Fitness evolution of a recombinant murine norovirus during serial passages in cell culture

Human norovirus (NoV) infections are among the most important causes of gastroenteritis in both children and adults and often occur as outbreaks which may be foodborne. Recombination can dramatically change virulence properties of the viruses and has been often evidenced in silico for different NoV strains. Recently, after in vitro coinfection of RAW264.7 cells with parental murine norovirus (MuNoV) strains CW1 and Wu20, we obtained a recombinant Wu20/CW1 strain (Mathijs et al., 2010). This recombinant strain showed reduced plaque size compared to the parental strains. The aim of the study was to observe and molecularly characterize the natural genetic evolution of the recombinant MuNoV strain across in vitro replications. The recombinant strain was serially replicated in vitro (up to 14 passages). Viral plaque diameters of early and late progenies were compared with the Image software. A significant difference was shown between them with the Mann and Whitney non parametric statistical test. The average size of plaques increased from the earlier to the later progenies (from 0.1 mm2 to around 0.5 mm2). Molecular investigations are currently performed in order to specify in which genetic region mutations occur and whether or not this could explain fitness modifications during in vitro evolution. In addition two other parameters of in vitro virulence modification will be investigated (i) virus production and (ii) one step growth kinetics. The data should provide interesting information about genetic evolution in the genus Norovirus, especially regarding recombination events and explain how a recombinant strain, first disadvantaged compared to its parental strains, could regain fitness by genetic evolution.

Mathijs, E., Muylkens, B., Mauroy, A., Ziant, D., Delwiche, T., Thiry, E., 2010. Experimental evidence of recombination in murine noroviruses. J Gen Virol 91, 2723-2733.