Graduation Year

2010

Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Marine Science

Major Professor

Mya Breitbart

Co-Major Professor

John Paul

Committee Member

David Mann

Committee Member

John Cannon

Committee Member

James Casey

Keywords

Animal Viruses, Plant Viruses, Insect Vectors, Metagenomics, Sequencing

Abstract

Understanding emerging viruses is critical for disease monitoring and prediction; however, surveys of novel viruses are hindered by the lack of a universal assay for viruses. Viral metagenomics, consisting of viral particle purification and shotgun sequencing, is a powerful technique for discovering viruses in a wide variety of sample types. However, current protocols are not effective on tissue samples (e.g., lungs, livers and tumors), where they are hindered by the high amount of host nucleic acids which limits the percentage of sequences that originate from viruses. In this dissertation, a modified viral metagenomics protocol was developed and utilized to effectively purify viruses from tissues, enabling the sequencing of novel viruses from animals, plants, and insect vectors.

Viral metagenomics performed directly on tissue samples enabled the discovery of novel vertebrate, plant, insect and bacterial viruses. From a sea turtle fibropapilloma, viral metagenomics revealed a novel tornovirus STTV1, which is only the second single-stranded DNA virus known in reptiles and is extremely different from any previously described viruses. Similarly, from the lung of a sea lion involved in a mortality event, viral metagenomics identified a novel sea lion anellovirus (ZcAV), which is the first anellovirus characterized from a marine animal. The STTV1 and ZcAV genomes were highly divergent from known viruses, to a degree that they could not have been detected by degenerate PCR assays or microarrays, demonstrating viral metagenomics as an effective method for characterizing novel viruses.

In addition to discovery of viruses in individual diseased animals, this dissertation pioneered a technique called vector-enabled metagenomics (VEM) to examine viruses present in insect vectors. VEM combines the power of metagenomics to sequence novel viruses with the ability of insect vectors to integrate viral diversity over space, time, and many host individuals and species. VEM allows for the investigation of viral diversity among the broad range of hosts that the insects feed on, providing an unprecedented snapshot of the viral diversity in natural reservoirs. This dissertation describes the first viral metagenome performed on mosquitoes and whiteflies, providing significant insights to the viral diversity in animal and plant reservoirs. Both animal and plant viruses were represented in the mosquito viromes, which likely originate from animal blood and plant nectar that the mosquitoes feed on. Mosquito viromes contained a diverse range of viruses, including vertebrate, insect, plant, and bacterial viruses, and almost all the viral sequences were novel, suggesting the pan-animal virome is largely uncharacterized. In contrast, only plant viruses were observed in the whitefly viromes because whiteflies feed solely on plants. Whitefly viromes contained known and novel viral sequences infecting crops, novel viral sequences infecting native plants, as well as novel satellites that were the first viral satellites to be documented in North America. Distinct viromes were found amongst the three mosquito samples as well as between the two whitefly samples, demonstrating the diverse and dynamic nature of the viruses in plant and animal reservoirs.

By enabling the discovery of virus in diseased organisms and in insect vectors, viral metagenomics is a powerful technique that will significantly enhance our fundamental scientific understanding of the diversity, transmission, biogeography, and emergence of viruses. The viral metagenomic approach described here has implications for surveillance of emerging viruses, prediction of viral epidemics, and proactive control of diseases.

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