An Optimized Reverse Genetics System Suitable for Efficient Recovery of Simian, Human, and Murine-Like Rotaviruses

S�nchez-Tacuba, Liliana; Feng, Ningguo; Meade, Nathan J.; Mellits, Kenneth H.; Ja�s, Philippe H.; Yasukawa, Linda L.; Resch, Theresa K.; Jiang, Baoming; L�pez, Susana; Ding, Siyuan; Greenberg, Harry B.

Abstract

Copyright © 2020 American Society for Microbiology. An entirely plasmid-based reverse genetics (RG) system was recently developed for rotavirus (RV), opening new avenues for in-depth molecular dissection of RV biology, immunology, and pathogenesis. Several improvements to further optimize the RG efficiency have now been described. However, only a small number of individual RV strains have been recovered to date. None of the current methods have supported the recovery of murine RV, impeding the study of RV replication and pathogenesis in an in vivo suckling mouse model. Here, we describe useful modifications to the RG system that significantly improve rescue efficiency of multiple RV strains. In addition to the 11 group A RV segment-specific (+)RNAs [(+)ssRNAs], a chimeric plasmid was transfected, from which the capping enzyme NP868R of African swine fever virus (ASFV) and the T7 RNA polymerase were expressed. Second, a genetically modified MA104 cell line was used in which several components of the innate immunity were degraded. Using this RG system, we successfully recovered the simian RV RRV strain, the human RV CDC-9 strain, a reassortant between murine RV D6/2 and simian RV SA11 strains, and several reassortants and reporter RVs. All these recombinant RVs were rescued at a high efficiency (≥80% success rate) and could not be reliably rescued using several recently published RG strategies (

Citation

Sánchez-Tacuba, L., Feng, N., Meade, N. J., Mellits, K. H., Jaïs, P. H., Yasukawa, L. L., Resch, T. K., Jiang, B., López, S., Ding, S., & Greenberg, H. B. (2020). An Optimized Reverse Genetics System Suitable for Efficient Recovery of Simian, Human, and Murine-Like Rotaviruses. Journal of Virology, 94(18), Article e01294. https://doi.org/10.1128/JVI.01294-20

• Journal Article Type: Article
• Acceptance Date: Jul 1, 2020
• Online Publication Date: Aug 31, 2020
• Publication Date: Aug 31, 2020
• Deposit Date: Sep 9, 2020
• Publicly Available Date: Mar 1, 2021
• Journal: Journal of virology
• Print ISSN: 0022-538X
• Electronic ISSN: 1098-5514
• Publisher: American Society for Microbiology
• Peer Reviewed: Peer Reviewed
• Volume: 94
• Issue: 18
• Article Number: e01294
• DOI: https://doi.org/10.1128/JVI.01294-20
• Keywords: Immunology; Insect Science; Microbiology; Virology
• Public URL: https://nottingham-repository.worktribe.com/output/4892472
• Publisher URL: https://jvi.asm.org/content/94/18/e01294-20/