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Unfortunately, the inclusion of original names (in non-Latin script) of the following authors caused problems with author name indexing in PubMed. Therefore, these original names were removed from XML data to correct the PubMed record. Mengji Cao, Yuya Chiaki, Hideki Ebihara, Jingjing Fu, George Fú Gāo, Tong Han, Jiang Hong, Ni Hong, Seiji Hongo, Masayuki Horie, Dàohóng Jiāng, Fujio Kadono, Hideki Kondō, Kenji Kubota, Shaorong Li, Longhui Li, Jiànróng Lǐ, Huazhen Liu, Tomohide Natsuaki, Sergey V. Netesov, Anna Papa, Sofia Paraskevopoulou, Liying Qi, Takahide Sasaya, Mang Shi, Xiǎohóng Shí, Zhènglì Shí, Yoshifumi Shimomoto, Jin‑Won Song, Ayato Takada, Shigeharu Takeuchi, Yasuhiro Tomitaka, Keizō Tomonaga, Shinya Tsuda, Changchun Tu, Tomio Usugi, Nikos Vasilakis, Jiro Wada, Lin‑Fa Wang, Guoping Wang, Yanxiang Wang, Yaqin Wang, Tàiyún Wèi, Shaohua Wen, Jiangxiang Wu, Lei Xu, Hironobu Yanagisawa, Caixia Yang, Zuokun Yang, Lifeng Zhai, Yong‑Zhen Zhang, Song Zhang, Jinguo Zhang, Zhe Zhang, Xueping Zhou. In addition, the publication call-out in the supplementary material was updated from issue 11 to issue 12. The original article has been corrected.

Following a request from the EU Commission, the Panel on Plant Health addressed the pest categorisation of the viruses and viroids of Vitis L. determined as being either non-EU or of undetermined standing in a previous EFSA opinion. These infectious agents belong to different genera and are heterogeneous in their biology. With the exclusion of grapevine virus 101-14.N.23.9.1/South Africa/2009 for which very limited information exists, the pest categorisation was completed for 30 viruses or viroids having acknowledged identities and available detection methods. All these viruses are efficiently transmitted by vegetative propagation techniques, with plants for planting representing the major pathway for long-distance dispersal and thus considered as the major pathway for potential entry. Depending on the virus, additional pathway(s) can also be seeds, pollen and/or vector(s). Most of the viruses categorised here are known to infect only one or few plant genera, but some of them have a wide host range, thus extending the possible entry pathways. Grapevine yellow speckle viroid 2, blueberry leaf mottle virus, grapevine Ajinashika virus, grapevine Anatolian ringspot virus, grapevine berry inner necrosis virus, grapevine deformation virus, grapevine fabavirus, grapevine red blotch virus, grapevine stunt virus, grapevine Tunisian ringspot virus, grapevine vein-clearing virus, temperate fruit decay-associated virus, peach rosette mosaic virus, tobacco ringspot virus, tomato ringspot virus meet all the criteria evaluated by EFSA to qualify as potential Union quarantine pests (QPs). With the exception of impact for the EU territory, on which the Panel was unable to conclude, blackberry virus S, grapevine geminivirus A, grapevine leafroll-associated virus 7, grapevine leafroll-associated virus 13, grapevine satellite virus, grapevine virus E, grapevine virus I, grapevine virus J, grapevine virus S, summer grape enamovirus, summer grape latent virus satisfy all the other criteria to be considered as potential Union QPs. Australian grapevine viroid, grapevine cryptic virus 1, grapevine endophyte endornavirus and wild vitis virus 1 do not meet all the criteria evaluated by EFSA to be regarded as potential Union QPs because they are not known to cause an impact on Vitis. For several viruses, especially those recently discovered, the categorisation is associated with high uncertainties mainly because of the absence of data on their biology, distribution and impact. Since this opinion addresses specifically non-EU viruses, in general these viruses do not meet the criteria assessed by EFSA to qualify as a potential Union regulated non-quarantine pests.

The Panel on Plant Health of EFSA conducted a pest categorisation of 17 viruses of Rubus L. that were previously classified as either non-EU or of undetermined standing in a previous opinion. These infectious agents belong to different genera and are heterogeneous in their biology. Blackberry virus X, blackberry virus Z and wineberry latent virus were not categorised because of lack of information while grapevine red blotch virus was excluded because it does not infect Rubus. All 17 viruses are efficiently transmitted by vegetative propagation, with plants for planting representing the major pathway for entry and spread. For some viruses, additional pathway(s) are Rubus seeds, pollen and/or vector(s). Most of the viruses categorised here infect only one or few plant genera, but some of them have a wide host range, thus extending the possible entry pathways. Cherry rasp leaf virus, raspberry latent virus, raspberry leaf curl virus, strawberry necrotic shock virus, tobacco ringspot virus and tomato ringspot virus meet all the criteria to qualify as potential Union quarantine pests (QPs). With the exception of impact in the EU territory, on which the Panel was unable to conclude, blackberry chlorotic ringspot virus, blackberry leaf mottle-associated virus, blackberry vein banding-associated virus, blackberry virus E, blackberry virus F, blackberry virus S, blackberry virus Y and blackberry yellow vein-associated virus satisfy all the other criteria to be considered as potential QPs. Black raspberry cryptic virus, blackberry calico virus and Rubus canadensis virus 1 do not meet the criterion of having a potential negative impact in the EU. For several viruses, the categorisation is associated with high uncertainties, mainly because of the absence of data on biology, distribution and impact. Since the opinion addresses non-EU viruses, they do not meet the criteria to qualify as potential Union regulated non-quarantine pests. (C) 2020 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.

