微生物学报

Types of reporter genes	Name	Core characteristics	Main application scenarios	Emission color	References
Fluorescent protein	Green fluorescent protein (GFP)	Common reporter for gene expression; tag for protein localization	Gene expression reporter; protein localization tag; organelle-specific marker	Green fluorescence (488-507 nm)	[31]
Enhanced green fluorescent protein (EGFP)	Optimized GFP mutant; enhanced fluorescence brightness; high expression efficiency; excellent photostability; most widely used green fluorescent protein variant	Gene expression reporter; protein localization tag; organelle marker; long-term live-cell imaging; quantitative fluorescence study	Green fluorescence (488-507 nm)	[31]
Red fluorescent protein (RFP, e.g., DsRed)	Derived from corals; low tissue scattering; high tissue penetration; low autofluorescence background; suitable for multicolor labeling and in vivo imaging	Multicolor fluorescence labeling; in vivo deep tissue imaging; protein localization tag	Red fluorescence (558-583 nm)	[32]
Monomeric red fluorescent protein (mCherry)	Optimized DsRed derivative; genuine monomer; rapid maturation; excellent photostability; widely used red fluorescent protein tag	Protein fusion and localization tag; long-term live-cell imaging; organelle and subcellular structure labeling	Red fluorescence (587-610 nm)	[33]
Far-red/near-infrared fluorescent protein (e.g., iRFP)	Derived from bacterial phytochromes; minimal tissue light scattering and absorption; deep penetration; ideal for in vivo imaging	Small animal in vivo whole-body imaging; multispectral fluorescence labeling; in vivo deep tissue imaging	Far-red/near-infrared light (690-713 nm)	[34]
Luciferase	Firefly luciferase	Most classic and widely used; substrate D-luciferin; ATP-dependent reaction; stable and long-lasting signal	Quantitative detection of gene expression in vitro; main reporter system for dual-reporter gene assays; detection of viral replication activity	Yellow-green light (560 nm)	[35]
Renilla luciferase	Substrate is coelenterazine; rapid flash signal; fast signal decay	Internal reference system for dual-reporter gene assays; transfection efficiency normalization; auxiliary quantitative detection of gene expression in vitro	Blue light (480 nm)	[35]
Gaussia luciferase (GLuc)	Naturally secreted; directly detectable in cell supernatant; real-time live-cell monitoring; high signal intensity	Real-time monitoring of gene expression in living cells; non-destructive detection in vitro; high-throughput screening of antiviral drugs	Blue light (480 nm)	[36]
NanoLuc luciferase (NLuc)	Engineered optimized probe; minimal size; ultra-strong signal; excellent stability; enhanced signal-to-noise ratio; substrate: furimazine	Ultra-high sensitivity in vitro detection; long-term live-cell monitoring; in vivo viral imaging; quantitative analysis of trace samples	Blue light (460 nm)	[37-38]

Types of reporter genes	Name	Core characteristics	Main application scenarios	Emission color	References
Fluorescent protein	Green fluorescent protein (GFP)	Common reporter for gene expression; tag for protein localization	Gene expression reporter; protein localization tag; organelle-specific marker	Green fluorescence (488-507 nm)	[31]
Enhanced green fluorescent protein (EGFP)	Optimized GFP mutant; enhanced fluorescence brightness; high expression efficiency; excellent photostability; most widely used green fluorescent protein variant	Gene expression reporter; protein localization tag; organelle marker; long-term live-cell imaging; quantitative fluorescence study	Green fluorescence (488-507 nm)	[31]
Red fluorescent protein (RFP, e.g., DsRed)	Derived from corals; low tissue scattering; high tissue penetration; low autofluorescence background; suitable for multicolor labeling and in vivo imaging	Multicolor fluorescence labeling; in vivo deep tissue imaging; protein localization tag	Red fluorescence (558-583 nm)	[32]
Monomeric red fluorescent protein (mCherry)	Optimized DsRed derivative; genuine monomer; rapid maturation; excellent photostability; widely used red fluorescent protein tag	Protein fusion and localization tag; long-term live-cell imaging; organelle and subcellular structure labeling	Red fluorescence (587-610 nm)	[33]
Far-red/near-infrared fluorescent protein (e.g., iRFP)	Derived from bacterial phytochromes; minimal tissue light scattering and absorption; deep penetration; ideal for in vivo imaging	Small animal in vivo whole-body imaging; multispectral fluorescence labeling; in vivo deep tissue imaging	Far-red/near-infrared light (690-713 nm)	[34]
Luciferase	Firefly luciferase	Most classic and widely used; substrate D-luciferin; ATP-dependent reaction; stable and long-lasting signal	Quantitative detection of gene expression in vitro; main reporter system for dual-reporter gene assays; detection of viral replication activity	Yellow-green light (560 nm)	[35]
Renilla luciferase	Substrate is coelenterazine; rapid flash signal; fast signal decay	Internal reference system for dual-reporter gene assays; transfection efficiency normalization; auxiliary quantitative detection of gene expression in vitro	Blue light (480 nm)	[35]
Gaussia luciferase (GLuc)	Naturally secreted; directly detectable in cell supernatant; real-time live-cell monitoring; high signal intensity	Real-time monitoring of gene expression in living cells; non-destructive detection in vitro; high-throughput screening of antiviral drugs	Blue light (480 nm)	[36]
NanoLuc luciferase (NLuc)	Engineered optimized probe; minimal size; ultra-strong signal; excellent stability; enhanced signal-to-noise ratio; substrate: furimazine	Ultra-high sensitivity in vitro detection; long-term live-cell monitoring; in vivo viral imaging; quantitative analysis of trace samples	Blue light (460 nm)	[37-38]

