Complete genome sequence of a carlavirus identified in grapevine (Vitis sp) in Greece.

Using high-throughput sequencing, we identified a novel carlavirus sequence in a 28-year-old 'Kotsifali' grapevine sample collected in Heraklion (Crete, Greece). Using RT-PCR and 5'/3' RACE together with Sanger sequencing, the complete genome sequence of 8299 nt was confirmed and found to contain five open reading frames (ORFs) but to lack an ORF6, which is present in some members of the genus Carlavirus. The novel sequence is most similar to those of two carlaviruses infecting caper, and taking into account the ICTV nomenclature, we propose the name "grapevine carlavirus 1" for this new virus.

Molecular diversity of yam virus Y and identification of banana mild mosaic virus isolates infecting yam (Dioscorea spp.).

Two members of the family Betaflexiviridae associated with yam (Dioscorea spp.) have been described so far: yam latent virus (YLV) and yam virus Y (YVY). However, their geographical distribution and molecular diversity remain poorly documented. Using a nested RT-PCR assay, we detected YVY in D. alata, D. bulbifera, D. cayenensis, D. rotundata, and D. trifida in Guadeloupe, and in D. rotundata in Côte d'Ivoire, thus extending the known host range of this virus and geographical distribution. Using amplicon sequencing, we determined that the molecular diversity of YVY in the yam samples analyzed in this work ranged between 0.0 and 29.1% and that this diversity is partially geographically structured. We also identified three isolates of banana mild mosaic virus (BanMMV) infecting D. alata in Guadeloupe, providing the first evidence for BanMMV infection in yam.

Identification and Molecular Characterization of a Novel Carlavirus Infecting Chrysanthemum morifolium in China.

Chrysanthemum (Chrysanthemum morifolium) is an important ornamental and medicinal plant suffering from many viruses and viroids worldwide. In this study, a new carlavirus, tentatively named Chinese isolate of Carya illinoinensis carlavirus 1 (CiCV1-CN), was identified from chrysanthemum plants in Zhejiang Province, China. The genome sequence of CiCV1-CN was 8795 nucleotides (nt) in length, with a 68-nt 5'-untranslated region (UTR) and a 76-nt 3'-UTR, which contained six predicted open reading frames (ORFs) that encode six corresponding proteins of various sizes. Phylogenetic analyses based on full-length genome and coat protein sequences revealed that CiCV1-CN is in an evolutionary branch with chrysanthemum virus R (CVR) in the Carlavirus genus. Pairwise sequence identity analysis showed that, except for CiCV1, CiCV1-CN has the highest whole-genome sequence identity of 71.3% to CVR-X6. At the amino acid level, the highest identities of predicted proteins encoded by the ORF1, ORF2, ORF3, ORF4, ORF5, and ORF6 of CiCV1-CN were 77.1% in the CVR-X21 ORF1, 80.3% in the CVR-X13 ORF2, 74.8% in the CVR-X21 ORF3, 60.9% in the CVR-BJ ORF4, 90.2% in the CVR-X6 and CVR-TX ORF5s, and 79.4% in the CVR-X21 ORF6. Furthermore, we also found a transient expression of the cysteine-rich protein (CRP) encoded by the ORF6 of CiCV1-CN in Nicotiana benthamiana plants using a potato virus X-based vector, which can result in a downward leaf curl and hypersensitive cell death over the time course. These results demonstrated that CiCV1-CN is a pathogenic virus and C. morifolium is a natural host of CiCV1.

Hop Latent Viroid: A Hidden Threat to the Cannabis Industry.

Hop latent viroid (HLVd) is the biggest concern for cannabis and hop growers worldwide. Although most HLVd-infected plants remain asymptomatic, research on hops has demonstrated a decrease in both the α-bitter acid and terpene content of hop cones, which affects their economic value. The HLVd-associated "dudding" or "duds" disease of cannabis was first reported in 2019 in California. Since then, the disease has become widespread in cannabis-growing facilities across North America. Although severe yield loss associated with duds disease has been recorded, little scientific information is available to growers in order to contain HLVd. Consequently, this review aims to summarise all of the scientific information available on HLVd so as to be able to understand the effect of HLVd on yield loss, cannabinoid content, terpene profile, disease management and inform crop protection strategies.

