RESUMO
Plant viruses are major restrictive pathogens to cucurbits production in Jordan. During field surveys conducted in September 2022 in the main cucurbit growing areas (desert area, Jordan Valley, and highlands), virus-like symptoms such as vein clearing, mosaic patterns, interveinal chlorosis, and fruit malformation were observed, in the presence of high whitefly populations (Bemisia tabaci MEAM1, Mdanat et al., 2022). A total of 80 leaf samples from different species [48 cucumber (Cucumis sativus), 11 squash (Cucurbita pepo), 14 melon (Cucumis melo) and 7 watermelon (Citrullus lanatus)], including 70 symptomatic and 10 asymptomatic samples, were collected for further investigations. Total RNA and DNA were extracted using RNeasy and DNeasy plant mini kit (QIAGEN), respectively, and molecular detection against an array of cucurbit-infecting viruses was conducted using protocols available in the literature (Suppl. Table). Squash vein yellowing virus (SqVYV; Potyviridae, Ipomovirus) was detected in combination with other cucurbit-infecting viruses, in 10 of 70 symptomatic samples with primers (SqYVV-v4762; SqYVV-c5512) targeting a portion of the cylindrical inclusion (CI) gene (Hernandez et al., 2021), including 3 cucumber, 4 squash and 3 melon samples, however, it was not detected in watermelon. Among other RNA viruses, cucumber green mottle mosaic virus, cucurbit yellow stunting disorder virus, cucurbit aphid-borne yellows virus, cucumber vein yellowing virus, and cucurbit chlorotic yellows virus were detected in 9, 34, 7, 18, and 23 samples, respectively. The DNA viruses, squash leaf curl virus and watermelon chlorotic stunt virus, were detected in 36 and 30 samples. None of the detected viruses were present in asymptomatic samples. All 80 samples were negative for watermelon mosaic virus and tomato leaf curl New Delhi virus. Cloning (pGEM T-Easy Vector; Promega), sequencing, and BLASTn analysis of 4 CI-specific cloned amplicons (~ 0.75 kb, GenBank Acc no. PP908660-PP908663) confirmed the identification of SqVYV, with highest BLASTn identity of ~91% and ~99% to isolates ESF3 from USA (MW584342) and SqVYV-IL from Israel (KT721735), respectively. To reconstruct the complete genome sequence of the SqVYV isolate from Jordan, total RNA from a pool of squash and melon plants was used to construct a cDNA library with the Illumina DNA Prep kit, which was sequenced on a NextSeq2000 instrument as paired reads (2x150 bp) at Leibniz Institute DSMZ, to generate 18,723,252 total reads. Bioinformatic analysis in Geneious (Biomatters) resulted in the assembly of a single genome sequence of 9,831 nt (GenBank Acc. no. PP916052), covered by 83,995 reads, with mean coverage of 1,197 (Geneious mapper, 10% maximum mismatches per read). The complete genome sequence and the deduced polyprotein sequence shared over 99% identity with SqVYV-IL from Israel. Mechanical inoculation of 10 cucumber (cv. Giant Global) and 10 squash (cv. Lebanese) plants with inoculum from infected cucumber, resulted in vein clearing and mosaic symptoms, after three weeks from inoculation, while no symptoms were observed in the six negative controls. Symptomatic plants were confirmed by PCR to be infected by SqVYV as described earlier. In this study, we report the occurrence of SqVYV for the first time in cucurbit crops in Jordan. SqVYV has been reported to cause large economic losses in cucurbits in USA and Israel, posing a major threat to watermelon growers (Adkins et al., 2007; 2008 a & b; 2013; Reingold et al., 2016; Webster et al., 2013). Our findings should encourage further studies on the incidence and prevalence of SqVYV in cucurbit and non-cucurbit crops, including weeds, to understand its epidemiology in Jordan, including its natural host range.
