Characterization of Recombinant Flaviviridae Viruses Possessing a Small Reporter Tag

The family consists of four genera, , , , and , and comprises important pathogens of human and animals. 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 genome...

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Published inJournal of virology Vol. 92; no. 2
Main Authors Tamura, Tomokazu, Fukuhara, Takasuke, Uchida, Takuro, Ono, Chikako, Mori, Hiroyuki, Sato, Asuka, Fauzyah, Yuzy, Okamoto, Toru, Kurosu, Takeshi, Setoh, Yin Xiang, Imamura, Michio, Tautz, Norbert, Sakoda, Yoshihiro, Khromykh, Alexander A, Chayama, Kazuaki, Matsuura, Yoshiharu
Format Journal Article
LanguageEnglish
Published United States American Society for Microbiology 15.01.2018
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Abstract The family consists of four genera, , , , and , and comprises important pathogens of human and animals. 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 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 Taken together, our findings indicate that the recombinant 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 viruses. 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 viruses carrying the 11-amino-acid subunit derived from NanoLuc luciferase and demonstrated that these viruses are applicable to and experiments, suggesting that these recombinant 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 viruses.
AbstractList The family consists of four genera, , , , and , and comprises important pathogens of human and animals. 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 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 Taken together, our findings indicate that the recombinant 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 viruses. 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 viruses carrying the 11-amino-acid subunit derived from NanoLuc luciferase and demonstrated that these viruses are applicable to and experiments, suggesting that these recombinant 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 viruses.
The family Flaviviridae consists of four genera, Flavivirus, Pestivirus, Pegivirus, and Hepacivirus, and comprises important pathogens of human and animals. 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 Flaviviridae 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 in vivo Taken together, our findings indicate that the recombinant Flaviviridae 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 Flaviviridae viruses.IMPORTANCE 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 Flaviviridae viruses carrying the 11-amino-acid subunit derived from NanoLuc luciferase and demonstrated that these viruses are applicable to in vitro and in vivo experiments, suggesting that these recombinant Flaviviridae 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 Flaviviridae viruses.
The family Flaviviridae consists of four genera, Flavivirus , Pestivirus , Pegivirus , and Hepacivirus , and comprises important pathogens of human and animals. 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 Flaviviridae 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 in vivo . Taken together, our findings indicate that the recombinant Flaviviridae 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 Flaviviridae viruses. IMPORTANCE 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 Flaviviridae viruses carrying the 11-amino-acid subunit derived from NanoLuc luciferase and demonstrated that these viruses are applicable to in vitro and in vivo experiments, suggesting that these recombinant Flaviviridae 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 Flaviviridae viruses.
Author Tamura, Tomokazu
Sakoda, Yoshihiro
Imamura, Michio
Sato, Asuka
Khromykh, Alexander A
Fukuhara, Takasuke
Kurosu, Takeshi
Ono, Chikako
Okamoto, Toru
Fauzyah, Yuzy
Setoh, Yin Xiang
Uchida, Takuro
Mori, Hiroyuki
Chayama, Kazuaki
Matsuura, Yoshiharu
Tautz, Norbert
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  givenname: Yoshihiro
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  organization: Laboratory of Microbiology, Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University, Hokkaido, Japan
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  organization: Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan fukut@biken.osaka-u.ac.jp matsuura@biken.osaka-u.ac.jp
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Citation Tamura T, Fukuhara T, Uchida T, Ono C, Mori H, Sato A, Fauzyah Y, Okamoto T, Kurosu T, Setoh YX, Imamura M, Tautz N, Sakoda Y, Khromykh AA, Chayama K, Matsuura Y. 2018. Characterization of recombinant Flaviviridae viruses possessing a small reporter Tag. J Virol 92:e01582-17. https://doi.org/10.1128/JVI.01582-17.
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Snippet The family consists of four genera, , , , and , and comprises important pathogens of human and animals. Although the construction of recombinant viruses...
The family Flaviviridae consists of four genera, Flavivirus , Pestivirus , Pegivirus , and Hepacivirus , and comprises important pathogens of human and...
The family Flaviviridae consists of four genera, Flavivirus, Pestivirus, Pegivirus, and Hepacivirus, and comprises important pathogens of human and animals....
SourceID pubmedcentral
proquest
crossref
pubmed
SourceType Open Access Repository
Aggregation Database
Index Database
SubjectTerms Animals
Antiviral Agents - pharmacology
Cell Line
Dose-Response Relationship, Drug
Drug Discovery
Drug Evaluation, Preclinical
Flaviviridae - drug effects
Flaviviridae - genetics
Gene Expression
Genes, Reporter
Genome, Viral
Hepacivirus - genetics
Humans
Mice
Mutagenesis, Insertional
Recombination, Genetic
Virus-Cell Interactions
Title Characterization of Recombinant Flaviviridae Viruses Possessing a Small Reporter Tag
URI https://www.ncbi.nlm.nih.gov/pubmed/29093094
https://search.proquest.com/docview/1959321974
https://pubmed.ncbi.nlm.nih.gov/PMC5752933
Volume 92
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