Virus taxonomy emerged as a discipline in the middle of the twentieth century. Traditionally, classification by virus taxonomists has been focussed on the grouping of relatively closely related viruses. However, during the past few years, the International Committee on Taxonomy of Viruses (ICTV) has recognized that the taxonomy it develops can be usefully extended to include the basal evolutionary relationships among distantly related viruses. Consequently, the ICTV has changed its Code to allow a 15-rank classification hierarchy that closely aligns with the Linnaean taxonomic system and may accommodate the entire spectrum of genetic divergence in the virosphere. The current taxonomies of three human pathogens, Ebola virus, severe acute respiratory syndrome coronavirus and herpes simplex virus 1 are used to illustrate the impact of the expanded rank structure. This new rank hierarchy of virus taxonomy will stimulate further research on virus origins and evolution, and vice versa, and could promote crosstalk with the taxonomies of cellular organisms., Here, the International Committee on Taxonomy of Viruses describe a new, expanded virus classification scheme with 15 ranks that closely aligns with the Linnaean taxonomic system and better encompasses viral diversity.

Following a request from the EU Commission, the Panel on Plant Health addressed the pest categorisation of the viruses and viroids of Fragaria L. determined as being either non‐EU or of undetermined standing in a previous EFSA opinion. These infectious agents belong to different genera and are heterogeneous in their biology. With the exclusion of strawberry latent virus and strawberry latent C virus for which very limited information exists, the pest categorisation was completed for 12 viruses having acknowledged identities and available detection methods. All these viruses are efficiently transmitted by vegetative propagation techniques, with plants for planting representing the major pathway for long‐distance dispersal and thus considered as the major pathway for entry. Depending on the virus, additional pathway(s) can also be Fragaria seeds, pollen and/or vector(s). Most of the viruses categorised here are known to infect only one or few plant genera, but some of them have a wide host range, thus extending the possible entry pathways. Strawberry chlorotic fleck‐associated virus, strawberry leaf curl virus, strawberry necrotic shock virus, strawberry pallidosis‐associated virus, strawberry vein banding virus (SVBV) and tomato ringspot virus meet all the criteria evaluated by EFSA to qualify as potential Union quarantine pests (QPs). For SVBV, the Panel considered that following its entry and establishment into the EU territory, an impact of uncertain magnitude is expected mainly because a synergistic effect may occur in strawberry in case of mixed infections with viruses already present in the EU . Strawberry crinivirus 3, strawberry crinivirus 4 and strawberry polerovirus 1 meet all criteria for being considered as potential Union QPs, except for the impact in the EU territory, on which the Panel was unable to conclude. Fragaria chiloensis cryptic virus, Fragaria chiloensis latent virus and strawberry pseudo mild yellow edge virus do not meet the criterion of having potential negative impact in the EU. For several viruses, especially those recently discovered, the categorisation is associated with high uncertainties mainly because of the absence of data on their biology, distribution and impact. Since this opinion addresses specifically the non‐EU viruses, in general, these viruses do not meet the criteria assessed by EFSA to qualify as potential Union regulated non‐quarantine pests., This publication is linked to the following EFSA Journal articles: http://onlinelibrary.wiley.com/doi/10.2903/j.efsa.2019.5501/full, http://onlinelibrary.wiley.com/doi/10.2903/j.efsa.2019.5590/full, http://onlinelibrary.wiley.com/doi/10.2903/j.efsa.2019.5735/full, http://onlinelibrary.wiley.com/doi/10.2903/j.efsa.2019.5769/full

