A virus was isolated from potted plants of an unidentified species of Aeonium (Crassulaceae), a succulent ornamental very common in Southern Italy, showing faint chlorotic spots and rings on both leaf surfaces. The virus was successfully transmitted by sap inoculation to a limited range of hosts, and propagated in Nicotiana benthamiana which was used for ultrastructural observations and virus purification. Virus particles are isometric, ca. 30 nm in diameter, have a single type of coat protein (CP) subunits 54 kDa in size, that encapsidate single-stranded positive-sense RNA species of 7,549 (RNA1) and 4,010 (RNA2) nucleotides. A third RNA molecule 3,472 nts in size entirely derived from RNA2 was also detected in infected Aeonium plants. The structural organization of both genomic RNAs and the cytopathological features were comparable to those of nepoviruses. In addition, amino acid sequence comparisons of CP and the Pro-Pol region (a sequence containing parts of the proteinase and polymerase) with those of other nepoviruses showed that the Aeonium virus belongs to the subgroup A of the genus Nepovirus and is phylogenetically close to Tobacco ringspot virus (TRSV). Comparison of each single domain of TRSV and the Aeonium virus polyproteins disclosed a relatively low percentage of identity throughout. Moreover, the Aeonium virus showed to be serologically distinct from TRSV. Based on the species demarcation criteria for the family Secoviridae, the virus under study appears to be a novel member of the genus Nepovirus for which the name of Aeonium ringspot virus (AeRSV) is proposed.

Tombusviruses may support the replication of satellite (sat) RNAs. In particular, two satRNAs, sat L and Cymsat RNAs, are replicated by carnation Italian ringspot (CIRV) and tomato bushy stunt (TBSV) virus, but not by cymbidium ringspot virus (CymRSV) in vitro transcripts unless they contain a poly(A) tail at the 3_ end. Conversely, the replication of both satRNAs was supported by virus particles or viral RNA ofthe original CymRSV inoculum even in the absence of the poly(A) tail. Sequence and mutational analyses revealed that the full-length infectious CymRSV clone contains one relevant sequence variation in the ORF 1-encoded protein (p33) compared with the original inoculum, i.e. a Ser19 TCC codon instead of a Phe19 TTC codon, which inhibited the replication of sat L and Cymsat RNAs. It is suggested that this amino acid is contained in a domain essential for the replication of some subviral RNAs

This article lists the changes to virus taxonomy approved and ratified by the International Committee on Taxonomy of Viruses in February 2018. A total of 451 species, 69 genera, 11 subfamilies, 9 families and one new order were added to the taxonomy. The current totals at each taxonomic level now stand at 9 orders, 131 families, 46 subfamilies, 803 genera and 4853 species. A change was made to the International Code of Virus Classification and Nomenclature to allow the use of the names of people in taxon names under appropriate circumstances. An updated Master Species List incorporating the approved changes was released in March 2018 (https://talk.ictvonline.org/taxonomy/).

A virus was isolated from potted plants of an unidentified species of Aeonium, a succulent ornamental very common in Southern Italy, showing chlorotic spots and rings on both leaf surfaces. It was successfully transmitted by sap inoculation to a limited range of hosts, including Nicotiana benthamiana which was used for ultrastructural observations and virus purification. Virus particles are isometric, ca. 30nm in diameter, have a single type of coat protein (CP) subunits 54kDa in size, that encapsidate single-stranded positive-sense RNA species of 7,549 (RNA1) and 4,010 (RNA2) nucleotides. A third RNA molecule 3,472 nts in size entirely derived from RNA2 was also found. The structural organization of both genomic RNAs and the cytopathological features were comparable to those of nepoviruses. In addition, amino acid sequence comparisons of CP and the Pro-Pol region (a sequence containing parts of the proteinase and polymerase) with those of other nepoviruses showed that the Aeonium virus belongs to the subgroup A of the genus Nepovirus and is phylogenetically close to, but serologically distinct from tobacco ringspot virus (TRSV). Based on the species demarcation criteria for the family Secoviridae, the virus under study appears to be a novel member of the genus Nepovirus for which the name of Aeonium ringspot virus (AeRSV) is proposed.