Genomic Analysis and Development of Infectious Clone of a Novel Carlavirus Infecting Blueberry.

A new blueberry virus was discovered using high-throughput sequencing. Using sequence identity values, phylogenetics, and serological and biological properties, we propose the virus, putatively named blueberry virus S (BluVS), to be a distinct species within the genus Carlavirus (family Betaflexiviridae). The genome was analyzed in depth, and an infectious clone was developed to initiate studies on virus pathogenicity. Agroinfiltration of the binary vector construct produced severe systemic symptoms in Nicotiana occidentalis. Back-inoculation using sap from agroinfiltrated N. occidentalis produced identical symptoms to the recipient plants (N. occidentalis), and virus purification yielded flexuous carlavirus-like particles. However, unlike blueberry scorch virus (BlScV), BluVS caused symptomless infection in Chenopodium quinoa and reacted weakly to BlScV antibodies in an enzyme-linked immunosorbent assay. Collectively, the results provide evidence for the distinct speciation of BluVS. The availability of an infectious clone provides tools for future studies on the biology of the virus.

Integrated next-generation sequencing and comparative transcriptomic analysis of leaves provides novel insights into the ethylene pathway of Chrysanthemum morifolium in response to a Chinese isolate of chrysanthemum virus B.

BACKGROUND: Chrysanthemum virus B (CVB), a key member of the genus Carlavirus, family Betaflexiviridae, causes severe viral diseases in chrysanthemum (Chrysanthemum morifolium) plants worldwide. However, information on the mechanisms underlying the response of chrysanthemum plants to CVB is scant. METHODS: Here, an integrated next-generation sequencing and comparative transcriptomic analysis of chrysanthemum leaves was conducted to explore the molecular response mechanisms of plants to a Chinese isolate of CVB (CVB-CN) at the molecular level. RESULTS: In total, 4934 significant differentially expressed genes (SDEGs) were identified to respond to CVB-CN, of which 4097 were upregulated and 837 were downregulated. Gene ontology and functional classification showed that the majority of upregulated SDEGs were categorized into gene cohorts involved in plant hormone signal transduction, phenylpropanoid and flavonoid biosynthesis, and ribosome metabolism. Enrichment analysis demonstrated that ethylene pathway-related genes were significantly upregulated following CVB-CN infection, indicating a strong promotion of ethylene biosynthesis and signaling. Furthermore, disruption of the ethylene pathway in Nicotiana benthamiana, a model plant, using virus-induced gene silencing technology rendered them more susceptible to cysteine-rich protein of CVB-CN induced hypersensitive response, suggesting a crucial role of this pathway in response to CVB-CN infection. CONCLUSION: This study provides evidence that ethylene pathway has an essential role of plant in response to CVB and offers valuable insights into the defense mechanisms of chrysanthemum against Carlavirus.

Subcellular localization and interactions among TGB proteins of cowpea mild mottle virus.

Cowpea mild mottle virus (CPMMV) is a flexuous filamentous virus that belongs to the genus Carlavirus (family Betaflexiviridae). The CPMMV genome contains six open reading frames (ORFs), among which the triple gene block (TGB), encoded by ORFs 2 to 4, has been reported to encode movement proteins for different viruses. The subcellular localization of the TGB proteins of CPMMV isolate CPMMV:BR:MG:09:2 was analysed by transient expression of each protein fused to a fluorophore. Overall, the accumulation pattern and interactions among CPMMV TGB proteins (TGBp) were similar to those of their counterparts from the potex-like group. Considering these similarities, we evaluated the potential interactions between the TGB proteins of CPMMV and of potato virus X, which could complement cell-to-cell movement. The TGBp2 and TGBp3 of PVX had an effect on CPMMV TGBp1, directing it to the plasmodesmata, but the reverse was not true.

Virus Elimination from Naturally Infected Field Cultivars of Potato (Solanum tuberosum) by Transgenic RNA Interference.

