Characterisation of the human gut virome in patients with liver steatosis in order to identify biomarkers for diagnosis and targets for drug development.

The community of viruses, mainly phages, inhabiting our gastrointestinal tract constitutes a crucial, yet understudied component of the complex human gut microbial ecosystem. While the significance of the gut virome in human health and disease is increasingly recognized, the precise involvement of these viruses in the development and progression of chronic liver disease (CLD), a major global health burden, remains a subject of scientific investigation. CLD is a complex and multifaceted group of conditions characterized by long-term progressive liver damage, affecting more than 1.5 billion people globally and contributing to 3.5% of all annual deaths. A disturbed gut-liver axis has emerged as a critical player in the progression of CLD. Chapter 1 provides a comprehensive introduction to the current knowledge of the gut virome, clinical aspects of CLD and the gut-liver axis.

Understanding how alterations in the gut virome relate to the presence, severity and outcomes of CLD, along with its interactions with the bacterial counterpart of the gut microbiome, is vital for unraveling the relationship of the gut virome with disease development and progression. This doctoral thesis aims to explore the gut virome in the context of health and CLD, more specifically focusing on patients with decompensated liver cirrhosis, acute-on-chronic liver failure (ACLF) and alcohol-related liver disease (ALD). Specific research objectives are outlined in Chapter 2. 

The characterization of the gut virome involved enriching fecal samples for viral-like particles followed by Illumina sequencing. Viral sequences were identified, taxonomically classified and a host and lifestyle were predicted for phages using bacterial metagenomic data from the same samples. 

In Chapter 3, a human gut viral genome and gene catalog was generated, shedding light on previously uncharacterized viral populations and their genes. Gut viromes from healthy children, adolescents and adults were characterized in terms of composition, diversity and function, demonstrating limited differences between them, with the exception of a decrease in temperate phage diversity from childhood into adulthood, aligning with previous reports in infants. The global prevalence of a subset of phage genomes which were highly prevalent in the catalog samples was determined, revealing several age-, geography- and disease-associated patterns. Interestingly, a previously undescribed phage, named LoVEphage, had a global prevalence of at least 14% and was predicted to temperately infect Bacteroides dorei. 

Chapter 4 described the gut virome in a longitudinal cohort of patients with decompensated liver cirrhosis and ACLF. Viromes from patients with ACLF were marked by an increased phage diversity compared to viromes from patients with decompensated liver cirrhosis. Associations between disease severity and abundance levels of temperate phages, support the previously raised hypothesis of inflammation-induced prophage induction. Lactococcus A phages were identified as potential predictors for ACLF development and associated with short-term mortality. Enterococcus B phages, and their bacteria, were linked to bacterial infection, an important precipitating event in pathogenesis of decompensated liver cirrhosis, as well as to short-term mortality. Their association with bacterial infection was confirmed in an external validation cohort. 

In Chapter 5, the gut virome was characterized in a large cross-sectional cohort of patients with ALD and healthy subjects. Lower alpha-diversity was associated with the presence of ALD, but its severity. Increased inter-individual variation was observed in patients with ALD compared to healthy individuals, and the inter-individual variation further associated with disease severity. Several phage genera were depleted in ALD. Flavonifractor phages were enriched in patients with ALD, while several other Firmicutes phages were depleted, some of which also negatively associated with fibrosis stage. Two gut viral community types could be identified in this cohort, associating with disease presence, severity and outcome. 

This research significantly advanced our understanding of the composition, diversity, function and dynamics of the gut virome in health and disease. The implications of these novel insights into the association of the gut virome with presence, severity and clinical outcomes of CLD are further discussed in Chapter 6. The identification of specific virome-based biomarkers holds promise for the development of novel clinical applications to improve patient outcomes. Although validation of these findings in other cohorts or animal models is essential before translation into the clinic. This research has paved to the way for future investigations aimed at further elucidating the role of the gut virome in the complex multidimensional microbial gut ecosystem, and its implications in human health and disease.