Following a request from the EU Commission, the Panel on Plant Health performed a pest categorisation of 17 viruses and viroids, herein called viruses, of Cydonia Mill., Malus Mill. and Pyrus L. determined as being either non‐EU or of undetermined standing in a previous EFSA opinion. These viruses belong to different genera and are heterogeneous in their biology. They can be detected by available methods and are efficiently transmitted by vegetative propagation techniques, with plants for planting representing a major long‐distance spread mechanism and, potentially, a major entry pathway. Depending on the viruses, additional pathway(s) can also be seed, pollen and/or vector transmission. Most of the viruses categorised here are known to infect only one of few related plant genera, but some of them have a wider host range, thus extending the possible entry pathways. Three viruses (apple necrotic mosaic virus, cherry rasp leaf virus, temperate fruit decay‐associated virus) and one viroid (apple fruit crinkle viroid) satisfy all the criteria to be considered as Union quarantine pests. Five viruses (apple green crinkle‐associated virus, blackberry chlorotic ringspot virus, eggplant mottled crinkle virus, tobacco ringspot virus and tomato ringspot virus) and one viroid (apple scar skin viroid), satisfy the criteria to be considered as Union quarantine pests with the possible exception of being absent from the EU territory or having a restricted presence and being under official control. The remaining six viruses (apple geminivirus, apple latent spherical virus, apple‐associated luteovirus, Pyrus pyrifolia cryptic virus, Pyrus pyrifolia partitivirus 2 and Tulare apple mosaic virus) and one viroid (apple hammerhead viroid) were not found to satisfy one or more of these criteria. The Panel highlights that for several viruses, especially those recently discovered, the categorisation is associated with high uncertainties mainly linked to the absence of data on biology and distribution. Since this opinion addresses specifically the non‐EU viruses, in general these viruses do not meet the criteria assessed by EFSA to qualify as a potential Union regulated non‐quarantine pests., This publication is linked to the following EFSA Journal articles: http://onlinelibrary.wiley.com/doi/10.2903/j.efsa.2019.5501/full, http://onlinelibrary.wiley.com/doi/10.2903/j.efsa.2019.5669/full, http://onlinelibrary.wiley.com/doi/10.2903/j.efsa.2019.5735/full, http://onlinelibrary.wiley.com/doi/10.2903/j.efsa.2019.5766/full

The Panel on Plant Health performed a listing of non-EU viruses and viroids (reported hereinafter as viruses) of Cydonia Mill., Fragaria L., Malus Mill., Prunus L., Pyrus L., Ribes L., Rubus L. and Vitis L. A systematic literature review identified 197 viruses infecting one or more of the host genera under consideration. Viruses were allocated into three categories (i) 86 non-EU viruses, known to occur only outside the EU or having only limited presence in the EU (i.e. reported in only one or few Member States (MSs), known to have restricted distribution, outbreaks), (ii) 97 viruses excluded at this stage from further categorisation efforts because they have significant presence in the EU (i.e. only reported so far from the EU or known to occur or be widespread in some MSs or frequently reported in the EU), (iii) 14 viruses with undetermined standing for which available information did not readily allow to allocate to one or the other of the two above groups. Comments provided by MSs during consultation phases were integrated in the opinion. The main knowledge gaps and uncertainties of this listing concern (i) the geographic distribution and prevalence of the viruses analysed, in particular when they were recently described; (ii) the taxonomy and biological status of a number of poorly characterised viruses; (iii) the host status of particular plant genera in relation to some viruses. The viruses considered as non-EU and those with undetermined standing will be categorised in the next steps to answer a specific mandate from the Commission to develop pest categorisations for non-EU viruses. This list does not imply a prejudice on future needs for a pest categorisation for other viruses which are excluded from the current categorisation efforts. (C) 2019 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.

"A universal feature of the replication of positive-strand RNA viruses is the association with intracellular membranes. Carnation Italian ringspot virus (CIRV) replication in plants occurs in vesicles derived from the mitochondrial outer membrane. The product encoded by CIRV ORF1, p36, is required for targeting the virus replication complex to the outer mitochondrial membrane both in plant and yeast cells. Here the yeast Saccharomyces cerevisiae was used as a model host to study the effect of CIRV p36 on cell survival and death. It was shown that p36 does not promote cell death, but decreases cell growth rate. In addition, p36 changed the nature of acetic acid-induced cell death in yeast by increasing the number of cells dying by necrosis with concomitant decrease of the number of cells dying by programmed cell death, as judged by measurements of phosphatidylserine externalization. The tight association of p36 to membranes was not affected by acetic acid treatment, thus confirming the peculiar and independent interaction of CIRV p36 with mitochondria in yeast. This work proved yeast as an invaluable model organism to study both the mitochondrial determinants of the type of cell death in response to stress and the molecular pathogenesis of (+)RNA viruses."

Contains fulltext : 202661.pdf (Publisher’s version ) (Closed access) This article reports the changes to virus taxonomy approved and ratified by the International Committee on Taxonomy of Viruses (ICTV) in October 2018. Of note, the ICTV has approved, by an absolute majority, the creation of additional taxonomical ranks above those recognized previously. A total of 15 ranks (realm, subrealm, kingdom, subkingdom, phylum, subphylum, class, subclass, order, suborder, family, subfamily, genus, subgenus, and species) are now available to encompass the entire spectrum of virus diversity. Classification at ranks above genus is not obligatory but can be used by the authors of new taxonomic proposals when scientific justification is provided.

International audience; 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.

Satellite viruses and satellite nucleic acids depend on their helper viruses for replication and other essential functions, but are dispensable for the virus infectious cycle. Satellites may influence the host range and the transmission of the helper virus, and by adaptation to different helpers, they may induce the outbreak of new devastating diseases. The most attractive feature of satellites stands in their ability to alter helper virus-induced symptoms, either attenuating them, or exacerbating the disease caused by the helper virus alone. In addition, some satellites may produce new symptoms, different from those incited by the cognate virus. In the relationship between satellites and their helper viruses, host plants play a major role in the elicitation of diseases.

