This document describes the development of a plasmid-based reverse genetics system for rotaviruses. Key aspects include: - Rotaviruses have a genome consisting of 11 segments of double-stranded RNA that encode 6 structural and 6 nonstructural proteins. - A plasmid-based system was developed to genetically engineer rotaviruses, including constructing recombinant viruses expressing reporter genes by inserting GFP or NLuc into the NSP1 gene. - Recombinant viruses lacking the NSP6 protein were generated to study its role, and growth curves and plaque formation of these mutant viruses were analyzed with wild-type viruses as controls.

1. Rotaviruses genetic engineering through a fully
recombinant plasmid-based reverse genetics system
Asha Ann Philip
Graduate Student
Department of Biology
10-16-2017

3. Rotavirus
 Member of the Reoviridae
 Genome consists of 11 segments of dsRNA
 Genome encodes 6 structural proteins (VP1-VP4, VP6-7) and 6 nonstructural proteins (NSP1-6)

4. Fig. 1. Development of a plasmid-based reverse genetics
system for RV.

5. Experimental designs
2 day incubation in serum-free media (SFM)
(RG4-T7) (RG4?) SA11-4F RG3 SA11-4F
RG3 SA11-4F

6. Fig. 1

7. Fig. 2

8. Fig. 3

9. Fig. 4. Generation of RVs expressing reporter genes.
Construction of NSP1 genes containing the split- GFP tag or NLuc gene.
Electropherotypes of rsSA11-GFP11 and rsSA11-NLuc.
Subcellular localization of NSP1-GFP11 fusion protein In infected cells.

10. Luciferase imaging of plaques from
cells infected with rsSA11-NLuc.
Replication kinetics of rsSA11-NLuc virus.
Effect of ribavirin on rsSA11-NLuc virus infection.

12. FIG 1. Generation of the rSA11-delNSP6 virus incapable of expressing the NSP6 protein.

13. FIG 2. Infectivity of the rSA11-delNSP6 virus lacking NSP6 expression.
(A) Single-step growth curves of rSA11 and rSA11-
delNSP6.
(B) Plaque formation by rSA11 and rSA11-delNSP6.

14. FIG 3. Generation of recombinant SA11-based monoreassortant viruses having a KU-
derived segment 11 capable or incapable of NSP6 expression.
(A) Schematic presentation of the
plasmids encoding KU segment 11
(NSP5/6 genes)
PAGE of viral dsRNAs Expression of the NSP6 and other
RVA proteins in MA104 cells

15. FIG 4. Infectivity of the rSA11-s11KU-delNSP6 monoreassortant virus
lacking NSP6 expression.
(A) Single-step growth curves of rSA11-s11KU and
rSA11-s11KU-delNSP6.
(B) Plaque formation by rSA11-s11KU and rSA11-s11KU-
delNSP6

16. FIG 5. Single-step growth curves of the NSP6-deficient viruses in IFN-deficient Vero E6 cells.
(A) Vero E6 cells were infected with rSA11 or rSA11-delNSP6. (B) rSA11-s11KU or rSA11-s11KU-delNSP6

17. The comparison of two Experimental designs
2 day incubation in serum-free media (SFM)