Engineering human/simian rotavirus VP7 reassortants in the absence of UTR sequence information

Recently developed plasmid-based reverse genetics systems for rotavirus A (RVA) enable rapid engineering of reassortants carrying human RVA antigens. However, complete genome segment sequences are required for successful generation of such reassortants, and sequencing of the untranslated regions (UTRs) of field strains is often not accomplished. To address this problem, we established a system that permits the generation of reassortants using only the open reading frame (ORF) nucleotide sequence information. Plasmids containing the VP7-ORF nucleotide sequence of six human RVA field strains (genotypes G2, G5, G8, G9, G12 and G29) derived from GenBank and flanked by the UTR sequences of simian RVA strain SA11 were constructed. Using these plasmids, four VP7 (G2, G5, G9 and G12) reassortants in an SA11 backbone were successfully generated. In contrast, the G8 and G29 reassortants were not viable. BLASTp search of the G8 and G29 sequences revealed an unusual amino acid substitution in each sequence, which was not present in related field strains. Site-directed reversion of the corresponding C656T mutation in G8 led to effective rescue of reassortant virus. However, reverting the G84C mutation in G29 did not result in replicating virus. The results suggest that most human RVA VP7 UTRs can be substituted with simian RVA UTRs. However, generation of reassortants might be impeded by potential sequencing errors or intrinsic reassortment limitations. The established system could help to broaden the antigenic repertoire for rapid engineering of potential novel RVA vaccine strains. KEY POINTS: • Generation of diverse rotavirus vaccine strains is impeded by missing UTR sequences. • UTRs from SA11 can be used instead of missing UTR sequences from field strains. • Human RVA reassortants of genotypes G2, G5, G8, G9, G12 were successfully rescued.