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White yam (Dioscorea rotundata) plants collected from farmers' fields and planted at the Areka Agricultural Research Center, Southern Ethiopia, displayed mosaic, mottling, and chlorosis symptoms. To determine the presence of viral pathogens, an investigation for virome characterization was conducted by Illumina high-throughput sequencing. The bioinformatics analysis allowed the assembly of five viral genomes, which according to the ICTV criteria were assigned to a novel potyvirus (3 genome sequences) and a novel crinivirus (2 genome sequences). The potyvirus showed ~ 66% nucleotide (nt) identity in the polyprotein sequence to yam mosaic virus (NC004752), clearly below the demarcation criteria of 76% identity. For the crinivirus, the RNA 1 and RNA 2 shared the highest sequence identity to lettuce chlorosis virus, and alignment of the aa sequence of the RdRp, CP and HSP70h (~ 49%, 45% and 76% identity), considered for the demarcation criteria, revealed the finding of a novel virus species. The names Ethiopian yam virus (EYV) and Yam virus 1 (YV-1) are proposed for the two tentative new virus species.
Assuntos
Crinivirus , Dioscorea , Genoma Viral , Filogenia , Doenças das Plantas , Potyvirus , Dioscorea/virologia , Potyvirus/genética , Potyvirus/isolamento & purificação , Potyvirus/classificação , Etiópia , Doenças das Plantas/virologia , Crinivirus/genética , Crinivirus/isolamento & purificação , Crinivirus/classificação , Genoma Viral/genética , RNA Viral/genética , Sequenciamento de Nucleotídeos em Larga Escala , Coinfecção/virologiaRESUMO
Strawberry (Fragaria × ananassa Duch.) was introduced in Sicily (Italy) in the 1930s in the small town of Maletto, on the slopes of Etna volcan, where it's currently cultivated in a total area of 30 ha. The French cv. 'Madame Moutot', appreciated for its unique flavor and intense fragrance, was there propagated vegetatively and after decades, the distinctive 'Etna ecotype' originated by adaptation to the peculiar environmental conditions of the area (Milella et al., 2006). In May 2023, in a 0.5 ha "Etna ecotype" strawberry field, virus-like symptoms were observed in approximately 50% of the plants. Symptoms included severe dwarfing, leaf cupping and chlorotic spotting which lead to decline of infected plants. To investigate the etiology of the disease, leaf samples were collected from eight symptomatic plants for analysis by High-Throughput Sequencing (HTS). To this aim, total RNAs were extracted by using the RNeasy PowerPlant Kit (Qiagen, Germany). The RNAs were pooled, depleted of ribosomal RNA (QIAseq FastSelect; Qiagen), and a library was prepared according to the Illumina DNA Prep Kit. Sequencing on a NextSeq2000 instrument at Leibniz Institute DSMZ (Braunschweig, Germany) generated 31,149,784 of paired-end reads (150 nt), which were further analyzed in Geneious Prime version 2023.2 (Biomatters) using a custom workflow for virus discovery and genome assembly. Analysis of the assembled contigs by local BLASTn and BLASTp alignments against a custom plant virus database of NCBI nuclear-core (NC) reference sequences assigned a number of contigs to accession NC_025435, strawberry polerovirus 1 (SPV-1). Reconstruction of the virus genome by assembly of contigs and reads alignment resulted in a nearly complete genome sequence of SPV-1 (GenBank Acc. No. OR989958) showing by BLASTn 98.69% identity to the SPV-1 NC reference sequence, and 98.99 % identity with an isolate from the Czech Republic (GenBank Acc. OL421571). To confirm the presence of SPV-1 in each sample, RT-PCR using specific primers designed in this study SPV-1-CP-1F (5'-TCGAGATACGTCTAGAACTGCAA-3') and SPV-1-CP-1R (5'-GAGAGGCCCCTTCTACCTATTTG-3') targeting the entire 623 bp coat protein (CP) gene was performed. Amplicons of the expected size were obtained in five samples and Sanger-sequenced. The resulting sequences shared 99.85% - 100% of identity to the HTS - derived sequence (GenBank Acc. No. OR989958) through BLASTn analysis. Strawberry mottle virus (SMoV), strawberry mild yellow edge virus (SMYEV) and strawberry crinkle virus (SCV) were detected in the same library in addition to SPV-1 and then confirmed by RT-PCR using specific primers (Martin & Tzanetakis 2013). Strawberry polerovirus 1, related to the genus Polerovirus in the family Solemoviridae, was first reported in strawberries in Canada (Xiang et al. 2015) and was thereafter detected in the United States (Thekke-Veetil & Tzanetakis 2016), Argentina (Luciani et al. 2016), and Nepal (Kuwak et al. 2022). To date, the virus has been reported in Europe only in the Czech Republic (Franova et al. 2021). To our knowledge, this is the first report of SPV-1 in strawberry plants in Italy. Although the correlation between SPV-1 and strawberry decline (SD) is still uncertain (Xiang et al. 2015) transmission of the virus via aphids has recently been demonstrated (Franova et al. 2021). Our report let to hypothesize that its dissemination in Europe can be considered as increasing.