Tissue culture methods enable virus elimination from vegetatively propagated crop plants but cannot prevent new infections. Here we used a tissue culture transgenic approach for curing field cultivars of Solanum tuberosum through the stimulation of RNA interference (RNAi)-based antiviral defenses. Expression cassettes carrying inverted repeats of potato virus S (PVS, genus Carlavirus) movement or coat protein sequences were used for the transformation of potato cultivars naturally infected with PVS and/or a related carlavirus potato virus M (PVM), without or with potato virus Y (PVY, genus Potyvirus). A high proportion of transformants PCR-positive for transgenes were cured from both carlaviruses and PVY. After 3-year field trials, 22 transgenic lines representing seven cultivars remained free of any virus or became infected only with PVY. Vegetative progenies of the transgenic lines of cultivar Zeren (initially coinfected with PVS, PVM, and PVY), sampled after in vitro propagation or field trials, and other field cultivars accumulated transgene-derived 21, 22, and 24 nt small interfering (si)RNAs almost exclusively from the PVS inverted repeats. Additionally, some field progenies accumulated 21-22 nt siRNAs from the entire PVY genome, confirming PVY infection. Taken together, transgenic RNAi is effective for virus elimination from naturally infected potato cultivars and their sequence-specific immunization against new infections.

Establishment of a one-step reverse transcription recombinase polymerase amplification assay for the detection of potato virus S.

Potato virus S (PVS) is a noteworthy threat to the propagation of healthy seed potatoes. Accurate and speedy detection is critical for effective PVS management. In the present study, an isothermal-based one-step reverse transcription-recombinase polymerase amplification (RT-RPA) approach was developed to detect PVS infection in potato leaves and tubers. A primer set based on the coat protein gene successfully amplified a 158 bp product out of three primer sets examined. The amplification reaction took less than 30 min to complete with no account of cross-reactivity with major potato viruses. Additionally, amplification of RT-RPA products was performed on the heating system and/or water bath at 38-42 °C. The results of sensitivity analysis revealed that one-step RT-RPA has shown 100 times higher sensitivity than routine RT-PCR for the detection of PVS in infected leaves. Furthermore, ten times higher sensitivity of RT-RPA was observed in infected tubers. The methodology was simplified further by the use of template RNA extracted using a cellular disc paper-based extraction method that detected the PVS more effectively than purified total RNA. PVS was detected in 175 samples (leaves and tubers each) of several potato varieties using this innovative technique. To our acquaintance, this is the first report of one-step RT-RPA using a basic RNA extract derived through cellular disc paper that is significantly sensitive and precise for PVS detection in potatoes. The advantages of one-step RT-RPA in terms of proficiency, robustness, and the availability of a highly pure RNA template make it an attractive choice for seed accreditation, resistance breeding, and field inspections.

Complete genome organization and characterization of Hippeastrum latent virus.

The complete genome sequences of two carlaviruses were determined by high-throughput sequencing of RNA extracted from ringspot and mosaic, disease symptoms on leaves of spider lily plants (Crinum asiaticum, family Amaryllidaceae) growing as landscape plants in Hawaii. One, named Nerine latent virus (NeLV)-Hawaii with a genome of 8281 nucleotide exhibited the highest nucleotide identity and amino acid similarity of 95.5% and 96.0%, respectively, to the genome sequence of an isolate of NeLV from Narcissus sp. in Australia (JQ395044). The second, named Hippeastrum latent virus (HiLV)-Hawaii with a genome of 8497 nucleotides exhibited the highest nucleotide identity and amino acid similarity, 84.3% and 88.7%, respectively, to the sequence of a previously uncharacterized HiLV isolate from a potted flowering plant, Amaryllis (Hippeastrum hybridum Hort) in Taiwan (DQ098905). The amino acid sequence similarities of replicase (Rep) and coat protein (CP) between HiLV-Hawaii and NeLV-Hawaii were 44.8% and 38.4%, respectively. Results of viral protein Rep and CP amino acid sequence comparisons from various carlaviruses provide evidence that HiLV and NeLV, previously classified as synonymous viruses are in fact unique viruses. This is the first report for the complete sequence, organization, and phylogenetic characterization of HiLV and the first detection of HiLV both in C. asiaticum and in the USA.

Highly divergent isolates of chrysanthemum virus B and chrysanthemum virus R infecting chrysanthemum in Russia.

