A novel rotavirus reverse genetics platform supports flexible insertion of exogenous genes and enables rapid development of a high-throughput neutralization assay

Despite the success of rotavirus vaccines, rotaviruses are still one of the leading causes of diarrheal diseases, resulting in significant childhood morbidity and mortality especially in low- and middle-income countries. Reverse genetics system enables the manipulation of the rotavirus genome and opens the possibility of using rotavirus as an expression vector for heterologous proteins such as vaccine antigens and therapeutic payloads. Here, we identified three positions in rotavirus genome, the C terminus of NSP1, NSP3 and NSP5, for reporter gene insertion. By using rotavirus expressing GFP, we developed a high-throughput neutralization assay and revealed the pre-existing immunity against rotavirus in human and other animal species. Our work shows the plasticity of rotavirus genome and establishes a high-throughput assay for interrogating humoral immune responses, benefiting the design of next-generation rotavirus vaccines and the development of rotavirus-based expression platforms.