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IMPORTANCE: We report here efforts to benchmark performance of two widespread approaches for virome analysis, which target either virion-associated nucleic acids (VANA) or highly purified double-stranded RNAs (dsRNAs). This was achieved using synthetic communities of varying complexity levels, up to a highly complex community of 72 viral agents (115 viral molecules) comprising isolates from 21 families and 61 genera of plant viruses. The results obtained confirm that the dsRNA-based approach provides a more complete representation of the RNA virome, in particular, for high complexity ones. However, for viromes of low to medium complexity, VANA appears a reasonable alternative and would be the preferred choice if analysis of DNA viruses is of importance. Several parameters impacting performance were identified as well as a direct relationship between the completeness of virome description and sample sequencing depth. The strategy, results, and tools used here should prove useful in a range of virome analysis efforts.
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Metagenômica , Biologia Sintética , Viroma , Vírus , Vírus de DNA/classificação , Vírus de DNA/genética , Metagenômica/métodos , Metagenômica/normas , Vírion/genética , Viroma/genética , Biologia Sintética/métodos , RNA de Cadeia Dupla/genética , Vírus/classificação , Vírus/genética , Vírus de Plantas/classificação , Vírus de Plantas/genéticaRESUMO
Clematis vitalba L. is a climbing shrub and a pioneer plant in abandoned orchards or vineyards that are widespread in temperate climate zones. In past years, several viruses infecting the Clematis species have been identified, including different ilarviruses. Prunus virus I (PrVI) is a recently described ilarvirus, which has been shown to infect sweet cherries and peaches in Greece. Moreover, its presence has been detected in ornamental Clematis in Russia. In the present work, we analyzed the virome of wildly growing C. vitalba plants from Hungary, Slovakia and Croatia showing different kinds of symptoms using high-throughput sequencing (HTS) of small RNAs or ribodepleted RNAs. Applying HTS enabled us to identify the presence of PrVI in C. vitalba, and the bioinformatic analyses were further validated with RT-PCR using PrVI-specific primers and Sanger dideoxy sequencing. Nearly full genome sequences of all three viral RNAs of one Hungarian, two Slovak and one Croatian isolate were determined. Their phylogenetic analysis showed high similarity to each other and to other PrVI isolates described from Central Europe. As the sampled plants were co-infected with other viruses, it is not possible to determine a direct correlation between the infection with PrVI and the observed symptoms. Analyses of different Prunus species in stock collection showed infection of several peach and sweet cherry varieties in Hungary. Our results expand the knowledge on the natural host range of PrVI and highlight the necessity to evaluate alternative plant hosts (even non-Prunus) of PrVI and the role of the virus in the etiology of the potential diseases.
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In the field of plant virology, the usage of reverse genetic systems has been reported for multiple purposes. One is understanding virus-host interaction by labelling viral cDNA clones with fluorescent protein genes to allow visual virus tracking throughout a plant, albeit this visualization depends on technical devices. Here we report the first construction of an infectious cDNA full-length clone of beet mosaic virus (BtMV) that can be efficiently used for Agrobacterium-mediated leaf inoculation with high infection rate in Beta vulgaris, being indistinguishable from the natural virus isolate regarding symptom development and vector transmission. Furthermore, the BtMV clone was tagged with the genes for the monomeric red fluorescent protein or the Beta vulgaris BvMYB1 transcription factor, which activates the betalain biosynthesis pathway. The heterologous expression of BvMYB1 results in activation of betalain biosynthesis genes in planta, allowing visualization of the systemic BtMV spread with the naked eye as red pigmentation emerging throughout beet leaves. In the case of BtMV, the BvMYB1 marker system is stable over multiple mechanical host passages, allows qualitative as well as quantitative virus detection and offers an excellent opportunity to label viruses in plants of the order Caryophyllales, allowing an in-depth investigation of virus-host interactions on the whole plant level.
