Metagenomic analysis of nepoviruses: diversity, evolution and identification of a genome region in members of subgroup A that appears to be important for host range.

Data mining and metagenomic analysis of 277 open reading frame sequences of bipartite RNA viruses of the genus Nepovirus, family Secoviridae, were performed, documenting how challenging it can be to unequivocally assign a virus to a particular species, especially those in subgroups A and C, based on some of the currently adopted taxonomic demarcation criteria. This work suggests a possible need for their amendment to accommodate pangenome information. In addition, we revealed a host-dependent structure of arabis mosaic virus (ArMV) populations at a cladistic level and confirmed a phylogeographic structure of grapevine fanleaf virus (GFLV) populations. We also identified new putative recombination events in members of subgroups A, B and C. The evolutionary specificity of some capsid regions of ArMV and GFLV that were described previously and biologically validated as determinants of nematode transmission was circumscribed in silico. Furthermore, a C-terminal segment of the RNA-dependent RNA polymerase of members of subgroup A was predicted to be a putative host range determinant based on statistically supported higher π (substitutions per site) values for GFLV and ArMV isolates infecting Vitis spp. compared with non-Vitis-infecting ArMV isolates. This study illustrates how sequence information obtained via high-throughput sequencing can increase our understanding of mechanisms that modulate virus diversity and evolution and create new opportunities for advancing studies on the biology of economically important plant viruses.

First Report of Grapevine Pinot gris virus (GPGV) in grapevine in France.

Grapevine Pinot gris virus (GPGV), belonging to the genus Trichovirus of the family Betaflexiviridae, was first identified by siRNA sequencing in northern Italy in 2012, in the grapevine varieties Pinot gris, Traminer, and Pinot Noir, which exhibited mottling and leaf deformation (1), and in asymptomatic vines, with a lower frequency. Since 2012, this virus has also been reported in South Korea, Slovenia, Greece (3), Czech Republic (2), Slovakia (2), and southern Italy (4). In 2014, GPGV was identified by Illumina sequencing of total RNAs extracted from leaves of the Merlot variety (Vitis vinifera) grafted onto Gravesac rootstock originated from a vineyard in the Bordeaux region of France. This Merlot plant exhibited fanleaf-like degeneration symptoms associated with Tomato black ring virus (TBRV) infection. Cuttings were collected in 2010 and maintained thereafter in a greenhouse. The full-length genome was assembled either de novo or by mapping of the Illumina reads on a reference GPGV genome (GenBank FR877530) using the CLC Genomics workbench software (CLC Bio, Qiagen, USA). The French GPGV isolate "Mer" (7,223 nucleotides, GenBank KM491305) is closely related to other European GPGV sequences; it exhibits 95.4% nucleotide identity with the reference Italian isolate (NC_015782) and 98 to 98.3% identity with Slovak isolates (KF134123 to KF134125). The higher divergence between French and Italian GPGV isolates was mainly due to differences in the 5' extremity of the genome, as already shown with the Slovak GPGV isolates. RNA extracted from phloem scrapings of 19 cv. Merlot vines from the same plot collected in 2014 were analyzed by RT-PCR using the specific primer pair Pg-Mer-F1 (5'-GGAGTTGCCTTCGTTTACGA-3') and Pg-Mer-R1 (5'-GTACTTGATTCGCCTC GCTCA-3'), designed on the basis of alignments of all available GPGV sequences from GenBank. The resulting amplicon of 770 bp corresponded to a fragment of the putative movement protein (MP) gene. Seven (35%) of the tested plants gave a strong positive amplification. Three RT-PCR products were directly sequenced and showed 99.3 to 99.5% identity within the MP gene of the GPGV-Mer isolate. Given the mixed viral infection status of the vines found infected by GPGV, it was not possible to associate a specific symptomatology with the presence of GPGV. Furthermore, similar RT-PCR tests were also performed on RNA extracts prepared from two plants of cv. Carignan that originated from a French grapevine collection, exhibiting fanleaf-like symptoms without any nepovirus detection. These samples similarly gave a strong positive amplification. The sequences obtained from the two Carignan vines showed 98.4 and 97.8% identity with the GPGV-Mer isolate. To our knowledge, this is the first report of GPGV in France. GPGV has been discovered in white and red berry cultivars, suggesting that its prevalence could be important in European vineyards (2). Further large-scale studies will be essential to determine the world prevalence of GPGV and to evaluate its potential effects on yield and on wine quality, as well as to shed light on GPGV epidemiology. Of particular concern is whether, like the other grapevine-infecting Trichovirus, Grapevine berry inner necrosis virus (GPGV) can be transmitted by the eryophid mite Colomerus vitis. References: (1) A. Giampetruzzi et al. Virus Res. 163: 262, 2012. (2) M. Glasa et al. Arch. Virol. 159: 2103, 2014. (3) G. P. Martelli, J. Plant Pathol. 96: S105, 2014. (4) M. Morelli et al. J. Plant Pathol. 96:431, 2014.

