The genus Betanucleorhabdovirus includes plant viruses with negative sense, non-segmented, single-stranded RNA genomes. Here, we characterized putative novel betanucleorhabdoviruses infecting a medically important plant, elderberry. Total RNA was purified from the leaves of several plants, ribodepleted and sequenced using the Illumina platform. Sequence data analysis led to the identification of thirteen contigs of approximately 13.5 kb, showing a genome structure (3'-N-P-P3-M-G-L-5') typical of plant rhabdoviruses. The detected isolates showed 69.4 to 98.9% pairwise nucleotide identity and had the highest identity among known viruses (64.7-65.9%) with tomato betanucleorhabdovirus 2. A detailed similarity analysis and a phylogenetic analysis allowed us to discriminate the elderberry isolates into five groups, each meeting the sequence-based ICTV demarcation criterion in the Betanucleorhabdovirus genus (lower than 75% identity for the complete genome). Hence, the detected viruses appear to represent five novel, closely related betanucleorhabdoviruses, tentatively named Sambucus betanucleorhabdovirus 1 to 5.
- Publikační typ
- časopisecké články MeSH
The genus Cytorhabdovirus includes plant viruses with an unsegmented, single-stranded, negative-sense RNA genome that infect various plant hosts. In this work, we report the detection of a new cytorhabdovirus infecting elderberry (Sambucus nigra L.). Total RNA was purified from infected leaves and, after ribodepletion, sequenced using an Illumina system. The RNA genome of viral isolate B15 is 12,622 nucleotides (nt) long, and that of isolate B42 is 12,621 nt long. A nearly complete sequence (12,592 nt) was also obtained for a third isolate (B160). The RNA genomes of all three isolates showed an organisation typical of cytorhabdoviruses, harbouring all six of the expected genes (3 ́ N-P-P3-M-G-L 5 ́), separated by intergenic regions. These isolates were closely related to each other (99.5-99.6% nt sequence identity) and showed the highest overall similarity to trichosanthes associated rhabdovirus 1 (63.5% identity) and Wuhan insect virus 5 (58% identity), and similar results were obtained when comparing individual coding sequences or proteins. Phylogenetic analysis confirmed that this elderberry virus, for which we propose the name "sambucus virus 1" belongs to the genus Cytorhabdovirus and fulfils the criteria to represent a novel species.
- MeSH
- bez černý * MeSH
- bez * genetika MeSH
- fylogeneze MeSH
- genom virový MeSH
- nemoci rostlin MeSH
- otevřené čtecí rámce MeSH
- Rhabdoviridae * MeSH
- RNA MeSH
- virové proteiny genetika MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Česká republika MeSH
Recent developments in high-throughput sequencing (HTS), also called next-generation sequencing (NGS), technologies and bioinformatics have drastically changed research on viral pathogens and spurred growing interest in the field of virus diagnostics. However, the reliability of HTS-based virus detection protocols must be evaluated before adopting them for diagnostics. Many different bioinformatics algorithms aimed at detecting viruses in HTS data have been reported but little attention has been paid thus far to their sensitivity and reliability for diagnostic purposes. Therefore, we compared the ability of 21 plant virology laboratories, each employing a different bioinformatics pipeline, to detect 12 plant viruses through a double-blind large-scale performance test using 10 datasets of 21- to 24-nucleotide small RNA (sRNA) sequences from three different infected plants. The sensitivity of virus detection ranged between 35 and 100% among participants, with a marked negative effect when sequence depth decreased. The false-positive detection rate was very low and mainly related to the identification of host genome-integrated viral sequences or misinterpretation of the results. Reproducibility was high (91.6%). This work revealed the key influence of bioinformatics strategies for the sensitive detection of viruses in HTS sRNA datasets and, more specifically (i) the difficulty in detecting viral agents when they are novel or their sRNA abundance is low, (ii) the influence of key parameters at both assembly and annotation steps, (iii) the importance of completeness of reference sequence databases, and (iv) the significant level of scientific expertise needed when interpreting pipeline results. Overall, this work underlines key parameters and proposes recommendations for reliable sRNA-based detection of known and unknown viruses.
