Reverse Genetics System for a Human Group A Rotavirus

概要

〔目的(Purpose)〕
Group A Rotavirus (RVA) is a major cause of acute gastroenteritis in infants and young children worldwide. Recently, we established the first entirely plasmid-based reverse genetics system for simian RV. Al though the system was robust enough to generate reassortant RV strain with human RV gene segments, a complete reverse genetics system for human RV strains is required to improve the current understanding of biological differences between RV strains in human and other animals. Therefore, we established a reverse genetic system for RVA human strain Odelia G4P [8] and used for NSP1 protein study.

〔方法ならびに成績(Method s/Results)〕
Eleven gene segments from Odelia strain were cloned between T7 promoter and hepatitis delta virus ribozyme sequence and transfected into BHK cells expressing T7 RNA polymerase, as well as expression plasmid encoding vaccinia capping enzyme subunits DIR and D12L. Nelson Bay reovirus fusion associated small transmembrane (FAST) protein, and SAI1 NSP2 and NSP5. Following transfection, cell lysates were transferred to MA 104 for rescue of recombinant virus.

〔総括(Conclusion)〕
A recombinant strain of human RVA Odelia strain (rsOdelia) was rescued. RNA electropherotypes of dsRNA genome and viral growth kinetics were indistinguishable between rsOdel ia and parental virus. To examine the versatility of the reverse genelics system, mono-reassortant viruses were generated in both the SA11 backbone and Odel ia backbone. Replication of some viruses was similar to rsOdelia, while the others showed lower replication. We generated an Odelia NSP1 that lacked 13 C-terminal amino acids (rsOdelia-NSP1-Δc13), thus abolishing the DSG(I)S motif. β-TrCP was found to be degraded by ful1length rsOdel ia-NSPl, but not by rsOdelia-NSP1-Ac13, implying that DSG(I)S is important for β-TrCP degradation. However, the deletion of DSG(I)S did not affect virus replication compared to rsOdelia. This result suggests that another interferon evasion mechanism may occur during human strain RVA infection, besides β-TrCP degradation.