Strategies	Application	Advantages	Limitations
High-resolution microscopy technology	Study of viral ultrastructure, universal visualization of pathogens	High spatiotemporal resolution, feasible for long-term observation, and capable of simultaneously acquiring fluorescent dynamics and ultrastructure	Complex sample preparation, expensive equipment, professional operation, and technique-induced sample degradation
Exogenous nanomaterial labeling	Single-particle tracing of the entire viral life cycle, tracing of multiple viral structures	Long-term multicolor tracing, accurate localization, in situ non-invasiveness, excellent photostability, study on early molecular mechanisms combined with super-resolution technology	Cytotoxicity, poor tissue penetration, interference with viral infection, low labeling efficiency, non-specific adsorption
Recombinant virus reporter	Drug screening, serological determination, study on virus-host interactions	Dynamic analysis of viral infection, adapt to in vivo applications, high-throughput sequencing (HTS) for antiviral drugs, neutralizing antibody detection	Impaired viral replication with inserted reporter gene, genome instability, loss of exogenous reporter genes limiting its use in diverse clinical samples or emerging viruses
Virus infection-inducible reporter system	RdRp activity detection, high-throughput screening, replication mechanism	No need for viral modification, especially suitable for clinical isolates, flexible replacement of reporter genes, HTS for RdRp inhibitors, virus-host interaction research	Complex design with certain background signal

Strategies	Application	Advantages	Limitations
High-resolution microscopy technology	Study of viral ultrastructure, universal visualization of pathogens	High spatiotemporal resolution, feasible for long-term observation, and capable of simultaneously acquiring fluorescent dynamics and ultrastructure	Complex sample preparation, expensive equipment, professional operation, and technique-induced sample degradation
Exogenous nanomaterial labeling	Single-particle tracing of the entire viral life cycle, tracing of multiple viral structures	Long-term multicolor tracing, accurate localization, in situ non-invasiveness, excellent photostability, study on early molecular mechanisms combined with super-resolution technology	Cytotoxicity, poor tissue penetration, interference with viral infection, low labeling efficiency, non-specific adsorption
Recombinant virus reporter	Drug screening, serological determination, study on virus-host interactions	Dynamic analysis of viral infection, adapt to in vivo applications, high-throughput sequencing (HTS) for antiviral drugs, neutralizing antibody detection	Impaired viral replication with inserted reporter gene, genome instability, loss of exogenous reporter genes limiting its use in diverse clinical samples or emerging viruses
Virus infection-inducible reporter system	RdRp activity detection, high-throughput screening, replication mechanism	No need for viral modification, especially suitable for clinical isolates, flexible replacement of reporter genes, HTS for RdRp inhibitors, virus-host interaction research	Complex design with certain background signal

流感病毒可视化报告系统的研究进展

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熊佳祺 ¹^,³ , 杨文广 ² , 陈月红 ³ , 李标 ⁴ , 冯烨 ³ , 姜涛 ¹^,³^,⁴

微生物学报 | 综述 2026,66(8): 3732-3744

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微生物学报 |综述 2026 , 66 (8) : 3732 -3744

流感病毒可视化报告系统的研究进展

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熊佳祺¹^,³, 杨文广², 陈月红³, 李标⁴, 冯烨³, 姜涛¹^,³^,⁴

作者信息

^1.牡丹江医科大学公共卫生学院，黑龙江牡丹江

^2.中国人民解放军南部战区海军第二医院，海南三亚

^3.军事医学研究院病原微生物生物安全全国重点实验室，北京

^4.安徽医科大学基础医学院，安徽合肥

通讯作者:

冯烨, 姜涛

作者简介:

作者贡献声明

熊佳祺：文献检索、初稿撰写及修改；杨文广：重要内容修改，内容核对；陈月红：论文整体框架讨论；李标：内容核对，文献检索；冯烨：论文审阅与整体框架修改；姜涛：论文大纲指导，核心内容补充、修正。

Research progress in visualization reporter systems for influenza virus

Jiaqi XIONG¹^,³, Wenguang YANG², Yuehong CHEN³, Biao LI⁴, Ye FENG³, Tao JIANG¹^,³^,⁴

Affiliations

^1.School of Public Health, Mudanjiang Medical University, Mudanjiang, Heilongjiang, China

^2.The Second Hospital of the Navy, Southern Theater Command of PLA, Sanya, Hainan, China

^3.State Key Laboratory of Pathogen and Biosecurity, Academy of Military Medical Sciences, Beijing, China

^4.School of Basic Medical Sciences, Anhui Medical University, Hefei, Anhui, China

Corresponding Author:

E-mail: JIANG Tao, jiangtao@bmi.ac.cn;

FENG Ye, fengye621@126.com

出版时间: 2026-08-04 doi: 10.13343/j.cnki.wsxb.20260130

文章导航

摘要

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流感病毒的持续进化与耐药性变异对其致病机制研究、抗病毒药物及疫苗研发提出了更高要求。病毒可视化报告系统作为实时监测病毒感染、复制与传播的关键工具，已发展出高分辨率显微镜成像、外源纳米材料标记、重组报告病毒、病毒感染诱导报告系统等多种策略。本文系统梳理流感病毒可视化技术的研究进展，阐述各技术的原理、核心突破与应用场景，分析当前技术体系存在的共性局限与个性化挑战，并结合研究前沿展望其发展方向，为流感病毒可视化研究的技术选择与创新应用提供参考。

关键词

流感病毒 / 病毒示踪 / 高分辨率显微技术 / 纳米材料标记 / 重组报告病毒 / 报告基因

Abstract

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The continuous evolution and drug-resistant mutations of influenza virus have placed higher demands on the study of its pathogenic mechanism, as well as the development of antiviral drugs and vaccines. As a key tool for real-time monitoring of viral infection, replication and transmission, viral visualization reporter systems have evolved into various strategies, including high-resolution microscopic imaging, exogenous nanomaterial labeling, recombinant reporter viruses, and virus infection-inducible reporter systems. This manuscript systematically reviewed the research progress of influenza virus visualization technologies, elaborated the principles, core breakthroughs, and application scenarios of each technique, analyzed the common limitations and specific problems existing in the current technical system, and prospected its development direction combined with cutting-edge research, providing a reference for technical selection and innovative applications in influenza virus visualization research.

