W. Ian Lipkin, M.D.
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 discovery.

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).

Contact Dr. Lipkin.