Phage stability in public transport, their diversity and self- assembly in spaceflight, and evaluation of copper surfaces for their antiviral potential

Viruses are the most diverse and numerous biological entities on Earth. They are a crucial part of any ecosystem and pose a range of health-related risks but also biotechnological opportunities. This is true for bacterial viruses (bacteriophages or phages), archaeal viruses, and eukaryotic viruses. Due to their appreciable diversity, it is complex to describe how their characteristics like infectivity or diversity change in a given environment. The research presented in this thesis was divided into four main chapters. In Chapter 1, different bacteriophages frequently used as surrogates for human/animal viruses were tested for their stability on glass surfaces after drying. The phages examined here were Enterobacteria Phage MS2, Enterobacteria Phage M13, Enterobacteria Phage PhiX174, Enterobacteria Phage T4, and Pseudomonas Phage Phi6. All the phages except PhiX174 were mixed with the host bacteria while their optical density at 400 nm (OD400) was measured over time. This was performed to try to corelate the Plaque-forming unit assays (PFU assays) to the effect of the host growth curve. However the growth curves showed significant variation, so PFU assays were chosen to determine the effect of drying. PFU assays were therefore performed to determine the infectivity of the bacteriophages after drying on glass for up to 1, 3 and 7 days at room temperature and fridge (approximately 4 °C). Additionally, the molecular detection through loop-mediated isothermal amplification (LAMP) was also performed on the dried phages. This experiment served to introduce the virus cultivation and molecular detection and demonstrate the infectivity and detectivity of viral surrogates after drying on glass surfaces. The results show significant variation in stability between different surrogates. Specifically, PFU assays showed that M13 and T4 both showed high stability at room temperature and low temperature (approximately 4 °C) with minimal reductions after 7 days. On the other hand, PhiX174 showed high stability in the fridge, but not at the room temperature – with infectivity near 100% after 7 days in the fridge but near no detection at RT. At the same time, MS2 and Phi6 showed low stability both at RT and in the fridge with infectivity approaching the limit of detection after 7 days. In addition, the phages were detected with LAMP after 1, 7 and 31 days. Despite some lacking infectivity, all of them were detected using LAMP after up to 7 days, and PhiX174 and T4 were only ones not detectable after 31 days. The experiments demonstrate the variation on infectivity of virus surrogates of different structures though molecular detection can be performed even long after some viruses are rendered non-infectious. The experiments also introduced and tested safe surrogate viruses for space and public transportation research. Chapter 2 explores the potential use of copper-based antiviral surfaces against human pathogenic viruses, Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-Cov-2) and Monkeypox Virus (MPXV) as well as one phage surrogate, Phi6. Three different variants ofSARS-CoV-2 (B.3, B.1.617.2, and XBB.1.5) were tested and all four MPXV clades (Ia, Ib, IIa, and IIb). For SARS-CoV-2, variant B.3 showed the lowest stability on copper, while XBB.1.5 showed the highest. B.3 fully inactivated after 10 minutes on copper surface and XBB.1.5 showed minimal reduction even after 15 minutes on copper. B.617.2 showed intermediate stability with almost full titer reduction after 10 minutes on copper. The Spike receptor trimer structure and electric potential distribution was determined and compared among the tested variants. It was shown that all variants possess differences, and XBB.1.5 showed the highest overall electric potential with the lowest amount of acidic amino acids. This could result in the least copper cation interaction of all other Spike variants. No genomic damage was observed through qPCR. For MPXV, it was shown that the clade Ia had the highest stability on copper, IIb and IIa intermediate stability, and Ib had the lowest stability. For each clade, the genome was assembled from Illumina sequencing data and genetic variants were determined. The variations in the surface glycosylated proteins were found for each clade. All the clades except Ia had substitution mutations in the heavily glycosylated surface protein OPG210. All the mutations could affect the protein structure or glycosylation effect. Additionally, novel 3D- printed antimicrobial surfaces containing copper microparticles and nanoparticles, respectively, were tested against SARS-CoV-2 and Phi6. The nanoparticle-containing 3D printed materials had a better antiviral effect against both SARS-CoV-2 and Phi6. Phi6 was shown to have low stability on copper compared to animal viruses, making it unsuitable surrogate when copper surface stability is considered. Chapter 3, in the context of environmental virology, explores the diversity of integrated prophages that are found within bacterial genomes of the International Space Station (ISS) microbiota. All known full genomic sequences from the isolates from the ISS were downloaded from NCBI. Their sequences were probed for prophage gene clusters. The found genomes were then analyzed taxonomically and functionally. This revealed a significant phage diversity of families Siphoviridae and Myoviridae. Their phylogenies were determined, together with their overall diversity and the diversity within host genera. It was shown that bacterial genus Stapylococcus contains by far the highest diversity of prophages inside their genomes. However, this could also be because they represented the highest number of genomes of all ISS isolates. Functional diversity of phage genomes was also determined and plotted. Among all the genomes, the antimicrobial resistance virulence resistance genes were identified. All those genes are available for potential spread within the bacterial population via prophage infection. Therefore, this study showed the potential genes that could be transmitted in this environment. The project was done on genomic data, but the same approach can be done with metagenomic sequences from sequencing samples of ISS or public transportation. Therefore, a similar approach could be used in the future for public transportation, which is especially interesting because of the spread of antimicrobial resistance and virulence genes among thepopulation. Also, the approach could be applied in the future for space station analysis of prophages. Finally, in Chapter 4, bacteriophage T7 virion assembly was explored in simulated microgravity through clinorotation in a transcription/translation solution. The virions were synthesized in a solution of phage genomic DNA and bacterial extract while on a rotation system often used to simulate microgravity on Earth, called clinorotation. Sampling the solution over time and performing PFU assays revealed that the endpoint-titer is higher and reached faster in the clinorotated solutions than the stationary controls. The effect on clinorotation on gene expression was explored with a GFP gene-containing control plasmid and measurement of fluorescence produced in clinorotation versus stationary control. In addition, the protein production was assessed with liquid chromatography mass spectrometry and dot blot. The replication of the viral DNA in the solution was also assessed through qPCR quantification. The protein production, gene expression and replication were not affected by clinorotation. Higher number of assembled virions was observed with transmission electron microscopy. Taken together, the results suggest a more efficient assembly of T7 virions in simulated microgravity. This is an important point for future of human space exploration. Taken together, this work explores the viral resistance to stressors on Earth and in space. This is done by means of four different parts, divided into chapters above. Desiccation stability is an important aspect of virus transmission on Earth and was therefore explored through its effect on different bacteriophage surrogate viruses. By comparing the virus surrogate desiccation stabilities among each other, a line can be drawn between virus type and their stability. The surrogate that is most structurally similar to human respiratory viruses, Phi6, was then compared to human virus stability, specifically, SARS-CoV-2 and MPOXV. Then, the research moves to space environment where the diversity of prophages on the International Space Station is characterized. The pipeline is established to determine the gene and species diversity aboard. Finally, the specific condition of spaceflight, microgravity is explored through its effect on self-assembly of bacteriophage T7. Overall, the stress environments on Earth and in space are evaluated here through their effects on viral stability, assembly and detection.