Omar J. Jabado

Panmicrobial Oligonucleotide Array for Diagnosis of Infectious Diseases

To facilitate rapid, unbiased, differential diagnosis of infectious diseases, we designed GreeneChipPm, a panmicrobial microarray comprising 29,455 sixty-mer oligonucleotide probes for vertebrate viruses, bacteria, fungi, and parasites. Methods for nucleic acid preparation, random primed PCR amplification, and labeling were optimized to allow the sensitivity required for application with nucleic acid extracted from clinical materials and cultured isolates. Analysis of nasopharyngeal aspirates, blood, urine, and tissue from persons with various infectious diseases confirmed the presence of viruses and bacteria identified by other methods, and implicated Plasmodium falciparum in an unexplained fatal case of hemorrhagic feverlike disease during the Marburg hemorrhagic fever outbreak in Angola in 2004–2005.

Impaired carbohydrate digestion and transport and mucosal dysbiosis in the intestines of children with autism and gastrointestinal disturbances.

Gastrointestinal disturbances are commonly reported in children with autism, complicate clinical management, and may contribute to behavioral impairment. Reports of deficiencies in disaccharidase enzymatic activity and of beneficial responses to probiotic and dietary therapies led us to survey gene expression and the mucoepithelial microbiota in intestinal biopsies from children with autism and gastrointestinal disease and children with gastrointestinal disease alone. Ileal transcripts encoding disaccharidases and hexose transporters were deficient in children with autism, indicating impairment of the primary pathway for carbohydrate digestion and transport in enterocytes. Deficient expression of these enzymes and transporters was associated with expression of the intestinal transcription factor, CDX2. Metagenomic analysis of intestinal bacteria revealed compositional dysbiosis manifest as decreases in Bacteroidetes, increases in the ratio of Firmicutes to Bacteroidetes, and increases in Betaproteobacteria. Expression levels of disaccharidases and transporters were associated with the abundance of affected bacterial phylotypes. These results indicate a relationship between human intestinal gene expression and bacterial community structure and may provide insights into the pathophysiology of gastrointestinal disturbances in children with autism.

Genome-wide mapping of methylated adenine residues in pathogenic Escherichia coli using single-molecule real-time sequencing

Single-molecule real-time (SMRT) DNA sequencing allows the systematic detection of chemical modifications such as methylation but has not previously been applied on a genome-wide scale. We used this approach to detect 49,311 putative 6-methyladenine (m6A) residues and 1,407 putative 5-methylcytosine (m5C) residues in the genome of a pathogenic Escherichia coli strain. We obtained strand-specific information for methylation sites and a quantitative assessment of the frequency of methylation at each modified position. We deduced the sequence motifs recognized by the methyltransferase enzymes present in this strain without prior knowledge of their specificity. Furthermore, we found that deletion of a phage-encoded methyltransferase-endonuclease (restriction-modification; RM) system induced global transcriptional changes and led to gene amplification, suggesting that the role of RM systems extends beyond protecting host genomes from foreign DNA.

Identification and remediation of biases in the activity of RNA ligases in small-RNA deep sequencing

Deep sequencing of small RNAs (sRNA-seq) is now the gold standard for small RNA profiling and discovery. Biases in sRNA-seq have been reported, but their etiology remains unidentified. Through a comprehensive series of sRNA-seq experiments, we establish that the predominant cause of the bias is the RNA ligases. We further demonstrate that RNA ligases have strong sequence-specific biases which distort the small RNA profiles considerably. We have devised a pooled adapter strategy to overcome this bias, and validated the method through data derived from microarray and qPCR. In light of our findings, published small RNA profiles, as well as barcoding strategies using adapter-end modifications, may need to be revisited. Importantly, by providing a wide spectrum of substrate for the ligase, the pooled-adapter strategy developed here provides a means to overcome issues of bias, and generate more accurate small RNA profiles.

Diagnostic system for rapid and sensitive differential detection of pathogens

Naturally emerging and deliberately released pathogens demand new detection strategies to allow early recognition and containment. We describe a diagnostic system for rapid, sensitive, multiplex discrimination of microbial gene sequences and report its application for detecting 22 respiratory pathogens in clinical samples.

