含有HiBiT荧光素酶标签的流感报告病毒：可用于稳定高效筛选抗流感药物和疫苗免疫评价

doi:10.1016/j.jia.2024.04.018

Journal of Integrative Agriculture ›› 2025, Vol. 24 ›› Issue (10): 4012-4025.DOI: 10.1016/j.jia.2024.04.018

含有HiBiT荧光素酶标签的流感报告病毒：可用于稳定高效筛选抗流感药物和疫苗免疫评价

收稿日期:2023-10-11 修回日期:2024-04-13 接受日期:2024-03-22 出版日期:2025-10-20 发布日期:2025-09-24

HiBiT-tagged influenza A virus: A stable and efficient tool for antiviral reagent screening and vaccine evaluation

Zhengxiang Wang^{1, 2}, Wentao Shen², Xuegang Zhang², Yanli Wei^{1, 2}, Yingying Du², Yingying Yu², Jing Wang⁴, Qiyun Zhu^{1, 2}, Qiaoying Zeng^1#, Shuai Xu^{2, 3#}

¹ College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730046, China
²State Key Laboratory for Animal Disease Control and Prevention, College of Veterinary Medicine, Lanzhou University/Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730000, China
³ Gansu Province Research Center for Basic Disciplines of Pathogen Biology, Lanzhou 730046, China
⁴ National Animal Husbandry Service, Beijing 100125, China

Received:2023-10-11 Revised:2024-04-13 Accepted:2024-03-22 Online:2025-10-20 Published:2025-09-24
About author:Zhengxiang Wang, E-mail: wzx4350@163.com; #Correspondence Qiaoying Zeng, E-mail: zengqy@gsau.edu.cn; Shuai Xu, E-mail: xushuai@caas.cn
Supported by:
This work was supported by the National Key R&D Program of China (2021YFD1800204); the National Natural Science Foundation of China (32172820 and 32272972), the Youth Innovation Program of Chinese Academy of Agricultural Sciences (CAAS) (Y2023QC30) and the Agricultural Science and Technology Innovation Program of CAAS, China (CAAS-ASTIP-JBGS-20210102).

摘要/Abstract

摘要：

【背景】A型流感病毒（Influenza A viruses， IAVs）是重要的人兽共患病病原，对养禽业造成了重大经济损失，也严重威胁公共卫生安全。疫苗和抗流感药物是流感防治的主要手段。但由于IAV持续发生变异和重组，导致疫苗有效性不断降低，现有抗流感药物的耐药毒株也接连出现。因此，科研人员需要更加高效稳定的方法用于疫苗株的不断更新以及新型抗流感药物的开发。【目的】建立一个稳定、高效的筛选工具，用于流感疫苗的免疫效果评价和抗流感病毒药物的筛选。【方法】本研究以H9N2亚型IAV作为研究模型，将来源于NanoLuc荧光素酶的仅含有11个氨基酸的小亚基HiBiT，插入到IAV基因组中NS1基因RNA结合域和效应域之间的柔性连接区域，替换NS1蛋白的74-84 aa片段。通过反向遗传技术拯救含有HiBiT荧光素酶标记的H9N2病毒。随后，对HiBiT标记病毒的遗传稳定性、复制能力和致病性等生物学特性进行检测，并探索该标记病毒在抗病毒药物筛选和疫苗免疫效果评价中的应用优势。【结果】成功拯救出含有HiBiT荧光素酶标记的H9N2病毒（H9N2-HiBiT）；HiBiT标记毒株与亲本毒株在哺乳动物细胞和禽源细胞上的复制能力一致，并且HiBiT标记病毒连续传代15代后仍可以保持很高的荧光素酶活性。HiBiT标记毒株对SPF鸡和Balb/c小鼠的致病性与亲本毒株一致。抗流感药物Baloxavir处理后，HiBiT标记病毒感染细胞中的荧光素酶活性呈剂量依赖性降低，且检测效率和通量较传统滴定方法均有提高。在流感疫苗免疫鸡后采用HiBiT标记病毒进行免疫效果评价，可快速准确地检测病毒排毒水平、中和抗体水平等指标。【结论】本研究构建的含有HiBiT荧光素酶标记的报告病毒是一种稳定方便的研究工具，可用于高效筛选抗流感病毒药物和流感疫苗免疫效果评价。

Abstract:

Influenza A viruses (IAVs) possess variable pathogenic potency causing great economic losses in the poultry industry worldwide and threatening public health. The control of IAV epidemics desperately necessitates an efficient platform for screening antiviral compounds and evaluating vaccine efficacy. In this study, we utilized the H9N2 subtype IAV as the working model. An 11-amino-acid HiBiT tag, derived from NanoLuc luciferase, was incorporated into the flexible linker region of the NS1 protein. Subsequently, the recombinant HiBiT-tagged virus was rescued. The recombinant virus exhibited high genetic stability and similar virological characteristics to the parental virus, both in vitro and in vivo. Particularly importantly, the replication profile of the HiBiT-tagged virus can be easily measured using the Nano-Glo assay system, achieving an efficient screening platform. Based on this platform, we have developed assays with both convenience and efficiency for screening antiviral reagents, evaluating immunization efficacy, and measuring neutralizing antibodies.

Key words: H9N2 IAV , luciferase tag , antiviral screening , vaccine evaluation

Zhengxiang Wang, Wentao Shen, Xuegang Zhang, Yanli Wei, Yingying Du, Yingying Yu, Jing Wang, Qiyun Zhu, Qiaoying Zeng, Shuai Xu. 含有HiBiT荧光素酶标签的流感报告病毒：可用于稳定高效筛选抗流感药物和疫苗免疫评价[J]. Journal of Integrative Agriculture, 2025, 24(10): 4012-4025.

Zhengxiang Wang, Wentao Shen, Xuegang Zhang, Yanli Wei, Yingying Du, Yingying Yu, Jing Wang, Qiyun Zhu, Qiaoying Zeng, Shuai Xu. HiBiT-tagged influenza A virus: A stable and efficient tool for antiviral reagent screening and vaccine evaluation[J]. Journal of Integrative Agriculture, 2025, 24(10): 4012-4025.

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含有HiBiT荧光素酶标签的流感报告病毒：可用于稳定高效筛选抗流感药物和疫苗免疫评价

HiBiT-tagged influenza A virus: A stable and efficient tool for antiviral reagent screening and vaccine evaluation

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