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Although the construction of recombinant viruses carrying reporter genes encoding fluorescent and bioluminescent proteins has been reported, the stable insertion of foreign genes into viral genomes retaining infectivity remains difficult. Here, we applied the 11-amino-acid subunit derived from NanoLuc luciferase to the engineering of the\n Flaviviridae<\/jats:named-content>\n viruses and then examined the biological characteristics of the viruses. We successfully generated recombinant viruses carrying the split-luciferase gene, including dengue virus, Japanese encephalitis virus, hepatitis C virus (HCV), and bovine viral diarrhea virus. The stability of the viruses was confirmed by five rounds of serial passages in the respective susceptible cell lines. The propagation of the recombinant luciferase viruses in each cell line was comparable to that of the parental viruses. By using a purified counterpart luciferase protein, this split-luciferase assay can be applicable in various cell lines, even when it is difficult to transduce the counterpart gene. The efficacy of antiviral reagents against the recombinant viruses could be monitored by the reduction of luciferase expression, which was correlated with that of viral RNA, and the recombinant HCV was also useful to examine viral dynamics\n in vivo<\/jats:italic>\n . Taken together, our findings indicate that the recombinant\n Flaviviridae<\/jats:named-content>\n viruses possessing the split NanoLuc luciferase gene generated here provide powerful tools to understand viral life cycle and pathogenesis and a robust platform to develop novel antivirals against\n Flaviviridae<\/jats:named-content>\n viruses.\n <\/jats:p>\n \n IMPORTANCE<\/jats:bold>\n The construction of reporter viruses possessing a stable transgene capable of expressing specific signals is crucial to investigations of viral life cycle and pathogenesis and the development of antivirals. However, it is difficult to maintain the stability of a large foreign gene, such as those for fluorescence and bioluminescence, after insertion into a viral genome. Here, we successfully generated recombinant\n Flaviviridae<\/jats:named-content>\n viruses carrying the 11-amino-acid subunit derived from NanoLuc luciferase and demonstrated that these viruses are applicable to\n in vitro<\/jats:italic>\n and\n in vivo<\/jats:italic>\n experiments, suggesting that these recombinant\n Flaviviridae<\/jats:named-content>\n viruses are powerful tools for increasing our understanding of viral life cycle and pathogenesis and that these recombinant viruses will provide a robust platform to develop antivirals against\n Flaviviridae<\/jats:named-content>\n viruses.\n <\/jats:p>","DOI":"10.1128\/jvi.01582-17","type":"journal-article","created":{"date-parts":[[2017,11,1]],"date-time":"2017-11-01T20:17:05Z","timestamp":1509567425000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":69,"title":["Characterization of Recombinant Flaviviridae Viruses Possessing a Small Reporter Tag"],"prefix":"10.1128","volume":"92","author":[{"given":"Tomokazu","family":"Tamura","sequence":"first","affiliation":[{"name":"Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Takasuke","family":"Fukuhara","sequence":"additional","affiliation":[{"name":"Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Takuro","family":"Uchida","sequence":"additional","affiliation":[{"name":"Department of Gastroenterology and Metabolism, Applied Life Sciences, Institute of Biomedical & Health Sciences, Hiroshima University, Hiroshima, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chikako","family":"Ono","sequence":"additional","affiliation":[{"name":"Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hiroyuki","family":"Mori","sequence":"additional","affiliation":[{"name":"Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Asuka","family":"Sato","sequence":"additional","affiliation":[{"name":"Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuzy","family":"Fauzyah","sequence":"additional","affiliation":[{"name":"Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Toru","family":"Okamoto","sequence":"additional","affiliation":[{"name":"Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Takeshi","family":"Kurosu","sequence":"additional","affiliation":[{"name":"Department of Virology I, National Institute of Infectious Diseases, Tokyo, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yin Xiang","family":"Setoh","sequence":"additional","affiliation":[{"name":"Australian Infectious Diseases Research Centre, School of Chemistry and Molecular Biosciences, University of Queensland, St. Lucia, Queensland, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michio","family":"Imamura","sequence":"additional","affiliation":[{"name":"Department of Gastroenterology and Metabolism, Applied Life Sciences, Institute of Biomedical & Health Sciences, Hiroshima University, Hiroshima, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Norbert","family":"Tautz","sequence":"additional","affiliation":[{"name":"Institute of Virology and Cell Biology, University of L\u00fcbeck, L\u00fcbeck, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yoshihiro","family":"Sakoda","sequence":"additional","affiliation":[{"name":"Laboratory of Microbiology, Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University, Hokkaido, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6206-6935","authenticated-orcid":false,"given":"Alexander A.","family":"Khromykh","sequence":"additional","affiliation":[{"name":"Australian Infectious Diseases Research Centre, School of Chemistry and Molecular Biosciences, University of Queensland, St. Lucia, Queensland, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kazuaki","family":"Chayama","sequence":"additional","affiliation":[{"name":"Department of Gastroenterology and Metabolism, Applied Life Sciences, Institute of Biomedical & Health Sciences, Hiroshima University, Hiroshima, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yoshiharu","family":"Matsuura","sequence":"additional","affiliation":[{"name":"Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_2_2_2","volume-title":"Fields virology","author":"Lindenbach BD MC","year":"2013","unstructured":"Lindenbach BD MC, Thiel HJ, Rice CM. 2013. 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