Key words

influenza virus / viral tracking / high-resolution microscopy technology / nanomaterial labeling / recombinant reporter virus / reporter gene

引用本文

熊佳祺, 杨文广, 陈月红, 李标, 冯烨, 姜涛. 流感病毒可视化报告系统的研究进展. 微生物学报, 2026 , 66 (8) : 3732 -3744 . DOI: 10.13343/j.cnki.wsxb.20260130

Jiaqi XIONG, Wenguang YANG, Yuehong CHEN, Biao LI, Ye FENG, Tao JIANG. Research progress in visualization reporter systems for influenza virus[J]. Acta Microbiologica Sinica, 2026 , 66 (8) : 3732 -3744 . DOI: 10.13343/j.cnki.wsxb.20260130

正文

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流感病毒(influenza virus)可感染人类及多种哺乳动物，引起人类季节性流感和周期性全球大流行，被世界卫生组织(World Health Organization, WHO)列为重点关注的病原体之一^[1]。随着流感病毒的持续进化和耐药毒株的出现，构建能够实时监测病毒感染和复制过程的研究工具，对深入理解其致病机制、加速药物与疫苗研发具有重要意义。

目前，流感病毒报告系统主要包含4种可视化策略：(1)利用高分辨率显微镜等物理手段直接观察病毒^[2]；(2)外源纳米材料标记的病毒颗粒示踪策略可实现病毒的亚细胞时空定位，追踪病毒颗粒的入侵与增殖过程^[3-5]；(3)表达报告蛋白的重组报告病毒策略，病毒复制增殖伴随荧光蛋白或荧光素酶的表达，可直接示踪病毒颗粒的入侵、复制与传播，尤其适用于活体动物模型中的动态示踪^[6-7]，其中基于复制缺陷设计的互补系统在维持报告功能的同时提升了生物安全性^[8-10]；(4)病毒感染诱导的报告系统策略，利用病毒自身高度保守的启动子元件，在细胞中构建病毒诱导型报告基因系统，实现高灵敏、高通量的间接可视化与定量检测^[11]。

本文概述了流感病毒可视化报告系统的研究进展，对比现有可视化报告系统，围绕使用不同标记物[如荧光蛋白、荧光素酶、量子点(quantum dots, QDs)等]标记病毒结构蛋白或宿主细胞的策略，阐述可视化系统在机制研究、药物筛选、抗体研发等方面的应用，并对病毒可视化报告系统的发展进行了展望。

1 基于高分辨率显微技术的流感病毒可视化

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高分辨率显微技术的病毒可视化是多技术互补、满足多种需求的体系。原子力显微镜(atomic force microscopy, AFM)聚焦近生理条件下的形态与力学特性^[2]。超分辨荧光显微镜(super-resolution fluorescence microscopy, SRFM)可在活细胞内实时追踪病毒动态，结合量子点、荧光蛋白等标记实现高分辨与高特异性的双重保障^[12]。SRFM涵盖多种技术，包括受激发射损耗显微镜(stimulated emission depletion microscopy, STED)、随机光学重建显微镜/光激活定位显微镜(stochastic optical reconstruction microscopy/photoactivated localization microscopy, STORM/PALM)、结构光照明显微镜(structured illumination microscopy, SIM)、最小光子通量显微镜(minimal photon flux microscopy, MINFLUX)等。其中STED是唯一能实现毫秒级快速成像的分支^[12]，与其他分支分工互补(如SIM侧重厚组织成像，STORM/PALM侧重单分子定位)^[13]。透射电子显微镜(transmission electron microscopy, TEM)聚焦病毒超微结构的原子级解析，为病毒结构特征、组装机制及纳米标记物结合位点验证提供直观的超微结构证据^[14]。扫描电子显微镜(scanning electron microscopy, SEM)则侧重病毒与细胞互作的表面形貌及三维宏观成像^[15-16]。这些技术不仅在流感病毒的感染机制、结构解析中发挥关键作用，更已广泛应用于严重急性呼吸综合征冠状病毒2 (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2)、乙肝病毒、疱疹病毒等多种病原体的研究，共同构成了病毒可视化的核心技术体系，克服了单一技术的局限性，为病毒学研究提供了多维度、多层次的可视化支撑。

在多种高分辨率显微技术中，原子力显微镜凭借其独特的成像优势，成为流感病毒动态机制研究的重要工具。原子力显微镜是一种基于探针与样品表面原子间相互作用力进行成像的纳米级尖端仪器，其核心原理是通过检测尖锐探针在扫描样品表面时产生的微小悬臂偏转，重构出样品表面的三维形貌及物理特性^[17-18]。与传统显微技术不同，AFM的显著优势在于成像的灵活性——它摆脱了传统电子显微镜对真空环境和复杂样品制备的依赖，允许在生理缓冲液或近似活体环境中直接对蛋白质、活细胞、病毒颗粒等进行高分辨成像与力学测量，这一能力使其成为连接微观形貌、纳米力学与生物功能的独特研究平台，在生命科学与材料科学领域发挥着不可替代的作用^[18]。在针对流感病毒的研究中，Yoshida等^[2]通过耦合荧光定位与AFM扫描技术，实现了对单个流感病毒颗粒入侵活细胞过程的动态追踪；该研究不仅直观揭示了病毒与细胞膜结合、内吞启动的形态学特征，更通过力学谱分析定量监测到病毒在酸性内体环境中囊膜硬度的动态变化，为阐明流感病毒脱壳与膜融合机制提供了直接的成像证据。这项技术将病毒学研究从静态的形态观察和群体的生化分析，推向了动态的、定量的、单个病毒颗粒研究的新维度，为流感病毒入侵机制研究和药物开发提供了有力工具。