First Report of Grapevine redglobe virus (GRGV) in Grapevine in France.

The isometric virus Grapevine redglobe virus (GRGV), was first described on grapevine cv. Red Globe in southern Italy in 2000 (3) and later in Greece and California. GRGV belongs to the genus Maculavirus in the family Tymoviridae. These viruses are thought to be disseminated through propagation and grafting, as no vectors or seed transmission are known to date. A partial sequence (2,006 nucleotides [nt]) encompassing the 3' end of the replicase, the coat protein, and P17 genes, was obtained in 2003 (1). GRGV infections are apparently symptomless (2). In 2014, GRGV was identified by Illumina sequencing of total RNAs extracted from a Vitis vinifera cv. Cabernet franc (CF) vine grafted onto Gravesac in a vineyard of the Bordeaux region in France. This Cabernet franc plant displayed fanleaf-like degeneration symptoms associated with Tomato black ring virus (TBRV) infection. It had been collected in 2010 and maintained since in a greenhouse. The partial contigs assembled from the Illumina reads (552 and 430 nt, both in the putative replicase gene, KM491303 and KM491304) showed 85.9 and 86.3% nt identity with the partial sequence of a GRGV Italian isolate (AF521577), respectively. Total RNA extracts from leaves of 18 plants of cv. Cabernet franc from the same plot, collected in 2014, were analyzed by RT-PCR using specific primers RG-CF-F1 (5'-GAATTCGCTGTCGGCCACTC-3') and RG-CF-R1 (5'-AGTGAGAGGAGAGATTCCATC-3') designed on the basis of the alignment of the partial sequences of GRGV-CF and the Italian isolate (AF521577). Fifteen (83%) of the plants gave strong positive amplification for GRGV. Given the mixed viral infection status of these vines, it was not possible to associate a specific symptomatology with the presence of GRGV. Two RT-PCR amplicons were directly sequenced and showed 91.5 and 91.7% identities, respectively, with the reference GRGV-CF sequence. To our knowledge, this is the first report of GRGV in France. Further studies will be necessary to determine the prevalence of GRGV in the French vineyards and varieties, including rootstocks, and its possible threat to the grapevine industry. Studies are also needed to assess the pathogenicity of GRGV. Similarly to its close relative, Grapevine fleck virus, does it induce latent or semi-latent infections in Vitis vinifera and rootstock hybrids, influencing vigor, rooting ability, and graft compatibility? References: (1) N. Abou Ghanem-Sabanadzovic et al. Virus Genes 27:11, 2003. (2) G. P. Martelli et al. Arch. Virol. 147:1847, 2002. (3) S. Sabanadzovic et al. Arch. Virol. 145:553, 2000.

Status and prospects of plant virus control through interference with vector transmission.

Most plant viruses rely on vector organisms for their plant-to-plant spread. Although there are many different natural vectors, few plant virus-vector systems have been well studied. This review describes our current understanding of virus transmission by aphids, thrips, whiteflies, leafhoppers, planthoppers, treehoppers, mites, nematodes, and zoosporic endoparasites. Strategies for control of vectors by host resistance, chemicals, and integrated pest management are reviewed. Many gaps in the knowledge of the transmission mechanisms and a lack of available host resistance to vectors are evident. Advances in genome sequencing and molecular technologies will help to address these problems and will allow innovative control methods through interference with vector transmission. Improved knowledge of factors affecting pest and disease spread in different ecosystems for predictive modeling is also needed. Innovative control measures are urgently required because of the increased risks from vector-borne infections that arise from environmental change.