BACKGROUND: Chrysanthemum is a popular ornamental and medicinal plant that suffers from many viruses and viroids. Among them, chrysanthemum virus B (CVB, genus Carlavirus, family Betaflexiviridae) is widespread in all chrysanthemum-growing regions. Another carlavirus, chrysanthemum virus R (CVR), has been recently discovered in China. Information about chrysanthemum viruses in Russia is very scarce. The objective of this work was to study the prevalence and genetic diversity of CVB and CVR in Russia. METHODS: We surveyed the chrysanthemum (Chrysanthemum morifolium Ramat.) germplasm collection in the Nikita Botanical Gardens, Yalta, Russia. To detect CVB and CVR, we used RT-PCR with virus-specific primers. To reveal the complete genome sequences of CVB and CVR isolates, metatransciptomic analysis of the cultivars Ribonette, Fiji Yellow, and Golden Standard plants, naturally co-infected with CVB and CVR, was performed using Illumina high-throughput sequencing. The recombination detection tool (RDP4) was employed to search for recombination in assembled genomes. RESULTS: A total of 90 plants of 23 local and introduced chrysanthemum cultivars were surveyed. From these, 58 and 43% plants tested positive for CVB and CVR, respectively. RNA-Seq analysis confirmed the presence of CVB and CVR, and revealed tomato aspermy virus in each of the three transcriptomes. Six near complete genomes of CVB and CVR were assembled from the RNA-Seq reads. The CVR isolate X21 from the cultivar Golden Standard was 92% identical to the Chinese isolate BJ. In contrast, genomes of the CVR isolates X6 and X13 (from the cultivars Ribonette and Fiji Yellow, respectively), were only 76% to 77% identical to the X21 and BJ, and shared 95% identity to one another and appear to represent a divergent group of the CVR. Two distantly related CVB isolates, GS1 and GS2, were found in a plant of the cultivar Golden Standard. Their genomes shared from 82% to 87% identity to each other and the CVB genome from the cultivar Fiji Yellow (isolate FY), as well as to CVB isolates from Japan and China. A recombination event of 3,720 nucleotides long was predicted in the replicase gene of the FY genome. It was supported by seven algorithms implemented in RDP4 with statistically significant P-values. The inferred major parent was the Indian isolate Uttar Pradesh (AM765837), and minor parent was unknown. CONCLUSION: We found a wide distribution of CVB and CVR in the chrysanthemum germplasm collection of the Nikita Botanical Gardens, which is the largest in Russia. Six near complete genomes of CVR and CVB isolates from Russia were assembled and characterized for the first time. This is the first report of CVR in Russia and outside of China thus expanding the information on the geographical distribution of the virus. Highly divergent CVB and CVR isolates have been identified that contributes the better understanding the genetic diversity of these viruses.

Identification and molecular characterization of a novel carlavirus infecting rose plants (Rosa chinensis Jacq.).

In the present study, the genome sequence of a potential novel virus, tentatively named "rose virus C" (RVC), was mined from publically available transcriptomic data from a Rosa chinensis plant. The complete genome sequence of RVC consists of 8,386 nt, excluding a 3' poly(A) tail, and contains five ORFs. Phylogenetic analysis showed that RVC clustered with members of the genus Carlavirus, family Betaflexiviridae. The replicase gene had 48.8-52.1% nt sequence identity to those of other carlaviruses, while the CP gene had 40.4-45.9% nt sequence identity, which is far below the species demarcation cutoff of 72%. The incidence of RVC in rose plants was low (5.4%). Overall, our data suggest that RVC is a novel atypical virus of the genus Carlavirus.

Complete genome sequence of a new isolate of caper latent virus in caper.

The genome of a new carlavirus isolate from asymptomatic wild Capparis spinosa L. plants in Sicily was sequenced via high-throughput sequencing (HTS) and 5'/3' RACE experiments. The complete genomic sequence was found to be 8,280 nt in length, excluding the poly(A) tail, and contained five putative open reading frames (ORFs). Molecular characterization revealed a close relationship to caper latent virus (CapLV), with 87% and 90% nucleotide sequence identity to available partial sequences of the ORFs encoding the replicase and coat protein of that virus. According to the molecular criteria for species demarcation, which is based on the ORF-1- and ORF-5-encoded proteins, the virus characterized in this study could be considered a variant of CapLV, and we have thus designated it as CapLV-W.