尽管各类高分辨率显微技术为流感病毒可视化提供了多维度支撑，但仍存在样品制备复杂、难以完全模拟生理动态环境、时空分辨率难以兼顾等局限，对病毒原位、精细结构的实时动态观测仍需进一步突破。

2 外源纳米材料标记的流感病毒颗粒示踪技术

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2.1 量子点标记的流感病毒荧光标记技术及单颗粒示踪技术

量子点是一种半导体纳米晶体，其微小尺寸引发量子限域效应，赋予其卓越的光学特性：当被光或电激发时，量子点可发射出波长精准、光谱狭窄且亮度极高的荧光^[19-20]。凭借超高的荧光亮度(比传统有机染料高10-100倍)、卓越的光稳定性(抗光漂白能力极强)以及狭窄对称的发射光谱，量子点能在复杂的活细胞微环境中对单个病毒粒子实现长达数十分钟至数小时的连续、多色、高分辨示踪成像^[21]。在甲型流感病毒可视化应用中，Qin等^[22]利用该特性开展了里程碑式研究，通过基因工程在流感病毒基质蛋白M1上引入特异性标签，经酶促反应实现量子点与病毒的稳定共价标记，再结合高速宽场荧光显微镜，对单个甲型流感病毒从黏附、内吞、内体转运至脱壳的全过程(约10 min)进行实时持续追踪，通过量子点荧光信号的突然淬灭精准定位病毒囊膜破裂、内部遗传物质复合体释放的关键时刻，首次精确测定脱壳发生的时间尺度(如进入内体后约3.5 min启动)，揭示脱壳是由特定细胞因子触发的分步精密生物学过程。更具突破性的是，Hong等^[23]开发了量子点双标记策略，通过电穿孔技术在病毒囊膜上形成瞬时可逆通道，将偶联核蛋白抗体的绿色量子点(green quantum dots conjugated with nucleoprotein antibody, GQDs-NPAb)递送至病毒内部，特异性标记核心的病毒核糖核蛋白复合体(viral ribonucleoprotein complex, vRNP) (标记效率34%，病毒感染性保留93%)，再通过温和的肼-醛偶联反应将红色量子点(red fluorescent quantum dots, RQDs)标记于病毒囊膜，实现内部核心与外部囊膜的同步示踪，双标记效率达11%且不影响病毒活性，该策略还适用于H9N2等多种甲型流感病毒亚型。Qin等^[24]通过生物素-链霉亲和素介导的脂质特异性标记策略，使量子点结合甲型流感病毒(influenza A virus, IAV)包膜，在不影响病毒生物学活性的前提下实现单个病毒粒子的特异性标记。Du等^[25]结合单病毒追踪(single virus tracking, SVT)技术与共聚焦成像系统，对病毒入侵宿主细胞的全过程进行长时间、实时、动态的荧光示踪，同步捕捉病毒与细胞骨架(肌动蛋白、微管)、关键调控蛋白(网格蛋白、肌球蛋白VI、Arp2/3复合物)的相互作用轨迹。这些应用不仅突破了传统标记仅能追踪病毒单一阶段或表面的局限，为深入解析甲型流感病毒感染全生命周期机制提供了直接可视化证据，更构建了单病毒水平动态追踪的研究范式，为开发靶向病毒脱壳、vRNP转运等关键过程的抗病毒策略提供了关键理论与技术支撑，凸显了量子点在甲型流感病毒机制研究、亚型普适性追踪及抗病毒靶点筛选中的核心价值。

此外，高分辨率显微技术为量子点标记病毒示踪提供了关键观测支撑，其中受激发射损耗显微镜(STED)凭借20-30 nm的超分辨能力，成为活细胞内量子点标记病毒动态追踪的核心工具。庞岱文教授团队在2025年的研究中，通过量子点分别标记IAV的包膜与衣壳，结合STED显微镜实时追踪，清晰解析了病毒通过网格蛋白介导内吞(clathrin-mediated endocytosis, CME)途径入侵的完整动态，即从病毒与细胞膜受体结合后招募网格蛋白形成包被小窝，到鞘磷脂-胆固醇微结构域稳定颈部结构、发动蛋白缩缢形成囊泡，每一步骤的时空坐标均通过量子点信号精准定位，同时量化了不同内吞途径的占比(CME约80%、非网格蛋白介导途径约20%)^[12]。这种量子点标记与STED成像的组合，首次实现了流感病毒入侵早期分子事件的动态可视化，为内吞机制研究提供了直接证据。

尽管如此，量子点标记技术在流感病毒单颗粒示踪与超分辨成像应用中仍存在一定局限，如传统含镉类量子点存在潜在重金属细胞毒性，长时间活细胞示踪时可能干扰宿主生理状态与病毒正常感染进程^[26]；量子点粒径(5-15 nm)与病毒颗粒尺寸接近，标记后易产生空间位阻，从而影响病毒黏附、内吞及膜融合等关键行为^[12]；同时标记效率与病毒感染性难以兼顾，内部或双标记策略常面临标记率偏低、表面非特异性吸附等问题，增加成像背景干扰^[23]；此外，量子点的荧光闪烁与宽激发光谱特性，在与STED等高分辨技术联用时易降低信号连续性并引发多色串扰，制约长时间、多靶点同步示踪的精度^[12,27]。

