Clark Tibbetts

Testing and Validation of High Density Resequencing Microarray for Broad Range Biothreat Agents Detection

Rapid and effective detection and identification of emerging microbiological threats and potential biowarfare agents is very challenging when using traditional culture-based methods. Contemporary molecular techniques, relying upon reverse transcription and/or polymerase chain reaction (RT-PCR/PCR) provide a rapid and effective alternative, however, such assays are generally designed and optimized to detect only a limited number of targets, and seldom are capable of differentiation among variants of detected targets. To meet these challenges, we have designed a broad-range resequencing pathogen microarray (RPM) for detection of tropical and emerging infectious agents (TEI) including biothreat agents: RPM-TEI v 1.0 (RPM-TEI). The scope of the RPM-TEI assay enables detection and differential identification of 84 types of pathogens and 13 toxin genes, including most of the class A, B and C select agents as defined by the Centers for Disease Control and Prevention (CDC, Atlanta, GA). Due to the high risks associated with handling these particular target pathogens, the sensitivity validation of the RPM-TEI has been performed using an innovative approach, in which synthetic DNA fragments are used as templates for testing the assays limit of detection (LOD). Assay specificity and sensitivity was subsequently confirmed by testing with full-length genomic nucleic acids of selected agents. The LOD for a majority of the agents detected by RPM-TEI was determined to be at least 104 copies per test. Our results also show that the RPM-TEI assay not only detects and identifies agents, but is also able to differentiate near neighbors of the same agent types, such as closely related strains of filoviruses of the Ebola Zaire group, or the Machupo and Lassa arenaviruses. Furthermore, each RPM-TEI assay results in specimen-specific agent gene sequence information that can be used to assess pathogenicity, mutations, and virulence markers, results that are not generally available from multiplexed RT-PCR/PCR-based detection assays.

Genomic and Bioinformatics Analysis of HAdV-4, a Human Adenovirus Causing Acute Respiratory Disease: Implications for Gene Therapy and Vaccine Vector Development

ABSTRACT Human adenovirus serotype 4 (HAdV-4) is a reemerging viral pathogenic agent implicated in epidemic outbreaks of acute respiratory disease (ARD). This report presents a genomic and bioinformatics analysis of the prototype 35,990-nucleotide genome (GenBank accession no. AY594253 ). Intriguingly, the genome analysis suggests a closer phylogenetic relationship with the chimpanzee adenoviruses (simian adenoviruses) rather than with other human adenoviruses, suggesting a recent origin of HAdV-4, and therefore species E, through a zoonotic event from chimpanzees to humans. Bioinformatics analysis also suggests a pre-zoonotic recombination event, as well, between species B-like and species C-like simian adenoviruses. These observations may have implications for the current interest in using chimpanzee adenoviruses in the development of vectors for human gene therapy and for DNA-based vaccines. Also, the reemergence, surveillance, and treatment of HAdV-4 as an ARD pathogen is an opportunity to demonstrate the use of genome determination as a tool for viral infectious disease characterization and epidemic outbreak surveillance: for example, rapid and accurate low-pass sequencing and analysis of the genome. In particular, this approach allows the rapid identification and development of unique probes for the differentiation of family, species, serotype, and strain (e.g., pathogen genome signatures) for monitoring epidemic outbreaks of ARD.

Single Assay for Simultaneous Detection and Differential Identification of Human and Avian Influenza Virus Types, Subtypes, and Emergent Variants

For more than four decades the cause of most type A influenza virus infections of humans has been attributed to only two viral subtypes, A/H1N1 or A/H3N2. In contrast, avian and other vertebrate species are a reservoir of type A influenza virus genome diversity, hosting strains representing at least 120 of 144 combinations of 16 viral hemagglutinin and 9 viral neuraminidase subtypes. Viral genome segment reassortments and mutations emerging within this reservoir may spawn new influenza virus strains as imminent epidemic or pandemic threats to human health and poultry production. Traditional methods to detect and differentiate influenza virus subtypes are either time-consuming and labor-intensive (culture-based) or remarkably insensitive (antibody-based). Molecular diagnostic assays based upon reverse transcriptase-polymerase chain reaction (RT-PCR) have short assay cycle time, and high analytical sensitivity and specificity. However, none of these diagnostic tests determine viral gene nucleotide sequences to distinguish strains and variants of a detected pathogen from one specimen to the next. Decision-quality, strain- and variant-specific pathogen gene sequence information may be critical for public health, infection control, surveillance, epidemiology, or medical/veterinary treatment planning. The Resequencing Pathogen Microarray (RPM-Flu) is a robust, highly multiplexed and target gene sequencing-based alternative to both traditional culture- or biomarker-based diagnostic tests. RPM-Flu is a single, simultaneous differential diagnostic assay for all subtype combinations of type A influenza viruses and for 30 other viral and bacterial pathogens that may cause influenza-like illness. These other pathogen targets of RPM-Flu may co-infect and compound the morbidity and/or mortality of patients with influenza. The informative specificity of a single RPM-Flu test represents specimen-specific viral gene sequences as determinants of virus type, A/HN subtype, virulence, host-range, and resistance to antiviral agents.

