Browsing by Subject "Nucleic acids"

Now showing 1 - 4 of 4
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    Open Access
    Appearances can be deceptive: revealing a hidden viral infection with deep sequencing in a plant quarantine context
    (Public Library of Science, 2014) Candresse, Thierry; Filloux, Denis; Muhire, Brejnev; Julian, Charlotte; Galzi, Serge; Fort, Guillaume; Bernardo, Pauline; Daugrois, Jean-Heindrich; Fernandez, Emmanuel; Martin, Darren P
    Comprehensive inventories of plant viral diversity are essential for effective quarantine and sanitation efforts. The safety of regulated plant material exchanges presently relies heavily on techniques such as PCR or nucleic acid hybridisation, which are only suited to the detection and characterisation of specific, well characterised pathogens. Here, we demonstrate the utility of sequence-independent next generation sequencing (NGS) of both virus-derived small interfering RNAs (siRNAs) and virion-associated nucleic acids (VANA) for the detailed identification and characterisation of viruses infecting two quarantined sugarcane plants. Both plants originated from Egypt and were known to be infected with Sugarcane streak Egypt Virus (SSEV; Genus Mastrevirus , Family Geminiviridae ), but were revealed by the NGS approaches to also be infected by a second highly divergent mastrevirus, here named Sugarcane white streak Virus (SWSV). This novel virus had escaped detection by all routine quarantine detection assays and was found to also be present in sugarcane plants originating from Sudan. Complete SWSV genomes were cloned and sequenced from six plants and all were found to share >91% genome-wide identity. With the exception of two SWSV variants, which potentially express unusually large RepA proteins, the SWSV isolates display genome characteristics very typical to those of all other previously described mastreviruses. An analysis of virus-derived siRNAs for SWSV and SSEV showed them to be strongly influenced by secondary structures within both genomic single stranded DNA and mRNA transcripts. In addition, the distribution of siRNA size frequencies indicates that these mastreviruses are likely subject to both transcriptional and post-transcriptional gene silencing. Our study stresses the potential advantages of NGS-based virus metagenomic screening in a plant quarantine setting and indicates that such techniques could dramatically reduce the numbers of non-intercepted virus pathogens passing through plant quarantine stations.
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    Open Access
    Biochemical characterization of the nucleic acids of some human and animal viruses
    (1982) Mew, Ronald Terence
    In Part I, the isolation and partial characterization of human polyomaviruses from a number of renal transplant patients is described. These isolates proved refractory to cell culture propagation by the methods used, and were thus extracted directly from large volumes of patient's urine. This approach has the advantage that the virus cannot undergo any possible genomic modification, as tends to occur during adaptation to cell culture. Human polyomavirus DNA is very susceptible to mutation during cell passage. Four isolates from different patients yielded sufficient DNA for limited restriction endonuclease characterization. Surprisingly, all four gave the same patterns with EcoRI, BamHI and HindIII. Two isolates that were also digested with PstI gave an identical pattern. These patterns are similar to, but distinct from, other strains of the human polyomavirus BK that have been described. Our isolates had a similar-sized genome to BK, but only 3 HindIII sites compared with 4 in the prototype, and 2 PstI sites compared with only 1 in the prototype. The quantity of DNA obtained directly from urine was usually very limited. In order to produce adequate DNA for complete analysis, viral DNA was recombined with· a bacterial vector (pBR322) and cloned into Escherichia coli strains HB101 and C600. Initially, the well-studied strain BK(MM) was successfully cloned. This clone was used to prepare radioactively-labelled DNA probes for the detection of BK-specific sequences in urine isolates and in subsequent recombinants with patient material. Such cloned material is easier to prepare in bulk than DNA from virus passaged in cell culture. Early attempts to clone DNA from clinical isolates failed, but BK-specific DNA from a patient (P.R.) has recently been cloned successfully. These clones are presently being used to investigate the differences in sequence between our isolates and the known strains of BK. It is hoped that this will shed light on the mechanisms of gene expression of these potentially oncogenic viruses. In Part II, the genomes of four rotaviruses were studied. "Simian agent 11" (SAll) and "offal agent" (OA) were cell culture-adapted strains, whereas "epizootic diarrhoea of infant mice virus" (EDIM) and "infantile gastroenteritis virus" (IGV) were isolated from stool specimens. Experiments were performed to confirm the double-stranded RNA (dsRNA) nature of the SAll genome. It ran at a characteristic density of l.595g/ml in caesium sulphate density gradients, and was resistant to DNase and RNase at high ionic strengths, but susceptible to RNase at low ionic strength. At the start of the project few or no polyacrylamide gel pictures of the nucleic acids of these viruses had been published, although it was known they resembled reovirus in consisting of segmented double-stranded RNA. Such pictures were obtained, and molecular weight estimations made by comparison with dsRNA markers of known MW from a cytoplasmic polyhedrosis virus (CPV) from Heliothis armigera (Harley, Rubenstein, Losman and Lutton, 1977. Virology 76: 210-216). The difficulties in obtaining precise MW values for rotavirus genome segments are discussed. All four genomes consist of 11 dsRNA segments. The pattern of bands produced by PAGE is very similar, and high-resolution gels are required to detect the small mobility differences between some segments. Gel systems were developed to improve on the resolution obtained in co-electrophoresis experiments. During attempts to culture SAll and OA viruses in cell culture, it was observed that treatment of the cells and/or virus with versene-trypsin solution during infection gave a marked increase in virus yield. While this effect was being investigated, reports appeared on the potentiating effect of trypsin on the cell culture of previously refractory rotaviruses. We confirmed that trypsin, when present in the culture medium, greatly increased the yield of progeny SAll virus.
