Rapid generation of rotavirus single-gene reassortants by means of eleven plasmid-only based reverse genetics

Abstract

Reassortment is an important mechanism in the evolution of group A rotaviruses (RVAs), yielding viruses with novel genetic and phenotypic traits. The classical methods for generating RVA reassortants with the desired genetic combinations are laborious and time-consuming because of the screening and selection processes required to isolate a desired reassortant. Taking advantage of a recently developed RVA reverse genetics system based on just 11 cloned cDNAs encoding the RVA genome (11 plasmid-only system), we prepared a panel of simian SA11-L2 virus-based single-gene reassortants, each containing 1 segment derived from human KU virus of the G1P[8] genotype. It was shown that there was no gene-specific restriction of the reassortment potential. In addition to these 11 single-gene reassortants, a triple-gene reassortant with KU-derived core-encoding VP1–3 gene segments with the SA11-L2 genetic background, which make up a virion composed of the KU-based core, and SA11-L2-based intermediate and outer layers, could also be prepared with the 11 plasmid-only system. Finally, for possible clinical application of this system, we generated a series of VP7 reassortants representing all the major human RVA G genotypes (G1–4, G9 and G12) efficiently. The preparation of each of these single-gene reassortants was achieved within just 2 weeks. Our results demonstrate that the 11 plasmid-only system allows the rapid and reliable generation of RVA single-gene reassortants, which will be useful for basic research and clinical applications.

Strategy for generation of replication–competent recombinant rotaviruses expressing multiple foreign genes

“…RVA replication kinetics was determined as described previously [26]. Briefly, monolayers of MA104 cells in 12-well plates (Falcon) were infected in triplicate with trypsin-pretreated recombinant RVAs at an MOI of 0.01, washed twice with incomplete medium, and then incubated in incomplete medium with trypsin (1 µg ml −1 ) for various numbers of hours.…”

“…A set of 11 rescue T7 plasmids for SA11-L2 virus had been prepared in our laboratory [5]. Recombinant RVAs entirely derived from cloned cDNAs were generated according to the protocol described for the 11 plasmid-only based reverse genetics system [5,6,26]. In brief, the procedure was as follows: monolayers of BHK/T7-9 cells in 6-well plates (Falcon) were cotransfected with 11 T7 plasmids, encoding the cloned cDNAs of the SA11-L2 11-segment genome, pT7/VP1SA11 (0.75 µg), pT7/VP2SA11 (0.75 µg), pT7/ VP3SA11 (0.75 µg), pT7/VP4SA11-ΔPstI (0.75 µg), pT7/ VP6SA11 (0.75 µg), pT7/VP7SA11 (0.75 µg), pT7/NSP2SA11 (2.25 µg), pT7/NSP3SA11 (0.75 µg), pT7/NSP4SA11 (0.75 µg), and pT7/NSP5SA11 (2.25 µg), in combination with pT7/NSP1SA11 (0.75 µg), pT7/NSP1-Nluc (0.75 µg), pT7/ NSP1(222)-Nluc-(222) (0.75 µg), or pT7/NSP1(222)-Nluc-EGFP-mCherry-(222) (0.75 µg).…”

Recent advances in rotavirus reverse genetics and its utilization in basic research and vaccine development

“…Prior to the development of an efficient plasmid-only-based RGS for RVs, studying the functions of viral proteins and addressing the mechanism of viral replication depended largely on gene reassortment experiments involving coinfection of the same cells with two parental RV strains. New RV reassortants were selected in a random fashion for further genotype-phenotype studies, which was labor-intensive and time-consuming [ 88 ]. In some cases, mono-gene-reassortant viruses were difficult to generate due to the different replication levels of the two parental viruses.…”

“…Recent RGSs for RVs have decreased the time and effort required to generate desirable reassortant viruses from months to weeks. Thus, RGSs are highly suitable for rapid generation of vaccine candidates in cases of emergence of novel RV strains of clinical significance [ 88 ]. An RV RGS has been successfully used to generate human RV reassortants.…”

“…Using 11 plasmids, a panel of 11 reassortant viruses, each having a single gene segment from human strain KU in the backbone of SA11, were generated. In addition to monogenic reassortant RVs, triple gene reassortant viruses (VP1, VP2, VP3) and multiple VP7 reassortant viruses belonging to genotypes G1-G4, G9, and G12 were also produced [ 88 ]. Diverse RVs harboring genetic segments from different species, generated from forced reverse genetics experiments as discussed above, can be utilized to study the replication fitness, host range restriction, and pathogenesis of RVs.…”