revealed infection with a distinct strain of Tobacco streak virus (TSV). TSV was one of the 15 viruses initially tested for using DIBA and had tested negative. TSV belongs to the genus Ilarvirus and has been reported as a causal agent of bud blight in soybean crops in Brazil and the United States. Out of 10 reported primer pairs for TSV reverse transcription-polymerase chain reaction (RT-PCR), only two had the potential, based on sequence similarity, to amplify part of the genome of the distinct strain of TSV found in Oklahoma and only one was actually able to amplify the region. In this study, a new primer pair, specific to all known TSV and capable of amplifying the Oklahoma strain (TSV-OK), was designed from a highly conserved region of coat protein (CP) sequences and end-point PCR and quantitative RT-PCR detection methods were developed and their sensitivity assayed. This is the first report of specific primers designed from this highly conserved region in the CP of TSV for detection of TSV. Twenty-three of the 57 DIBA soybean samples that initially tested negative were retested with the new specific end-point PCR method and found positive for TSV infection.

Pollen transmission of asparagus virus 2 (AV-2) may facilitate mixed infection by two AV-2 isolates in asparagus plants.

Asparagus virus 2 (AV-2) is a member of the genus Ilarvirus and thought to induce the asparagus decline syndrome. AV-2 is known to be transmitted by seed, and the possibility of pollen transmission was proposed 25 years ago but not verified. In AV-2 sequence analyses, we have unexpectedly found mixed infection by two distinct AV-2 isolates in two asparagus plants. Because mixed infections by two related viruses are normally prevented by cross protection, we suspected that pollen transmission of AV-2 is involved in mixed infection. Immunohistochemical analyses and in situ hybridization using AV-2-infected tobacco plants revealed that AV-2 was localized in the meristem and associated with pollen grains. To experimentally produce a mixed infection via pollen transmission, two Nicotiana benthamiana plants that were infected with each of two AV-2 isolates were crossed. Derived cleaved-amplified polymorphic sequence analysis identified each AV-2 isolate in the progeny seedlings, suggesting that pollen transmission could indeed result in a mixed infection, at least in N. benthamiana.

Rapid detection of Prunus necrotic ringspot virus using magnetic nanoparticle-assisted reverse transcription loop-mediated isothermal amplification.

Prunus necrotic ringspot virus (PNRSV) has seriously reduced the yield of Prunus species worldwide. In this study, a highly efficient and specific two-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed to detect PNRSV. Total RNA was extracted from sweet cherry leaf samples using a commercial kit based on a magnetic nanoparticle technique. Transcripts were used as the templates for the assay. The results of this assay can be detected using agarose gel electrophoresis or by assessing in-tube fluorescence after adding SYBR Green I. The assay is highly specific for PNRSV, and it is more sensitive than reverse-transcription polymerase chain reaction (RT-PCR). Restriction enzyme digestion verified further the reliability of this RT-LAMP assay. To our knowledge, this is the first report of the application of RT-LAMP to PNRSV detection in Prunus species.