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    Open Access
    Detection of Streptococcus pneumoniae from different types of nasopharyngeal swabs in children
    (Public Library of Science, 2013) Dube, Felix S; Kaba, Mamadou; Whittaker, Elizabeth; Zar, Heather J; Nicol, Mark P
    BACKGROUND: A better understanding of the epidemiology of nasopharyngeal carriage of Streptococcus pneumoniae is important to assess the impact of vaccination and the pathogenesis of pneumococcal disease. We compared the recovery of S. pneumoniae from nylon flocked, Dacron and rayon swabs. METHODS: The recovery of S. pneumoniae from mocked specimens using flocked, Dacron and rayon swabs were compared by culture. The yield from paired nasopharyngeal (NP) samples obtained from healthy children sampled with flocked and Dacron swabs was also determined using culture and lytA -targeted real-time polymerase chain reaction (qPCR). RESULTS: Using mock specimen, the percentage recovery of S. pneumoniae ATCC 49619 (serotype 19F) strain from the flocked swabs was 100%, while it was 41% from Dacron swabs and 7% from rayon swabs. Similar results were observed for S. pneumoniae serotypes 1 and 5. S. pneumoniae was cultured from 18 of 42 (43%) paired NP samples from the healthy children (median age 8 [interquartile range (IQR) 5-16] months). The median number of colony-forming units (CFU) recovered from flocked swabs was two-fold higher (8.8×10 4 CFU/mL [IQR, 2.0×10 2 - 4.0×10 5 CFU/mL]) than Dacron swabs (3.7×10 4 CFU/mL [IQR, 4.0×10 2 -3.2×10 5 CFU/mL], p  = 0.17). Using lytA -targeted qPCR from paired NP samples, the median copy number of S. pneumoniae detected from flocked swabs was significantly higher than from Dacron swabs (3.0×10 5 genome copies/mL [IQR, 1.3×10 2 −1.8×10 6 ] vs. 9.3×10 4 genome copies/mL [IQR, 7.0×10 1 −1.1×10 6 ]; p  = 0.005). CONCLUSION: Flocked swabs released more S. pneumoniae compared to both Dacron and rayon swabs from mock specimens. Similarly, higher bacterial loads were detected by qPCR from flocked swabs compared with Dacron swabs from healthy children.
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    Open Access
    DNA-mediated biomineralization of a new planar Pt-complex
    (2006) Klump, H H; Koch, K; Lin, C T
    The crystal growth morphology of a coordination complex of Pt(II) that crystallizes from solution can be controlled by using a second molecular species such as peptides or other organic compounds. Examples of crystal growth controlled by nucleic acids are few. In this article we describe the use of branched three-way junction (3WJ) DNA to influence the crystal growth of a planar platinum compound, cis-[(2, 2′-bipyridyl)N,N-di(2-hydroxyethyl)-N′-benzoylthioureatoplatinum(II)]chloride. Platinum complexes with extended planar aromatic residues are capable of stacking in the absence as well as in the presence of linear DNA double helices. This feature is based on the interaction of the compound with DNA through intercalation, resulting in the prevention of binding of DNA polymerase. Microscopic one-dimensional crystals were observed under these conditions. In the presence of the branched 3WJ DNA, however, additional nucleation sites are present, resulting in extended crystal growth of unique Pt compounds. At least two different crystal modifications were observed using transmission electron microscopy.