The infectivity of small circular satellite RNAs (sc-satRNAs) depends on a helper virus. However, sc-satRNAs resemble viroids in several features, including the small (220!257 nt) and single-stranded genome, the accumulation in the infected host as covalently-closed circular RNAs, the replication through rolling-circle mechanisms based on RNA intermediates only, and the inability to code for proteins, with one exception. In addition, sc-satRNAs are endowed with self-cleaving (and some with self-ligation) activity mediated by ribozymes, another feature shared with some viroids, thus reinforcing the hypothesis that these two groups of subviral infectious RNAs may have a monophyletic origin (Elena et al., 2001). However, a key biological difference exists between sc-satRNAs and viroids regarding the molecular machinery needed for replication, which is supplied by the helper virus and the host in the first case, but only by the host in the second. In addition, in contrast to viroids, sc-satRNAs are encapsidated by the helper virus coat protein. This chapter focuses on the main structural and biological features of sc-satRNAs, pointing out the role of hammerhead and hairpin ribozymes in their replication. Readers looking for additional information on sc-satRNAs may check previous reviews

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 of the 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.

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.

Yeast cells co-expressing the replication proteins p36 and p95 of Carnation Italian ringspot virus (CIRV) support the RNA-dependent replication of several defective interfering (DI) RNAs derived from either the genome of CIRV or the related Cymbidium ringspot virus (CymRSV), but not the replication of a satellite RNA (sat RNA) originally associated with CymRSV. DI, but not sat RNA, was replicated in yeast cells co-expressing both DI and sat RNA. Using transgenic Nicotiana benthamiana plants constitutively expressing CymRSV replicase proteins (p33 and p92), or transiently expressing either these proteins or CIRV p36 and p95, it was shown that expression of replicase proteins alone was also not sufficient for the replication of sat RNA in plant cells. However, it was also shown that replicating CIRV genomic RNA deletion mutants encoding only replicase proteins could sustain replication of sat RNA in plant cells. These results suggest that sat RNA has a replication strategy differing from that of genomic and DI RNAs, for it requires the presence of a cis-replicating genome acting as a trans-replication enhancer.

The p36 and p95 proteins of Carnation Italian ringspot virus (CIRV), when expressed in Saccharomyces cerevisiae, supported the replication of defective interfering (DI) RNA. Double-label confocal immunofluorescence showed that both proteins localized to mitochondria, independently of each other. DI RNA progeny was localized by in situ hybridization both to mitochondria and to their proximity. Fractionation of cell extracts showed that replicase proteins associated with membranes with a consistent portion of DI RNA. DI RNA transcripts were stabilized more efficiently when co-expressed with both p36 and p95 than with either protein alone. By using the copper-inducible CUP1 promoter, p36 was shown to have an effect on DI RNA stability only above a threshold concentration, suggesting an ‘all-or-none’ behaviour. Conversely, the stabilizing activity of p95 was proportional to protein concentration in the range examined. Similarly, DI RNA replication level was proportional to p95 concentration and depended on a threshold concentration of p36.

Infection ofNicotiana benthamianacells with cymbidium ringspot (CymRSV) and carnation Italian ringspot (CIRV) viruses results in the formation of conspicuous membranous bodies [multivesicular bodies (MVBs)], which develop from modified peroxisomes or mitochondria, respectively. The organelle targeting signal is located in the proteins of 33 kDa (CymRSV) or 36 kDa (CIRV) encoded by ORF 1, which contain an N-terminal hydrophilic portion followed by two predicted hydrophobic transmembrane segments. Biochemical analysis showed that the 33- and 36-kDa proteins are integral membrane proteins. By exchanging small portions of the ORF 1 sequence between the infectious full-length clones of the two viruses, hybrid constructs were obtained of which thein vitrosynthesized RNA was inoculated toN. benthamianaplants and protoplasts. The structure of infectious clones suggested that both the N-terminal hydrophilic region and the transmembrane segments of the ORF 1-encoded proteins specify which organelle is involved in the synthesis of MVBs. Mutational analysis of the CIRV 36-kDa protein also suggested the presence of an internal mitochondrial targeting sequence similar to that found in several normal host proteins that are synthesized in the cytoplasm and transported to mitochondria. The CymRSV 33-kDa protein did not contain the obvious consensus signals thought to be characteristic of proteins targeted to peroxisomes, and an mitochondrial targeting sequence motif was not evident.

