Hill, Jack Douglas
(2024)
A three-pronged approach to virus discovery.
PhD thesis, University of Nottingham.
Abstract
Rodents and bats are two key groups of host species for viral diversity and zoonotic transmission to humans. Whilst the use of NGS and PCR based virus discovery is improving our collective knowledge of the virome, very little is known about the virome overall, and even less is known about the specific virome of these mammalian hosts. Similarly, information is lacking regarding the prevalence of viruses within UK rodents. As climate change progresses and rodents are driven into closer proximity to humans and livestock, the risk of rodent zoonotic transmission is increasing, increasing the risk of transmission of viruses with pandemic potential. Evolutionary information about key viruses may help to improve pandemic preparedness, but the field of historic virology is still relatively underdeveloped, particularly regarding historic RNA virus investigations. Therefore, this project was designed to take a three-pronged approach to investigating virus diversity within UK rodents via degenerate PCR and NGS screening, and to advance the field of historic RNA virus discovery.
140 modern rodents from 5 species were screened by degenerate PCR for the presence of adenoviruses, hantaviruses, coronaviruses, Rotaviruses and Rubiviruses. 2 adenoviruses and 1 hantavirus were identified, and all other viruses were not found in. This screening was complemented by unbiased NGS sequencing of these rodents and analysis of the NGS data. The metagenomics screening yielded a total of 216 viral hits across 19 viral genera, including potentially important viruses such as Cardioviruses, Hepaciviruses and hantaviruses, amongst others. These viruses were PCR confirmed, and expanded the known viral diversity of bank voles, field voles, wood mice and yellow-necked mice to include Picobirnaviruses, Kunsaigiviruses, Rosaviruse, Pegiviruses, Bocaparvoviruses and polyomaviruses. Hepacivirus F, Rosaviruses and Orbiviruses were also found in the UK for the first time, and accurate abundance estimates for 19 viral genera were quantified, ranging from 0.7%-67.1%. Collectively, this drastically improves our collective knowledge of the UK virome.
This project also advanced the techniques used for historic RNA virus discovery and manipulation. A protocol for historic RNA extraction from preserved animals ranging from at least 35-156 years old was developed with over 90% extraction success. NGS library preparation processes were improved to yielded functional NGS libraries, albeit with substantial adaptor dimer contamination. qPCR screening methods for historic coronaviruses were also developed, and historic coronaviruses were found in 3 samples. Collectively, this advances the methodology and techniques of historic RNA virus discovery, and shows as a proof of concept that conventional screening methods can be used for the discovery of historic viruses in well preserved samples.
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