Transmission of six ampeloviruses and two vitiviruses to grapevine by Phenacoccus aceris.

Grapevine leafroll disease is caused by grapevine leafroll-associated viruses (GLRaVs). These viruses are common in vineyards worldwide and often associated with vitiviruses that are involved in the rugose wood complex of grapevine. Ten mealybug species are known as vectors of one or several of these grapevine viruses, including the apple mealybug Phenacoccus aceris which is widespread in Holarctic regions and able to transmit Grapevine leafroll-associated virus-1 and -3 (GLRaV-1 and -3). Our aim was to characterize the transmission features of leafroll viruses by Phenacoccus aceris in order to better understand the contribution of this mealybug to leafroll epidemics. Results showed that Phenacoccus aceris is able to transmit GLRaV-1, -3, -4, -5, -6, and -9 to grapevine but not GLRaV-7. This is the first report of GLRaV-6 transmission by a mealybug. Also, for the first time it was shown that Phenacoccus aceris could vector vitiviruses Grapevine virus A (GVA) and Grapevine virus B (GVB). First instar nymphs were the most efficient stage in transmitting GLRaV-1, -3, and GVA. This research sheds light on the transmission biology of grapevine viruses by Phenacoccus aceris and represents a step forward to leafroll disease management.

[Pelvic and spinal MR follow-up of multiple myeloma patients after high-dose chemotherapy and autologous peripheral blood stem cell transplant]. / Suivi par IRM pelvi-rachidienne d'une cohorte de patients atteints de myélome multiple, traités par chimiothérapie à haute dose et autogreffe de cellules souches périphériques.

PURPOSE: To evaluate the changes of bone marrow lesions on pelvic and spinal MR in patients with multiple myeloma after high-dose chemotherapy and autologous peripheral blood stem cell transplant. PATIENTS AND METHODS: Pelvic and spinal MR examinations were obtained at presentation (myeloma diagnosis) and 1 year after transplant in 20 patients that were part of a group of 39 patients enrolled in a prospective study. The type of marrow replacement (classified in stages with stage 0: normal; stage 1: salt and pepper; stage 2: focal infiltration; stage 3: diffuse infiltration), the number and size of marrow lesions and the number of vertebral compression fractures were recorded. We have compared the findings prior to and following transplant, with correlation to the response to treatment and the use of biphosphonates. RESULTS: The type of marrow replacement was improved following transplant in 65% of patients (not statistically significant). The number and size of nodules > 20 mm showed significant reduction (p = 0.0224 and p = 0.0237 respectively). Lesions on MR improved in 50% of patients with good response and 75% of patients with poor response to treatment. Patients receiving biphosphonates showed more vertebral compression fractures. CONCLUSION: The evolution of marrow replacing lesions on MR is discordant compared to the biological and clinical response to treatment. Pelvic and spinal MR evaluation at the time of diagnosis does not appear to be a good predictive factor of response to treatment. Biphosphonates do not appear to prevent new vertebral compression fractures. Pelvic and spinal MR provides interesting data in the follow-up of patients with myeloma following autologous transplant, especially in the local evolution of marrow replacing lesions, but our results do not justify its use in routine clinical practice.

Distal Madelung-Launois-Bensaude disease: an unusual differential diagnosis of acromelic arthritis.

Madelung-Launois-Bensaude (MLB) disease is uncommon and probably under-diagnosed. It is defined as a symmetrical proximal accumulation of fat. Its physiopathology is obscure, but favouring factors, like alcohol or corticosteroids, are well known. We describe, for the first time, an unusual case of distal form of the disease, involving hands and feet simultaneously. Moreover, this phenotype was associated with a proximal form of MLB, and new localizations in the elbows and knees have been identified. We review the literature concerning distal forms of MLB, and compare the features. This disease is an interesting new differential diagnosis of acromelic arthritis.

