CRISPR/Cas9-based generation of a recombinant double-reporter pseudorabies virus and its characterization in vitro and in vivo
Abstract Pseudorabies, caused by pseudorabies virus (PRV) variants, has broken out among commercial PRV vaccine-immunized swine herds and resulted in major economic losses to the pig industry in China since late 2011. However, the mechanism of virulence enhancement of variant PRV is currently unclea...
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| Format: | Article |
| Language: | English |
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BMC
2021-06-01
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| Series: | Veterinary Research |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s13567-021-00964-4 |
| _version_ | 1819083840729120768 |
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| author | Peng-Fei Fu Xuan Cheng Bing-Qian Su Li-Fang Duan Cong-Rong Wang Xin-Rui Niu Jiang Wang Guo-Yu Yang Bei-Bei Chu |
| author_facet | Peng-Fei Fu Xuan Cheng Bing-Qian Su Li-Fang Duan Cong-Rong Wang Xin-Rui Niu Jiang Wang Guo-Yu Yang Bei-Bei Chu |
| author_sort | Peng-Fei Fu |
| collection | DOAJ |
| description | Abstract Pseudorabies, caused by pseudorabies virus (PRV) variants, has broken out among commercial PRV vaccine-immunized swine herds and resulted in major economic losses to the pig industry in China since late 2011. However, the mechanism of virulence enhancement of variant PRV is currently unclear. Here, a recombinant PRV (rPRV HN1201-EGFP-Luc) with stable expression of enhanced green fluorescent protein (EGFP) and firefly luciferase as a double reporter virus was constructed on the basis of the PRV variant HN1201 through CRISPR/Cas9 gene-editing technology coupled with two sgRNAs. The biological characteristics of the recombinant virus and its lethality to mice were similar to those of the parental strain and displayed a stable viral titre and luciferase activity through 20 passages. Moreover, bioluminescence signals were detected in mice at 12 h after rPRV HN1201-EGFP-Luc infection. Using the double reporter PRV, we also found that 25-hydroxycholesterol had a significant inhibitory effect on PRV both in vivo and in vitro. These results suggested that the double reporter PRV based on PRV variant HN1201 should be an excellent tool for basic virology studies and evaluating antiviral agents. |
| first_indexed | 2024-12-21T20:38:58Z |
| format | Article |
| id | doaj.art-738a57ecbdce484fa74c5192696d2ab6 |
| institution | Directory Open Access Journal |
| issn | 1297-9716 |
| language | English |
| last_indexed | 2024-12-21T20:38:58Z |
| publishDate | 2021-06-01 |
| publisher | BMC |
| record_format | Article |
| series | Veterinary Research |
| spelling | doaj.art-738a57ecbdce484fa74c5192696d2ab62022-12-21T18:51:01ZengBMCVeterinary Research1297-97162021-06-0152111510.1186/s13567-021-00964-4CRISPR/Cas9-based generation of a recombinant double-reporter pseudorabies virus and its characterization in vitro and in vivoPeng-Fei Fu0Xuan Cheng1Bing-Qian Su2Li-Fang Duan3Cong-Rong Wang4Xin-Rui Niu5Jiang Wang6Guo-Yu Yang7Bei-Bei Chu8College of Veterinary Medicine, Henan Agricultural UniversityCollege of Veterinary Medicine, Henan Agricultural UniversityCollege of Veterinary Medicine, Henan Agricultural UniversityCollege of Veterinary Medicine, Henan Agricultural UniversityCollege of Veterinary Medicine, Henan Agricultural UniversityCollege of Veterinary Medicine, Henan Agricultural UniversityCollege of Veterinary Medicine, Henan Agricultural UniversityCollege of Veterinary Medicine, Henan Agricultural UniversityCollege of Veterinary Medicine, Henan Agricultural UniversityAbstract Pseudorabies, caused by pseudorabies virus (PRV) variants, has broken out among commercial PRV vaccine-immunized swine herds and resulted in major economic losses to the pig industry in China since late 2011. However, the mechanism of virulence enhancement of variant PRV is currently unclear. Here, a recombinant PRV (rPRV HN1201-EGFP-Luc) with stable expression of enhanced green fluorescent protein (EGFP) and firefly luciferase as a double reporter virus was constructed on the basis of the PRV variant HN1201 through CRISPR/Cas9 gene-editing technology coupled with two sgRNAs. The biological characteristics of the recombinant virus and its lethality to mice were similar to those of the parental strain and displayed a stable viral titre and luciferase activity through 20 passages. Moreover, bioluminescence signals were detected in mice at 12 h after rPRV HN1201-EGFP-Luc infection. Using the double reporter PRV, we also found that 25-hydroxycholesterol had a significant inhibitory effect on PRV both in vivo and in vitro. These results suggested that the double reporter PRV based on PRV variant HN1201 should be an excellent tool for basic virology studies and evaluating antiviral agents.https://doi.org/10.1186/s13567-021-00964-4Pseudorabies virusFirefly luciferaseEGFPCRISPR/Cas9Imaging in vivo25-hydroxycholesterol |
| spellingShingle | Peng-Fei Fu Xuan Cheng Bing-Qian Su Li-Fang Duan Cong-Rong Wang Xin-Rui Niu Jiang Wang Guo-Yu Yang Bei-Bei Chu CRISPR/Cas9-based generation of a recombinant double-reporter pseudorabies virus and its characterization in vitro and in vivo Veterinary Research Pseudorabies virus Firefly luciferase EGFP CRISPR/Cas9 Imaging in vivo 25-hydroxycholesterol |
| title | CRISPR/Cas9-based generation of a recombinant double-reporter pseudorabies virus and its characterization in vitro and in vivo |
| title_full | CRISPR/Cas9-based generation of a recombinant double-reporter pseudorabies virus and its characterization in vitro and in vivo |
| title_fullStr | CRISPR/Cas9-based generation of a recombinant double-reporter pseudorabies virus and its characterization in vitro and in vivo |
| title_full_unstemmed | CRISPR/Cas9-based generation of a recombinant double-reporter pseudorabies virus and its characterization in vitro and in vivo |
| title_short | CRISPR/Cas9-based generation of a recombinant double-reporter pseudorabies virus and its characterization in vitro and in vivo |
| title_sort | crispr cas9 based generation of a recombinant double reporter pseudorabies virus and its characterization in vitro and in vivo |
| topic | Pseudorabies virus Firefly luciferase EGFP CRISPR/Cas9 Imaging in vivo 25-hydroxycholesterol |
| url | https://doi.org/10.1186/s13567-021-00964-4 |
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