A research group led by Assistant Professor Tomohiro Kotaki, Assistant Professor Yuki Akieda, Professor Tohru Ishitani, and Professor Takeshi Kobayashi from the University of Osaka's Research Institute for Microbial Diseases, in collaboration with Wakayama Medical University and the Osaka Institute of Public Health, has announced the successful artificial synthesis of human norovirus using zebrafish. This was achieved using zebrafish embryos (eggs). The group confirmed that serial passage is possible and that mutations can be introduced at will. This also marks the first demonstration that human pathogenic viruses can be artificially synthesized in fish embryos. The findings are expected to serve as a foundation for understanding infection mechanisms and vaccine development. The results were published in the Proceedings of the National Academy of Sciences (PNAS) December 4 issue.
Provided by the University of Osaka's Research Institute for Microbial Diseases
Norovirus is a highly infectious RNA virus that causes food poisoning resulting in diarrhea and vomiting, and urgent public health measures are needed from the perspective of medical costs. With many genetic groups and genotypes, it infects 700 million people worldwide annually, causing approximately 200,000 deaths each year, mainly in developing countries. However, research on norovirus has not progressed due to the absence of both established culture systems and artificial synthesis methods.
Generally, cultured cells are used for virus replication in research, however norovirus does not replicate in cultured cells. Intestinal organoids are the only system capable of replicating it, but they are costly and replication ceases after several passages. Although replication in zebrafish embryos was reported about 10 years ago, it was unclear whether serial passage was possible, whether infectivity was maintained, and whether it could be used as a research platform.
RNA virus artificial synthesis technology is widely used in virus research. It introduces virus genome-derived nucleic acids into cultured cells to synthesize infectious recombinant viruses.
The research group has previously developed artificial synthesis technologies for RNA viruses, including rotavirus.
During this study, they first verified whether cDNA derived from three norovirus genotypes (GII.4, GII.2, and GII.17) would replicate when injected into zebrafish embryos using microinjection methods.
As a result, efficient virus replication was confirmed in all strains. In the case of the GII.17 genotype, it was also found that edema occurred in hatched larvae. When viruses obtained from crushing hatched larvae were serially passaged using embryos, it was verified that virus replication and at least five serial passages were possible.
Next, the researchers considered an artificial synthesis method combining cultured cells and zebrafish embryos.
They confirmed that recombinant viruses could be produced by introducing plasmids containing GII.17 norovirus cDNA (cDNA clones) into cultured cells, concentrating virus particles, and injecting them into embryos by microinjection. The resulting recombinant viruses were found to be similarly capable of serial passage and infecting intestinal organoids.
The researchers also succeeded in creating luminescent viruses by introducing foreign genes; it confirmed that evaluation of replication capacity and antiviral drugs is possible.
They even examined further efficiency improvements. The results revealed recombinant viruses can be efficiently produced by directly injecting norovirus cDNA clones into zebrafish embryos by microinjection without going through cultured cells.
This research also succeeded in producing viruses of the GII.4 genotype, which is the predominant epidemic type in humans. It was confirmed that the virus begins replication within 2 days of injection and that serial passage is possible.
Kobayashi stated: "It is conceivable that our success in artificially synthesizing norovirus using zebrafish presents a new area of application in artificially creating viruses in small fish research. Furthermore, this technology is expected to contribute broadly to elucidating norovirus replication mechanisms and developing vaccines and therapeutic drugs. By advancing the development of norovirus therapeutics and vaccines, we hope to develop research that can contribute to people's health and welfare in the future."
Journal Information
Publication: PNAS
Title: Recovery of infectious recombinant human norovirus using zebrafish embryos
DOI: 10.1073/pnas.2526726122
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