First Report on the Natural Occurrence of Beet chlorosis virus in Poland.

Yellowing symptoms on sugar beet (Beta vulgaris L.) are caused by several viruses, especially those belonging to the genus Polerovirus of the family Luteoviridae, including Beet mild yellowing virus (BMYV) and Beet western yellows virus (BWYV), and recently, a new species, Beet chlorosis virus (BChV), was reported (2). To identify Polerovirus species occurring in beet crops in Poland and determine their molecular variability, field surveys were performed in the summer and autumn of 2005. Leaves from symptomatic beet plants were collected at 26 localities in the main commercial sugar-beet-growing areas in Poland that included the Bydgoszcz, Kutno, Lublin, Poznan, Olsztyn, and Warszawa regions. Enzyme-linked immunosorbent assay (ELISA) tests (Loewe Biochemica GmbH, Sauerlach, Germany) detected poleroviruses in 23 of 160 samples (approximately 20 samples from each field). Multiplex reverse-transcription polymerase chain reaction (RT-PCR) (1) (GE Healthcare S.A.-Amersham Velizy, France) confirmed the presence of poleroviruses in 13 of 23 samples. Nine of twenty sugar beet plants gave positive reactions with BChV-specific primers and three with primers specific to the BMYV P0 protein. Two isolates reacted only with primer sets CP+/CP, sequences that are highly conserved for all beet poleroviruses. Leaf samples collected from three plants infected with BChV were used as inoculum sources for Myzus persicae in transmission tests to suitable indicator plants including sugar beet, red beet (Beta vulgaris L. var. conditiva Alef.), and Chenopodium capitatum. All C. capitatum and beet plants were successfully infected with BChV after a 48-h acquisition access period and an inoculation access period of 3 days. Transmission was confirmed by the presence of characteristic symptoms and by ELISA. Amino acid sequences obtained from each of four purified (QIAquick PCR Purification kit, Qiagen S.A., Courtaboeuf, France) RT-PCR products (550 and 750 bp for CP and P0, respectively) were 100% identical with the CP region (GenBank Accession No. AAF89621) and 98% identical with the P0 region (GenBank Accession No. NP114360) of the French isolate of BChV. To our knowledge, this is the first report of BChV in Poland. References: (1) S. Hauser et al. J. Virol. Methods 89:11, 2000. (2) M. Stevens et al. Mol. Plant Pathol. 6:1, 2005.

Characterisation of a new potyvirus species infecting meadow saffron Colchicum autumnale).

The alkaloids contained in Colchicum autumnale seeds are used in numerous medicines. Good quality seeds are difficult to obtain from this undomesticated plant. Therefore, a research program was set up aiming to cultivate C. autumnale in order to improve alkaloid contents and seed yields. In this context, a collection was established in 1999 by transplanting corms from twelve different locations in Eastern France. However, serious symptoms of necrosis and decay have appeared in this collection since 2001. Electron microscopic observations of plants showing symptoms revealed the presence of filamentous particles and pinwheel-like structures characteristic of the Potyviridae family. Leaves and corms from symptomatic plants were assayed with potyvirus-specific Enzyme-Linked Immunosorbent Assay test. Positive reactions were obtained with plants from all the geographic origins, which exhibited flower breaking symptoms on petals. RT-PCR tests with family Potyviridae-specific primers confirmed the ELISA results and showed that the virus can be detected in corms, roots and flowers of symptomatic plants. The 3' region of the genome was cloned, sequenced and compared to other potyvirus species. Phylogenetic analyses suggest the presence of a new viral species tentatively named Meadow saffron breaking virus (MSBV) in C. autumnale.

Biological properties and molecular characterization of beet chlorosis virus (BChV).

