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

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

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

Visual inspections of elm trees in south Moravia in 1997-2007 revealed a rare occurrence of plants with smaller and cowl-forming leaves on some twigs, i.e. a feature resembling witches'-broom disease observed on the end of twigs. The presence of phytoplasma-like bodies was observed by transmission electron microscopy of phloem tissue. On the other hand, no phytoplasmas were found in asymptomatic trees. Nucleic acids extracted from these plants were used in nested-PCR assays with primers amplifying 16S rRNA sequences specific for phytoplasmas. Sequence analyses of the 16S-23S ribosomal operon (1852 bp) allowed for the classification of the detected phytoplasmas in the elm yellows group, but its position remained on the boundary of the 16SrV-A and 16SrV-C ribosomal subgroups. Sequence analyses of the ribosomal protein of the rpl22-rps3 and secY genes lead to further classification and revealed the phytoplasmas' affiliations to the 'Candidates Phytoplasma ulmi'. Some exceptions in unique oligonucleotide sequences defined for 'Ca. Phytoplasma ulmi' were found in the Czech isolate. This is the northernmost confirmed occurrence of phytoplasma on elm trees within Europe.

• MeSH
• bakteriální proteiny genetika   MeSH
• DNA bakterií genetika   MeSH
• elektronová mikroskopie MeSH
• fylogeneze MeSH
• molekulární sekvence - údaje MeSH
• nemoci rostlin mikrobiologie   MeSH
• Phytoplasma MeSH
• ribozomální DNA genetika   MeSH
• RNA ribozomální 16S genetika   MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• sekvenční analýza DNA MeSH
• Ulmus mikrobiologie   MeSH
• Geografické názvy
• Česká republika MeSH