Human Rotavirus Reverse Genetics Systems to Study Viral Replication and Pathogenesis

Figure 1. Schematic representation of the 11 plasmid-only reverse genetics system (Komoto’s reverse genetics system). BHK-T7 cells are transfected with a set of 11 rescue T7 plasmids encoding an RVA genome with 3-fold increased proportions of T7 plasmids for the NSP2 and NSP5 genes compared to the other nine T7 plasmids. Next day, the transfected BHK-T7 cells are overlaid with RVA-susceptible cells (CV-1 or MA104 cells) and then co-cultured for 3 days for amplification of recombinant RVAs. Infectious recombinant RVAs can be recovered from the co-cultures. PT7 and Rib denote the T7 RNA polymerase promoter and HDV ribozyme sequence, respectively.

Figure 2. Schematic representation of Sánchez-Tacuba’s reverse genetics system. BHK-T7 cells are transfected with a set of 11 rescue T7 plasmids encoding an RVA genome with 3-fold increased proportions of T7 plasmids for the NSP2 and NSP5 genes compared to the other nine T7 plasmids, as well as a chimeric helper expression plasmid for the African swine fever virus-capping enzyme NP868R and T7 RNA polymerase. Next day, the transfected BHK-T7 cells are overlaid with genetically modified MA104N*V cells with impaired innate immune responses and then co-cultured for 3 days for amplification of recombinant RVAs. Infectious recombinant RVAs can be rescued from the co-cultures. PT7, Rib, and PCMV denote the T7 RNA polymerase promoter, HDV ribozyme, and cytomegalovirus promoter sequence, respectively.

Figure 3. Schematic representation of Kawagishi’s reverse genetics system. BHK-T7 cells are transfected with a set of 11 rescue T7 plasmids encoding an RVA genome as well as five helper expression plasmids for the NSP2, NSP5, VV-capping enzyme, and small membrane fusion proteins. After 2 days, the transfected BHK-T7 cells are overlaid with RVA-susceptible cells (MA104 cells) and then co-cultured for 5 days for amplification of recombinant RVAs. Recombinant RVAs can be rescued from the co-cultures. PT7, Rib, PCAG, and FAST denote the T7 RNA polymerase promoter sequence, HDV ribozyme sequence, CAG promoter sequence, and small membrane fusion protein, respectively.