Iain G. Old

Phi29 polymerase based random amplification of viral RNA as an alternative to random RT-PCR

BackgroundPhi29 polymerase based amplification methods provides amplified DNA with minimal changes in sequence and relative abundance for many biomedical applications. RNA virus detection using microarrays, however, can present a challenge because phi29 DNA polymerase cannot amplify RNA nor small cDNA fragments (<2000 bases) obtained by reverse transcription of certain viral RNA genomes. Therefore, ligation of cDNA fragments is necessary prior phi29 polymerase based amplification. We adapted the QuantiTect Whole Transcriptome Kit (Qiagen) to our purposes and designated the method as Whole Transcriptome Amplification (WTA).ResultsWTA successfully amplified cDNA from a panel of RNA viruses representing the diversity of ribovirus genome sizes. We amplified a range of genome copy numbers from 15 to 4 × 107 using WTA, which yielded quantities of amplified DNA as high as 1.2 μg/μl or 1010 target copies. The amplification factor varied between 109 and 106. We also demonstrated that co-amplification occurred when viral RNA was mixed with bacterial DNA.ConclusionThis is the first report in the scientific literature showing that a modified WGA (WTA) approach can be successfully applied to viral genomic RNA of all sizes. Amplifying viral RNA by WTA provides considerably better sensitivity and accuracy of detection compared to random RT-PCR.

Conservation of gene arrangement and an unusual organization of rRNA genes in the linear chromosomes of the Lyme disease spirochaetes Borrelia burgdorferi, B. garinii and B. afzelii

Physical maps of the chromosomes of the Lyme disease spirochaetes Borrelia garinii and Borrelia afzelii have been elucidated for the enzymes CspI, SgrAI, I-CeuI, SmaI, EagI, BssHII, MluI and ApaI by two-dimensional pulsed-field gel electrophoresis techniques. The maps contain 42 sites for B. garinii and 32 for B. afzelii. The mapping studies showed that the two chromosomes are linear DNA molecules of 953 and 948 kbp, respectively. A comparison of the physical maps of B. garinii and B. afzelii and the published map of the other Lyme disease spirochaete, Borrelia burgdorferi [Davidson, B.E., MacDougall, J. & Saint Girons, I. (1992) J Bacteriol 174, 3766-3774] revealed that the three chromosomes have few endonuclease sites in common, apart from a cluster in rrl (encoding 23S rRNA) and rrs (encoding 16S rRNA). Cloned borrelial genes were used as specific hybridization probes to construct genetic maps, using the physical maps as a basis. The resulting maps contain 41 genetic loci for B. burgdorferi, 39 for B. garinii, and 33 for B. afzelii. In contrast to the physical maps, the three genetic maps are closely related, with no detectable differences in gene order along the entire length of the chromosome. It is concluded that the chromosomes of these three borrelial species have undergone no major rearrangements, deletions or insertions during their evolution from a common ancestor. Detailed mapping of the region of the B. garinii and B. afzelii chromosomes that encodes rRNA revealed that each chromosome contains one copy of rrs separated by 5 kbp from two copies each of rrl and rrf (encoding 5S rRNA). (ABSTRACT TRUNCATED AT 250 WORDS)

Massively parallel pathogen identification using high-density microarrays.

Identification of microbial pathogens in clinical specimens is still performed by phenotypic methods that are often slow and cumbersome, despite the availability of more comprehensive genotyping technologies. We present an approach based on whole‐genome amplification and resequencing microarrays for unbiased pathogen detection. This 10 h process identifies a broad spectrum of bacterial and viral species and predicts antibiotic resistance and pathogenicity and virulence profiles. We successfully identify a variety of bacteria and viruses, both in isolation and in complex mixtures, and the high specificity of the microarray distinguishes between different pathogens that cause diseases with overlapping symptoms. The resequencing approach also allows identification of organisms whose sequences are not tiled on the array, greatly expanding the repertoire of identifiable organisms and their variants. We identify organisms by hybridization of their DNA in as little as 1–4 h. Using this method, we identified Monkeypox virus and drug‐resistant Staphylococcus aureus in a skin lesion taken from a child suspected of an orthopoxvirus infection, despite poor transport conditions of the sample, and a vast excess of human DNA. Our results suggest this technology could be applied in a clinical setting to test for numerous pathogens in a rapid, sensitive and unbiased manner.

Application of Broad-Spectrum Resequencing Microarray for Genotyping Rhabdoviruses

ABSTRACT The rapid and accurate identification of pathogens is critical in the control of infectious disease. To this end, we analyzed the capacity for viral detection and identification of a newly described high-density resequencing microarray (RMA), termed PathogenID, which was designed for multiple pathogen detection using database similarity searching. We focused on one of the largest and most diverse viral families described to date, the family Rhabdoviridae. We demonstrate that this approach has the potential to identify both known and related viruses for which precise sequence information is unavailable. In particular, we demonstrate that a strategy based on consensus sequence determination for analysis of RMA output data enabled successful detection of viruses exhibiting up to 26% nucleotide divergence with the closest sequence tiled on the array. Using clinical specimens obtained from rabid patients and animals, this method also shows a high species level concordance with standard reference assays, indicating that it is amenable for the development of diagnostic assays. Finally, 12 animal rhabdoviruses which were currently unclassified, unassigned, or assigned as tentative species within the family Rhabdoviridae were successfully detected. These new data allowed an unprecedented phylogenetic analysis of 106 rhabdoviruses and further suggest that the principles and methodology developed here may be used for the broad-spectrum surveillance and the broader-scale investigation of biodiversity in the viral world.