First report of a resistance-greaking strain of Tomato spotted wilt virus from Gerbera jamesonii in Apulia, Southern Italy

Agroinfiltration of Nicotiana benthamiana leaves with a construct expressing the coat protein of Cymbidium ringspot virus (CyRSV) resulted in the production of virus-like particles (VLPs) which showed a preferential localization within mitochondria with an apparently intact bounding membrane. VLPs were either non penetrated or penetrated by the negative stain. The former had the same outward aspect and size of wild type CymRSV and were assumed to encapsidate the CP messanger RNA, which was recovered from virus preparations purified from agroinfiltrated tissues. In addition, VLPs were shown to be able to encapsidate tombusviral satellite RNAs. Immunoblot analysis of dissociated VLPs showed that they were made up of a protein indistinguishable from the native CymRSV CP. A mitochondrial targeting signal was identified in the N-terminal region of CymRSV CP at amino acid (aa) position 10-27. The fusion protein CPMyc, containing the 10-aa Myc epitope fused to the CymRSV CP C-terminus, formed VLPs that were decorated by a Myc-specific antiserum.

Positive-strand RNA [(+)RNA] viruses, the largest class of viruses, include many important pathogens of humans, animals and plants, sharing common replication mechanisms. A highly conserved feature of (+)RNA virus replication is the association of the viral replication complex with specific intracellular membranes, which are induced to proliferate and are extensively rearranged to form vesicles (or spherules). These partially closed vesicular enclaves constitute the confined environment in which virus and host factors concentrate to allow for a productive viral RNA synthesis, under conditions protected from host defense reactions. Virus-encoded proteins are responsible for the intracellular localization of the replication complex and for the formation of spherules. The association of viral replicase proteins with the outer membrane of mitochondria has been studied in details with Carnation Italian ringspot virus (CIRV, genus Tombusvirus, family Tombusviridae), a virus with a (+)RNA genome 4.8 kb in size, containing five ORFs. In infected plants, CIRV replication takes place in membranous structures originating from vesiculation of the mitochondrial outer membrane. The signals targeting and anchoring CIRV replication complex to the mitochondrial membrane are contained in the 36-kDa product of ORF1 (p36). Most traits of CIRV replication can be reconstituted in Saccharomyces cerevisiae cells, thus representing a good model for virus-host interaction studies. Heterologous expression of p36 protein fused or not to GFP localizes to mitochondria in yeast cells and causes organelle and membrane proliferation. To gain insights into the interaction between p36 and mitochondria, the effects were studied of p36 heterologous expression on yeast cell viability as well as on programmed cell death induced by acetic acid. It was shown that p36 affects cell viability and seems to exert an inhibitory effect on the nature of acetic acid-induced cell death. Due to the conservation of replication mechanisms between (+)RNA viruses, data obtained with simple model viruses, like CIRV, in a simple eukaryotic host, could be extended to pathogens of higher eukaryotes.

