UvA-DARE (Digital Academic Repository) Computational discovery of viruses and their hosts Kinsella, C. Publication date 2023 Document Version Final published version Link to publication Citation for published version (APA): Kinsella, C. Computational discovery of viruses and their hosts. General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons).
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UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.nl) Download date:31 Aug 2023 Computational discovery of viruses and their hosts ? x Cormac M. Kinsella Computational discovery of viruses and their hosts Cormac M. Kinsella ISBN: 978-94-6483-273-0 © 2023 Cormac M. Kinsella Layout and cover design: Cormac M.
Kinsella Chapter facing art: Kristel Parv Kinsella, inspired by the works of J. Tolkien Printing: Ridderprint, the Netherlands The research reported in this doctoral thesis received financial assistance from the European Union’s Horizon 2020 research and innovation programme, under the Marie Skłodowska-Curie Actions grant agreement no. Financial support for the printing of this thesis was kindly provided by the Amsterdam UMC. Computational discovery of viruses and their hosts ACADEMISCH PROEFSCHRIFT ter verkrijging van de graad van doctor aan de Universiteit van Amsterdam op gezag van de Rector Magnificus prof.
Verbeek ten overstaan van een door het College voor Promoties ingestelde commissie, in het openbaar te verdedigen in de Agnietenkapel op maandag 11 september 2023, te 14.00 uur door Cormac Michael Kinsella geboren te Harrow Promotiecommissie Promotor: dr. van der Hoek AMC-UvA Copromotores: prof. Berkhout AMC-UvA dr. Bart Tergooi Ziekenhuis Overige leden: prof.
de Jong AMC-UvA prof. Russell AMC-UvA prof. Koopmans Erasmus Universiteit Rotterdam dr. Krupovic Institut Pasteur dr.
Matthijnssens KU Leuven Faculteit der Geneeskunde Table of contents Chapter 1 General introduction and scope of this thesis 7 2 Enhanced bioinformatic profiling of VIDISCA libraries 19 for virus detection and discovery (Virus Research, 2019) 3 Entamoeba and Giardia parasites implicated as hosts of 33 CRESS viruses (Nature Communications, 2020) 4 Host prediction for disease-associated gastrointestinal 57 cressdnaviruses (Virus Evolution, 2022) 5 Vertebrate-tropism of a cressdnavirus lineage implicated 85 by poxvirus gene capture (PNAS, 2023) 6 Human clinical isolates of pathogenic fungi are host to 115 diverse mycoviruses (Microbiology Spectrum, 2022) 7 General discussion 135 Addendum Summary 146 Samenvatting 148 Author affiliations 150 Author contributions 152 About the author 153 PhD portfolio 154 List of publications 158 Acknowledgements 161 Chapter 1 General introduction and scope of this thesis Chapter 1 The discovery of viruses, a distinct class of disease agents ‘Virus’, derived from a Latin word meaning poison, has been used to non-specifically describe infectious disease agents for centuries1. When scientists in the 1800s came to understand that some microbes could cause disease, a flurry of cellular pathogens were isolated in pure culture by growing them on nutrient-rich matrices, allowing their associations to disease to be directly tested under experimental conditions2. An assumption that culturable bacteria, fungi, and protists caused all infectious diseases took root. Usage of the term ‘virus’ remained non-specific into the early 1900s, with apparent oxymorons such as ‘bacterial viruses’ appearing3 – meaning ‘bacterial agents of disease’ – not ‘viruses infecting bacteria’ as we might now understand it.
However, in 1898 a key conceptual leap was made that would shape the modern conception of viruses, namely that a category of disease agents distinct from bacteria existed. First, work by Friedrich Loeffler and Paul Frosch showed that the causative agent of foot and mouth disease could pass through filters capable of holding back all known bacterial cells4. They postulated a very small, particulate agent of disease that was capable of replication (i. Secondly, Dutch microbiologist Martinus Beijerinck showed that the agent causing tobacco mosaic disease could also pass filters5.
Beijerinck proposed a non-bacterial identity for the agent, though he considered it to be liquid-like, or as he called it: “contagious living fluid”. A new class of agents known as ‘filterable viruses’ were thus recognised, and over the following decades non-specific usage of the terminology faded, until ‘filterable’ was also eventually dropped. What defines a virus? We now understand that viruses are not liquid-like, instead they are made up of infectious particles called virions. The small size of most virions explains why they can pass fine filters, though size does not define them.
In fact, so-called ‘giant viruses’ have been found that are larger than the smallest bacteria6,7. More fundamentally, viruses are acellular but require cells to replicate, as they lack some of the necessary machinery for producing further generations. They are thus obligate intracellular parasites of host replication machinery, and must transmit between host cells to gain access to this. Virions represent individual virus units, such that in some cases a single virion can produce a new infection.
At the least, virions possess a genome or genome segment of RNA or DNA, and some proteins encoded by that genome. While these features define most known viruses, biological discoveries regularly complicate attempts at an all-encompassing yet restrictive definition. For example, one definition8 splits biological entities into either ribosome- encoding or capsid-encoding forms, i., cellular life and viruses respectively. However, viruses that lack capsids and encode other proteins are now known9, excluding them from this definition, and also from the viroids (virus-like elements that do not encode protein).
