High throughput sequencing analysis reveals genetic variability and selection pressure in different murine norovirus genomic regions during in vitro replication

Murine norovirus (MuNoV), a single stranded positive sense RNA virus belonging to the Caliciviridae family, is considered as a representative model for human norovirus infections, one of the most important etiological cause of both epidemic and sporadic gastroenteritis cases worldwide. Four open reading frames are described into its genome: ORF1 codes the non-structural (NS) proteins, including the viral RNA dependent RNA polymerase (RdRp); ORF2 codes the single capsid protein (VP1), wherein two domains are present: a relatively conserved domain (“shell”) and a more variable domain (“protruding”); ORF3 codes a minor structural protein; and ORF4, currently only found in viruses genetically related to MuNoV codes a virulence factor. In this study, we demonstrated by high throughput sequencing that, during serial passages of MuNoV in cell culture, the substitution rates, estimated by Bayesian inferences, did not significantly differ across the five targeted genomic regions except one. These rates were similar in four genomic regions encompassing partial non-structural 1-2 protein (NS1-2)-, NS5-, NS6-, NS7 (RdRp)- and VP1-coding sequences (coding the conserved part of the protein also including the ORF4 region). In the partial minor structural protein-coding region, this substitution rate was however estimated to be at least one log higher when expressed as substitution/site/day. The precise localisation of the detected nucleotide point mutations (substitution, deletion and insertion) were reported as well as the quantitative increase or decrease of the sequences harbouring them along ten cell culture passages. The non-silent amino acid mutations were also depicted in 3D models for four out of the five studied regions. These results have important implications for different norovirus research fields, especially in terms of diagnosis, classification methodology and genetic evolution.