An improved reverse genetics system for rotavirus vaccine strain LLR using five plasmid vectors

and contain a genome of 11 segmented dsRNA segments, are a leading cause of severe acute gastroenteritis in infants and children younger than 5 years of age. We previously developed a strategy to recover rotavirus vaccine strain LLR from 11 cloned plasmids. Here, we report an improved reverse genetics system for LLR by combining two or three transcriptional cassettes in a single plasmid, which substantially enhances rescue efficiency from 66.7% (8/12) to 91.7 % (11/12). Furthermore, the recombinant LLR stably expressing NLuc was rescued based on the five-plasmid reverse genetics system. Improvements to the rotavirus reverse genetics system will enhance its applicability for studies of rotavirus biology and clinical use.