E-Predict: a computational strategy for species identification based on observed DNA microarray hybridization patterns

DNA microarrays may be used to identify microbial species present in environmental and clinical samples. However, automated tools for reliable species identification based on observed microarray hybridization patterns are lacking. We present an algorithm, E-Predict, for microarray-based species identification. E-Predict compares observed hybridization patterns with theoretical energy profiles representing different species. We demonstrate the application of the algorithm to viral detection in a set of clinical samples and discuss its relevance to other metagenomic applications

Experimental induction of proventricular dilatation disease in cockatiels (Nymphicus hollandicus) inoculated with brain homogenates containing avian bornavirus 4

<p>Abstract</p> <p>Background</p> <p>Proventricular dilatation disease (PDD) is a fatal disorder of psittacine birds worldwide. The disease is characterized by lymphoplasmacytic infiltration of the central and peripheral nervous systems, leading to gastrointestinal motility and/or central nervous system dysfunction. Recently, we detected a significant association between avian bornavirus (ABV) infection and clinical signs of PDD in psittacines. However, it remains unclear whether ABV infection actually causes PDD. To address this question, we examined the impact of ABV inoculation on the cockatiel (<it>Nymphicus hollandicus</it>).</p> <p>Results</p> <p>Five cockatiels were inoculated via multiple routes (intramuscular, intraocular, intranasal, and oral) with a brain homogenate derived from either a PDD(+) avian bornavirus 4 (ABV4) (+) case (n = 3 inoculees) or from a PDD(-) ABV(-) control (n = 2 inoculees). The control birds remained free of clinical or pathological signs of PDD, and tested ABV(-) by RT-PCR and immunohistochemistry (IHC). In contrast, all three cockatiels inoculated with ABV4(+) brain homogenate developed gross and microscopic PDD lesions, and two exhibited overt clinical signs. In numerous tissues, ABV RT-PCR and sequence analysis demonstrated the presence of ABV4 RNA nearly identical to that in the inoculum. ABV was detected in the central nervous system of the three ABV-inoculees by IHC. Pyrosequencing to investigate the viral flora in the ABV4(+) inoculum uncovered 7 unique reads sharing 73–100% nucleotide sequence identity with previously identified ABV sequences and 24 reads sharing 40–89% amino acid sequence identity with viruses in the <it>Retroviridae </it>and <it>Astroviridae </it>families. Of these candidate viral species, only ABV RNA was recovered from tissues of the inoculated birds.</p> <p>Conclusion</p> <p>In this study, the clinical and pathological manifestations of PDD were induced by inoculation of cockatiels with brain homogenates containing avian bornavirus 4. By using high throughput pyrosequencing an in-depth view of the viral content of the inoculum was achieved, revealing that of 3 candidate virus families detected, only the presence of ABV RNA correlated with the development of PDD. This study provides evidence of a causal association between ABV4 infection and PDD in cockatiels.</p

Recovery of divergent avian bornaviruses from cases of proventricular dilatation disease: Identification of a candidate etiologic agent

<p>Abstract</p> <p>Background</p> <p>Proventricular dilatation disease (PDD) is a fatal disorder threatening domesticated and wild psittacine birds worldwide. It is characterized by lymphoplasmacytic infiltration of the ganglia of the central and peripheral nervous system, leading to central nervous system disorders as well as disordered enteric motility and associated wasting. For almost 40 years, a viral etiology for PDD has been suspected, but to date no candidate etiologic agent has been reproducibly linked to the disease.</p> <p>Results</p> <p>Analysis of 2 PDD case-control series collected independently on different continents using a pan-viral microarray revealed a bornavirus hybridization signature in 62.5% of the PDD cases (5/8) and none of the controls (0/8). Ultra high throughput sequencing was utilized to recover the complete viral genome sequence from one of the virus-positive PDD cases. This revealed a bornavirus-like genome organization for this agent with a high degree of sequence divergence from all prior bornavirus isolates. We propose the name avian bornavirus (ABV) for this agent. Further specific ABV PCR analysis of an additional set of independently collected PDD cases and controls yielded a significant difference in ABV detection rate among PDD cases (71%, n = 7) compared to controls (0%, n = 14) (P = 0.01; Fisher's Exact Test). Partial sequence analysis of a total of 16 ABV isolates we have now recovered from these and an additional set of cases reveals at least 5 distinct ABV genetic subgroups.</p> <p>Conclusion</p> <p>These studies clearly demonstrate the existence of an avian reservoir of remarkably diverse bornaviruses and provide a compelling candidate in the search for an etiologic agent of PDD.</p

