Life science
Medical & healthcare, Vaccine

Reverse Genetics System for a Human Group A Rotavirus
Department of Virology, Research Institute for Microbial Diseases
Professor Takeshi Kobayashi
Researchmap https://researchmap.jp/virology

Life science

Abstract
Group A rotaviruses (RVs) are one of the etiological agents thatcause severe diarrhea in mammals and avian host species. In humans, RVs are a leading cause of acute gastroenteritis in young
children under the age of 5 years. RV infection kills approximately
215,000 infants and young children annually, particularly in developing countries. In 2017, we established an entirely plasmid-based
reverse genetics system for simian RV. Although the simian RV
reverse genetics system is robust enough to generate reassortant
RVs and enable better understanding of the biological differences
between animal and human RV strains, a complete reverse genetics system for human RV strains is desirable. Here, we establisheda plasmid-based reverse genetics system for human RV.

Figure 1. Human rotavirus reverse genetics system▍ Background & Results
Recently, we developed an entirely plasmid-based reverse genetics system for simian RV strain SA11, thereby enabling manipulation of all 11 RV gene segments to expand our understandingof RV biology. However, the biological diversity among humanand animal RVs means that this system is limited in terms of understanding the characteristics of human RVs. Thus, a reverse
genetics system for human RV is needed. To establish the system,
cDNAs encoding each of the human RV strain Odelia 11 dsRNA
gene segments were introduced into plasmids at sites flanked by
the T7 promoter and hepatitis delta virus ribozyme sequences.
Recombinant human RV strain Odelia was generated by transfection of the 11 Odelia cDNAs and polymerase II promoter-driven
expression plasmids encoding Nelson Bay reovirus FAST protein,
vaccinia virus capping enzyme (D1R and D12L), and RV NSP2
and NSP5 proteins into baby hamster kidney cells expressing T7
RNA polymerase (Figure 1). This technology was used to generatea panel of monoreassortant viruses between human and simian RV
strains for all of the 11 gene segments. Furthermore, we generated
recombinant viruses lacking the C-terminal region of the viral nonstructural protein NSP1 and used it to define the biological functionof NSP1 (Figure 2). The truncation mutant lacking the C-terminal
166 amino acids of NSP1 replicated poorly, suggesting that theC-terminal region of NSP1 plays critical roles in viral replication(Figure 2).

▍ Significance of the research and Future perspective
Reverse genetics, an approach used to generate viruses fromcloned cDNA, has increased our understanding of virus biology.
Here, we developed a reverse genetics system for the human RV
strain Odelia, which replicates efficiently and is suitable for in vitro
molecular studies. The system will allow generation of engineered
recombinant virus harboring desired mutations, increase our understanding of the molecular biology of human RV, and facilitate
development of novel therapeutics and vaccines.

Figure 2. Generation of recombinant viruses lacking C-terminal region of NSP1. (A)Schematic presentation of the plasmid encoding the NSP1 gene used for recovery of recombinant virus. (B) Electropherotype of rsOdelia-NSP1-Δ166. (C) Viral
replication of NSP1 mutant in MA104 cells and HT29 cells.

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P a t e n t Japanese Patent No.6762070, No.6944213
Kawagishi, Takahiro et al. Reverse genetics system for a human group A rotavirus. J Virol. 2020 Jan 6; 94(2): e00963-19. doi: 10.1128/JVI.00963-19.
T r e a t i s e Kanai, Yuta et al. Entirely plasmid-based reverse genetics system for rotaviruses. Proc Natl Acad Sci USA. 2017 Feb 28; 114(9): 2349-2354. doi: 10.1073/pnas.1618424114.
URLK e y w o r d rotavirus, vaccine, reverse genetics system