Assuntos
Beta vulgaris , Potyvirus , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Betalaínas , Beta vulgaris/metabolismo , DNA Complementar/genética , Potyvirus/genética , Doenças das PlantasRESUMO
Two new umbravirus-like associated RNAs (ulaRNAs) were found, respectively, in maize and Johnsongrass samples from Ecuador. The complete sequences consist of 3,053 and 3,025 nucleotides, respectively, and contain four open reading frames (ORFs). Their genome sequences were 58% identical to each other and 28 to 60% identical to the most closely related viruses. Phylogenetic analysis using full genome sequences and amino acid sequence of the RNA-dependent-RNA polymerase (RdRp) placed both sequences in a clade sharing the most recent common ancestor with ulaRNAs from sugarcane and maize, suggesting that they belong to a monophyletic grass-infecting lineage. Their terminal regions exhibit features common to umbraviruses and ulaRNAs.
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Sorghum , Tombusviridae , Equador , Genoma Viral , Fases de Leitura Aberta , Filogenia , RNA , RNA Viral/genética , Tombusviridae/genética , Zea maysRESUMO
The family Alphaflexiviridae includes viruses with flexuous filamentous virions that are 470-800 nm in length and 12-13 nm in diameter. Alphaflexiviruses have a single-stranded, positive-sense RNA genome of 5.5-9 kb. They infect plants and plant-infecting fungi. They share a distinct lineage of alphavirus-like replication proteins that is unusual in lacking any recognized protease domain. With a single exception, cell-to-cell and long-distance movement is facilitated by triple gene block proteins in plant-infecting genera. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Alphaflexiviridae, which is available at www.ictv.global/report/alphaflexiviridae.
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Flexiviridae/classificação , Flexiviridae/fisiologia , Flexiviridae/ultraestrutura , Regulação Viral da Expressão Gênica , Genoma Viral , Especificidade de Hospedeiro , Filogenia , Vírion/ultraestrutura , Replicação ViralRESUMO
Viruses in the family Closteroviridae have a mono-, bi- or tripartite positive-sense RNA genome of 13-19 kb, and non-enveloped, filamentous particles 650-2200 nm long and 12 nm in diameter. They infect plants, mainly dicots, many of which are fruit crops. This is a summary of the ICTV Report on the family Closteroviridae, which is available at ictv.global/report/closteroviridae.
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Closteroviridae/genética , Closteroviridae/metabolismo , Closteroviridae/ultraestrutura , Genoma Viral , Filogenia , Vírion/genética , Vírion/ultraestrutura , Replicação ViralRESUMO
Using next-generation sequencing to characterize agents associated with a severe stunting disease of parsley from Germany, we identified a hitherto undescribed virus. We sequenced total RNA and rolling-circle-amplified DNA from diseased plants. The genome sequence of the virus shows that it is a member of the genus Nanovirus, but it lacks DNA-U4. In addition to the seven genomic DNAs of the virus, we identified a second DNA-R and seven distinct alphasatellites associated with the disease. We propose the name "parsley severe stunt associated virus" (PSSaV) for this novel nanovirus.
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DNA Viral/genética , Nanovirus/genética , Nanovirus/isolamento & purificação , Petroselinum/virologia , Doenças das Plantas/virologia , Sequência de Bases , DNA Circular/genética , DNA Satélite/genética , DNA de Cadeia Simples/genética , Genoma Viral/genética , Alemanha , Sequenciamento de Nucleotídeos em Larga Escala , Nanovirus/classificaçãoRESUMO
Virus discovery based on high-throughput sequencing relies on enrichment for virus sequences prior to library preparation to achieve a sufficient number of viral reads. In general, preparations of double-stranded RNA or total RNA preparations treated to remove rRNA are used for sequence enrichment. We used virus-specific antibodies to immunocapture virions from plant sap to conduct cDNA synthesis, followed by library preparation and HTS. For the four potato viruses PLRV, PVY, PVA and PYV, template preparation by virion immunocapture provided a simpler and less expensive method than the enrichment of total RNA by ribosomal depletion. Specific enrichment of viral sequences without an intermediate amplification step was achieved, and this high coverage of sequences across the viral genomes was important to identify rare sequence variations. Using this approach, the first complete genome sequence of a potato yellowing virus isolate (PYV, DSMZ PV-0706) was determined in this study. PYV can be confidently assigned as a distinct species in the genus Ilarvirus.
