A dual-reporter HCoV-OC43 for coronavirus biology and countermeasure development

IF 4.3 2区医学 Q1 PHARMACOLOGY & PHARMACY

Antiviral research Pub Date : 2025-12-01 Epub Date: 2025-11-04 DOI:10.1016/j.antiviral.2025.106306

Xiangxue Deng , Jing Zou , Zhenjie Liang , Ping Ren , Pei-Yong Shi , Vineet D. Menachery , Xuping Xie

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Abstract

A reliable experimental system is essential for advancing coronavirus biology and expediting countermeasure development. In this study, we report the construction of an infectious clone of human coronavirus OC43 (HCoV-OC43) using an in vitro ligation strategy. The clone-derived virus faithfully recapitulates the replication characteristics of the parental isolate in vitro. Leveraging this platform, we have developed a dual-reporter virus, OC43-mNG-Nluc, by replacing the viral accessory gene ns2 with a gene cassette encoding both nanoluciferase and fluorescent protein mNeonGreen. This construct enables dual-mode detection via chemiluminescence and fluorescence within a single assay. OC43-mNG-Nluc maintains replication kinetics identical to wild-type HCoV-OC43 in standard immortalized cell lines but exhibits marked attenuation in primary human airway epithelial cultures, underscoring the critical role of ns2 in viral replication within physiologically relevant systems. The dual-reporter virus also demonstrates excellent genetic and phenotypic stability following fifteen passages in vitro. Furthermore, we validate the proof-of-concept utility of OC43-mNG-Nluc in high-throughput antiviral screening, showcasing its advantages in streamlining and facilitating hit identification, triaging, and prioritization. Collectively, this study provides valuable insights into HCoV-OC43 biology and introduces a versatile and robust tool for coronavirus research and therapeutic development.

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新型冠状病毒生物学双报告型HCoV-OC43及其对策研究

可靠的实验系统是推进冠状病毒生物学和加快对策制定的必要条件。在这项研究中，我们报告了使用体外结扎策略构建人类冠状病毒OC43的感染性克隆。克隆衍生病毒在体外忠实地再现了亲本分离物的复制特性。利用这一平台，我们开发了一种双报告病毒，oc43 - mg - nluc，通过用编码纳米荧光素酶和荧光蛋白mNeonGreen的基因盒取代病毒的辅助基因ns2。这种结构使双模式检测通过化学发光和荧光在一个单一的分析。在标准永生化细胞系中，OC43- mg - nluc保持与野生型OC43相同的复制动力学，但在原代人气道上皮培养物中表现出明显的衰减，强调了ns2在生理相关系统中病毒复制中的关键作用。在体外15次传代后，双报告病毒也表现出良好的遗传和表型稳定性。此外，我们验证了OC43-mNG-Nluc在高通量抗病毒筛选中的概念验证效用，展示了其在简化和促进命中识别、分诊和优先排序方面的优势。总的来说，这项研究为HCoV-OC43生物学提供了有价值的见解，并为冠状病毒的研究和治疗开发引入了一个多功能和强大的工具。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Antiviral research 医学-病毒学

CiteScore

17.10

自引率

3.90%

发文量

157

审稿时长

34 days

期刊介绍： Antiviral Research is a journal that focuses on various aspects of controlling viral infections in both humans and animals. It is a platform for publishing research reports, short communications, review articles, and commentaries. The journal covers a wide range of topics including antiviral drugs, antibodies, and host-response modifiers. These topics encompass their synthesis, in vitro and in vivo testing, as well as mechanisms of action. Additionally, the journal also publishes studies on the development of new or improved vaccines against viral infections in humans. It delves into assessing the safety of drugs and vaccines, tracking the evolution of drug or vaccine-resistant viruses, and developing effective countermeasures. Another area of interest includes the identification and validation of new drug targets. The journal further explores laboratory animal models of viral diseases, investigates the pathogenesis of viral diseases, and examines the mechanisms by which viruses avoid host immune responses.