University of California, Irvine
Pandora’s Box Project.
The goal of this project is to establish rapid, sensitive methods for virus detection,
and apply them in global networks for infectious disease surveillance and pathogen
Genome projects and high throughput methods for profiling gene statement using
cDNAs and oligonucleotides have revolutionized biology by providing tools for
simultaneous assessment of thousands of nucleic acid sequences. Although these
methods have been extended to analysis of polymorphisms and gene statement
patterns in single viral systems (for example, HIV, CMV), they have not been
employed to define complex viral flora or to implicate specific viruses in disease.
There are still no high throughput molecular systems with which to pursue viral
surveillance. Furthermore, for many viral taxa, we lack the information required to
define broad hybridization targets that will comprise the foundation of such
systems. The Pandora’s Box Project will address these needs by expanding viral
sequence databases and designing microarray, flow cytometric and multiplex PCR
assays for rapid molecular detection and quantitation of known, novel, and
bioengineered viral pathogens.
Methods for cloning nucleic acids of microbial pathogens directly from clinical
specimens offer new opportunities to investigate microbial associations in diseases
predicted to have an infectious basis. The power of these methods is that they can
succeed where methods for pathogen identification through serology or cultivation
may fail due to absence of specific reagents or fastidious requirements for agent
replication. Over the past decade the application of molecular pathogen discovery
methods resulted in identification of novel agents including Borna disease virus,
Hepatitis C virus, Sin Nombre virus, HHV-8, Bartonella henselae, and
Tropherema whippeli, and facilitated implication of Nipah virus and West Nile
virus as significant causes of human morbidity and mortality.
To establish and implement nucleic acid-based methods for rapid, high throughput
virus identification, characterization, and quantitation we will: (1) generate software
programs for automated retrireview and curation of nucleic acid sequence data for
individual viral families from public and proprietary databases; (2) design and
implement algorithms with which to identify conserved nucleic acid sequences in
viral taxa; (3) create virtual libraries of viral sequences; (4) create plasmid libraries
of viral nucleic acids representing potential hybridization targets; (5) establish viral
DNA microarrays and flow cytometric bead-based assays; and (6) establish
PCR-based assays for identification and quantitation of viral nucleic acid targets.
The initial application of this technology will be to investigation of encephalitis,
where despite use of a wide range of diagnostic tools (culture, serology, PCR), an
infectious agent is identified in less than 40% of cases. Once proof of principle is
established, we will be positioned to secure support to distribute reagents and
assays to an international network focused on global public health and microbial
ecology. High throughput methods will also facilitate investigation of chronic
disorders (neurodegenerative, neurodevelopmental, immune, and neoplastic)
where infectious agents may be primary or co-factors. Here we will examine not
only tissues and fluids collected after onset of disease, but also samples collected
prospectively during gestation and childhood (two birth cohorts, approx. 100,000
individuals each). Such studies could elucidate relationships between timing of
exposure to microbes and statement of specific diseases (e.g., diabetes mellitus,
multiple sclerosis, neuropsychiatric disorders).