Middle East Respiratory Syndrome Coronavirus Quasispecies That Include Homologues of Human Isolates Revealed through Whole-Genome Analysis and Virus Cultured from Dromedary Camels in Saudi Arabia

ABSTRACT Complete Middle East respiratory syndrome coronavirus (MERS-CoV) genome sequences were obtained from nasal swabs of dromedary camels sampled in the Kingdom of Saudi Arabia through direct analysis of nucleic acid extracts or following virus isolation in cell culture. Consensus dromedary MERS-CoV genome sequences were the same with either template source and identical to published human MERS-CoV sequences. However, in contrast to individual human cases, where only clonal genomic sequences are reported, detailed population analyses revealed the presence of more than one genomic variant in individual dromedaries. If humans are truly infected only with clonal virus populations, we must entertain a model for interspecies transmission of MERS-CoV wherein only specific genotypes are capable of passing bottleneck selection. IMPORTANCE In most cases of Middle East respiratory syndrome (MERS), the route for human infection with the causative agent, MERS coronavirus (MERS-CoV), is unknown. Antibodies to and viral nucleic acids of MERS-CoV have been found in dromedaries, suggesting the possibility that they may serve as a reservoir or vector for human infection. However, neither whole viral genomic sequence nor infectious virus has been isolated from dromedaries or other animals in Saudi Arabia. Here, we report recovery of MERS-CoV from nasal swabs of dromedaries, demonstrate that MERS-CoV whole-genome consensus sequences from dromedaries and humans are indistinguishable, and show that dromedaries can be simultaneously infected with more than one MERS-CoV. Together with data indicating widespread dromedary infection in the Kingdom of Saudi Arabia, these findings support the plausibility of a role for dromedaries in human infection. In most cases of Middle East respiratory syndrome (MERS), the route for human infection with the causative agent, MERS coronavirus (MERS-CoV), is unknown. Antibodies to and viral nucleic acids of MERS-CoV have been found in dromedaries, suggesting the possibility that they may serve as a reservoir or vector for human infection. However, neither whole viral genomic sequence nor infectious virus has been isolated from dromedaries or other animals in Saudi Arabia. Here, we report recovery of MERS-CoV from nasal swabs of dromedaries, demonstrate that MERS-CoV whole-genome consensus sequences from dromedaries and humans are indistinguishable, and show that dromedaries can be simultaneously infected with more than one MERS-CoV. Together with data indicating widespread dromedary infection in the Kingdom of Saudi Arabia, these findings support the plausibility of a role for dromedaries in human infection.

Detection of Respiratory Viruses and Subtype Identification of Influenza A Viruses by GreeneChipResp Oligonucleotide Microarray

ABSTRACT Acute respiratory infections are significant causes of morbidity, mortality, and economic burden worldwide. An accurate, early differential diagnosis may alter individual clinical management as well as facilitate the recognition of outbreaks that have implications for public health. Here we report on the establishment and validation of a comprehensive and sensitive microarray system for detection of respiratory viruses and subtyping of influenza viruses in clinical materials. Implementation of a set of influenza virus enrichment primers facilitated subtyping of influenza A viruses through the differential recognition of hemagglutinins 1 through 16 and neuraminidases 1 through 9. Twenty-one different respiratory virus species were accurately characterized, including a recently identified novel genetic clade of rhinovirus.

A Murine Model for Neuropsychiatric Disorders Associated with Group A β-Hemolytic Streptococcal Infection

A syndrome of motoric and neuropsychiatric symptoms comprising various elements, including chorea, hyperactivity, tics, emotional lability, and obsessive-compulsive symptoms, can occur in association with group A β-hemolytic streptococcal (GABHS) infection. We tested the hypothesis that an immune response to GABHS can result in behavioral abnormalities. Female SJL/J mice were immunized and boosted with a GABHS homogenate in Freunds adjuvant, whereas controls received Freunds adjuvant alone. When sera from GABHS-immunized mice were tested for immunoreactivity to mouse brain, a subset was found to be immunoreactive to several brain regions, including deep cerebellar nuclei (DCN), globus pallidus, and thalamus. GABHS-immunized mice having serum immunoreactivity to DCN also had increased IgG deposits in DCN and exhibited increased rearing behavior in open-field and hole-board tests compared with controls and with GABHS-immunized mice lacking serum anti-DCN antibodies. Rearing and ambulatory behavior were correlated with IgG deposits in the DCN and with serum immunoreactivity to GABHS proteins in Western blot. In addition, serum from a GABHS mouse reacted with normal mouse cerebellum in nondenaturing Western blots and immunoprecipitated C4 complement protein and α-2-macroglobulin. These results are consistent with the hypothesis that immune response to GABHS can result in motoric and behavioral disturbances and suggest that anti-GABHS antibodies cross-reactive with brain components may play a role in their pathophysiology.

