Visualizing Sacbrood Virus of Honey Bees via Transformation and Coupling with Enhanced Green Fluorescent Protein
Sacbrood virus (SBV) of honey bees is a picornavirus in the genus <i>Iflavirus</i>. Given its relatively small and simple genome structure, single positive-strand RNA with only one ORF, cloning the full genomic sequence is not difficult. However, adding nonsynonymous mutations to the bee...
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MDPI AG
2020-02-01
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| Series: | Viruses |
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| Online Access: | https://www.mdpi.com/1999-4915/12/2/224 |
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| author | Lang Jin Shahid Mehmood Giikailang Zhang Yuwei Song Songkun Su Shaokang Huang Heliang Huang Yakun Zhang Haiyang Geng Wei-Fone Huang |
| author_facet | Lang Jin Shahid Mehmood Giikailang Zhang Yuwei Song Songkun Su Shaokang Huang Heliang Huang Yakun Zhang Haiyang Geng Wei-Fone Huang |
| author_sort | Lang Jin |
| collection | DOAJ |
| description | Sacbrood virus (SBV) of honey bees is a picornavirus in the genus <i>Iflavirus</i>. Given its relatively small and simple genome structure, single positive-strand RNA with only one ORF, cloning the full genomic sequence is not difficult. However, adding nonsynonymous mutations to the bee iflavirus clone is difficult because of the lack of information about the viral protein processes. Furthermore, the addition of a reporter gene to the clones has never been accomplished. In preliminary trials, we found that the site between 3′ untranslated region (UTR) and poly(A) can retain added sequences. We added enhanced green fluorescent protein (EGFP) expression at this site, creating a SBV clone with an expression tag that does not affect virus genes. An intergenic region internal ribosome entry site (IRES) from Black queen cell virus (BQCV) was inserted to initiate EGFP expression. The SBV-IRES-EGFP clone successfully infected <i>Apis cerana</i> and <i>Apis mellifera</i>, and in <i>A. cerana</i> larvae, it was isolated and passaged using oral inoculation. The inoculated larvae had higher mortality and the dead larvae showed sacbrood symptoms. The added IRES-EGFP remained in the clone through multiple passages and expressed the expected EGFP in all infected bees. We demonstrated the ability to add gene sequences in the site between 3′-UTR and poly(A) in SBV and the potential to do so in other bee iflaviruses; however, further investigations of the mechanisms are needed. A clone with a desired protein expression reporter will be a valuable tool in bee virus studies. |
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| format | Article |
| id | doaj.art-6d92991a795248c6a67a11d8ff2337f5 |
| institution | Directory Open Access Journal |
| issn | 1999-4915 |
| language | English |
| last_indexed | 2024-04-13T21:04:41Z |
| publishDate | 2020-02-01 |
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| series | Viruses |
| spelling | doaj.art-6d92991a795248c6a67a11d8ff2337f52022-12-22T02:30:00ZengMDPI AGViruses1999-49152020-02-0112222410.3390/v12020224v12020224Visualizing Sacbrood Virus of Honey Bees via Transformation and Coupling with Enhanced Green Fluorescent ProteinLang Jin0Shahid Mehmood1Giikailang Zhang2Yuwei Song3Songkun Su4Shaokang Huang5Heliang Huang6Yakun Zhang7Haiyang Geng8Wei-Fone Huang9College of Animal Science (College of Bee Science), Fujian Agriculture and Forestry University, Fuzhou 350002, ChinaCollege of Animal Science (College of Bee Science), Fujian Agriculture and Forestry University, Fuzhou 350002, ChinaCollege of Animal Science (College of Bee Science), Fujian Agriculture and Forestry University, Fuzhou 350002, ChinaCollege of Animal Science (College of Bee Science), Fujian Agriculture and Forestry University, Fuzhou 350002, ChinaCollege of Animal Science (College of Bee Science), Fujian Agriculture and Forestry University, Fuzhou 350002, ChinaCollege of Animal Science (College of Bee Science), Fujian Agriculture and Forestry University, Fuzhou 350002, ChinaCollege of Animal Science (College of Bee Science), Fujian Agriculture and Forestry University, Fuzhou 350002, ChinaCollege of Animal Science (College of Bee Science), Fujian Agriculture and Forestry University, Fuzhou 350002, ChinaCollege of Animal Science (College of Bee Science), Fujian Agriculture and Forestry University, Fuzhou 350002, ChinaCollege of Animal Science (College of Bee Science), Fujian Agriculture and Forestry University, Fuzhou 350002, ChinaSacbrood virus (SBV) of honey bees is a picornavirus in the genus <i>Iflavirus</i>. Given its relatively small and simple genome structure, single positive-strand RNA with only one ORF, cloning the full genomic sequence is not difficult. However, adding nonsynonymous mutations to the bee iflavirus clone is difficult because of the lack of information about the viral protein processes. Furthermore, the addition of a reporter gene to the clones has never been accomplished. In preliminary trials, we found that the site between 3′ untranslated region (UTR) and poly(A) can retain added sequences. We added enhanced green fluorescent protein (EGFP) expression at this site, creating a SBV clone with an expression tag that does not affect virus genes. An intergenic region internal ribosome entry site (IRES) from Black queen cell virus (BQCV) was inserted to initiate EGFP expression. The SBV-IRES-EGFP clone successfully infected <i>Apis cerana</i> and <i>Apis mellifera</i>, and in <i>A. cerana</i> larvae, it was isolated and passaged using oral inoculation. The inoculated larvae had higher mortality and the dead larvae showed sacbrood symptoms. The added IRES-EGFP remained in the clone through multiple passages and expressed the expected EGFP in all infected bees. We demonstrated the ability to add gene sequences in the site between 3′-UTR and poly(A) in SBV and the potential to do so in other bee iflaviruses; however, further investigations of the mechanisms are needed. A clone with a desired protein expression reporter will be a valuable tool in bee virus studies.https://www.mdpi.com/1999-4915/12/2/224chinese sacbroodiflaviruscloned virusexpression tag3′-utr |
| spellingShingle | Lang Jin Shahid Mehmood Giikailang Zhang Yuwei Song Songkun Su Shaokang Huang Heliang Huang Yakun Zhang Haiyang Geng Wei-Fone Huang Visualizing Sacbrood Virus of Honey Bees via Transformation and Coupling with Enhanced Green Fluorescent Protein Viruses chinese sacbrood iflavirus cloned virus expression tag 3′-utr |
| title | Visualizing Sacbrood Virus of Honey Bees via Transformation and Coupling with Enhanced Green Fluorescent Protein |
| title_full | Visualizing Sacbrood Virus of Honey Bees via Transformation and Coupling with Enhanced Green Fluorescent Protein |
| title_fullStr | Visualizing Sacbrood Virus of Honey Bees via Transformation and Coupling with Enhanced Green Fluorescent Protein |
| title_full_unstemmed | Visualizing Sacbrood Virus of Honey Bees via Transformation and Coupling with Enhanced Green Fluorescent Protein |
| title_short | Visualizing Sacbrood Virus of Honey Bees via Transformation and Coupling with Enhanced Green Fluorescent Protein |
| title_sort | visualizing sacbrood virus of honey bees via transformation and coupling with enhanced green fluorescent protein |
| topic | chinese sacbrood iflavirus cloned virus expression tag 3′-utr |
| url | https://www.mdpi.com/1999-4915/12/2/224 |
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