Genome-wide diversity and selective pressure in the human rhinovirus

BACKGROUND: The human rhinoviruses (HRV) are one of the most common and diverse respiratory pathogens of humans. Over 100 distinct HRV serotypes are known, yet only 6 genomes are available. Due to the paucity of HRV genome sequence, little is known about the genetic diversity within HRV or the forces driving this diversity. Previous comparative genome sequence analyses indicate that recombination drives diversification in multiple genera of the picornavirus family, yet it remains unclear if this holds for HRV. RESULTS: To resolve this and gain insight into the forces driving diversification in HRV, we generated a representative set of 34 fully sequenced HRVs. Analysis of these genomes shows consistent phylogenies across the genome, conserved non-coding elements, and only limited recombination. However, spikes of genetic diversity at both the nucleotide and amino acid level are detectable within every locus of the genome. Despite this, the HRV genome as a whole is under purifying selective pressure, with islands of diversifying pressure in the VP1, VP2, and VP3 structural genes and two non-structural genes, the 3C protease and 3D polymerase. Mapping diversifying residues in these factors onto available 3-dimensional structures revealed the diversifying capsid residues partition to the external surface of the viral particle in statistically significant proximity to antigenic sites. Diversifying pressure in the pleconaril binding site is confined to a single residue known to confer drug resistance (VP1 191). In contrast, diversifying pressure in the non-structural genes is less clear, mapping both nearby and beyond characterized functional domains of these factors. CONCLUSION: This work provides a foundation for understanding HRV genetic diversity and insight into the underlying biology driving evolution in HRV. It expands our knowledge of the genome sequence space that HRV reference serotypes occupy and how the pattern of genetic diversity across HRV genomes differs from other picornaviruses. It also reveals evidence of diversifying selective pressure in both structural genes known to interact with the host immune system and in domains of unassigned function in the non-structural 3C and 3D genes, raising the possibility that diversification of undiscovered functions in these essential factors may influence HRV fitness and evolution

Rapid DNA mapping by fluorescent single molecule detection

DNA mapping is an important analytical tool in genomic sequencing, medical diagnostics and pathogen identification. Here we report an optical DNA mapping strategy based on direct imaging of individual DNA molecules and localization of multiple sequence motifs on the molecules. Individual genomic DNA molecules were labeled with fluorescent dyes at specific sequence motifs by the action of nicking endonuclease followed by the incorporation of dye terminators with DNA polymerase. The labeled DNA molecules were then stretched into linear form on a modified glass surface and imaged using total internal reflection fluorescence (TIRF) microscopy. By determining the positions of the fluorescent labels with respect to the DNA backbone, the distribution of the sequence motif recognized by the nicking endonuclease can be established with good accuracy, in a manner similar to reading a barcode. With this approach, we constructed a specific sequence motif map of lambda-DNA. We further demonstrated the capability of this approach to rapidly type a human adenovirus and several strains of human rhinovirus

Multiplex primer prediction software for divergent targets

We describe a Multiplex Primer Prediction (MPP) algorithm to build multiplex compatible primer sets to amplify all members of large, diverse and unalignable sets of target sequences. The MPP algorithm is scalable to larger target sets than other available software, and it does not require a multiple sequence alignment. We applied it to questions in viral detection, and demonstrated that there are no universally conserved priming sequences among viruses and that it could require an unfeasibly large number of primers (∼3700 18-mers or ∼2000 10-mers) to generate amplicons from all sequenced viruses. We then designed primer sets separately for each viral family, and for several diverse species such as foot-and-mouth disease virus (FMDV), hemagglutinin (HA) and neuraminidase (NA) segments of influenza A virus, Norwalk virus, and HIV-1. We empirically demonstrated the application of the software with a multiplex set of 16 short (10 nt) primers designed to amplify the Poxviridae family to produce a specific amplicon from vaccinia virus