2.2 基于原位生物正交代谢标记的流感病毒示踪技术

原位生物正交代谢标记技术为流感病毒活细胞与活体水平的高保真可视化提供了重要手段。该技术借助宿主细胞天然代谢过程，将叠氮等生物正交反应基团原位、非破坏性地整合至病毒结构中，再通过无铜点击化学反应实现荧光探针或量子点的特异性共价标记，能够在不影响病毒结构完整性与感染活性的前提下对单个流感病毒颗粒进行长时程动态追踪。Pan等^[28]率先利用脂质代谢前体将叠氮基团标记于流感病毒囊膜，并在小鼠肺部实现原位生物正交偶联与荧光成像，首次完成了流感病毒在活体动物内的单颗粒动态示踪，为体内感染过程研究建立了低干扰、高特异性的可视化范式。在此基础上，Liu等^[29]进一步发展脂质介导的生物合成标记策略，结合量子点探针实现禽流感病毒入侵途径的实时单颗粒追踪，清晰揭示了病毒吸附、内吞、内体转运及脱壳等连续动态事件。此外，Carvalho等^[30]通过非天然氨基酸代谢掺入策略，完成流感病毒样颗粒HA囊膜蛋白的位点特异性生物正交修饰，搭建起通用的定点荧光标记体系，为包膜型病毒样颗粒疫苗的体外功能修饰提供了可靠的技术手段。上述研究共同推动了原位生物正交代谢标记从体外单颗粒示踪走向活体动态成像，为解析流感病毒感染机制、筛选抗病毒靶点与评估药物效应提供了可靠的可视化技术支撑。

原位生物正交代谢标记技术在流感病毒可视化中仍有局限：非天然代谢前体易干扰宿主代谢与病毒活性，标记位点及修饰程度不可控、成像背景较高，活体层面组织穿透性与反应速率有限，且依赖宿主代谢，对不同流感亚型的通用性不足^[28]。

3 基于重组报告病毒的流感病毒可视化技术

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流感报告病毒是基于反向遗传学技术，通过在流感病毒基因组特定位置定向插入荧光素酶、荧光蛋白(表1)等报告基因构建的重组病毒，可实现病毒生命活动的可视化监测。依据其复制特性，该类重组病毒可分为常规报告病毒与复制限制型报告病毒2类。

3.1 常规报告病毒

常规报告病毒是重组报告病毒的基础类型，通过在病毒基因组中插入报告基因实现病毒生命活动的可视化，其核心研究脉络围绕优化插入位点、平衡报告基因表达与病毒生物学特性展开，是活体示踪流感病毒感染的开创性工具。

早期研究受限于对流感病毒基因组包装信号复杂性的认识，通常选择非必需片段[如非结构蛋白(non-structural protein, NS)片段]作为外源基因的插入位点，为确保报告基因的有效表达并尽可能降低对病毒复制与致病性的影响，常规策略是将报告基因编码序列与病毒基因的调控元件相连，进而取代该病毒基因原有的编码区^[38-40]，以最大限度维持病毒的复制能力和致病性。例如，2010年Manicassamy等^[41]利用T2A肽连接策略，首次在PR8毒株的NS1基因中成功整合了绿色荧光蛋白(green fluorescent protein, GFP)基因，构建出NS片段携带报告基因的重组甲型流感病毒，为活体示踪病毒感染提供了开创性工具。随着对病毒复制与包装机制理解的深化，报告基因的插入位点已扩展至PB2、PB1、PA、NA^[9,38,42-45]等多个基因片段(图1)，并且利用报告病毒发现了插入外源基因影响最小的PA片段^[44]。

目前，基于报告系统的流感病毒研究主要包括感染与致病机制解析、抗病毒药物筛选和中和抗体检测等，这些应用充分发挥了报告系统可视化、定量化及高通量的技术优势。在揭示免疫应答启动机制方面，DiPiazza等^[46]利用表达Venus荧光蛋白的H1N1报告病毒，在体内感染模型中系统性鉴定了能够呈递病毒抗原的MHC II类分子阳性细胞；他们首次发现，除抗原呈递细胞外，肺泡上皮细胞在感染初期也能有效呈递病毒抗原，进而解析了流感病毒在体内感染初期建立免疫识别的关键细胞特征，为理解免疫应答的启动机制提供了关键信息。在阐释病毒蛋白功能方面，报告系统有助于明确特定基因片段的功能。例如，Nogales等^[40]发现敲除NS片段的报告病毒在33 ℃下复制能力减弱，证实了NS片段在病毒耐热性中的作用；Avilov等^[47]利用报告系统可视化了PB2蛋白从细胞质向细胞核的转运过程，为理解流感病毒聚合酶复合物的组装、入核机制及其与宿主因子的互作提供了直观证据。Yan等^[48]构建了分别表达萤火虫荧光素酶(firefly luciferase, FLuc)和海肾荧光素酶(Renilla luciferase, RLuc)的流感病毒和呼吸道合胞病毒(respiratory syncytial virus, RSV)报告系统，可实现同一孔板中2种共感染病毒的同步检测，通过双荧光素酶信号的差异化分析，同步完成对广谱抗病毒化合物及亚型选择性抑制剂的高效筛选，大幅提升了药物筛选的通量与效率，尤其适用于靶向宿主通用机制药物的筛选。此外，报告病毒在高通量与实时动力学监测方面也表现出显著优势。Eckert等^[49]构建的GLuc流感报告病毒可在96或384孔板中进行药物筛选，通过连续监测上清中的GLuc活性能够实时、无创地评估病毒复制动力学及化合物在不同时间点的抑制效果，满足工业化药物筛选对通量和动态数据的需求。Creanga等^[10]构建了包含19种具有完全复制能力的甲型流感(H1N1、H3N2)和乙型流感(Victoria、Yamagata谱系)报告病毒的平台，通过在病毒NS基因片段中稳定插入Nluc荧光素酶报告基因形成了流感报告病毒的高通量中和抗体深度分析平台；该平台不仅能将抗体的中和活性精确映射到病毒蛋白的特定结构区域，定义其功能表位，还能系统性地揭示此前未知的、可导致抗体失效的病毒氨基酸逃逸突变位点，为理解病毒在免疫压力下的变异与进化提供了新见解。

