Production of OSU G5P Porcine Rotavirus Expressing a Fluorescent Reporter by Reverse Genetics

Abstract

Rotaviruses are a significant cause of severe, potentially life-threatening gastroenteritis in the young of many economically important animals. Although vaccines against porcine rotavirus exist, both live oral and inactivated, their effectiveness in preventing gastroenteritis is less than ideal. Thus, a need remains for the development of new generations of porcine rotavirus vaccines. The Ohio State University (OSU) rotavirus strain represents a Rotavirus A species with a G5P[7] genotype, the genotype most frequently associated with rotavirus disease in piglets. Using complete genome sequences that were determined by Nanopore sequencing, we developed a robust reverse genetics system enabling recovery of recombinant (r)OSU rotavirus. Although rOSU grew to high titers ([~]107 plaque-forming units/ml), its growth kinetics were modestly decreased in comparison to laboratory-adapted OSU virus. The reverse genetics system was used to generate rOSU rotavirus that served as an expression vector for foreign protein. Specifically, by engineering a fused NSP3-2A-UnaG open reading frame into the segment 7 RNA, we produced genetically stable rOSU that expressed the fluorescent UnaG protein as a functional separate product. Together, these findings raise the possibility of producing improved live oral porcine rotavirus vaccines through reverse genetics-based modification or combination porcine rotavirus vaccines that can express neutralizing antigens of other porcine enteric diseases.