Aureusvirus is a new genus of plant viruses typified by pothos latent virus (PoLV) and comprising cucumber leaf spot virus (CLSV), previously classified as definitive species in the genus Carmovirus. Aureusviruses are soil-borne viruses readily transmitted by sap inoculation to a moderate range of hosts. Natural transmissions of CLSV is by the chytrid fungus Olpidium bornovanus, whereas PoLV infects the host without the apparent intervention of a vector. Aureusviruses have isometric particles with size (c. 30 nm) and structure similar to those of the family Tombusviridae, to which the genus belongs. The genome consists of a molecule of single-stranded, positive-sense RNA c. 4.4 kb in size comprising five ORFs. The structural organization (i.e. number and order of genes) is virtually identical to that of members of the genus Tombusvirus. However, the aureusvirus genome has a smaller size and shows distinct differences in the amino acid sequence of some of the ORFs. ORF 1 encodes a 25 kDa product and terminates with a leaky amber codon the readthrough of which results in a 84 kDa protein (ORF 2) with the conserved motifs of RNA dependent RNA polymerase. ORF 3 encodes the coat protein (40-41 kDa), ORF 4 the movement protein (27 kDa), and ORF 5 a 14-17 kDa product responsible for symptom severity. Virions accumulate in great quantity in the cytoplasm, often forming crystalline aggregates, and in bubble-like evaginations of the tonoplast protruding into the vacuole. Replication is likely to occur in the cytoplasm with a stategy based on direct expression of the 5' proximal ORF and expression of downstream ORFs through subgenomic RNAs.

Replication of positive-strand RNA viruses is commonly associated with extensive cell membrane proliferation and rearrangement and/or modification of some organelles of the infected hosts. These changes, which are induced by nonstructural proteins, provide the sites (e.g., vesicles or membrane invaginations) in which virus replication occurs. In plants infected by members of the genus Tombusvirus, this function is played by peculiar cytopathologic structures, called ‘multivesicular bodies’ (MVBs), that originate from heavily modified peroxisomes or mitochondria. The genesis, structural organization, and role of MVBs in the economy of tombusvirus infections are reviewed and commented upon.

The complete sequences of genomic and defective interfering (DI) RNAs of carnation Italian ringspot tombusvirus (CIRV) were determined. The genome (4760 nt) has an organization identical to that reported for other tombusviruses except that the pre-readthrough domain of the viral replicase encoded by the 5'-proximal open reading frame (ORF) is larger. In particular, the N-terminal region of this protein differs from the corresponding region of the other members of the genus Tombusvirus and Carmovirus. Two DI RNAs were found in infected tissues whose sequences were completely derived from genomic RNA. The smaller molecule (474 nt) is contained completely within the larger molecule (656 nt), which suggests that it is derived from the larger one.

Cymbidium ringspot tombusvirus (CyRSV) supports the replication of an RNA molecule known to be a satellite (sat-RNA). Sequence analysis of wild-type sat-RNA showed that the 3' terminus is heterogeneous and terminates with one, two, or three C residues. Transcripts from mutant clones which lacked up to eight nucleotides at the 3' end were biologically active and yielded progeny RNA that had the 3' end restored as in wild-type RNA. Deletions or substitutions at the 5' end produced nonviable molecules. Studies with mutants containing deletions in internal regions showed that replication was affected essentially by the location of deletions; viable mutants were much less encapsidated than the wild type, showing that size also may be important in the encapsidation and survival of CyRSV sat-RNA.

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.

An antiserum was raised against the 33 K protein encoded by the 5' proximal gene of cymbidium ringspot tombusvirus RNA. This antiserum reacts specifically with the 33 K and 92 K proteins, which constitute the viral replicase, in CyRSV-infected Nicotiana benthamiana plants and in transgenic plants transformed with the full-length replicase gene. In inoculated leaves of infected plants, synthesis of the 33 K/92 K proteins stops ten days after inoculation, whereas in newly produced systemically infected leaves there was continuous production of these proteins. In transgenic plants, both proteins were detected showing that readthrough of the termination codon of the 33 K protein does not depend on the presence of the replicating virus. The subcellular localization of the 33 K/92 K proteins is similar in infected and transgenic plants. No correlation was found between the level of expression of integrated virus gene and level of resistance to the challenging virus.

Genomic RNA of cymbidium ringspot tombusvirus (CyRSV) contains five large open reading frames (ORFs). The two 5′ proximal ORFs encode proteins of 33 and 92 kDa and the three 3′ proximal ORFs encode proteins of 41,22, and 19 kDa. In addition, a small reading frame encoding a protein of 4 kDa was recently observed by computer analysis of the 3′ nontranslated region of CyRSV and other tombusvirus RNAs (ORF 6). The function of putative gene products was investigated with the use of several mutants. Mutants in ORFs 1 and 2 were not viable indicating that both 33- and 92-kDa proteins are required for replication. Deletion of a large portion of the coat protein gene, encoding a 41-kDa protein, did not prevent replication of viral RNA and cell-to-cell movement, but interfered severely with long-distance translocation. No virus particles or viral RNA could be detected in inoculated or upper leaves of plants inoculated with transcripts obtained from mutants not expressing the 22-kDa protein. However, replication and encapsidation occurred normally in inoculated protoplasts indicating that this gene product is a transport protein. Conversely, no role could be assigned to putative gene products of ORF 5 (19-kDa protein) and QRF 6. It was also shown that factors other than gene expression can influence the replication of RNA mutants, probably due to instability of RNA molecules.

