A HiBiT-Tagged influenza A virus reporter system reveals cytokine-inhibitory micropeptide 53 as a modulator of viral infection and immunopathology

Shujie Ma; Sha Liu; Conghui Zhao; Wenjun Shi; Chunping Zhang; Jiacheng Huang; Yang Wang; Xiaoxuan Zhang; Haoxi Qiang; Huanhuan Wang; Peilin Li; Xinhui Chen; Ziyi Zhang

doi:10.1016/j.virs.2026.09.003

October 2026

Citation: Shujie Ma, Sha Liu, Conghui Zhao, Wenjun Shi, Chunping Zhang, Jiacheng Huang, Yang Wang, Xiaoxuan Zhang, Haoxi Qiang, Huanhuan Wang, Peilin Li, Xinhui Chen, Ziyi Zhang. A HiBiT-Tagged influenza A virus reporter system reveals cytokine-inhibitory micropeptide 53 as a modulator of viral infection and immunopathology .VIROLOGICA SINICA, 2026, 41(5) : 1106-1120. http://dx.doi.org/10.1016/j.virs.2026.09.003

A HiBiT-Tagged influenza A virus reporter system reveals cytokine-inhibitory micropeptide 53 as a modulator of viral infection and immunopathology

Shujie Ma ^{a,c,d
,,} ,
Sha Liu ^a,c,d ,
Conghui Zhao ^a,c,d ,
Wenjun Shi ^b ,
Chunping Zhang ^a,c,d ,
Jiacheng Huang ^a,c,d ,
Yang Wang ^a,c,d ,
Xiaoxuan Zhang ^a,c,d ,
Haoxi Qiang ^a,c,d ,
Huanhuan Wang ^a,c,d ,
Peilin Li ^a,c,d ,
Xinhui Chen ^a,c,d ,
Ziyi Zhang ^a,c,d

a. Joint Laboratory of Animal Pathogen Prevention and Control of Fujian-Nepal, College of Animal Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China;
b. Animal Quarantine Laboratory, Technology Center of Qingdao Customs, Qingdao 266000, China;
c. Key Laboratory of Animal Pathogen Infection and Immunology of Fujian Province, College of Animal Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China;
d. Key Laboratory of Fujian-Taiwan Animal Pathogen Biology, College of Animal Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China

Corresponding author: Shujie Ma, mashujie@fafu.edu.cn
Received Date: 03 June 2026
Accepted Date: 03 September 2026
Available online: 05 September 2026

Abstract

Influenza A virus (IAV) remains a major threat to human and animal health, highlighting the need for efficient approaches to identify host factors and antivirals. Reporter viruses are critical tools for these efforts, but incorporating large reporter genes often compromises viral fitness and genetic stability. Here, we developed a replication competent reporter IAV by fusing an 11-amino acid HiBiT tag into the nonstructural protein 1 (NS1) of the A/WSN/1933 (H1N1) backbone. The modified virus retained parental virion morphology, comparable replication kinetics, and in vivo tissue tropism, while enabling the highly sensitive, rapid, and quantitative detection of viral replication. Using this reporter virus, we established a robust high-throughput screening (HTS) platform with excellent assay quality and validated its capability to identify host regulators of IAV infection. Application of this platform to host-encoded micropeptides (miPEPs) led to the identification of cytokine-inhibitory micropeptide 53 (CIM53) as a novel regulator of IAV replication. Mechanistically, CIM53 promoted IAV replication by intrinsically suppressing antiviral innate immune responses. Furthermore, although CIM53 enhanced viral replication, its immunomodulatory activity contributed to reduced lung injury and improved survival when combined with Oseltamivir (OSV) treatment in infected mice. Our study establishes a highly practical screening platform for virological research and highlights CIM53 as a potential host-directed adjunct therapy for severe IAV infection.
- Influenza A virus (IAV)
- , HiBiT tag
- , NS1
- , Reporter virus
- , Cytokine-inhibitory micropeptide 53 (CIM53)

Electronic Supplementary Material

10.1016j.virs.2026.09.003-ESM.docx
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