Positive-strand RNA viruses constitute a large group of infectious agents causing major plant, animal and human diseases. Genome replication occurs in association with host cell membrane structures derived from the endoplasmic reticulum (ER) (picornaviruses, potyviruses, comoviruses, nepoviruses and bromoviruses) or from the limiting membrane of organelles such as lysosomes or endosomes (alphaviruses), vacuoles (cucumoviruses), mitochondria (nodaviruses, some tombusviruses, carmoviruses, ampeloviruses and maculaviruses), peroxisomes (several tombusviruses) and chloroplasts (tymoviruses and some marafiviruses). Viral proteins are involved in targeting the replication complex to the specific intracellular membranes. Intracellular membranes are normally modified to form vesicular structures with a narrow neck through which the interior of the vesicles communicates with the cytosol. A variety of observations indicates that, indeed, virus replication takes place in the closed environment of the vesicles, including co-localization of virus replicase and virus RNA progeny with cell membranes and strong dependance of viral synthesis on lipid metabolism. Confinement of the virus replication complexes in closed environments represents an advantage for the viral RNA, which is protected from degradation of host ribonucleases and recognition of host defence reactions. Vesiculation of the target cellular membrane in natural hosts and in the yeast Saccharomyces cerevisisae, an alternative model host for studying virus replication, is well documented for nodaviruses (animal viruses) and bromo- and tombusviruses (plant viruses). Flock house virus (FHV, genus Nodavirus, family Nodaviridae) protein A is a transmembrane protein that contains N-terminal signals targeting the outer membrane of mitochondria and elicits the formation of vesicular structures. Brome mosaic virus (BMV, genus Bromovirus, family Bromoviridae) replication occurs on the ER membranes, in spherules containing genomic RNA, the 2a replicase protein and the 1a virus RNA replication factor. The 1a multifunctional protein has RNA capping and helicase functions, and directs targeting and assembly of the replication complex on the ER membranes. The replication of members of the genus Tombusvirus (family Tombusviridae) has been studied in plant and yeast cells. Carnation Italian ringspot virus p36 protein contains the determinants for targeting the replication complex to the outer membrane of mitochondria; the p33 of several other tombusviruses contains sequences necessary to localize virus replication on the limiting membrane of peroxisomes.

Positive-strand RNA [(+)RNA] viruses constitute the largest class of infectious agents causing major plant, animal and human diseases. (+)RNA viruses share common replication mechanisms; in particular, a highly conserved feature is the association of the genome replication with host cell membrane structures derived from the endoplasmic reticulum (ER) (picornaviruses, potyviruses, comoviruses, nepoviruses and bromoviruses) or from the limiting membrane of organelles such as lysosomes or endosomes (alphaviruses), vacuoles (cucumoviruses), mitochondria (nodaviruses, some tombusviruses, carmoviruses, ampeloviruses and maculaviruses), peroxisomes (several tombusviruses) and chloroplasts (tymoviruses and some marafiviruses). Virus-encoded proteins are responsible for the intracellular localization of the viral replication complex. Cell membranes are modified to form vesicular structures in which virus replication takes place, protected from degradation of host ribonucleases and recognition of host defence reactions. Vesiculation of the target cellular membrane in natural hosts and in the yeast Saccharomyces cerevisisae, an alternative model host for studying virus replication, is well documented for nodaviruses (animal viruses) and bromo- and tombusviruses (plant viruses). An overview of virus-induced cell membrane modifications in plant and yeast cells will be given.

In March 2011 plants of Aeonium spp., family Crassulaceae, showing chlorotic spots and rings on both leaf surfaces were observed in a private garden in the vicinity of Salerno (southern Italy). Electron microscope observations of leaf dips from several of these plants revealed the presence of isometric virus-like particles ca. 30 nm in diameter, some of which were partially or completely penetrated by the negative stain, as if they were devoid of nucleic acid in part or totally. A number of herbaceous hosts were successfully infected after mechanical inoculation with sap expressed from symptomatic Aeonium plants. For example, Nicotiana benthamiana and Lycopersicon esculentum (tomato) were systemically invaded and reacted with mottling and deformation of the leaves and yellowish concentric rings and line patterns, respectively.A virus with isometric particles indistinguishable from those seen in leaf dips was readily purified from symptomatic N. benthamiana leaves. RNA extracted from virus particles and analyzed in ethidium bromide-permeated agarose gels migrated as two separate bands. These were recovered and used as template for synthesizing cDNAs, which were cloned and partially sequenced.The viral genome was confirmed to consist of two distinct RNA species which were molecularly similar, but not identical, to RNA-1 and RNA-2 of Tobacco ringspot virus (TRSV) (Sanfaçon et al., 2012). TRSV has been intercepted in Italy in imported gladiolus bulbs (Bellardi and Marani, 1985) but, to our knowledge, has never been found in a cultivated plant. Its potential danger to economical crops like tomato is to be taken into account, especially should the presence of a nematode vector be ascertained.