The replication ofCymbidiumringspot virus (CymRSV) defective interfering (DI) RNA in cells of the yeastSaccharomyces cerevisiaenormally takes place in association with the peroxisomal membrane, thus paralleling the replication events in infected plant cells. However, previous results with a peroxisome-deficient mutant strain of yeast had suggested that the presence of peroxisomes is not a strict requirement for CymRSV DI RNA replication. Thus, a novel approach was used to study the putative alternative sites of replication by usingS. cerevisiaestrain YPH499 which does not contain normal peroxisomes. In this strain, CymRSV p33 and p92 accumulated over portions of the nuclear membrane and on membranous overgrowths which were identified as endoplasmic reticulum (ER) strands, following immunofluorescence and immunoelectron microscope observations. The proteins were not released by high-pH treatment, but were susceptible to proteolytic digestion, thus indicating peripheral and not integrated association. ER-associated p33 and p92 proteins supportedin transthe replication of DI RNA. The capacity of plus-strand RNA viruses to replicate in association with different types of cell membranes was thus confirmed.

The replicase proteins p33 and p92 of Cymbidium ringspot virus (CymRSV) were found to support the replication of defective interfering (DI) RNA in Saccharomyces cerevisiae cells. Two yeast strains were used, differing in the biogenesis of peroxisomes, the organelles supplying the membranous vesicular environment in which CymRSV RNA replication takes place in infected plant cells. Double-labelled immunofluorescence showed that both p33 and p92 replicase proteins localized to peroxisomes, independently of one another and of the presence of the replication template. It is suggested that these proteins are sorted initially from the cytosol to the endoplasmic reticulum and then to peroxisomes. However, only the expression of p33, but not p92, increased the number of peroxisomes and induced membrane proliferation. DI RNA replication occurred in yeast cells, as demonstrated by the presence of monomers and dimers of positive and negative polarities. Labelling with BrUTP showed that peroxisomes were the sites of nascent viral synthesis, whereas in situ hybridization indicated that DI RNA progeny were diffused throughout the cytoplasm. DI RNA replication also took place in yeast cells devoid of peroxisomes. It is suggested that replication in these cells was targeted to the endoplasmic reticulum.

Open reading frame 1 in the viral genome of Cymbidium ringspot virus encodes a 33-kDa protein (p33), which was previously shown to localize to the peroxisomal membrane in infected and transgenic plant cells. To determine the sequence requirements for the organelle targeting and membrane insertion, the protein was expressed in the yeast Saccharomyces cerevisiae in native form (33K) or fused to the green fluorescent protein (33KGFP). Cell organelles were identified by immunolabeling of marker proteins. In addition, peroxisomes were identified by simultaneous expression of the red fluorescent protein DsRed containing a peroxisomal targeting signal and mitochondria by using the dye MitoTracker. Fluorescence microscopy showed the 33KGFP fusion protein concentrated in a few large bodies colocalizing with peroxisomes. These bodies were shown by electron microscopy to be composed by aggregates of peroxisomes, a few mitochondria and endoplasmic reticulum (ER) strands. In immunoelectron microscopy, antibodies to p33 labeled the peroxisomal clumps. Biochemical analysis suggested that p33 is anchored to the peroxisomal membrane through a segment of ca. 7 kDa, which corresponds to the sequence comprising two hydrophobic transmembrane domains and a hydrophilic interconnecting loop. Analysis of deletion mutants confirmed these domains as essential components of the p33 peroxisomal targeting signal, together with a cluster of three basic amino acids (KRR). In yeast mutants lacking peroxisomes p33 was detected in the ER. The possible involvement of the ER as an intermediate step for the integration of p33 into the peroxisomal membrane is discussed.

Two plasmids from which the sequences coding for the 36- and 95-kDa proteins of Carnation Italian ringspot virus (CIRV) could be transcribed in vivo in the yeast Saccharomyces cerevisiae under the control of the ADH1 promoter and terminator were constructed. The two proteins, which constitute the viral replicase, were correctly translated and integrated into membranes of the yeast cells. An additional plasmid was introduced in yeasts expressing the CIRV replicase, from which a defective interfering (DI) RNA (DI-7 RNA) could be transcribed under the control of the GAL1 promoter and terminated by the Tobacco ringspot virus satellite ribozyme, which cleaved 19 nucleotides downstream of the 3′ end of DI RNA. The DI-7 RNA transcripts were amplified by the viral replicase as demonstrated by the restoration of the authentic 3′ end, the requirement of a specific cis -acting signal at this terminus, the preferential accumulation of molecules with the authentic 5′ terminus (AGAAA), the synthesis of head-to-tail dimers, the presence of negative strands, and the incorporation of 5-bromo-UTP. Additionally, transformation with a dimeric construct of DI-7 RNA led to the synthesis of monomers, mimicking the activity of the viral replicase in plant cells.