The genus Bromovirus currently contains six species whose members have relatively narrow host ranges. In the present work, a new bromovirus infecting elderberry (Sambucus nigra L.) is reported. dsRNA was purified and sequenced by next-generation sequencing, and with minimal additional completion by Sanger sequencing, the full tripartite genome was obtained. RNA1 is 3241 nt long and contains ORF1 (1a protein), RNA2 is 2810 nt long and contains ORF2 (2a protein), and RNA3 is 2244 nt long and contains ORF3a (movement protein) and ORF3b (coat protein, CP), separated by an intercistronic poly(A) stretch. Proteins 1a and 2a showed highest sequence identity (69.9% and 69.4%) to the corresponding proteins of melandrium yellow fleck virus. The coat protein showed highest sequence identity (67.9%) to that of brome mosaic virus. The genome shows a typical bromovirus organisation comprising of all the conserved protein domains within the genus. Phylogenetic analysis supports the assignment of this virus as a new member of the genus Bromovirus, for which the name "sambucus virus S" (SVS) is proposed.
- MeSH
- bez černý virologie MeSH
- Bromovirus klasifikace genetika izolace a purifikace MeSH
- fylogeneze MeSH
- genom virový * MeSH
- molekulární sekvence - údaje MeSH
- nemoci rostlin virologie MeSH
- otevřené čtecí rámce MeSH
- RNA virová genetika MeSH
- sekvence nukleotidů MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Česká republika MeSH
A novel virus infecting elderberry was identified by high-throughput Illumina sequencing of double strand RNAs isolated form elderberry leaves. The complete genome sequence obtained (4512 nucleotides in length) shows an organization typical for aureusviruses, with five open reading frames (ORFs) and the typical ORF1-RT expression by the readthrough of an amber stop codon. The analysis of the RNA-dependent RNA polymerase (RdRp) and coat protein (CP) sequences showed the highest identity (respectively 75.7% and 55%) with the corresponding amino acid sequences of Pothos latent virus. These two values, below the species demarcation criteria for the genus, indicate that the detected virus is a new member of genus Aureusvirus, family Tombusviridae, with the proposed name Elderberry aureusvirus 1 (ElAV1). A survey confirmed the wide distribution of ElAV1 in elderberry in the Czech Republic. Phylogenetic analyses of RdRp and CP sequences showed distinct microevolution of geographically separated isolates, with a tendency for isolates coming from close localities or from the same region to cluster together but heterogeneity of viral populations down to a local scale was also observed. The symptomatology of the new virus is not fully clear, but many infected trees were either asymptomatic or showed mild chlorotic mosaics. More severe symptoms, potentially impacting yields of flowers or berries, were observed in plants with mixed infections of ElAV1 and other elderberry viruses. Further efforts are now needed to determine ElAV1 prevalence outside the Czech Republic and to unravel its epidemiology.
Pea seed-borne mosaic virus (PSbMV) significantly reduces yields in a broad spectra of legumes. The eukaryotic translation initiation factor has been shown to confer resistance to this pathogen, thus implying that translation and proteome dynamics play a role in resistance. This study presents the results of a proteome-wide analysis of Pisum sativum L. response to PSbMV infection. LC-MS profiling of two contrasting pea cultivars, resistant (B99) and susceptible (Raman) to PSbMV infection, detected >2300 proteins, 116 of which responded to PSbMV ten and/or twenty days post-inoculation. These differentially abundant proteins are involved in number of processes that have previously been reported in the plant-pathogen response, including protein and amino acid metabolism, stress signaling, redox homeostasis, carbohydrate metabolism, and lipid metabolism. We complemented our proteome-wide analysis work with targeted analyses of free amino acids and selected small molecules, fatty acid profiling, and enzyme activity assays. Data from these additional experiments support our findings and validate the biological relevance of the observed proteome changes. We found surprising similarities in the resistant and susceptible cultivars, which implies that a seemingly unaffected plant, with no detectable levels of PSbMV, actively suppresses viral replication. BIOLOGICAL SIGNIFICANCE: Plant resistance to PSbMV is connected to translation initiation factors, yet the processes involved are still poorly understood at the proteome level. To the best of our knowledge, this is the first survey of the global proteomic response to PSbMV in plants. The combination of label-free LC-MS profiling and two contrasting cultivars (resistant and susceptible) provided highly sensitive snapshots of protein abundance in response to PSbMV infection. PSbMV is a member of the largest family of plant viruses and our results are in accordance with previously characterized potyvirus-responsive proteomes. Hence, the results of this study can further extend our knowledge about these pathogens. We also show that even though no viral replication is detected in the PSbMV-resistant cultivar B99, it is still significantly affected by PSbMV inoculation.