Nucleotide sequence of the metH gene of Escherichia coli K-12 and comparison with that of Salmonella typhimurium LT2.

The Escherichia coli K-12 metH gene, encoding the vitamin B12-dependent homocysteine transmethylase, is located between iclR and lysC in the 91-min region of the chromosome. The metH gene has been sequenced and reveals an open reading frame of 3600 bp encoding a polypeptide of 1200 amino acids (aa) with a calculated Mr of 132 628. The first 414 aa of the deduced polypeptide sequence are 92% identical to the 414 aa deduced from the partially sequenced Salmonella typhimurium LT2 metH gene. In-frame fusions of metH to lacZ were used to confirm the reading frame of the metH gene and to study its regulation. metH was repressed tenfold, presumably indirectly, by L-methionine and the metJ gene product, while vitamin B12 did not induce de novo synthesis of MetH.

FliH and FliI of Borrelia burgdorferi are similar to flagellar and virulence factor export proteins of other bacteria

Two motility genes (fliH and fliI) of the Lyme disease spirochete Borrelia burgdorferi were cloned, physically mapped and sequenced, FliH and FliI showed extensive homology to the proteins involved in the export of flagellar components and to virulence factors found in both animal and plant bacterial pathogens. The results suggest that the flagellar apparatus and associated protein export pathway are well conserved in evolution.

Microbial genes homologous to the peptidyl-tRNA hydrolase-encoding gene of Escherichia coli

We have cloned and determined the nucleotide (nt) sequences of the genes encoding peptidyl-tRNA hydrolase (Pth) homologues of Salmonella typhi (St) and the Lyme disease spirochaete, Borrelia burgdorferi (Bb). We also completed the nt sequence of a pth homologous gene contained in a Chlamydia trachomatis (Ct) clone identified in the databanks. The open reading frames (ORFs) of the Pth homologues encode putative polypeptides of 194 (St), 188 (Bb) and 194 (Ct) amino acids exhibiting significant identity with Escherichia coli (Ec) Pth. Together with the products of two previously unidentified ORFs from Bacillus subtilis and Saccharomyces cerevisiae, and the recently recognized Haemophilus influenzae and Mycoplasma genitalium pth genes, these seven putative polypeptides and the Ec Pth form a group of homologous basic proteins spanning eubacteria and eukaryota which can be defined by at least three conserved regions. Previously known Ec pth mutations were located in highly conserved residues.

A cheA cheW operon in Borrelia burgdorferi, the agent of Lyme disease

Borrelia burgdorferi sensu stricto homologues of cheA and cheW were cloned and characterized. A combination of strategies such as polymerase chain reaction (PCR) using degenerate primers, random-primed gene walking PCR and construction of a lamda library were used to identify the putative cheA gene. Sequence analysis of the DNA fragments obtained from the CT strain identified a 2,592-bp open reading frame (ORF) encoding an 864-amino-acid protein with significant similarity (53-64.6% identical residues) to the CheA of several genera of eubacteria. In particular, hallmarks of a histidine kinase family were found such as the location of the histidine autophosphorylation domain very close to the NH2 terminus and the nucleotide-binding site. A second ORF located immediately downstream from the putative borrelial cheA gene encoded a 195-amino-acid protein which displayed a high level of similarity to bacterial CheW. Using reverse transcription PCR, we demonstrated that cheA and cheW form an operon with an upstream, unidentified ORF. The cheA and cheW homologues were located at 722-737 kbp, 738-768 kbp and 743-824 kbp on the linear chromosomes of B. burgdorferi sensu stricto, B. garinii and B. afzelii, respectively. Identification of cheA and cheW is the first step toward elucidation of a possible role of chemotaxis in virulence of the Lyme disease borreliae.

Use of consensus sequences for the design of high density resequencing microarrays: the influenza virus paradigm

BackgroundA resequencing microarray called PathogenID v2.0 has been developed and used to explore various strategies of sequence selection for its design. The part dedicated to influenza viruses was based on consensus sequences specific for one gene generated from global alignments of a large number of influenza virus sequences available in databanks.ResultsFor each HA (H1, H2, H3, H5, H7 and H9) and NA (N1, N2 and N7) molecular type chosen to be tested, 1 to 3 consensus sequences were computed and tiled on the microarray. A total of 12 influenza virus samples from different host origins (humans, pigs, horses and birds) and isolated over a period of about 50 years were used in this study. Influenza viruses were correctly identified, and in most cases with the accurate information of the time of their emergence.ConclusionsPathogenID v2.0 microarray demonstrated its ability to type and subtype influenza viruses, often to the level of viral variants, with a minimum number of tiled sequences. This validated the strategy of using consensus sequences, which do not exist in nature, for our microarray design. The versatility, rapidity and high discriminatory power of the PathogenID v2.0 microarray could prove critical to detect and identify viral genome reassortment events resulting in a novel virus with epidemic or pandemic potential and therefore assist health authorities to make efficient decisions about patient treatment and outbreak management.

Physical and Genetic Maps of the Borrelia Burgdorferi Sensu Lato Chromosomes

Physical and genetic maps of the three species of Borrelia burgdorferi sensu lato associated with Lyme borreliosis were constructed. The ribosomal genes which are not organised as trancriptional units, were mapped at the centre of the linear chromosome. The region spanning dnoA encoding the initiatior protein for chromosome replication was found to be atypical. dnaA and genes normally associated with the origin of replication in eubacteria were found very close to the ribosomal RNA genes.