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Anticorpos Antivirais , Vírus de Plantas/genética , Vírus de Plantas/imunologia , Vírion/genética , Vírion/imunologia , Animais , Especificidade de Anticorpos , Carlavirus/genética , Carlavirus/imunologia , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Luteoviridae/genética , Luteoviridae/imunologia , Filogenia , Vírus de Plantas/isolamento & purificação , Potyvirus/genética , Potyvirus/imunologia , RNA Viral/genética , Análise de Sequência de RNA , Solanum tuberosum/virologia , Vírion/isolamento & purificaçãoRESUMO
Celery latent virus (CeLV) is an incompletely described plant virus known to be sap and seed transmissible and to possess flexuous filamentous particles measuring about 900 nm in length, suggesting it as a possible member of the family Potyviridae. Here, an Italian isolate of CeLV was transmitted by sap to a number of host plants and shown to have a single-stranded and monopartite RNA genome being 11â519 nucleotides (nts) in size and possessing some unusual features. The RNA contains a large open reading frame (ORF) that is flanked by a short 5' untranslated region (UTR) of 13 nt and a 3' UTR consisting of 586 nt that is not polyadenylated. CeLV RNA shares nt sequence identity of only about 40â% with other members of the Potyviridae (potyvirids). The CeLV polyprotein is notable in that it starts with a signal peptide, has a putative P3N-PIPO ORF and shares low aa sequence identity (about 18â%) with other potyvirids. Although potential cleavage sites were not identified for the N-terminal two-thirds of the polyprotein, the latter possesses a number of sequence motifs, the identity and position of which are characteristic of other potyvirids. Attempts at constructing an infectious full-length cDNA clone of CeLV were successful following Rhizobium radiobacter infiltration of Nicotiana benthamiana and Apium graveolens. CeLV appears to have the largest genome of all known potyvirids and some unique genome features that may warrant the creation of a new genus, for which we propose the name 'celavirus'.
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Apium/virologia , DNA Complementar , Potyviridae/crescimento & desenvolvimento , Potyviridae/genética , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Agrobacterium tumefaciens/genética , Vetores Genéticos , Itália , Fases de Leitura Aberta , Doenças das Plantas/virologia , Poliproteínas/genética , Potyviridae/isolamento & purificação , RNA Viral/genética , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Nicotiana , Proteínas Virais/genéticaRESUMO
Recent metagenomic studies have provided an unprecedented wealth of data, which are revolutionizing our understanding of virus diversity. A redrawn landscape highlights viruses as active players in the phytobiome, and surveys have uncovered their positive roles in environmental stress tolerance of plants. Viral infectious clones are key tools for functional characterization of known and newly identified viruses. Knowledge of viruses and their components has been instrumental for the development of modern plant molecular biology and biotechnology. In this review, we provide extensive guidelines built on current synthetic biology advances that streamline infectious clone assembly, thus lessening a major technical constraint of plant virology. The focus is on generation of infectious clones in binary T-DNA vectors, which are delivered efficiently to plants by Agrobacterium. We then summarize recent applications of plant viruses and explore emerging trends in microbiology, bacterial and human virology that, once translated to plant virology, could lead to the development of virus-based gene therapies for ad hoc engineering of plant traits. The systematic characterization of plant virus roles in the phytobiome and next-generation virus-based tools will be indispensable landmarks in the synthetic biology roadmap to better crops.
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Biotecnologia , Metagenômica , Patologia Vegetal , Vírus de Plantas , Biologia Sintética , Biotecnologia/tendências , Humanos , Patologia Vegetal/tendências , Vírus de Plantas/genética , Vírus de Plantas/fisiologia , Plantas/virologia , Biologia Sintética/tendênciasRESUMO
The complete genome sequence of an isolate of euphorbia ringspot virus (EuRSV) was determined by deep sequencing and rapid amplification of cDNA ends (RACE) RT-PCR. It has an RNA genome of 10,154 nucleotides in size, excluding the poly(A) tail, and encodes a polyprotein of 3265 amino acids. Phylogenetic analysis from this study supports the earlier taxonomic assignment to the genus Potyvirus; however, a gene encoding the HAM1h protein, inserted between NIb and CP of the EuRSV genome, which was previously only observed for cassava brown streak virus and Ugandan cassava brown streak virus of the genus Ipomovirus, is an unusual feature of this potyvirus, which otherwise has typical potyvirus genome features.