One-step real-time multiplex reverse transcription-polymerase chain reaction assay with melt curve analysis for detection of potato leafroll virus, potato virus S, potato virus X, and potato virus Y.

BACKGROUND: Certification of seed potato as free of viruses is essential for stable potato production. Among more than 30 virus species infecting potato, potato leafroll virus (PLRV), potato virus S (PVS), potato virus X (PVX), and potato virus Y (PVY) predominate worldwide and should be the targets of a high-throughput detection protocol for seed potato quarantine. RESULTS: We developed an assay based on one-step real-time multiplex reverse transcription-polymerase chain reaction (mRT-PCR) with melt curve analysis for the four viruses and one internal control, potato elongation factor 1 alpha gene (EF1α). Virus-specific primers were derived from conserved regions among randomly selected representatives considering viral genomic diversity. Our assay simultaneously detected representative Japanese isolates of PLRV, O lineage of PVS, PVX, and NTN strain of PVY. The variability of melting temperature (Tm) values for each virus was confirmed using Japanese isolates, and virus species could be identified by the values of 87.6 for PLRV, 85.9 for PVX, 82.2 (Ordinary lineage) to 83.1 (Andean lineage) for PVS, and 79.4 (NA-N strain) to 80.5 (O strain and NTN strain) for PVY on average. The reliability of calculation was validated by comparing the calculated Tm values and measured Tm values and the values had a strong linear correlation (correlation of determination: R2 = 0.9875). Based on the calculated Tm values, representative non-Japanese isolates could also be identified by our assay. For removing false positives, two criteria were set for the evaluation of result; successful amplification was considered as 30.0 ≥ threshold cycle value, and the virus-specific peak higher than the EF1α-specific peak was considered as positive. According to these criteria, our assay could detect PLRV and PVS from 100-fold dilution of potato leaf homogenate and PVX and PVY from 1000-fold in a model assay. CONCLUSION: This new high-throughput detection protocol using one-step real-time mRT-PCR was sensitive enough to detect viruses in a 100-fold dilution of singly-virus contaminated homogenate in a model assay. This protocol can detect the four viruses in one assay and yield faster results for a vast number of samples, and greatly save the labor for seed potato quarantine and field surveys.

Occurrence and distribution of viruses infecting potato in Russia.

Potato viral disease has been a major problem in potato production worldwide including Russia. Here, we detected Potato Virus M (PVM), P (PVP), S (PVS), Y (PVY), and X (PVX) and Potato Leaf Roll Virus (PLRV) by RT-PCR on potato leaves and tubers from the Northwestern (NW), Volga (VF), and Far Eastern (FE) federal districts of Russia. Each sample was co-infected with up to five viruses. RT-PCR disclosed all six viruses in NW, three in VF, and five in FE. Phylogenetic analyses of PVM and PVS strains resolved all PVM isolates in Group O (ordinary) and all PVS isolates in Group O. Seven PVY strains were detected, and they included only recombinants. PVY recombinants were thus the dominant potato virus strains in Russia, although they widely varied among the regions. Our research provides insights into the geographical distribution and genetic variability of potato viruses in Russia.

Experimental evolution of cowpea mild mottle virus reveals recombination-driven reduction in virulence accompanied by increases in diversity and viral fitness.

Major themes in pathogen evolution are emergence, evolution of virulence, host adaptation and the processes that underlie them. RNA viruses are of particular interest due to their rapid evolution. The in vivo molecular evolution of an RNA plant virus was demonstrated here using a necrotic isolate of cowpea mild mottle virus (CPMMV) and a susceptible soybean genotype submitted to serial inoculations. We show that the virus lost the capacity to cause necrosis after six passages through the host plant. When a severe bottleneck was imposed, virulence reduction occurred in the second passage. The change to milder symptoms had fitness benefits for the virus (higher RNA accumulation) and for its vector, the whitefly Bemisia tabaci. Genetic polymorphisms were highest in ORF1 (viral replicase) and were independent of the symptom pattern. Recombination was a major contributor to this diversity - even with the strong genetic bottleneck, recombination events and hot spots were detected within ORF1. Virulence reduction was associated with different sites in ORF1 associated to recombination events in both experiments. Overall, the results demonstrate that the reduction in virulence was a consequence of the emergence of new variants, driven by recombination. Besides providing details of the evolutionary mechanisms behind a reduction in virulence and its effect under viral and vector fitness, we propose that this recombination-driven switch in virulence allows the pathogen to rapidly adapt to a new host and, potentially, switch back.

