Department of Biology, Indiana University, 212 S. Hawthorne Drive, Simon Hall 011, Bloomington, IN 47405, USA.
Department of Electron Microscopy and Histopathology, Noguchi Memorial Institute for Medical Research, College of Health Sciences, University of Ghana, Accra 00233, Ghana.
Viruses. 2024 Mar 7;16(3):411. doi: 10.3390/v16030411.
Rotaviruses are a significant cause of severe, potentially life-threatening gastroenteritis in infants and 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, there is a need for the development of new generations of porcine rotavirus vaccines. The Ohio State University (OSU) rotavirus strain represents a species with a G5P[7] genotype, the genotype most frequently associated with rotavirus disease in piglets. Using complete genome sequences that were determined via Nanopore sequencing, we developed a robust reverse genetics system enabling the recovery of recombinant (r)OSU rotavirus. Although rOSU grew to high titers (~10 plaque-forming units/mL), its growth kinetics were modestly decreased in comparison to the laboratory-adapted OSU virus. The reverse genetics system was used to generate the rOSU rotavirus, which served as an expression vector for a foreign protein. Specifically, by engineering a fused NSP3-2A-UnaG open reading frame into the segment 7 RNA, we produced a genetically stable rOSU virus 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 for other porcine enteric diseases.
轮状病毒是导致婴儿和许多经济重要动物幼崽严重、潜在致命性胃肠炎的主要原因。虽然存在针对猪轮状病毒的疫苗，包括活口服疫苗和灭活疫苗，但它们预防胃肠炎的效果并不理想。因此，需要开发新一代的猪轮状病毒疫苗。俄亥俄州立大学（OSU）轮状病毒株代表一种 G5P[7]基因型的病毒，该基因型与仔猪轮状病毒病的关联最为密切。我们使用通过纳米孔测序确定的完整基因组序列，开发了一种强大的反向遗传学系统，能够恢复重组（r）OSU 轮状病毒。尽管 rOSU 能够达到高滴度（~10 噬菌斑形成单位/mL），但其生长动力学与实验室适应的 OSU 病毒相比略有降低。反向遗传学系统用于生成 rOSU 轮状病毒，它可作为外源蛋白的表达载体。具体来说，通过在第 7 个 RNA 片段中构建融合的 NSP3-2A-UnaG 开放阅读框，我们生产了一种遗传稳定的 rOSU 病毒，该病毒可表达荧光 UnaG 蛋白作为一种功能独立的产物。这些发现共同表明，通过基于反向遗传学的修饰或组合猪轮状病毒疫苗，有可能生产出改进的活口服猪轮状病毒疫苗，这些疫苗可以表达针对其他猪肠道疾病的中和抗原。
