FITNESS EVALUATION OF A RECOMBINANT MURINE NOROVIRUS DURING SERIAL PASSAGES IN CELL CULTURE

Noroviruses are single stranded positive sense RNA viruses which can infect human and different animal species. Human norovirus (NoV) infections are among the most important causes of gastroenteritis in both children and adults. Infections often occur as outbreaks which may be foodborne. Due to the lack of an efficient cell culture system as well as a workable animal model, many aspects of the NoV infection in human are still poorly understood. The murine norovirus (MuNoV) grows easily in cell culture in contrast to the Human NoV, and constitutes an excellent animal model. Recombination can dramatically change virulence properties of the viruses and has been evidenced in silico for different human NoV strains isolated from clinical cases. Recently, after in vitro coinfection of RAW264.7 cells with parental MuNoV strains CW1 and Wu20, we obtained a recombinant Wu20/CW1 strain. 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. Viral fitness is a complex concept. Here we defined this fitness as the ability of a viral population to adapt to the cell culture system. Thus, the recombinant strain was serially replicated in vitro in RAW264.7 cells (up to 14 passages). Viral plaque sizes of early and late progenies were compared with the Image J software. A significant difference was shown between them with the Mann and Whitney non parametric statistical test. Afterwards, viruses from different cell passages were cloned and sequenced. The average plaque size 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.