常规报告病毒虽为流感病毒研究提供了直观的可视化工具，但仍存在诸多局限性。外源报告基因的插入易破坏病毒基因组的天然结构，导致病毒复制能力与毒力下降，难以完全模拟野生毒株的生物学特性^[40]。报告基因mRNA易形成复杂二级结构，阻碍病毒聚合酶的识别与转录，且外源基因在传代过程中易丢失，造成基因组不稳定、示踪信号中断^[50]。同时，其构建依赖反向遗传学技术，流程相对复杂、实验条件要求较高^[51]。

3.2 复制限制型报告病毒

常规报告病毒虽能实现病毒的全程示踪，但源自高致病性流感病毒的报告病毒仍存在生物安全风险，需要BSL-3/ABSL-3级实验室条件。复制限制型报告病毒通过病毒基因缺失与细胞互补策略，规避产生感染性子代病毒，从而降低高致病性流感病毒实验操作的生物安全等级要求。Harit等^[9]和Creanga等^[10]开发了更安全的病毒复制限制型报告系统，该策略采用双向控制的设计思路，首先对病毒进行改造，敲除流感病毒的关键基因(如PB1或HA)，并用荧光报告基因替代，构建出自身复制限制型报告病毒；其次设计能够稳定表达所缺失病毒蛋白的互补细胞系，只有当改造后的病毒感染了特定的互补细胞系时，病毒缺失的功能才能得到反式补充，从而完成单轮复制并产生报告信号；这种基于病毒基因改造和细胞功能互补的双向控制策略，通过荧光信号监测病毒活性，极大提升了生物安全性；此外，该方法能在BSL-2实验室条件下进行高通量中和抗体深度分析、评估抗神经氨酸酶抗体的抑制活性及检测疫苗临床试验的血清效价，有效克服了传统中和试验的局限性。

无论完整的报告病毒还是复制限制型报告病毒均需对病毒进行重组改构。该策略的核心问题在于如何平衡报告基因的高效稳定表达与病毒自身的复制力及致病性。外源报告基因的插入会破坏流感病毒基因组的天然结构，降低病毒复制能力与毒力，难以完全模拟野生毒株的真实生物学特性^{[38,40,49,52-53]}。报告基因的mRNA序列若形成复杂二级结构，可能阻碍病毒聚合酶的识别与转录，或影响核糖体的有效加载^[54]。外源基因在传代中易丢失，导致基因组不稳定与示踪信号中断^[55]。为缓解这一矛盾，研究人员通过优化连接肽(如2A肽)和利用蛋白水解酶(如caspase)识别位点等策略^[38]有效协调了报告蛋白与病毒蛋白的表达，使构建的报告病毒能在实验室条件下保持与野生型病毒相似的生物学特性，实现稳定的信号输出。

4 病毒感染诱导的报告系统

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病毒感染诱导的报告系统，不对病毒进行修饰重组，仅通过改造宿主细胞，利用流感病毒保守的启动子序列实现病毒感染的间接可视化，在保留病毒天然特性的同时实现高通量检测，已成为流感病毒筛查与药物筛选的重要工具。

病毒感染诱导的报告系统的核心原理在于高度保守的顺式作用启动子元件可作为RNA病毒的特异性识别元件。以甲型流感病毒(IAV)为例，其启动子序列位于各基因组片段两端的非编码区(non-coding region, NCR)，在病毒亚型间高度保守^[56-58]。Lutz等^[59]利用这一特性，将报告基因置于含有病毒启动子序列的框架中；当IAV感染细胞后，其进入细胞核的病毒RNA聚合酶复合体(由PA、PB1、PB2及NP蛋白组成)能特异性识别并转录这段人工构建的报告RNA，从而驱动下游报告基因的高水平表达，这一策略将病毒自身复制的核心机制——vRNP的转录活性直接转化为可定量读出的光学信号，实现了对病毒复制事件的“信号放大”与间接可视化。基于此原理，后续研究开发了多种优化的报告系统。如Hossain等^[60]成功构建了基于马-达二氏犬肾细胞(madin-darby canine kidney, MDCK)细胞的稳定报告细胞系Luc9.1，该系统将海肾荧光素酶报告基因置于细胞RNA聚合酶I启动子控制下，并在其两侧引入IAV NP基因的NCR序列；感染后，该体系能灵敏地检测病毒活性，已成功应用于雪貂恢复期血清中和抗体效价的测定(与传统方法吻合)及抗病毒药物(如金刚烷胺)的敏感性评价，在简化操作流程的同时保持了结果的可靠性。Chen等^[11]开发的基于分泌型GLuc的系统，则进一步专注于监测IAV RNA依赖性RNA聚合酶(RNA-dependent RNA polymerase, RdRp)的活性，展现出对多种IAV亚型的广泛兼容性、跨细胞系的适用性及长期传代的稳定性，凸显了其作为高通量筛选工具的潜力。

病毒诱导的报告系统虽在灵敏性、便捷性和高通量应用上优势显著，但存在固有技术局限：其性能依赖病毒聚合酶功能的完整性，不同毒株聚合酶活性差异易导致检测灵敏度不一致；且报告信号是病毒复制的间接读数，并非直接反映病毒粒子产量。该特性不仅使其结果与传统病毒滴度测定结果的相关性需重新校准，还会在抗病毒药物筛选中产生系统性偏差——对靶向聚合酶的药物异常敏感，而对抑制病毒进入、组装等环节的药物，测得的半数抑制浓度(half maximal inhibitory concentration, IC₅₀)与经典方法结果存在差异。因此，该系统在毒株评价、药物筛选等研究中应用时需结合其工作原理考量适用范围与解读局限^[11,60]。为更直观地比较不同病毒可视化与示踪策略的应用场景、优势及局限性，现将上述几类典型技术归纳对比，如表2所示。