Si riferisce sui risultati di una indagine condotta su piante di gerbera coltivate nel Nord barese ed in Provincia di Lecce interessate, rispettivamente, da estese necrosi delle foglie,degli steli e dei fiori e da virescenza e fillodia a carico dei soli fiori. L'agente causale dei sintomi necrotici è stato identificato in un ceppo 'resistance breaking' (RB) di Tomato spotted wilt virus (TSWV) mentre nel caso della virescenza e fillodia dei fiori la malattia è stata associata alla presenza di 'Candidatus Phytoplasma asteris'. Entrambi i patogeni sono stati già segnalati sulle coltivazioni di gerbera presenti in altre regioni italiane ma questa è la prima segnalazione della presenza di un ceppo RB di TSWV e la prima segnalazione pugliese della presenza del fitoplasma sulla ornamentale. Viene nuovamente sottolineata l'importanza del ritrovamento dei ceppi RB di TSWV anche nei confronti di colture orticole e sono fornite indicazioni sulla eco-epidemiologia delle infezioni da fitoplasmi.

The biological and molecular properties of a new satellite RNA (satRNA L) associated with Tomato bushy stunt virus (TBSV) are described. satRNA L consists of a linear single-stranded RNA 615 nucleotides in size, lacks significant open reading frames and has no sequence homology with the helper genome other than in the 5’-proximal seven nucleotides and in a central region that is also conserved in all tombusvirus genomic, defective interfering (DI) and satellite RNAs. Secondary structure analysis showed the presence of high order domains similar to those described for other tombusvirus RNAs. Shorter-than-unit-length molecules were shown not to be related to a silencing mechanism. satRNA L did not modify the symptoms induced by TBSV at all temperature conditions tested. A full-length cDNA clone was constructed and used in coinoculations with transcripts of Carnation Italian ringspot virus (CIRV) and Cymbidium ringspot virus (CymRSV). CIRV, but not CymRSV, supported the replication of satRNA L. Using CIRV/CymRSV hybrid infectious clones, two regions were identified as possible determinants of the different ability to support satRNA L replication. The first region is in the 5’-untranslated region, which folds differently in CymRSV in comparison with CIRV and TBSV; the second region is in the ORF 1-encoded protein where a more efficient satRNA L binding domain is suggested to be present in CIRV.

Positive-strand RNA virus replication always occurs in association with rearranged host cell membranes. In infected plants, replication of tombusviruses takes place in membranous structures, known as multivesicular bodies (MVBs) which originate from vesiculation of the limiting membrane of peroxisomes or of the mitochondrial outer membrane. We investigated the mechanism of vesicle formation on mitochondria in Carnation Italian ringspot virus (CIRV) infections. The genome of CIRV consists of a 4.8 kb single-stranded RNA molecule encapsidated in icosahedral particles. The genome lacks a 5' cap structure, is not polyadenylated at the 3' end and contains five open reading frames (ORFs). ORF1- and ORF2-encoded p36 and p95 proteins are essential for viral replication. In particular, p95 contains the conserved motifs of RNA-dependent RNA polymerases and p36 the motifs for viral RNA binding and recruitment to replication sites. ORF 3 codes for the p41 coat protein, ORFs 4 and 5 encode p22, required for cell-to-cell movement of the virus in infected plants, and p19, which is a suppressor of virus induced gene silencing, respectively. The requirements for the formation of MVBs were studied in cells infected by cis replicating wild type or defective CIRV genomes, or expressing the p36 and p95 replicase proteins from a non replicatable genome but able to support in trans the replication of defective interfering RNAs. It was ascertained that MVB developed in cells transfected with CIRV defective genomes that, although expressing only p36 and p95, were able to replicate.