Dropping the capsid requirement of the definition opens the door to other selfish genetic elements usually considered distinct from viruses, such as some transposons or plasmids. A clean definition is likely elusive, and given that viruses are a polyphyletic group (i., they did not all evolve from a single common ancestor) this should be expected. Individual 8 General introduction and scope of this thesis discoveries should therefore be evaluated in terms of how much their genetic relationships and biological behaviours overlap with those considered typically viral. The development of virus discovery techniques The visible effects of viruses have long been readily apparent to humans10,11, likely since our origin12.
Experimentation with viruses also began before their nature was understood, for example Edward Jenner’s work on smallpox vaccination in the 1700s 13. Virus discovery as a field arguably began with Loeffler, Frosch, and Beijerinck’s conclusions regarding filterable viruses4,5. By 1912, application of filtration techniques resulted in the discovery of at least 17 distinct viruses14,15, though detection and study was only possible via the diseases they induced. The subsequent development of virus discovery was tied to technological innovations enabling deeper characterisation and thus categorisation of filterable agents.
Key early advances were the 1935 crystallisation of tobacco mosaic virus (TMV)16, the 1937 discovery of viral nucleic acids17, the 1939 electron microscope analysis of TMV18, and the 1941 application of X-ray crystallography techniques19. These enabled analysis of virus biochemistry and morphology. Viruses only replicate in host cells, so early attempts to produce pure virus cultures in nutrient media were unsuccessful. Early propagation was done in whole organisms or eggs, and this had multiple drawbacks including bacterial contamination of stocks20.
It was during a negative experiment aiming to grow pure vaccinia virus that Frederick Twort inadvertently established the first virus culture, though it was not vaccinia. Reporting in 191521, Twort noticed that colonies of growing bacterial contaminants were killed off by a filterable, dilutable, infectious agent that could be propagated between colonies. Subsequent work from 1917 by Félix d'Hérelle named the ‘bacteriophages’ and properly established virus culture in bacterial cells, and specifically the plaque assay, as vital tools in virus research and discovery22. As eukaryotic tissue and cell culture techniques developed later in the 1900s, many viruses were discovered by inoculating cultures with infectious material and isolating agents23–25.
Cell, tissue, or host tropism could also be tested using panels of different cell cultures25, something that Twort already comprehended in 1915 when testing bacteriophage host tropism21. With advances in immunology, the possibility to characterise isolated viruses by their antigenic or serological properties also developed26, and with this came the ability to test for viruses using immunoassays25,27. While two agents may share similar morphology and cytopathic effects, different responses to antibodies could distinguish ‘serotypes’. By the 1970s scientists already had powerful tools to find and characterise new pathogenic viruses, but a revolution in molecular biology was underway.
Restriction enzymes that cut DNA in specific locations had been isolated28, vital components of molecular cloning techniques that enabled amplification of specific nucleic acids29. In 1977 Frederick Sanger refined a technique for DNA sequencing and the first ever virus genome sequence was published, φX17430,31. This would eventually allow determination of comparative virus 9 Chapter 1 relationships, but did not immediately overhaul virus discovery methods, as it required pure input DNA at high copy number, and was therefore limited to viruses established in culture or cloned fragments. In the 1980s the polymerase chain reaction (PCR) method was developed32,33, which enabled amplification of specific DNA sequences via multiple cycles of in vitro reactions.
Because PCR utilises ‘primer’ sequences that match sections of a target, it could also be used to detect closely related targets34. Primers designed to target sequences highly conserved across an entire viral lineage have often been used to detect unknown members of the group35. However, detection range is limited by design, and more divergent viruses will not be found. To solve this, advanced molecular biology techniques agnostic to virus sequence were applied.
These included shotgun cloning, wherein total DNA from a sample was randomly sheared, and fragments were then cloned and Sanger sequenced 36,37. As this could be applied to mixed samples containing nucleic acids from multiple organisms, it became known as ‘metagenomics’37. Representational difference analysis was another approach38, which disproportionately amplified nucleic acids found in one sample but not another (i., a virus found in a test sample, but not in a control sample). Similarly, techniques such as sequence-independent single primer amplification (SISPA) and virus discovery based on cDNA-amplified fragment length polymorphism (VIDISCA) used restriction enzymes to digest nucleic acids in control and test samples before amplification, with different nucleic acid fragments then visualised by gel electrophoresis39,40.
Samples containing a new virus displayed unique nucleic acid fragments, which were then excised from the gel, cloned, and sequenced. Inclusion of a reverse transcription step converting RNA virus genomes to DNA enabled detection of either genome type, and further laboratory techniques could non- specifically enrich virus nucleic acids relative to background. These included centrifugation of samples to remove heavier cell debris, filtration of supernatants to remove other large particles, treatment with nucleases such as DNase to digest naked host chromosomal DNA, and use of selective primers during reverse transcription to reduce host ribosomal RNA levels39–42. Virus discovery with high-throughput sequencing Despite the maturation of virology during the 1900s, key issues remained at the turn of the millennium.
One of these, discussed by Twort even in 1915 21, was efficient identification of viruses that do not cause visible disease or cytopathic effect, and relatedly, how to find viruses infecting host species difficult to isolate in cell culture. While molecular techniques offered promising solutions, they remained low-throughput and logistically complex36,38–40. It would be the development of high-throughput sequencing (HTS) platforms in the 2000s43 that precipitated a major leap forward for virus discovery. Also known as massively parallel sequencing or next-generation sequencing, HTS techniques allow simultaneous sequencing of millions of DNA fragments in a processed sample known as a ‘library’.
As the fragments overlap in their sequence content, they can be computationally ‘assembled’ together into longer sequences44, including whole virus genomes.