Universal Detection and Identification of Avian Influenza Virus by Use of Resequencing Microarrays

ABSTRACT Zoonotic microbes have historically been, and continue to emerge as, threats to human health. The recent outbreaks of highly pathogenic avian influenza virus in bird populations and the appearance of some human infections have increased the concern of a possible new influenza pandemic, which highlights the need for broad-spectrum detection methods for rapidly identifying the spread or outbreak of all variants of avian influenza virus. In this study, we demonstrate that high-density resequencing pathogen microarrays (RPM) can be such a tool. The results from 37 influenza virus isolates show that the RPM platform is an effective means for detecting and subtyping influenza virus, while simultaneously providing sequence information for strain resolution, pathogenicity, and drug resistance without additional analysis. This study establishes that the RPM platform is a broad-spectrum pathogen detection and surveillance tool for monitoring the circulation of prevalent influenza viruses in the poultry industry and in wild birds or incidental exposures and infections in humans.

Broad Spectrum Respiratory Pathogen Analysis of Throat Swabs from Military Recruits Reveals Interference Between Rhinoviruses and Adenoviruses

Military recruits experience a high incidence of febrile respiratory illness (FRI), leading to significant morbidity and lost training time. Adenoviruses, group A Streptococcus pyogenes, and influenza virus are implicated in over half of the FRI cases reported at recruit training center clinics, while the etiology of the remaining cases is unclear. In this study, we explore the carriage rates and disease associations of adenovirus, enterovirus, rhinovirus, Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria meningitidis in military recruits using high-density resequencing microarrays. The results showed that rhinoviruses, adenoviruses, S. pneumoniae, H. influenzae, and N. meningitidis were widely distributed in recruits. Of these five agents, only adenovirus showed significant correlation with illness. Among the samples tested, only pathogens associated with FRI, such as adenovirus 4 and enterovirus 68, revealed strong temporal and spatial clustering of specific strains, indicating that they are transmitted primarily within sites. The results showed a strong negative association between adenoviral FRI and the presence of rhinoviruses in recruits, suggesting some form of viral interference.

Resequencing microarray probe design for typing genetically diverse viruses: human rhinoviruses and enteroviruses

BackgroundFebrile respiratory illness (FRI) has a high impact on public health and global economics and poses a difficult challenge for differential diagnosis. A particular issue is the detection of genetically diverse pathogens, i.e. human rhinoviruses (HRV) and enteroviruses (HEV) which are frequent causes of FRI. Resequencing Pathogen Microarray technology has demonstrated potential for differential diagnosis of several respiratory pathogens simultaneously, but a high confidence design method to select probes for genetically diverse viruses is lacking.ResultsUsing HRV and HEV as test cases, we assess a general design strategy for detecting and serotyping genetically diverse viruses. A minimal number of probe sequences (26 for HRV and 13 for HEV), which were potentially capable of detecting all serotypes of HRV and HEV, were determined and implemented on the Resequencing Pathogen Microarray RPM-Flu v.30/31 (Tessarae RPM-Flu). The specificities of designed probes were validated using 34 HRV and 28 HEV strains. All strains were successfully detected and identified at least to species level. 33 HRV strains and 16 HEV strains could be further differentiated to serotype level.ConclusionThis study provides a fundamental evaluation of simultaneous detection and differential identification of genetically diverse RNA viruses with a minimal number of prototype sequences. The results demonstrated that the newly designed RPM-Flu v.30/31 can provide comprehensive and specific analysis of HRV and HEV samples which implicates that this design strategy will be applicable for other genetically diverse viruses.

