A Novel Rotavirus Reverse Genetics Platform Supports Flexible Insertion of Exogenous Genes and Enables Rapid Development of a High-Throughput Neutralization Assay
Jiajie Wei,
Scott Radcliffe,
Amanda Pirrone,
Meiqing Lu,
Yuan Li,
Jason Cassaday,
William Newhard,
Gwendolyn J. Heidecker,
William A. Rose II,
Xi He,
Daniel Freed,
Michael Citron,
Amy Espeseth,
Dai Wang
Affiliations
Jiajie Wei
Department of Infectious Diseases and Vaccines, Merck & Co., Inc., West Point, PA 19486, USA
Scott Radcliffe
Department of Quantitative Biosciences, Merck & Co., Inc., West Point, PA 19486, USA
Amanda Pirrone
Department of Infectious Diseases and Vaccines, Merck & Co., Inc., West Point, PA 19486, USA
Meiqing Lu
Department of Infectious Diseases and Vaccines, Merck & Co., Inc., West Point, PA 19486, USA
Yuan Li
Department of Infectious Diseases and Vaccines, Merck & Co., Inc., West Point, PA 19486, USA
Jason Cassaday
Department of Infectious Diseases and Vaccines, Merck & Co., Inc., West Point, PA 19486, USA
William Newhard
Department of Infectious Diseases and Vaccines, Merck & Co., Inc., West Point, PA 19486, USA
Gwendolyn J. Heidecker
Department of Infectious Diseases and Vaccines, Merck & Co., Inc., West Point, PA 19486, USA
William A. Rose II
Department of Quantitative Biosciences, Merck & Co., Inc., West Point, PA 19486, USA
Xi He
Department of Infectious Diseases and Vaccines, Merck & Co., Inc., West Point, PA 19486, USA
Daniel Freed
Department of Infectious Diseases and Vaccines, Merck & Co., Inc., West Point, PA 19486, USA
Michael Citron
Department of Infectious Diseases and Vaccines, Merck & Co., Inc., West Point, PA 19486, USA
Amy Espeseth
Department of Infectious Diseases and Vaccines, Merck & Co., Inc., West Point, PA 19486, USA
Dai Wang
Department of Infectious Diseases and Vaccines, Merck & Co., Inc., West Point, PA 19486, USA
Despite the success of rotavirus vaccines, rotaviruses remain one of the leading causes of diarrheal diseases, resulting in significant childhood morbidity and mortality, especially in low- and middle-income countries. The 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 demonstrate that three positions in rotavirus genome—the C terminus of NSP1, NSP3 and NSP5—can tolerate the insertion of reporter genes. By using rotavirus expressing GFP, we develop a high-throughput neutralization assay and reveal the pre-existing immunity against rotavirus in humans and other animal species. Our work shows the plasticity of the 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.