5 结语

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流感病毒可视化技术历经数十年发展已形成成熟的技术体系。高分辨率显微技术、外源纳米材料标记、重组报告病毒、病毒感染诱导报告系统四大策略各有优势，为从分子、细胞到活体水平动态解析病毒生命周期、病毒与宿主互作及免疫应答的诱导提供了关键工具，深度推动了流感病毒致病传播机制的研究，也为抗病毒药物与疫苗研发提速赋能。然而，现有技术仍存在诸多局限，如病毒基因组改造易改变其天然生物学特性^[49,55]；外部标记技术难以兼顾高保真示踪与普适性；部分系统检测存在灵敏度偏差或操作繁琐等问题^[22,29]，难以满足流感病毒持续进化、新型毒株不断涌现带来的快速精准评估需求。

上述可视化技术的构建策略与应用范式虽以流感病毒为切入点加以阐述，但其核心原理与技术框架已跨领域延伸至其他病毒研究中，并展现出广阔的通用应用前景。其中，高分辨率显微技术凭借超微结构解析、近生理条件动态观测的优势，已成功应用于SARS-CoV-2、乙型肝炎病毒、疱疹病毒等病原体研究，为观察各类病毒的颗粒形态、阐明入侵机制及解析病毒与宿主互作提供了通用可视化支撑，成为病毒学领域解析微观机制的基础工具^[61]。外源纳米材料标记的单颗粒示踪策略可通过适配不同病毒的结构特征优化标记方法，已为登革病毒、寨卡病毒、呼吸道合胞病毒等包膜病毒感染全生命周期的动态追踪提供了重要技术参考，其双标、多色示踪技术更可助力解析各类病毒的亚细胞转运与组装机制^[62]。重组报告病毒技术基于反向遗传学的改造思路，通过插入报告基因构建的重组报告病毒已实现SARS-CoV-2、副黏病毒等分节段或单链RNA病毒体内外感染与复制过程的实时示踪^[63]，而复制限制型报告病毒的设计策略更能为埃博拉病毒、马尔堡病毒等高致病性病毒的安全研究提供关键技术范式^[64]。病毒感染诱导的报告系统则可依托不同病毒的保守启动子或复制关键元件构建特异性报告细胞系，目前已在SARS-CoV-2、诺如病毒等研究中实现高通量筛查与药物活性评估，其无需改造病毒、高通量检测的核心优势，可适配各类病毒的大规模药物筛选与毒株快速鉴定需求^[65]。

未来，病毒可视化报告系统的研发将聚焦非侵入式、高保真的标记技术，实现病毒生命周期关键过程的零干扰标记与超高分辨率成像；同时推动多维度数据的整合分析，构建从病毒快速初筛到致病传播机制解析的完整技术方案，以此加速广谱抗病毒药物与通用疫苗的筛选评价，为病毒变异监测和流行风险预警提供核心技术支撑。

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2026年第66卷第8期

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doi: 10.13343/j.cnki.wsxb.20260130

接收时间：2026-02-12
首发时间：2026-08-21
出版时间：2026-08-04

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收稿日期：2026-02-12
录用日期：2026-03-28

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^1.牡丹江医科大学公共卫生学院，黑龙江牡丹江

^2.中国人民解放军南部战区海军第二医院，海南三亚

^3.军事医学研究院病原微生物生物安全全国重点实验室，北京

^4.安徽医科大学基础医学院，安徽合肥

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2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 total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
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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Types of reporter genes	Name	Core characteristics	Main application scenarios	Emission color	References
Fluorescent protein	Green fluorescent protein (GFP)	Common reporter for gene expression; tag for protein localization	Gene expression reporter; protein localization tag; organelle-specific marker	Green fluorescence (488-507 nm)	[31]
Enhanced green fluorescent protein (EGFP)	Optimized GFP mutant; enhanced fluorescence brightness; high expression efficiency; excellent photostability; most widely used green fluorescent protein variant	Gene expression reporter; protein localization tag; organelle marker; long-term live-cell imaging; quantitative fluorescence study	Green fluorescence (488-507 nm)	[31]
Red fluorescent protein (RFP, e.g., DsRed)	Derived from corals; low tissue scattering; high tissue penetration; low autofluorescence background; suitable for multicolor labeling and in vivo imaging	Multicolor fluorescence labeling; in vivo deep tissue imaging; protein localization tag	Red fluorescence (558-583 nm)	[32]
Monomeric red fluorescent protein (mCherry)	Optimized DsRed derivative; genuine monomer; rapid maturation; excellent photostability; widely used red fluorescent protein tag	Protein fusion and localization tag; long-term live-cell imaging; organelle and subcellular structure labeling	Red fluorescence (587-610 nm)	[33]
Far-red/near-infrared fluorescent protein (e.g., iRFP)	Derived from bacterial phytochromes; minimal tissue light scattering and absorption; deep penetration; ideal for in vivo imaging	Small animal in vivo whole-body imaging; multispectral fluorescence labeling; in vivo deep tissue imaging	Far-red/near-infrared light (690-713 nm)	[34]
Luciferase	Firefly luciferase	Most classic and widely used; substrate D-luciferin; ATP-dependent reaction; stable and long-lasting signal	Quantitative detection of gene expression in vitro; main reporter system for dual-reporter gene assays; detection of viral replication activity	Yellow-green light (560 nm)	[35]
Renilla luciferase	Substrate is coelenterazine; rapid flash signal; fast signal decay	Internal reference system for dual-reporter gene assays; transfection efficiency normalization; auxiliary quantitative detection of gene expression in vitro	Blue light (480 nm)	[35]
Gaussia luciferase (GLuc)	Naturally secreted; directly detectable in cell supernatant; real-time live-cell monitoring; high signal intensity	Real-time monitoring of gene expression in living cells; non-destructive detection in vitro; high-throughput screening of antiviral drugs	Blue light (480 nm)	[36]
NanoLuc luciferase (NLuc)	Engineered optimized probe; minimal size; ultra-strong signal; excellent stability; enhanced signal-to-noise ratio; substrate: furimazine	Ultra-high sensitivity in vitro detection; long-term live-cell monitoring; in vivo viral imaging; quantitative analysis of trace samples	Blue light (460 nm)	[37-38]