Tombusvirus infections are often associated with satellite (sat) RNAs, which are subviral molecules sharing no significant sequence homology with the helper genome other than some specific nucleotide stretches. We have characterized a new satRNA (satRNA L) associated with Tomato bushy stunt virus (TBSV). satRNA L consists of a single-stranded RNA 615 nucleotides in size, lacks significant open reading frames and has no sequence homology with the helper genome other than in the 5'-proximal seven nucleotides and in a central region that is also conserved in all tombusvirus genomic, defective interfering and satellite RNAs. Secondary structure analysis shows the presence of high order domains similar to those described for other tombusvirus RNAs. satRNA L does not modify the symptoms induced by TBSV. The accumulation of satRNA L is dependent on the temperature. siRNAs originate from both positive and negative polarities and may have a role in satRNA accumulation, whereas shorter-than-unit-length molecules are not related to a silencing mechanism. Coinoculation of satRNA L with transcripts of Carnation Italian ringspot virus (CIRV) and Cymbidium ringspot virus (CymRSV) shows that CIRV, but not CymRSV, supports satRNA L replication. The sequence region responsible for supporting satRNA L replication maps within the 5'-UTR and the ORF1 N-terminal region of CIRV genome. satRNA L can be replicated if coinoculated with CymRSV virus particles, suggesting that the coat protein has a role in the early stages of replication. The different intracellular replication site of CIRV and CymRSV may account for their diverse capability to replicate satRNA L.

Tepovirus is a new monotypic genus of plant viruses typified by potato virus T (PVT), a virus with helically constructed filamentous particles that are 640 nm long, previously classified as unassigned species in the family Betaflexiviridae. Virions have a single-stranded positive-sense polyadenylated RNA genome that is 6.5 kb in size, and a single type of coat protein with a size of 24 kDa. The viral genome contains three slightly overlapping ORFs encoding, respectively, the replication-related proteins (ORF1), a putative movement protein of the 30 K type (ORF2) and the coat protein (ORF3). Its structure and organization (number and order of genes) resembles that of trichoviruses and of citrus leaf blotch virus (CLBV, genus Citrivirus) but has a smaller size. Besides potato, the primary host, PVT can experimentally infect herbaceous hosts by mechanical inoculation. No vector is known, and transmission is through propagating material (tubers), seeds and pollen. PVT has a number of biological, physical and molecular properties that differentiate it from betaflexiviruses with a 30K-type movement protein. It is phylogenetically distant from all these viruses, but least so from grapevine virus A (GVA), the type member of the genus Vitivirus, with which it groups in trees constructed using the sequences of all of the genes.

The replication of positive-strand RNA [(+)RNA] viruses is always associated with specific cell membranes. The association of viral replicase proteins with mitochondria has been studied with Carnation Italian ringspot virus (CIRV), a (+)RNA plant virus replicating in membranous structures originating from vesiculation of the mitochondrial outer membrane. The replication-associated protein p36 is responsible for targeting and anchoring CIRV replication complex to the mitochondrial membrane. Yet, the mechanisms by which (+)RNA viruses control host cell fate are still unknown.Programmed cell death (PCD) is a primordial response of virus-infected cells, whose mechanism is conserved from yeast to mammals.Taking advantage of the successful CIRV replication in the yeast Saccharomyces cerevisiae,we investigated whether and how the heterologous expression of the mitochondria-targeted CIRV p36 alone or in combination with the replicase protein p95 and DI RNA could affect yeast cell viability or stress-induced PCD.S. cerevisiae YPH499 cells were transformed with plasmids containing the CIRV p36 sequence, fused or not to GFP, cloned under the control of the inducible GAL1 promoter. Acetic acid-induced PCD (AA-PCD) was triggered and cell viability measured as in Giannattasio et al. (2005).The type of yeast cell death was analyzed by measuring plasma membrane integrity and PS externalization by propidium iodide and FITC-annexin V staining, respectively, and fluorescence microscopy.Heterologous expression of p36/p95 did not affect yeast cell viability up to 72 h whereas p36 alone slowed down cell growth after 24 h expression compared to control cells, with virtually no loss in plasma membrane integrity. Interestingly,after AA-PCD induction, p36-expressing cells lost viability at the same rate as the controls but showed a change in the type of cell death judged by apoptotic and necrotic marker analysis.S. cerevisiae is a powerful model organism for virus-host interaction studies. Due to the conservation of replication mechanisms among (+)RNA viruses, the data obtained with plain model viruses in this simple eukaryotic host can potentially be transferred to pathogens of higher eukaryotes. In addition, this system can be extended to investigate cellular pathways such as PCD.