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Genoma Viral , Potyvirus/genética , Potyvirus/isolamento & purificação , RNA Viral/genética , Análise de Sequência de DNA , Análise por Conglomerados , Euphorbia/virologia , Ordem dos Genes , Sequenciamento de Nucleotídeos em Larga Escala , Filogenia , Doenças das Plantas/virologia , Poliproteínas/genética , Potyvirus/classificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de SequênciaRESUMO
Luffa aphid-borne yellows virus (LABYV) was proposed as the name for a previously undescribed polerovirus based on partial genome sequences obtained from samples of cucurbit plants collected in Thailand between 2008 and 2013. In this study, we determined the first full-length genome sequence of LABYV. Based on phylogenetic analysis and genome properties, it is clear that this virus represents a distinct species in the genus Polerovirus. Analysis of sequences from sample TH24, which was collected in 2010 from a luffa plant in Thailand, reveals the presence of two different full-length genome consensus sequences.
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Genoma Viral , Luffa/virologia , Luteoviridae/genética , Luteoviridae/isolamento & purificação , Sequência de Bases , Sequência Consenso , Luteoviridae/classificação , Dados de Sequência Molecular , Fases de Leitura Aberta , Filogenia , TailândiaRESUMO
Etiological studies of a recently emerged bushy top disease of tobacco in Ethiopia indicated that a ~4.5-kb dsRNA from infected plants represents an umbravirus, whereas a smaller band (~0.5 kb) is that of a new satellite RNA. Potato leafroll virus was also consistently associated with the disease. The three agents, whose experimental host ranges are restricted to members of the family Solanaceae, always occurred together in field samples and are transmitted together by the aphid Myzus persicae nicotianae. The umbravirus, which represents a new species, is most closely related to groundnut rosette virus, and the name Ethiopian tobacco bushy top virus is proposed.
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Luteoviridae/isolamento & purificação , Nicotiana/virologia , Doenças das Plantas/virologia , Vírus de Plantas/isolamento & purificação , RNA Satélite/isolamento & purificação , Animais , Afídeos/virologia , Análise por Conglomerados , Etiópia , Luteoviridae/genética , Dados de Sequência Molecular , Filogenia , Vírus de Plantas/genética , RNA Satélite/genética , RNA Viral/genética , Análise de Sequência de DNA , Homologia de SequênciaRESUMO
The complete genome of a tomato mild mottle virus (ToMMV) isolate was analysed, and some biological features were characterized. The ssRNA genome of ToMMV from Ethiopia encompasses 9283 nucleotides (excluding the 3' poly(A) tail) and encodes a polyprotein of 3011 amino acids. Phylogenetic and pairwise comparisons with other members of the family Potyviridae revealed that ToMMV is the most divergent member of the genus Ipomovirus, with a genome organization similar to that of members of the species Sweet potato mild mottle virus, the type species of the genus. In contrast to earlier reports, ToMMV isolates from Yemen and Ethiopia were not transmitted by the aphid Myzus persicae, but they were transmitted very erratically by the whitefly Bemisia tabaci. A comparison of the 3'-proximal sequences of different isolates provided evidence for geographically associated genetic variation.
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Evolução Molecular , Genoma Viral , Doenças das Plantas/virologia , Potyviridae/genética , Solanum lycopersicum/virologia , Sequência de Aminoácidos , Sequência de Bases , Dados de Sequência Molecular , Filogenia , Poliproteínas/genética , Potyviridae/classificação , Potyviridae/isolamento & purificaçãoRESUMO
The complete nucleotide sequence of an Albanian isolate of grapevine leafroll-associated virus 7 (GLRaV-7-Alb) was determined. The viral genome consists of 16,404 nucleotides and has nine open reading frames (ORFs) that potentially encode proteins, most of which are typical for members of the family Closteroviridae. Only the 25-kDa (ORF8) and 27-kDa (ORF9) proteins had no apparent similarity to other viral proteins in the sequence databases. The genome structure of GLRaV-7-Alb closely resembles that of little cherry virus 1 and cordyline virus 1. In phylogenetic trees constructed with HSP70h sequences, these three viruses cluster together in a clade next to that comprising members of the genus Crinivirus, to which they are more closely related than to the clostero- and ampeloviruses. The molecular properties of these three viruses differ sufficiently from those of members of the three extant genera of the family Closteroviridae to warrant their classification in a novel genus.