Blood Systems Research Institute, San Francisco, California
Department of Laboratory Medicine, University of California, San Francisco, California
Discovery of novel viral genomes has been a challenge due to the difficulty to culture the virus or perform PCR on highly divergent genomes. To circumvent these difficulties, metagenomic approach combining virus purification and high-throughput sequencing can provide a powerful tool for the discovery of previously unknown viral pathogens. We performed several high-throughput metagenomic studies on viral particles purified from human, animal and sewage samples, resulting in the discoveries of several novel viral species, as well as prototypes of novel viral genera/families.
Viral particles were enriched from the samples, and the viral nucleic acids were randomly amplified and sequenced with “next generation” 454 and Solexa sequencing (1-3). More than half a million reads were generated for each sample, which were analyzed using BLASTx to reveal known and novel viral sequences. For novel viral sequences, PCR and RACE amplifications were performed to obtain complete viral genomes, as well as investigating prevalence, geography and tissue tropism of the novel viruses.
High-throughput sequencing of human, animal and sewage samples reveals numerous highly divergent viruses. Besides identifying known human and animal pathogens, several novel viruses were discovered. Novel genomes belonging to enteric viruses such as kobuvirus, salivirus and sapovirus were characterized in human sewage. In addition, a novel astrovirus phylogenetically basal to those infecting mammals and birds was identified in sewage, potentially representing a novel astrovirus genus. A highly divergent virus with phylogenetic position near the root of the picornavirus-like superfamily was identified, which is tentatively named Picalivirus and potentially representing a novel viral family.
Several deeply branched ssRNA virus genomes were characterized, improving our understanding of emerging viruses affecting humans and animals, as well as expanding the field of virus taxonomy. Our results demonstrated the usefulness of high throughput sequencing in monitoring emerging viruses from a large cohort of human and animal samples.
1. Kapoor, A., J. Victoria, P. Simmonds, E. Slikas, T. Chieochansin, A. Naeem, S. Shaukat, S. Sharif, M. M. Alam, M. Angez, C. L. Wang, R. W. Shafer, S. Zaidi, and E. Delwart. 2008. A highly prevalent and genetically diversified Picornaviridae genus in South Asian children. Proceedings of the National Academy of Sciences of the United States of America 105:20482-20487.
2. Ng, T. F. F., C. Manire, K. Borrowman, T. Langer, L. Ehrhart, and M. Breitbart. 2009. Discovery of a novel single-stranded DNA virus from a sea turtle fibropapilloma by using viral metagenomics. Journal of Virology 83:2500-2509.
3. Ng, T. F. F., D. L. Willner, Y. W. Lim, R. Schmieder, B. Chau, C. Nilsson, S. Anthony, Y. Ruan, F. Rohwer, and M. Breitbart. 2011. Broad surveys of DNA viral diversity obtained through viral metagenomics of mosquitoes. PLoS ONE 6:e20579.
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