Generation of Infectious Recombinant Human Rotaviruses from Just 11 Cloned cDNAs Encoding the Rotavirus Genome

Abstract

The generation of recombinant group A rotaviruses (RVAs) entirely from cloned cDNAs has been described only for a single animal RVA strain, simian SA11-L2. We recently developed an optimized RVA reverse genetics system based on only RVA cDNAs (11-plasmid system), in which the concentration of cDNA plasmids containing the NSP2 and NSP5 genes is 3- or 5-fold increased in relation to that of the other plasmids. Based on this approach, we generated a recombinant human RVA (HuRVA)-based monoreassortant virus containing the VP4 gene of the simian SA11-L2 virus using the 11-plasmid system. In addition to this monoreassortant virus, authentic HuRVA (strain KU) was also generated with the 11-plasmid system with some modifications. Our results demonstrate that the 11-plasmid system involving just RVA cDNAs can be used for the generation of recombinant HuRVA and recombinant HuRVA-based reassortant viruses. IMPORTANCE Human group A rotavirus (HuRVA) is a leading pathogen causing severe diarrhea in young children worldwide. In this paper, we describe the generation of recombinant HuRVA (strain KU) from only 11 cloned cDNAs encoding the HuRVA genome by reverse genetics. The growth properties of the recombinant HuRVA were similar to those of the parental RVA, providing a powerful tool for better understanding of HuRVA replication and pathogenesis. Furthermore, the ability to manipulate the genome of HuRVAs “to order” will be useful for next-generation vaccine production for this medically important virus and for the engineering of clinical vectors expressing any foreign genes.

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

“…To generate constructs for the rescue of VP7 reassortant viruses, T7-driven plasmids pT7/VP7S2, pT7/VP7YO, pT7/ VP7Hosokawa and pT7/VP7L26, which encode the fulllength VP7 segment of strains S2, YO, Hosokawa, and L26, respectively, each cDNA was amplified by reverse transcription-PCR (RT-PCR) from an individual genomic dsRNA with ReverTra Ace reverse transcriptase (Toyobo), PrimeStar HS DNA polymerase (TaKaRa Bio) and specific primers. As described previously [30,[35][36][37], the forward primers contain the T7 RNA polymerase promoter sequence and a sequence corresponding to the 5′ terminus of each viral segment. To create a rescue T7 plasmid, pT7/VP7WI61, which encodes the full-length VP7 segment of strain WI61, the full-length cDNA fragment of its VP7 segment was artificially synthesized by Eurofins Genomics.…”

“…After digestion with restriction enzymes, the prepared cDNAs for strains S2, YO, Hosokawa, WI61 and L26 were ligated into the corresponding restriction enzyme sites of T7 expression plasmids. As T7 expression plasmids, we employed a modified pX8dT vector [36,37,53] (for strains S2, YO, Hosokawa, and L26) and a pEXR vector [36] (for strain WI61). The constructed T7 rescue plasmids, pT7/ VP7S2, pT7/VP7YO, pT7/VP7Hosokawa, pT7/VP7WI61, and pT7/VP7L26, contain the full-length cDNAs of individual VP7 segments of strains S2, YO, Hosokawa, WI61, and L26, respectively (GenBank/EMBL/DDBJ accession nos LC482501-482505), flanked by the T7 RNA polymerase promoter and HDV ribozyme sequences [29], followed by the T7 RNA polymerase terminator sequence.…”

“…However, these helper virus-dependent reverse genetics systems require strong selection conditions to isolate a recombinant reassortant virus from the majority of helper viruses, and so their use has been restricted to only 3 of the 11 segments (VP4, NSP2 and NSP3). On the other hand, entirely plasmid-based RVA reverse genetics systems, developed recently, allow the generation of recombinant reassortants without the need for helper virus infection [34][35][36][37][38].…”

“…A primary system, developed for simian SA11-L2 virus [39], which grows to high titres, requires 3 additional helper expression plasmids (encoding the vaccinia virus-capping enzyme and small membrane fusion protein) other than the 11 RVA cDNA plasmids [34]. Shortly after the establishment of this initial method, a simple and highly efficient reverse genetics system based solely on RVA cDNAs (11 plasmid-only system), which does not require the use of any helper plasmids, was developed [36,37]. This system greatly improved our ability to manipulate the RVA genome.…”

“…It also allowed the engineering of HuRVA VP7 genes into single-gene reassortants that have much higher infectivity titres in vitro than the parental HuRVAs [40]. This 11 plasmid-only system even allowed the rescue of recombinant HuRVAs with much lower infectivity compared to that of animal RVAs [37].…”