Human Norovirus Replication in Temperature-Optimized MDCK Cells by Forkhead Box O1 Inhibition

Human noroviruses (HuNoVs) are a leading cause of gastroenteritis outbreaks worldwide. However, the paucity of appropriate cell culture model for HuNoV replication has prevented developing effective anti-HuNoV therapy. In this study, first, the replication of the virus at various temperatures in different cells was compared, which showed that lowering the culture temperature from 37°C significantly increased virus replication in Madin-Darby canine kidney (MDCK) cells. Second, the expression levels of autophagy-, immune-, and apoptosis-related genes at 30°C and 37°C were compared to explore factors affecting HuNoV replication. HuNoV cultured at 37°C showed significantly increased autophagy- (ATG5 and ATG7) and immune- (IFNA, IFNB, ISG15, and NFKB) related genes compared to mock. However, the virus cultured at 30°C showed significantly decreased expression of autophagy- (ATG5 and ATG7) and not significantly different in major immune- (IFNA, ISG15, and NFKB) related genes compared to mock. Importantly, expression of the transcription factor FOXO1, which controls autophagy- and immune-related gene expression, was significantly lower at 30°C. Moreover, FOXO1 inhibition in temperature-optimized MDCK cells enhanced HuNoV replication, highlighting FOXO1 inhibition as an approach for successful virus replication. In the temperature-optimized cells, various HuNoV genotypes were successfully replicated, with GI.8 showing the highest replication levels followed by GII.1, GII.3, and GII.4. Furthermore, ultrastructural analysis of the infected cells revealed functional HuNoV replication at low temperature, with increased cellular apoptosis and decreased autophagic vacuoles. In conclusion, temperature-optimized MDCK cells can be used as a convenient culture model for HuNoV replication by inhibiting FOXO1, providing adaptability to different genotypes.