Genomic and Bioinformatics Analyses of HAdV-4vac and HAdV-7vac, Two Human Adenovirus (HAdV) Strains That Constituted Original Prophylaxis against HAdV-Related Acute Respiratory Disease, a Reemerging Epidemic Disease

ABSTRACT Vaccine strains of human adenovirus serotypes 4 and 7 (HAdV-4vac and HAdV-7vac) have been used successfully to prevent adenovirus-related acute respiratory disease outbreaks. The genomes of these two vaccine strains have been sequenced, annotated, and compared with their prototype equivalents with the goals of understanding their genomes for molecular diagnostics applications, vaccine redevelopment, and HAdV pathoepidemiology. These reference genomes are archived in GenBank as HAdV-4vac (35,994 bp; AY594254 ) and HAdV-7vac (35,240 bp; AY594256). Bioinformatics and comparative whole-genome analyses with their recently reported and archived prototype genomes reveal six mismatches and four insertions-deletions (indels) between the HAdV-4 prototype and vaccine strains, in contrast to the 611 mismatches and 130 indels between the HAdV-7 prototype and vaccine strains. Annotation reveals that the HAdV-4vac and HAdV-7vac genomes contain 51 and 50 coding units, respectively. Neither vaccine strain appears to be attenuated for virulence based on bioinformatics analyses. There is evidence of genome recombination, as the inverted terminal repeat of HAdV-4vac is initially identical to that of species C whereas the prototype is identical to species B1. These vaccine reference sequences yield unique genome signatures for molecular diagnostics. As a molecular forensics application, these references identify the circulating and problematic 1950s era field strains as the original HAdV-4 prototype and the Greider prototype, from which the vaccines are derived. Thus, they are useful for genomic comparisons to current epidemic and reemerging field strains, as well as leading to an understanding of pathoepidemiology among the human adenoviruses.

Polar encapsidation of adenovirus DNA: Evolutionary variants reveal dispensable sequences near the left ends of Ad3 genomes

Repeated passage of adenovirus type 3 in HeLa cells has led to a novel stock of variant genomes, characterized by deletions and substitutions of DNA sequences within the left-end 750 base pairs. This heterogeneous stock retains few if any parental genomes--the majority of variants appear viable. Analysis of viable variants with deleted sequences reveals the 182 nucleotides proximal to the left-end inverted terminal repeat (136-318 bp) are not required for Ad3 infectivity in cultured human cell lines nor for maintenance of viral DNA encapsidation polarity.

Natural variants of human adenovirus type 3 provide evidence for relative genome stability across time and geographic space

Human adenovirus type 3 (HAdV-B3) has an apparently stable genome yet remains a major circulating and problematic respiratory pathogen. Comparisons of the prototype genome to genomes from three current field strains, including two isolated from epidemics, and a laboratory strain, yielded small-scale nucleotide variations across 50 years of time and space (U.S. and China). This is in contrast to the recombination events that have been reported recently for HAdV genomes. Recombinant genomes have been identified in emergent HAdV pathogens and is a pathway for the molecular evolution of types. These two contrasting views of HAdV genome stability have repercussions in the development and use of vaccines for countering HAdV-B3, as well as in the continued effectiveness of vaccines developed against earlier and current circulating types of HAdV.

Genomic and bioinformatics analyses of HAdV-14p, reference strain of a re-emerging respiratory pathogen and analysis of B1/B2

Unlike other human adenovirus (HAdV) species, B is divided into subspecies B1 and B2. Originally this was partly based on restriction enzyme (RE) analysis. B1 members, except HAdV-50, are commonly associated with respiratory diseases while B2 members are rarely associated with reported respiratory diseases. Recently two members of B2 have been identified in outbreaks of acute respiratory disease (ARD). One, HAdV-14, has re-emerged after an apparent 52-year absence. Genomic analysis and bioinformatics data are reported for HAdV-14 prototype for use as a reference and to understand and counter its re-emergence. The data complement and extend the original criteria for subspecies designation, unique amongst the adenoviruses, and highlight differences between B1 and B2, representing the first comprehensive analysis of this division. These data also provide finer granularity into the pathoepidemiology of the HAdVs. Whole genome analysis uncovers heterogeneous identity structures of the hexon and fiber genes amongst the HAdV-14 and the B1/B2 subspecies, which may be important in prescient vaccine development. Analysis of cell surface proteins provides insight into HAdV-14 tropism, accounting for its role as a respiratory pathogen. This HAdV-14 prototype genome is also a reference for applications of B2 adenoviruses as vectors for vaccine development and gene therapy.