Rescue a Reassortant Human-Bovine Rotavirus and a Recombinant NSP3-EGFP Rotavirus Serotype G1

Human group A rotaviruses (RVs), with an 11-segmented double-stranded RNA genome, are a cause of viral gastroenteritis in infants and young children. Despite the availability of effective live-attenuated vaccines, RV infection is still a global health challenge. Hence, a reverse genetics system enabling the manipulation of RV segments, such as genome reassortment, is essential for developing new RV vaccines. Herein, a reverse genetics system was used to rescue a reassortant human-bovine RV, incorporating the VP7 gene segment from the human RV strain Wa (serotype G1) and a recombinant RV.

To rescue a reassortant human-bovine RV, we used a reverse genetics system in which 13 plasmid constructs were co-transfected into BHK-T7 cells. In this method, 10 plasmids were derived from bovine RV, one encoded the human RV VP7 segment of serotype G1, and two expressed the capping enzyme subunits of vaccinia virus. We also created a recombinant RV featuring enhanced green fluorescent protein (EGFP) by replacing the bovine NSP3 plasmid with an NSP3-EGFP construct, where EGFP expression was regulated by an internal ribosomal entry site.

Our results showed successful rescue of the reassortant human-bovine RV and the recombinant strain, with the NSP3-EGFP construct facilitating EGFP translation across multiple RV passages. Replacing the NSP3 gene segment of bovine RV with the release of a recombinant RV enabled EGFP expression in several RV passages.

Our study establishes the feasibility of using a reverse genetics system to generate a live-attenuated RV vaccine candidate, potentially enhancing future control efforts against RV infections.