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Development and in vitro application of a recombinant influenza A virus expressing enhanced green fluorescent protein
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Jia LI1, 2, Donghong WANG2, Yujie JIANG1, 2, Ruiwen HAN1, 2, Qiaohong CHU2, Tangqi WANG1, 2, Guanya LIU2, 3, Xuejie ZHANG2, 4, Baoying HUANG2, Yao DENG2, Wenjie TAN1, 2
Acta Microbiologica Sinica | 2025, 65(10) : 4607 - 4620
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Acta Microbiologica Sinica | 2025, 65(10): 4607-4620
• Research Article •
Development and in vitro application of a recombinant influenza A virus expressing enhanced green fluorescent protein
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Jia LI1, 2, Donghong WANG2, Yujie JIANG1, 2, Ruiwen HAN1, 2, Qiaohong CHU2, Tangqi WANG1, 2, Guanya LIU2, 3, Xuejie ZHANG2, 4, Baoying HUANG2, Yao DENG2, Wenjie TAN1, 2
Affiliations
  • 1Zhejiang Provincial Key Laboratory of Medical Genetics, School of Laboratory Medicine and Life Science, Wenzhou Medical University, Wenzhou, Zhejiang, China
  • 2National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases (NITFID), NHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China
  • 3School of Public Health, Baotou Medical College, Baotou, Inner Mongolia, China
  • 4School of Public Health, Xinxiang Medical University, Xinxiang, Henan, China
Published: 2025-09-04 doi: 10.13343/j.cnki.wsxb.20250234
Outline
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[Objective] To develop a recombinant influenza A virus (IAV) expressing a reporter (enhanced green fluorescent protein, EGFP), study its biological characteristics in vitro, and explore its application in antiviral drug screening and neutralizing antibody (nAb) detection. [Methods] The EGFP gene was inserted into the C-terminus of NA (derived from H1N1-PR8) by reverse genetics of influenza virus, and the recombinant IAV was successfully constructed, rescued, and named PR8NAEGFP/WSN. The EGFP insertion position and genetic stability were analyzed by RT-PCR and sequencing. PR8NAEGFP/WSN and the control virus (PR8NA/WSN, without EGFP insertion) were identified based on EGFP reporter gene expression, replication kinetics, and plaque morphology. The in vitro antiviral efficacy of favipiravir, a positive drug for influenza virus, was evaluated by focus-forming assay (FFA), qPCR, and EGFP fluorescence detection. The focus reduction neutralization test (FRNT) and reporter gene activity detection were conducted for IAV nAb detection. [Results] PR8NAEGFP/WSN remained genetically stable after five passages in chicken embryos. Compared with PR8NA/WSN, PR8NAEGFP/WSN showed a slightly declined titer at the time point of 48 h and similar crystal violet plaque morphology. The EC50 of favipiravir measured with the recombinant virus is consistent with that with the control virus, and the EC50 values obtained through various detection methods, including FFA, qPCR, and EGFP fluorescence, show good correlations. The correlation coefficient r of the nAb titer determined by FRNT and EGFP fluorescence was 0.930 4, which indicated good consistency. [Conclusion] PR8NAEGFP/WSN, a recombinant IAV carrying the reporter gene, might be used as a real-time visualization tool for the basic research on IAV and the evaluation of antivirals and vaccines for IAV.

influenza A virus  /  reverse genetics  /  reporter gene  /  in vitro application  /  antiviral drugs  /  neutralizing antibodies
Jia LI, Donghong WANG, Yujie JIANG, Ruiwen HAN, Qiaohong CHU, Tangqi WANG, Guanya LIU, Xuejie ZHANG, Baoying HUANG, Yao DENG, Wenjie TAN. Development and in vitro application of a recombinant influenza A virus expressing enhanced green fluorescent protein[J]. Acta Microbiologica Sinica, 2025 , 65 (10) : 4607 -4620 . DOI: 10.13343/j.cnki.wsxb.20250234
Funding
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  • the National Key Research and Development Program of China(2021YFA1201003)
Appendix
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Year 2025 volume 65 Issue 10
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Article Info
doi: 10.13343/j.cnki.wsxb.20250234
  • Receive Date：2025-03-24
  • Online Date：2025-11-03
  • Published：2025-09-04
Article Data
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History
  • Received：2025-03-24
  • Accepted：2025-05-02
Funding
the National Key Research and Development Program of China(2021YFA1201003)
Affiliations
    1Zhejiang Provincial Key Laboratory of Medical Genetics, School of Laboratory Medicine and Life Science, Wenzhou Medical University, Wenzhou, Zhejiang, China
    2National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases (NITFID), NHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China
    3School of Public Health, Baotou Medical College, Baotou, Inner Mongolia, China
    4School of Public Health, Xinxiang Medical University, Xinxiang, Henan, China

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表12种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
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Genus		 种数
Number of
species		 占总种数比例
Percentage of total
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鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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