title: Norovirus Evolution: Understanding and characterising the emergence of novel strains in the population.
Creators: name: Kelly, D
Publisher: name: University of Liverpool Repository
Year: 2018

description: Human noroviruses (HuNoVs) are distributed globally, affect all age groups and place a significant burden upon health services. The diversity of this RNA virus is thought to play a significant role in the persistence of HuNoVs as the main cause of non-bacterial gastroenteritis globally. Molecular diagnostics have been critical for understanding the epidemiology of outbreaks and sporadic cases, and to design and implement effective intervention strategies and disease control measures. Immunocompromised individuals are widely considered to be a reservoir for epidemic variants of HuNoV and whilst there are studies investigating the emergence of novel strains in an immunocompetent general population, reports at the the individual level are scarce. Three separate methodologies were developed to characterise HuNoV persistence in acute convalescent and chronic infection. First, a standardised quantification method to accurately quantify the most prevalent HuNoV genogroup. Second, a PGM-MB capture method to select HuNoV prior to massively parallel sequencing (MPS). Third, an assay to measure host specific coproantibody responses to three epidemic variants from different epochs. Quantification of longitudinal samples from individuals with acute or chronic HuNoV infection showed the virus distribution was homogenous in stool and an RNA external standard, in contrast to DNA, did not underestimate virus titre. HuNoV PGM-MB capture meant near complete viral genomes could be recovered at variable mean coverage. A bioinformatics pipeline demonstrated over the course of chronic infection allele frequencies were much more variable. In acute infection, minor alleles were present at a much lower frequency, but potential immune escape mutants were present. Immune escape mutants existed as minority variants or conserved mutations in the consensus sequence, and were in the presence of HuNoV specific-coproantibody, which were mapped to the protein surface. In HuNoV chronic infection, immune pressure is variable or non-existent, and therefore epidemic variants could emerge over long periods of infection by random chance. However, under immune pressure exerted by coproantibodies, escape variants may be seen. In three individuals, acute HuNoV symptomatic infection occurred despite the presence of specific secretory Ab responses to the VLP classed as the closest phylogenetic relative. The closest relative (Sydney 2012), differed at two amino acids, one of which has been previously described (A340T) as belonging to an epitope, and another which can be classed as having a potential role in immune escape (A323T). A single individual with acute HuNoV infection established a more prominent response to an earlier strain of HuNoV, rather than two contemporary strains, which proposes a role for Original Antigenic Sin (OAS) or Antigenic Seniority in the secretory Ab immunity. Finally, the use of MPS in outbreak tracking was assessed and compared to the currently used amplicon and Sanger based method. Overall both methods showed significant correlation. However, MPS provided greater depth and the ability to identify variants among samples within an outbreak that represented consensus changes in one or more samples from the same outbreak. This meant that the MPS data would have been able to link all the samples into a single outbreak or transmission network, where the current Sanger sequencing may not have been able to link them all.