Viral Discovery and Sequence Recovery Using DNA Microarrays

TLDR

Emerging infectious diseases pose a constant threat, and existing pathogen‑identification methods are limited, creating a demand for new viral discovery technologies. The authors present a DNA microarray platform designed to identify and characterize novel viruses. The platform employs a 70mer microarray containing conserved sequences from all fully sequenced viral genomes to hybridize viral RNA, then clones ~1 kb of recovered sequences from array elements for further analysis. During the 2003 SARS outbreak, the array detected an uncharacterized coronavirus, sequencing confirmed it as a new coronavirus, and the approach proved effective for rapid identification of emerging viruses.

Abstract

Because of the constant threat posed by emerging infectious diseases and the limitations of existing approaches used to identify new pathogens, there is a great demand for new technological methods for viral discovery. We describe herein a DNA microarray-based platform for novel virus identification and characterization. Central to this approach was a DNA microarray designed to detect a wide range of known viruses as well as novel members of existing viral families; this microarray contained the most highly conserved 70mer sequences from every fully sequenced reference viral genome in GenBank. During an outbreak of severe acute respiratory syndrome (SARS) in March 2003, hybridization to this microarray revealed the presence of a previously uncharacterized coronavirus in a viral isolate cultivated from a SARS patient. To further characterize this new virus, approximately 1 kb of the unknown virus genome was cloned by physically recovering viral sequences hybridized to individual array elements. Sequencing of these fragments confirmed that the virus was indeed a new member of the coronavirus family. This combination of array hybridization followed by direct viral sequence recovery should prove to be a general strategy for the rapid identification and characterization of novel viruses and emerging infectious disease.