Replication of the Carnation Italian ringspot virus genomic RNA in plant cells occurs in multivesicular bodies which develop from the mitochondrial outer membrane during infection. ORF1 in the viral genome encodes a 36-kDa protein, while ORF2 codes for the 95-kDa replicase by readthrough of the ORF1 stop codon. We have shown previously that the N-terminal part of ORF1 contains the information leading to vesiculation of mitochondria and that the 36-kDa protein localizes to mitochondria. Using infection, in vivo expression of green fluorescent protein fusions in plant and yeast cells, and in vitro mitochondrial integration assays, we demonstrate here that both the 36-kDa protein and the complete replicase are targeted to mitochondria and anchor to the outer membrane with the N terminus and C terminus on the cytosolic side. Analysis of deletion mutants indicated that the anchor sequence is likely to correspond approximately to amino acids 84 to 196, containing two transmembrane domains. No evidence for a matrix-targeting presequence was found, and the data suggest that membrane insertion of the viral proteins is mediated by an import receptor-independent signal-anchor mechanism relying on the two transmembrane segments and multiple recognition signals present in the N-terminal part of ORF1.

The localization of the 36 kDa (‘36K’) protein encoded by open reading frame 1 of Carnation Italian ringspot virus was studied in infected cells and in cells transiently expressing the 36K protein fused to green fluorescent protein (GFP). Subcellular fractionation demonstrated that the 36K protein accumulated in fractions containing mostly mitochondria. Fluorescence microscopy of transiently transformed cells showed that the 36K–GFP fusion protein accumulated in structures which could be stained with the mitochondrial-specific dye MitoTracker. However, these structures were larger than normal mitochondria and were irregular in shape and distribution in the cytoplasm. Electron microscopy showed severe alterations of mitochondria, which were often clumped. The stroma was more electron-opaque, the cristae were irregularly shaped, the intermembrane space was enlarged and the outer membrane was covered with an electron-dense amorphous material whose nature could not be determined. The organelle-targeted 36K protein seems to promote the overgrowth of the mitochondrial outer membrane.

The complete genomic sequence of galinsoga mosaic virus (GaMV) was determined. The genome consists of 3 803 nucleotides and has five open reading frames (ORFs). The 5′ ORF (ORF 1) encodes a protein with predicted molecular mass of 23 kDa and readthrough of its amber stop codon probably yields a 82 kDa protein (ORF 2). ORFs 3 and 4 encode two polypeptides with molecular masses of 8 and 7 kDa, respectively. ORF 5 encodes the 36 kDa capsid protein. Amino acid sequence comparisons revealed that the nonstructural proteins encoded by ORFs 1, 3, and 4 were more similar to the corresponding gene products of tobacco necrosis virus, strain A, than to those of carmoviruses. Conversely, the coat protein was more similar to that of tombusviruses. The readthrough region of the viral replicase (ORF 2) had high sequence homology with that of carmo-, tombus-, and necroviruses. Computer analysis of the protein encoded by ORF 1 as well as of the corresponding product of turnip crinkle (TCV) and melon necrotic spot (MNSV) carmoviruses revealed the presence of a sequence with local hydrophobicity and hydrophobic moment characteristic of mitochondrial targeting sequence which may explain the origin of the carmovirus-induced multivesicular bodies from mitochondria.

International audience; White stripe is a disease affecting leek in France with which an isometric virusc. 30 nm in diameter is associated. The most evident symptom is the presence of white stripes on the leaves extending to the stem. Attempts to demonstrate transmission through the soil by sowing or transplanting leek in contaminated soil were unsuccessful. The virus was transmitted by sap inoculation to a narrow range of herbaceous hosts, all of which were infected only locally. Virus purification was from infected leek tissues, where it accumulated in large amounts, as demonstrated by ultrastructural observations. RNA was extracted from purified virus preparations and cDNA clones were prepared. The complete nucleotide sequence of the viral RNA was determined: The genome is 3662 nucleotides long and contains five open reading frames (ORFs). The first (ORF 1) encodes a putative translation product of Mr 23803 (p24) and read through of its amber stop codon results in a protein of Mr 82625 (p83) (ORF 2). ORF 3 and ORF 4 encode two small polypeptides of Mr 11280 (p11) and Mr 6261 (p6), respectively. ORF 5 encodes the capsid protein of Mr 27460 (p27). The genome organization and sequence alignments with the corresponding products of necroviruses suggest that the virus isolated from leek is a new species in the genusNecrovirus, for which the name of leek white stripe virus (LWSV) is proposed.

The multimeric forms of cymbidium ringspot tombusvirus (CyRSV) satellite (sat) RNA were analysed. Attempts to amplify the putative junction region of oligomers using the polymerase chain reaction (PCR) failed, indicating the absence of such structures. SatRNA-related species having the size double than the unit length were shown to be double-stranded monomers and not single-stranded dimers. Similarly, satRNA species of a size four times the unit length were shown to be constituted by aggregates of double-stranded monomers. The absence of single-stranded CyRSV satRNA oligomers indicates that the formation of multimers is not a step in the replication of this RNA molecule.

Transgenic Nicotiana benthamiana plants expressing the coat protein gene of cymbidium ringspot virus (CyRSV) were tested for resistance against infection with CyRSV. Transgenic plants showed resistance to infection only when the purified virions concentration in the inoculum was as low as 0.05 micrograms/ml. No protection was observed in transgenic plants inoculated with virion concentrations of 0.5 and 5.0 micrograms/ml or when the inoculum was in vitro synthesized genomic RNA.

