Development of an infectious clone and replicon system of norovirus GII.4.

Human norovirus (HuNoV) is one of the main causes of acute gastroenteritis worldwide and is responsible for at least 20% of all cases. The detailed molecular mechanism of this norovirus remains unknown due to the lack of a suitable in vitro culturing system. An infectious clone of HuNoV would be a useful tool for elucidating the processes of viral infection and the mechanisms of replication. We developed an infectious cDNA clone of HuNoV using the rapid technique of Gibson Assembly. The complete genome of the HuNoV GII.4 Sydney subtype was cloned into a previously modified pcDNA3.1-based plasmid vector downstream from a cytomegaloviral promoter. We monitored the viral infection in vitro by inserting the reporter gene of the green fluorescent protein (GFP) between the NTPase and p22 genes, also by Gibson Assembly, to construct a HuNoV-GFP replicon. Human Caco-2 cells were transfected with the full-length genomic clone and the replicon containing GFP. The gene encoding the VP1/VP2 capsid protein was expressed, which was indirect evidence of the synthesis of subgenomic RNAs and thus the negative strand of the genome. We successfully constructed the infectious clone and its replicon containing GFP for the HuNoV GII.4 Sydney subtype, a valuable tool that will help the study of noroviral infection and replication.