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

Autor: Linda L. Yasukawa, Baoming Jiang, Philippe H. Jais, Susana López, Kenneth H. Mellits, Liliana Sánchez-Tacuba, Nathan Meade, Harry B. Greenberg, Theresa K. Resch, Siyuan Ding, Ningguo Feng
Rok vydání: 2020
Předmět:
Gene Expression Regulation
Viral
RNA Caps
Rotavirus
Immunology
Biology
Transfection
Virus Replication
medicine.disease_cause
Microbiology
African swine fever virus
law.invention
Mice
Viral Proteins
03 medical and health sciences
Plasmid
law
Virology
Chlorocebus aethiops
medicine
Animals
Humans
Vero Cells
030304 developmental biology
0303 health sciences
Expression vector
Attenuated vaccine
030306 microbiology
DNA-Directed RNA Polymerases
biology.organism_classification
African Swine Fever Virus
Nucleotidyltransferases
Immunity
Innate
Recombinant Proteins
Reverse Genetics
In vitro
Reverse genetics
Genome Replication and Regulation of Viral Gene Expression
STAT1 Transcription Factor
Insect Science
Host-Pathogen Interactions
Interferon Regulatory Factors
Recombinant DNA
Reassortant Viruses
Plasmids
Zdroj: J Virol
ISSN: 1098-5514
0022-538X
DOI: 10.1128/jvi.01294-20
Popis: 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 (
Databáze: OpenAIRE
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