First complete genome sequence of carnation latent virus, the type member of the genus Carlavirus.

The genus Carlavirus (family Betaflexiviridae, order Tymovirales) currently includes 53 species recognized by the ICTV. The NCBI GenBank database has 43 full-length carlavirus genome sequences (7,890 to 9,073 nt). Surprisingly, the type species Carnation latent virus is not associated with a complete genome sequence of a carnation latent virus (CLV) isolate; GenBank only has accessions with 1313 or fewer nucleotides. The goal of this study was to determine the full-length genome sequence of CLV. Naturally infected greenhouse-grown 'Kiwi Lace' carnation plants that tested positive for CLV by ELISA and RT-PCR were used as source plants for high-throughput sequencing, completed by 5' and 3' RACE and validated by Sanger sequencing of CLV-specific RT-PCR-generated amplicons. The complete CLV-KL sequence (MN450069) was determined to be 8,513 nt in length. In pairwise analysis, the genome shares 40-46% identity with recognized carlaviruses and the six in silico-translated proteins have 15-62% amino acid sequence identity to their respective carlavirus orthologs. The CLV-KL coat protein shares 98.4% identity with the NCBI reference sequence CLV-UK. In phylogenetic analysis, CLV clusters with butterbur mosaic virus, coleus vein necrosis virus, and garlic common latent virus. This is the first report of the full genome sequence of an isolate of carnation latent virus, the type member of the genus Carlavirus.

Complete nucleotide sequence of a new carlavirus infecting Aconitum carmichaelii in China.

A novel virus was identified in aconite (Aconitum carmichaelii Debx.) in China by high-throughput sequencing (HTS) and tentatively named "aconite virus A" (AcVA). The genomic RNA of AcVA consists of 8,844 nucleotides, excluding the poly(A) at the 3' end. Analysis of the genomic organization of AcVA indicated that it possesses a genomic structure that is typical of carlaviruses and contains six putative open reading frames (ORFs). Pairwise analysis revealed that the replicase and coat protein of AcVA share the highest amino acid sequence identity (43.78% and 57.01%) with those of coleus vein necrosis virus (CVNV) and butterbur mosaic virus (ButMV), respectively. Based on the current classification criteria for carlaviruses, AcVA should be considered a distinct member of the genus Carlavirus.

Development of RT-qPCR assays for the detection and quantification of three carlaviruses infecting hop.

American hop latent virus (AHLV), hop latent virus (HLV) and hop mosaic virus (HMV) infect members of the Humulus genus worldwide, but very little is known of the biology and etiology of these viruses. A better understanding of these viruses from the molecular level to their economic impact relies on efficient diagnostic assays. Therefore, in this study we developed reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays for the detection of AHLV, HLV, and HMV through an alignment of representative sequences from the National Center for Biotechnology Information (NCBI) database. These assays demonstrated unambiguously their high sensitivity by detecting the respective targets from as low as 102 copies of transcripts per reaction without any amplification from non-targets.

Detection and characterization of a second carlavirus in Rosa sp.

A new virus resembling members in the genus Carlavirus was identified in an Out of Yesteryear rose (Rosa sp.) by high-throughput sequencing. The virus was discovered during the screening of a rose virus collection belonging to Foundation Plant Services (UC-Davis). The full genome of the virus is 8825 nt long, excluding a poly(A) tail, and includes six predicted genes coding for replicase, triple gene block, coat protein (CP), and nucleic acid binding protein. The closest relative of the putative virus is rose virus A (RVA; genus Carlavirus), with 75% and 78% aa sequence identity in the replicase and CP, respectively. The relationship with RVA and other carlaviruses was supported by phylogenetic analyses using replicase and CP sequences. Based on genome organization, sequence identity, and phylogenetic analysis, the virus found in the Out of Yesteryear plant represents a new member of the genus Carlavirus and is provisionally named "rose virus B" (RVB). Further testing by reverse transcription PCR confirmed the presence of RVB in the original source and seven additional rose selections from the same collection.