Two distinct viruses belonging to the Polerovirus genus, in the family Luteoviridae, have been described as being able to induce mild yellowing on sugar beet: Beet mild yellowing virus (BMYV) and more recently, beet chlorosis virus (BChV). We have analysed biological properties and molecular organisation of two strains of BChV, one collected in England and the second from California. The biological data suggested that BChV displayed a narrower host range compared to BMYV and Beet western yellows virus lettuce isolate (BWYV). The complete genomic RNA sequence of the American isolate BChV-California and the European isolate BChV-2a showed a genetic organisation and expression typical of other Polerovirus members including 6 open reading frames (ORFs). Interspecific and intraspecific phylogenetic studies suggested that BChV arose by recombination events between a Polerovirus-like ancestor donating P0 and the replicase complex and either a BMYV or a BWYV progenitor providing the 3' ORFs [3, 4 and 5]. The 5'- and 3'-parts of the BChV genome have evolved differently in the two continents, possibly due to different selection pressures to allow adaptation to the various environments, hosts and vectors. BChV is a distinct species of the Polerovirus genus.

Improved detection and differentiation of poleroviruses infecting beet or rape by multiplex RT-PCR.

Three distinct species of virus inducing yellowing of beet, Beet mild yellowing virus (BMYV), Brassica yellows virus (BrYV, synonym BWYV) and Beet chlorosis virus (BChV) have been characterised from the genus Polerovirus. Until recently, no available tools were available to allow accurate and reliable distinction of the three species. Based on previous nucleotide sequence alignments and phylogenetic studies, we show that the use of molecular methods enabled the discrimination of these three beet Polerovirus species, but with differences in efficiency and specificity. Primers CP+ and CP- encompassing ORF-3, which encodes the coat protein, allowed the amplification by RT-PCR of a fragment of 563 bp for all isolates. Molecular methods such as SSCP or RFLP were able to discriminate these fragments by utilizing the differences in sequence. However, SSCP is a highly sensitive technique and was not suitable for the distinction of the Polerovirus species, because all isolates tested displayed a unique pattern. Analysis of the ORF3 RT-PCR products, digested with SmaI, RsaI and AccI restriction enzymes revealed four distinct patterns specific for the three species. However, point mutations can alter the RFLP patterns, making the interpretation of the results difficult. Primers were designed to amplify specifically sequences corresponding to ORF-0 of the three viral species. By using the three new sets of ORF-0 specific primers and CP+/CP- primers in a single multiplex RT-PCR, the detection and discrimination of the three beet Polerovirus species was possible in infected plants. The multiplex RT-PCR method provides a reliable and highly sensitive method to detect and identify viral species and will be of great interest for epidemiological studies of beet poleroviruses.

Improved detection of Beet necrotic yellow vein virus in a DAS ELISA by means of antibody single chain fragments (scFv) which were selected to protease-stable epitopes from phage display libraries.

The detection of Beet necrotic yellow vein virus (BNYVV) in stored sugar beets by means of monoclonal antibodies or antibody single chain fragments (scFv) often poses problems, because the immunodominant C-terminal epitope of the viral coat protein is readily lost due to proteolysis. Clones which produce scFv specific for protease-stable BNYVV epitopes were selected from two naive phage display libraries. Fusion proteins of the scFv with a human IgG kappa chain (expressed from the newly designed vector pCL) or with alkaline phosphatase,respectively, allow the ELISA detection of BNYVV even in stored sugar beets with a sensitivity which was comparable or often higher than that achieved with polyclonal antibodies.

Biological, serological, and molecular variability suggest three distinct polerovirus species infecting beet or rape.

Yellowing diseases of sugar beet can be caused by a range of strains classified as Beet mild yellowing virus (BMYV) or Beet western yellows virus (BWYV), both belonging to the genus Polerovirus of the family Luteoviridae. Host range, genomic, and serological studies have shown that isolates of these viruses can be grouped into three distinct species. Within these species, the coat protein amino acid sequences are highly conserved (more than 90% homology), whereas the P0 sequences (open reading frame, ORF 0) are variable (about 30% homology). Based on these results, we propose a new classification of BMYV and BWYV into three distinct species. Two of these species are presented for the first time and are not yet recognized by the International Committee on Taxonomy of Viruses. The first species, BMYV, infects sugar beet and Capsella bursa-pastoris. The second species, Brassica yellowing virus, does not infect beet, but infects a large number of plants belonging to the genus Brassica within the family Brassicaceae. The third species, Beet chlorosis virus, infects beet and Chenopodium capitatum, but not Capsella bursa-pastoris.

