Generation of Recombinant Rotaviruses Expressing Fluorescent Proteins by Using an Optimized Reverse Genetics System

An entirely plasmid-based reverse genetics system for rotaviruses was established very recently. We improved the reverse genetics system to generate recombinant rotavirus by transfecting only 11 cDNA plasmids for its 11 gene segments under the condition of increasing the ratio of the cDNA plasmids f...

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Bibliographic Details
Published in:Journal of virology 2018-07, Vol.92 (13)
Main Authors: Komoto, Satoshi, Fukuda, Saori, Ide, Tomihiko, Ito, Naoto, Sugiyama, Makoto, Yoshikawa, Tetsushi, Murata, Takayuki, Taniguchi, Koki
Format: Article
Language:English
Subjects:
Animals
Cells, Cultured
Cricetinae
Genome and Regulation of Viral Gene Expression
Green Fluorescent Proteins - genetics
Green Fluorescent Proteins - metabolism
Haplorhini
Kidney - metabolism
Kidney - virology
Plasmids
Reverse Genetics
RNA, Viral
Rotavirus - physiology
Rotavirus Infections - metabolism
Rotavirus Infections - virology
Viral Proteins - metabolism
Virus Replication
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Summary:An entirely plasmid-based reverse genetics system for rotaviruses was established very recently. We improved the reverse genetics system to generate recombinant rotavirus by transfecting only 11 cDNA plasmids for its 11 gene segments under the condition of increasing the ratio of the cDNA plasmids for NSP2 and NSP5 genes. Utilizing this highly efficient system, we then engineered infectious recombinant rotaviruses expressing bioluminescent (NanoLuc luciferase) and fluorescent (enhanced green fluorescent protein [EGFP] and mCherry) reporters. These recombinant rotaviruses expressing reporters remained genetically stable during serial passages. Our reverse genetics approach and recombinant rotaviruses carrying reporter genes will be great additions to the tool kit for studying the molecular virology of rotavirus and for developing future next-generation vaccines and expression vectors. Rotavirus is one of the most important pathogens causing severe gastroenteritis in young children worldwide. In this paper, we describe a robust and simple reverse genetics system based on only rotavirus cDNAs and its application for engineering infectious recombinant rotaviruses harboring bioluminescent (NanoLuc) and fluorescent (EGFP and mCherry) protein genes. This highly efficient reverse genetics system and recombinant group A rotaviruses expressing reporters could be powerful tools for the study of different aspects of rotavirus replication. Furthermore, they may be useful for next-generation vaccine production for this medically important virus.
ISSN:0022-538X
1098-5514
DOI:10.1128/jvi.00588-18