Off-campus UMass Amherst users: To download campus access dissertations, please use the following link to log into our proxy server with your UMass Amherst user name and password.

Non-UMass Amherst users: Please talk to your librarian about requesting this dissertation through interlibrary loan.

Dissertations that have an embargo placed on them will not be available to anyone until the embargo expires.

ORCID

https://orcid.org/0000-0003-3085-5993

Access Type

Open Access Thesis

Document Type

thesis

Degree Program

Molecular & Cellular Biology

Degree Type

Master of Science (M.S.)

Year Degree Awarded

2023

Month Degree Awarded

September

Abstract

Viruses hold our attention for the horrific impact they have on human health and welfare. However, viruses are a critical part of our ecosystem and facilitate the cycling of carbon and other important nutrients. The cycle of virus infection, followed by host resistance and the subsequent evolution of new strains enables adaptation to changing hosts and the environment. Giant viruses, some with particle sizes large enough to be visible in light microscopes and their bewildering array of accessory genes, have captivated scientists and the general public since their discovery two decades ago. Giant viruses are part of the Nucleocytoviricota (NCV) whose members include both harmful agents (such as the causative agents of smallpox and swine hemorrhagic fever) and beneficial ones (such as those that provide biocontrol of insects, mitigation of toxic algal blooms and enzymes for biotechnology). Most of the giant viruses discovered, to date, are from marine and freshwater ecosystems where their hosts are abundant. In terrestrial soils, very few giant viruses have been revealed because of challenges in shifting through the astounding microbial genetic diversity in soil to assemble genomes from metagenomic data. Currently there is a lack of knowledge about abundance and genetic diversity of giant viruses in terrestrial soils, knowledge about their hosts and their influence on biogeochemical cycling.

In 2018, giant viruses were discovered in the Barre Woods experimental warming plots at Harvard Forest. (Schulz et al 2018 Nature Communications). A novel environmental genomics approach involving filtration and fluorescence activating cell-sorting (FACS) was used to discover 16 Nucleocytoviricota (NCVs) in just a few grams of Harvard Forest soil. All these newly discovered viruses represent distinct lineages (new species, genera, and families). This experiment involved just two soil cores (1 warming and 1 control) and a single time point at Harvard Forest. There is much to learn about the terrestrial giant virus genetic biodiversity as these same viruses have not yet been discovered at other sites around the world. My research will focus on a genus of giant viruses with only three known representatives, all from Harvard Forest. They are Hyperionvirus (with the world’s 2nd largest virus genome at 2.4 MBp), Terrestrivrus the 10 th largest genome at 1.8 MBp), and Harvfovirus (the 15 th largest genome at 1.6 MBp).

In the experimental warming plots the relative amount of bacteria to fungi has increased. We hypothesize that the relative increase in bacteria has led to an increase in protists, which feed on the bacteria, which in turn has led to an increase in giant viruses, which infect the protists. Because of the high genetic diversity in viruses and the lack of ribosomal genes, it is not possible to create primers that span the entire Nucleocytoviricota phylum or even at the family level. To test our hypothesis, we designed degenerate PCR primers that detect and quantify members of the genus containing the 3 giant Harvard Forest viruses. DNA was extracted from soil samples the soil (stored at -80C) from the 2017 temperature toggle experiment at Barre Woods in which the power to the warmed plots was turned off from late May until early September were used. The giant viruses were originally discovered in the sample just prior to turning off the power. We used 4 time points spanning the experiment with 8 samples from each the warmed and control plots (4 x 16 = 16 samples total). The primers were designed based on five hallmark genes that are present in most members of the Nucleocytoviricota. After amplification, the amount of DNA would be quantified and normalized. We expect to better understand the genetic diversity of this genus of giant viruses in the soil including the possibility of detecting new species in this genus.

DOI

https://doi.org/10.7275/35937575.0

First Advisor

Jeffrey Blanchard

Second Advisor

Mandy Muller

Third Advisor

Yasu Morita

Download

Share

COinS