Agroinfection as an alternative to insects for infecting plants with beet western yellows luteovirus.

Beet western yellows luteovirus, like other luteoviruses, cannot be transmitted to host plants by mechanical inoculation but requires an aphid vector, a feature that has heretofore presented a serious obstacle to the study of such viruses. In this paper we describe use of agroinfection to infect hosts with beet western yellows virus without recourse to aphids. Agroinfection is a procedure for introducing a plant virus into a host via Agrobacterium tumefaciens harboring a Ti plasmid, which can efficiently transfer a portion of the plasmid (T-DNA) to plant cells near a wound. The viral genome must be inserted into the T-DNA in such a way that it can escape and begin autonomous replication, a requirement that has, so far, limited agroinfection to pathogens with a circular genome. We have cloned cDNA corresponding to the complete beet western yellows virus RNA genome between the cauliflower mosaic virus 35S promoter and the nopaline synthase transcription termination signal. In one construct, a self-cleaving (ribozyme) sequence was included so as to produce a transcript in planta with a 3' extremity almost identical to natural viral RNA. When inoculated mechanically to host plants, the naked plasmid DNA was not infectious but, when introduced into T-DNA and agroinfected to plants, both the construct with and without the ribozyme produced an infection. This approach should be applicable to virtually any plant virus with a linear plus-strand RNA genome.

Trisomy 16q23----qter arising from a maternal t(13;16)(p12;q23): case report and evidence of the reciprocal balanced maternal rearrangement by the Ag-NOR technique.

We describe a female new-born with partial trisomy of the long arm of chromosome 16. The chromosome anomaly was the result of an unbalanced segregation of a maternal translocation t(13;16)(p12;q23). Dynamic (RBG, GBG) banding and the Ag-NOR technique ascertained the reciprocal balanced maternal translocation between the 16q23----qter and 13q12----pter segments because nucleolar organizers were present on the tip of long arms of the derivative 16 maternal chromosome. As monosomy 13p has little or no deleterious effect we consider our case as exhibiting the phenotype of trisomy 16q23----qter free from any monosomic feature. Clinical effects are of less consequence as compared with previously published cases of partial trisomy 16q.

Comparison of enzyme-linked immunosorbent assay (ELISA) with dot hybridization using 32P- or 2-acetylaminofluorene (AAF)-labelled cDNA probes for the detection and characterization of beet necrotic yellow vein virus.

Beet Necrotic Yellow Vein Virus (BNYVV) was detected by enzyme-linked immunosorbent assay (ELISA) and RNA/DNA dot hybridization using either radiolabelled or non-radioactive probes. Dot hybridization specifically distinguished isolates that could not be distinguished by ELISA. The detection thresholds for ELISA, hybridization with non-radioactive probes and hybridization with radiolabelled probes were 2 ng, 0.2 ng, 0.02 ng of purified virus, respectively. Dot hybridization with non-radioactive probes could be performed on crude infected beet root extracts, thus providing a useful tool for monitoring BNYVV infection and for routine testing in plant breeding programs.

Effect of beet necrotic yellow vein virus RNA composition on transmission by Polymyxa betae.

Beet necrotic yellow vein virus (BNYVV) is naturally transmitted by the soil-borne fungus Polymyxa betae and usually remains confined to the roots of infected sugarbeets. In naturally infected sugarbeets the virion RNA always consists of four components which are uniform in size in different isolates but when BNYVV is propagated by mechanical inoculation to leaves of Chenopodium quinoa the two smallest RNA components, RNA-3 and -4, may undergo deletion or disappear from the isolate, suggesting that they are only essential for the natural mode of infection. To test this hypothesis, several C. quinoa isolates of BNYVV with different RNA-3 and -4 contents have been retransmitted to sugarbeet root via P. betae. The results show that the two isolates containing no detectable full-length RNA-3 and -4 are poorly transmitted and that cases of successful infection are associated with the reappearance of full-length RNA-3 and -4.