In vitro study of coinfection/superinfection parameters which can influence recombination events in noroviruses

Noroviruses (NoVs) are non-enveloped, single-stranded, positive-sense RNA viruses. They are important causes of acute non-bacterial gastroenteritis in humans worldwide but their study is currently yet hampered by the lack of a cell culture system. NoVs genetically evolve by both point mutations and recombination and the murine norovirus (MuNoV) is considered as the best model for human NoVs. The aim of this study was to develop an experimental model based on the MuNoV in order to investigate coinfection/superinfection parameters that could impact recombination events. Monolayers of RAW264.7 cells were coinfected or superinfected with two MuNoV strains (CW1 and WU20) using different multiplicity of infection (0.1/1; 1/1 and 10/1 for CW1 and Wu20 , respectively) and time delays (0h; 0.5h; 1h; 2h; 4h; 8h; 12h and 24h) for infection. Supernatants were collected at 24 and 48 hours post-infection. Genomic copies of both viruses were first quantified by RT-QPCR. Then, viruses from the supernatants were plaque purified (36 clones per condition) and their recombinant status was checked by a real-time PCR discriminating method using primers targeting both extremity of the MuNoV genome. Results of quantitative and plaque picking assays are compared. Together, the results confirm that recombination does not frequently occur, at least in vitro and raise the issue on why these events are however so usual with in silico detection methods. The data also showed that superinfection exclusion seems to be triggered from 4h post infection with the first MuNoV. The mechanisms of the later should be still studied.