- MeSH
- chromatografie kapalinová MeSH
- eukaryotické iniciační faktory MeSH
- hrách setý virologie MeSH
- interakce hostitele a patogenu * MeSH
- nemoci rostlin virologie MeSH
- odolnost vůči nemocem genetika MeSH
- Potyvirus patogenita MeSH
- proteomika metody MeSH
- rostlinné viry MeSH
- tandemová hmotnostní spektrometrie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND AND AIMS: Haemophilus influenzae new strain acquisition has been demonstrated to increase the relative risk of acute exacerbation fourfold in contrast to colonisation or chronic infection by the same strain in chronic obstructive pulmonary disease (COPD). Unfortunately, molecular typing techniques are not suitable for routine use due to cost, labour-intensity and need for special expertise. We tested two techniques potentially useful for routine typing, namely the newly available MALDI-TOF MS and the modified McRAPD compared to MLST as the gold standard. METHODS: In 10 patients (10.8%) suffering from COPD or cystic fibrosis, H. influenzae isolates were recovered repeatedly at different timepoints from the same patient during the study period. This allowed for thirteen pairwise comparisons of typing results in isolates recovered consecutively from the same patient to test the ability of the techniques to uncover new strain acquisition. RESULTS: MLST detected 9 cases of new strain acquisition among the 13 pairwise comparisons. However, MALDI-TOF MS reported all 13 pairs as different and thus new. In contrast, McRAPD was able to differentiate all the new strain acquisitions from pre-existing ones, both by visual inspection of melting profiles and by Relative Significant Difference values. CONCLUSIONS: Unlike MALDI-TOF MS, McRAPD appears to be a suitable candidate for routine discrimination of new strain acquisitions because of its accuracy and, rapid, easy and economic performance.
- MeSH
- chronická obstrukční plicní nemoc diagnóza MeSH
- cystická fibróza diagnóza MeSH
- diferenciální diagnóza MeSH
- Haemophilus influenzae izolace a purifikace MeSH
- hemofilové infekce diagnóza MeSH
- lidé MeSH
- senzitivita a specificita MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody normy MeSH
- techniky typizace bakterií metody normy MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
Creeping thistle [Cirsium arvense (L.) Scop.] and dahlia (Dahlia sp.) plants showing typical symptoms of phytoplasma infection including yellowing, stunting, inflorescence and proliferation, were sampled; the presence of phytoplasma was confirmed by standard PCR using universal primers. RFLP analysis allowed classification of the detected phytoplasma strains CirYS, CirYS1 and DahlP within the 16SrXI group, the unique restriction profile F2nR2 fragment obtained in silico by iPhyClassifier indicated that they belong to the new 16SrXI-E subgroup. Genetic analysis of the 16S rRNA gene revealed that the studied strains shared less than 97.5% similarity with all of the previously described 'Candidatus Phytoplasma' species. The closest relatives are 'Candidatus Phytoplasma cynodontis' and 'Candidatus Phytoplasma oryzae' with 96.8% and 96.6% similarity. All strains studied bear three specific regions in the 16S rRNA gene, discriminating them from the other phytoplasma species. Phylogenetic analysis of the 16S rRNA and secA genes confirmed this specificity, as the creeping thistle and dahlia phytoplasma strains clustered in a distinguishable lineage group. The uniqueness of the genetic analysis agrees with the biological characterization of the studied phytoplasma strains, their host range, and geographical distribution. The strains only infect dicotyledonous plants in Europe, contrary to their closest relatives. Based on their unique properties, it could be concluded that the studied phytoplasma strains represent a discrete group that is proposed as a novel taxon 'Candidatus Phytoplasma cirsii', with strain CirYS as a reference strain.