The two satellite RNAs associated with CYMV infections were sequenced. The larger (sCYMV-L1) has only linear molecules 1145 nucleotides long, a poly(A) tail, a long open reading frame (ORF) coding for a protein of Mr 39,636 resembling in composition those of other large nepovirus satellite RNAs, a 5' leader sequence of 16 nucleotides and a 3' non-coding region of 40 nucleotides. In vitro translation of sCYMV-L1 yielded a protein product with a size that corresponded to that predicted from the sequence. The smaller satellite (sCYMV-S1) is 457 nucleotides long, has no ORF of significant length and no in vitro messenger activity. Both linear and circular forms of this satellite RNA were detected in infected tissues. Comparison of the sCYMV-S1 primary structure with the sequences of other small nepoviral satellites reveals large regions of homology. Analysis of the secondary structures derived from the sequences of the plus and minus strands suggests possible consensus sequences for their self-cleavage.

Pothos latent virus (PoLV) is an isometric virus with a positive-sense, single-stranded RNA genome of 4415 nt. The genome contains five open reading frames (ORF), coding for five proteins with approximate molecular masses of 25, 84, 40, 27 and 14 kDa, respectively. In vitro synthesized PoLV RNA was infectious to Nicotiana benthamiana plants and protoplasts, but could not support replication of the defective interfering (DI) and satellite RNAs associated with Cymbidium ringspot tombusvirus. No DI RNA related to PoLV was generated after repeated passaging with infected sap. Mutagenesis studies defined the role of the ORF 4 product (27 kDa) as a movement protein, and the ORF 5 product (14 kDa) as being responsible for symptom severity. Moreover, it was shown that the coat protein (CP) is important in regulating synthesis of the 14 kDa protein, excess production of which is lethal to infected plants. CP mutants defective in capsid formation infected plants systemically and induced severe necrotic symptoms. Conversely, CP mutants able to form apparently normal virus particles induced symptoms indistinguishable from those elicited by wild-type virus.

Carnation Italian ringspot tombusvirus encodes a protein, referred to as 36K, that possesses a mitochondrial targeting signal and two transmembrane segments which are thought to anchor this protein to the outer membrane of the mitochondrial envelope of infected plant cells. To determine the topology of the virus protein inserted in the cell membrane, as well as the sequence requirements for targeting and insertion, anin vivosystem was set up in which this could be analysed in the absence of productive virus infection. The 36K protein was expressed in the yeastSaccharomyces cerevisiaein native form or fused to the green fluorescent protein. Using a fluorescence microscope, large green-fluorescing cytoplasmic aggregates were visible which stained red when cells were treated with the vital stain MitoTracker, which is specific for mitochondria. These aggregates were shown by electron microscopy to be composed of either mitochondria or membranes. The latter type was particularly abundant for the construct in which the green fluorescent protein was fused at the N terminus of the 36K protein. Immunoelectron microscopy demonstrated that the viral protein is present in the anomalous aggregates and Western blot analysis of protein extracts showed 36K to be resistant to alkaline, urea or salt extraction, a property of integral membrane proteins.

The complete genomic sequence of pothos latent virus (PoLV) has been determined. The genome organization is very similar to that of tombusviruses. The genome is 4415 nucleotides long and contains five ORFs. The 5′ ORF (ORF 1) encodes a protein with a predicted molecular mass of 25 kDa and readthrough of its amber stop codon results in an 84 kDa protein (ORF 2). ORF 3 encodes the 40 kDa capsid protein. Two nested ORFs (ORFs 4 and 5) in different reading frames encode 27 and 14 kDa proteins, respectively. Amino acid sequence alignments revealed significant similarities between the readthrough portion of ORF 2 and the coat protein (ORF 3) of PoLV and the corresponding proteins of several tombusviruses. Conversely, the predicted products of ORFs 1, 4 and 5 showed only limited similarity to the equivalent tombusvirus proteins. These results support the conclusion that PoLV is a new but atypical member of the family Tombusviridae.

Multivesicular bodies (MVB) are membranous cytoplasmic inclusions that are invariably associated with tombusvirus infections regardless of the virus species, the host, or the tissue examined. MVB are virus-induced structures since they are absent from tissues of healthy plants and are always present both in infected plants and protoplasts. MVB derive from peroxisomes in cells infected by a number of tombusviruses including cymbidium ringspot virus (CymRSV) and from mitochondria in cells infected by another tombusvirus, carnation Italian ringspot virus (CIRV). By using common restriction sites in full-length infectious clones, hybrid clones of these two viruses were constructed. In addition, a mutant of CIRV was prepared in which the protein encoded by the first open reading frame was shortened by 22 amino acids. All mutant transcripts were viable and infected Nicotiana benthamiana plants. Infected leaf tissue samples were collected, processed for thin sectioning, and observed in the electron microscope. The origin of MVB was shown to be under the control of the 5′ region of the viral genome. A sequence as short as about 600 nucleotides in ORF 1 contained the determinants for formation of MVB from peroxisomes or mitochondria.