Abstract :
[en] Noroviruses (genus Norovirus, family Caliciviridae) are recognised as the major global cause of sporadic and epidemic non-bacterial gastroenteritis in humans. Recombination and the accumulation of point mutations are key mechanisms in the evolution and diversity of noroviruses; increasing evidence indicates that recombination shapes norovirus pathogenesis and fitness and drives the evolution of emerging human norovirus strains.
The understanding of human norovirus biology in general and norovirus recombination in particular has lagged behind that of other viruses due to the difficulties historically associated with robust in vitro human norovirus propagation. While recently developed in vivo and in vitro human norovirus assays have provided invaluable tools to dissect the norovirus life cycle, significant questions remain unanswered due to the technical limitations of many of these experimental systems. The genetically and biologically closely related murine norovirus combines the advantages of easy in vivo infection of a genetically tractable native host, efficient and robust in vitro culture, and availability of tools for genetic manipulation and thus remains the model of choice for many norovirus studies.
In the context of this thesis, the various norovirus recombination checkpoints, namely host coinfection, single cell coinfection, recombination, and functional selection, are examined and their drivers and constraints are discussed.
The review “Norovirus recombinants: recurrent in the field, recalcitrant in the lab – a scoping review of recombination and recombinant types of noroviruses” (Ludwig-Begall et al., 2018) provides a comprehensive overview of norovirus recombination and its role in norovirus molecular evolution and identifies knowledge gaps pertaining to prerequisite processes both directly prior to and post actual recombination in sensu stricto; in investigating conditions governing cell coinfection and functional selection, respectively, experimental studies 1 and 2 provide novel insights into these crucial steps.
In vivo, synchronous single-cell coinfection by multiple viruses, the ultimate prerequisite to viral recombination, is likely to be a rare event and delayed secondary infections are a more probable occurrence. Study 1 determines the effect of a temporal separation of in vitro infections with the two homologous parental murine norovirus strains MNV-1 WU20 and CW1 on the composition of murine norovirus populations. WU20 and CW1 were either synchronously inoculated onto murine macrophage cell monolayers (coinfection) or asynchronously applied (superinfection with varying titres of CW1 at half-hour to 24-hour delays). Twenty-four hours after initial co- or superinfection, quantification of genomic copy numbers and discriminative screening of plaque picked infectious progeny viruses demonstrated a time-dependent predominance of primary infecting WU20 in the majority of viral progenies. Our results indicate that a time interval from one to two hours onwards between two consecutive norovirus infections allows establishment of a barrier that reduces or prevents super-infection; this first demonstration of time-dependent viral interference for noroviruses has clear implications for norovirus epidemiology, risk assessment, and potentially treatment.
Study 2 examines the processes directly following recombination and aims to characterise the adaptive capacity of previously in vitro-generated WU20-CW1 recombinant murine norovirus RecMNV, thus investigating how the accumulation of point mutations through successive viral passaging may compensate for initial replicative fitness losses incurred during deleterious recombination processes. By comparing the replicative fitness and genetic characteristics of RecMNV progenies at early and late stages of an adaptation experiment, replicative fitness regain of the recombinant was demonstrated between viral progenies prior to and post serial in vitro passaging and observable phenotypic profiles of viral fitness were associated to population-level genetic modifications. To investigate the effect of genomic changes separately and in combination in the context of an infectious lab-generated inter-murine norovirus chimera, mutations were introduced into a recombinant WU20-CW1 cDNA for subsequent DNA-based reverse genetics recovery. Fitness loss of RecMNV was thus linked to a C7245T mutation and functional minor capsid protein (open reading frame 3) truncation; individual and cumulative compensatory effects of one synonymous major capsid protein (open reading frame 2) and two non-synonymous non-structural protein 1/2 (open reading frame 1) consensus-level mutations acquired during successive rounds of in vitro replication were demonstrated, suggesting that interactions of viral proteins and/or RNA secondary structures of different open reading frames may play a role in the regulation of replicative fitness after a recombination event. This in vitro proof-of-concept study thus simulates successful adaptation (genetic drift) of a nascent norovirus after recombination (genetic shift) and serves to conceptualise how the emergence of recombinant human norovirus field strains, held to represent an adapted and functionally selected subset of all generated recombinants, may be regulated by an interplay between the two evolutionary processes of recombination and point mutation accumulation.
This thesis serves to provide a comprehensive overview of the recombination checkpoints to be bypassed and, in investigating both superinfection exclusion and functional selection, provides novel insights into prerequisite processes both before and after generation of a recombinant norovirus genome.