- MeSH
- Cirsium mikrobiologie MeSH
- DNA bakterií genetika MeSH
- fylogeneze * MeSH
- molekulární sekvence - údaje MeSH
- nemoci rostlin mikrobiologie MeSH
- Phytoplasma klasifikace genetika izolace a purifikace MeSH
- polymorfismus délky restrikčních fragmentů MeSH
- RNA ribozomální 16S genetika MeSH
- sekvenční analýza DNA MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Evropa MeSH
Adequate treatment of microbial infections requires rapid and accurate identification of the etiological agent. In routine diagnostics, identification of bacteria conventionally relies on phenotypic testing, which can be hindered by phenotypic variations. Therefore, genotyping techniques should perform faster and more accurately. Recently, the technique of high-resolution melting analysis (HRMA) of PCR amplicons promises to provide a convenient and economic tool of genotypic identification. In our study, we performed prospective routine testing of a PCR-HRMA system that was recently published in a proof-of-the-principle study. The system was evaluated by analysing 275 clinical isolates of bacteria acquired from 65 patients suffering from cystic fibrosis or chronic obstructive pulmonary disease. Our results show that its routine use may result in partial worsening of its discriminatory power; however, it still outmatched conventional phenotyping in the group of non-fermentative Gram-negative rods. Moreover, when supplemented by rapid, simple and economic oxidase test, it can be even simplified for more economic performance.
- MeSH
- bakteriologické techniky metody MeSH
- chronická obstrukční plicní nemoc komplikace MeSH
- cystická fibróza komplikace MeSH
- diagnostické techniky molekulární metody MeSH
- DNA bakterií chemie genetika MeSH
- gramnegativní aerobní tyčinky a koky genetika izolace a purifikace MeSH
- gramnegativní bakteriální infekce diagnóza mikrobiologie MeSH
- infekce dýchací soustavy mikrobiologie MeSH
- lidé MeSH
- polymerázová řetězová reakce metody MeSH
- prospektivní studie MeSH
- tranzitní teplota MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
BACKGROUND: The eukaryotic translation initiation factor 4E was shown to be involved in resistance against several potyviruses in plants, including pea. We combined our knowledge of pea germplasm diversity with that of the eIF4E gene to identify novel genetic diversity. METHODOLOGY/PRINCIPAL FINDINGS: Germplasm of 2803 pea accessions was screened for eIF4E intron 3 length polymorphism, resulting in the detection of four eIF4E(A-B-C-S) variants, whose distribution was geographically structured. The eIF4E(A) variant conferring resistance to the P1 PSbMV pathotype was found in 53 accessions (1.9%), of which 15 were landraces from India, Afghanistan, Nepal, and 7 were from Ethiopia. A newly discovered variant, eIF4E(B), was present in 328 accessions (11.7%) from Ethiopia (29%), Afghanistan (23%), India (20%), Israel (25%) and China (39%). The eIF4E(C) variant was detected in 91 accessions (3.2% of total) from India (20%), Afghanistan (33%), the Iberian Peninsula (22%) and the Balkans (9.3%). The eIF4E(S) variant for susceptibility predominated as the wild type. Sequencing of 73 samples, identified 34 alleles at the whole gene, 26 at cDNA and 19 protein variants, respectively. Fifteen alleles were virologically tested and 9 alleles (eIF4E(A-1-2-3-4-5-6-7), eIF4E(B-1), eIF4E(C-2)) conferred resistance to the P1 PSbMV pathotype. CONCLUSIONS/SIGNIFICANCE: This work identified novel eIF4E alleles within geographically structured pea germplasm and indicated their independent evolution from the susceptible eIF4E(S1) allele. Despite high variation present in wild Pisum accessions, none of them possessed resistance alleles, supporting a hypothesis of distinct mode of evolution of resistance in wild as opposed to crop species. The Highlands of Central Asia, the northern regions of the Indian subcontinent, Eastern Africa and China were identified as important centers of pea diversity that correspond with the diversity of the pathogen. The series of alleles identified in this study provides the basis to study the co-evolution of potyviruses and the pea host.
- MeSH
- alely MeSH
- eukaryotický iniciační faktor 4E genetika fyziologie MeSH
- hrách setý genetika metabolismus virologie MeSH
- nemoci rostlin virologie MeSH
- Potyvirus patogenita MeSH
- rostlinné proteiny genetika fyziologie MeSH
- zeměpis MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