NS1 Protein Secretion during the Acute Phase of West Nile Virus Infection

ABSTRACT The West Nile virus (WNV) nonstructural protein NS1 is a protein of unknown function that is found within, associated with, and secreted from infected cells. We systematically investigated the kinetics of NS1 secretion in vitro and in vivo to determine the potential use of this protein as a diagnostic marker and to analyze NS1 secretion in relation to the infection cycle. A sensitive antigen capture enzyme-linked immunosorbent assay (ELISA) for detection of WNV NS1 (polyclonal-ACE) was developed, as well as a capture ELISA for the specific detection of NS1 multimers (4G4-ACE). The 4G4-ACE detected native NS1 antigens at high sensitivity, whereas the polyclonal-ACE had a higher specificity for recombinant forms of the protein. Applying these assays we found that only a small fraction of intracellular NS1 is secreted and that secretion of NS1 in tissue culture is delayed compared to the release of virus particles. In experimentally infected hamsters, NS1 was detected in the serum between days 3 and 8 postinfection, peaking on day 5, the day prior to the onset of clinical disease; immunoglobulin M (IgM) antibodies were detected at low levels on day 5 postinfection. Although real-time PCR gave the earliest indication of infection (day 1), the diagnostic performance of the 4G4-ACE was comparable to that of real-time PCR during the time period when NS1 was secreted. Moreover, the 4G4-ACE was found to be superior in performance to both the IgM and plaque assays during this time period, suggesting that NS1 is a viable early diagnostic marker of WNV infection.

Virome Capture Sequencing Enables Sensitive Viral Diagnosis and Comprehensive Virome Analysis

ABSTRACT  Insensitivity and technical complexity have impeded the implementation of high-throughput nucleic acid sequencing in differential diagnosis of viral infections in clinical laboratories. Here, we describe the development of a virome capture sequencing platform for vertebrate viruses (VirCapSeq-VERT) that increases the sensitivity of sequence-based virus detection and characterization. The system uses ~2 million probes that cover the genomes of members of the 207 viral taxa known to infect vertebrates, including humans. A biotinylated oligonucleotide library was synthesized on the NimbleGen cleavable array platform and used for solution-based capture of viral nucleic acids present in complex samples containing variable proportions of viral and host nucleic acids. The use of VirCapSeq-VERT resulted in a 100- to 10,000-fold increase in viral reads from blood and tissue homogenates compared to conventional Illumina sequencing using established virus enrichment procedures, including filtration, nuclease treatments, and RiboZero rRNA subtraction. VirCapSeq-VERT had a limit of detection comparable to that of agent-specific real-time PCR in serum, blood, and tissue extracts. Furthermore, the method identified novel viruses whose genomes were approximately 40% different from the known virus genomes used for designing the probe library. The VirCapSeq-VERT platform is ideally suited for analyses of virome composition and dynamics. IMPORTANCE VirCapSeq-VERT enables detection of viral sequences in complex sample backgrounds, including those found in clinical specimens, such as serum, blood, and tissue. The highly multiplexed nature of the system allows both the simultaneous identification and the comprehensive genetic characterization of all known vertebrate viruses, their genetic variants, and novel viruses. The operational simplicity and efficiency of the VirCapSeq-VERT platform may facilitate transition of high-throughput sequencing to clinical diagnostic as well as research applications. VirCapSeq-VERT enables detection of viral sequences in complex sample backgrounds, including those found in clinical specimens, such as serum, blood, and tissue. The highly multiplexed nature of the system allows both the simultaneous identification and the comprehensive genetic characterization of all known vertebrate viruses, their genetic variants, and novel viruses. The operational simplicity and efficiency of the VirCapSeq-VERT platform may facilitate transition of high-throughput sequencing to clinical diagnostic as well as research applications.