Types of reporter genes

Name

Core characteristics

Main application scenarios

Emission color

References

Fluorescent protein

Green fluorescent protein (GFP)

Common reporter for gene expression; tag for protein localization

Gene expression reporter; protein localization tag; organelle-specific marker

Green fluorescence (488-507 nm)

[31]

Enhanced green fluorescent protein (EGFP)

Optimized GFP mutant; enhanced fluorescence brightness; high expression efficiency; excellent photostability; most widely used green fluorescent protein variant

Gene expression reporter; protein localization tag; organelle marker; long-term live-cell imaging; quantitative fluorescence study

Green fluorescence (488-507 nm)

[31]

Red fluorescent protein (RFP, e.g., DsRed)

Derived from corals; low tissue scattering; high tissue penetration; low autofluorescence background; suitable for multicolor labeling and in vivo imaging

Multicolor fluorescence labeling; in vivo deep tissue imaging; protein localization tag

Red fluorescence (558-583 nm)

[32]

Monomeric red fluorescent protein (mCherry)

Optimized DsRed derivative; genuine monomer; rapid maturation; excellent photostability; widely used red fluorescent protein tag

Protein fusion and localization tag; long-term live-cell imaging; organelle and subcellular structure labeling

Red fluorescence (587-610 nm)

[33]

Far-red/near-infrared fluorescent protein (e.g., iRFP)

Derived from bacterial phytochromes; minimal tissue light scattering and absorption; deep penetration; ideal for in vivo imaging

Small animal in vivo whole-body imaging; multispectral fluorescence labeling; in vivo deep tissue imaging

Far-red/near-infrared light (690-713 nm)

[34]

Luciferase

Firefly luciferase

Most classic and widely used; substrate D-luciferin; ATP-dependent reaction; stable and long-lasting signal

Quantitative detection of gene expression in vitro; main reporter system for dual-reporter gene assays; detection of viral replication activity

Yellow-green light (560 nm)

[35]

Renilla luciferase

Substrate is coelenterazine; rapid flash signal; fast signal decay

Internal reference system for dual-reporter gene assays; transfection efficiency normalization; auxiliary quantitative detection of gene expression in vitro

Blue light (480 nm)

[35]

Gaussia luciferase (GLuc)

Naturally secreted; directly detectable in cell supernatant; real-time live-cell monitoring; high signal intensity

Real-time monitoring of gene expression in living cells; non-destructive detection in vitro; high-throughput screening of antiviral drugs

Blue light (480 nm)

[36]

NanoLuc luciferase (NLuc)

Engineered optimized probe; minimal size; ultra-strong signal; excellent stability; enhanced signal-to-noise ratio; substrate: furimazine

Ultra-high sensitivity in vitro detection; long-term live-cell monitoring; in vivo viral imaging; quantitative analysis of trace samples

Blue light (460 nm)

[37-38]

Strategies	Application	Advantages	Limitations
High-resolution microscopy technology	Study of viral ultrastructure, universal visualization of pathogens	High spatiotemporal resolution, feasible for long-term observation, and capable of simultaneously acquiring fluorescent dynamics and ultrastructure	Complex sample preparation, expensive equipment, professional operation, and technique-induced sample degradation
Exogenous nanomaterial labeling	Single-particle tracing of the entire viral life cycle, tracing of multiple viral structures	Long-term multicolor tracing, accurate localization, in situ non-invasiveness, excellent photostability, study on early molecular mechanisms combined with super-resolution technology	Cytotoxicity, poor tissue penetration, interference with viral infection, low labeling efficiency, non-specific adsorption
Recombinant virus reporter	Drug screening, serological determination, study on virus-host interactions	Dynamic analysis of viral infection, adapt to in vivo applications, high-throughput sequencing (HTS) for antiviral drugs, neutralizing antibody detection	Impaired viral replication with inserted reporter gene, genome instability, loss of exogenous reporter genes limiting its use in diverse clinical samples or emerging viruses
Virus infection-inducible reporter system	RdRp activity detection, high-throughput screening, replication mechanism	No need for viral modification, especially suitable for clinical isolates, flexible replacement of reporter genes, HTS for RdRp inhibitors, virus-host interaction research	Complex design with certain background signal

Strategies

Application

Advantages

Limitations

High-resolution microscopy technology

Study of viral ultrastructure, universal visualization of pathogens

High spatiotemporal resolution, feasible for long-term observation, and capable of simultaneously acquiring fluorescent dynamics and ultrastructure

Complex sample preparation, expensive equipment, professional operation, and technique-induced sample degradation

Exogenous nanomaterial labeling

Single-particle tracing of the entire viral life cycle, tracing of multiple viral structures

Long-term multicolor tracing, accurate localization, in situ non-invasiveness, excellent photostability, study on early molecular mechanisms combined with super-resolution technology

Cytotoxicity, poor tissue penetration, interference with viral infection, low labeling efficiency, non-specific adsorption

Recombinant virus reporter

Drug screening, serological determination, study on virus-host interactions

Dynamic analysis of viral infection, adapt to in vivo applications, high-throughput sequencing (HTS) for antiviral drugs, neutralizing antibody detection

Impaired viral replication with inserted reporter gene, genome instability, loss of exogenous reporter genes limiting its use in diverse clinical samples or emerging viruses

Virus infection-inducible reporter system

RdRp activity detection, high-throughput screening, replication mechanism

No need for viral modification, especially suitable for clinical isolates, flexible replacement of reporter genes, HTS for RdRp inhibitors, virus-host interaction research

Complex design with certain background signal