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

. doi: 10.1016/j.virs.2026.09.003

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

HiBiT标记的流感病毒报告系统揭示细胞因子抑制性微肽53调控病毒感染与免疫病理

马树杰 ^a,c,d,, ,
刘莎 ^a,c,d ,
赵聪慧 ^a,c,d ,
石文军 ^b ,
张春萍 ^a,c,d ,
黄佳承 ^a,c,d ,
王洋 ^a,c,d ,
张晓璇 ^a,c,d ,
强皓溪 ^a,c,d ,
王欢欢 ^a,c,d ,
李沛霖 ^a,c,d ,
陈欣慧 ^a,c,d ,
张子艺 ^a,c,d

a. 福建农林大学, 动物科学学院/福建省-尼泊尔畜禽重大疫病防控联合实验室;
b. 青岛海关技术中心, 动物检疫实验室;
c. 福建农林大学, 动物科学学院/福建省畜禽病原感染与免疫学重点实验室;
d. 福建农林大学, 动物科学学院/闽台动物病原生物学福建省高校重点实验室

通讯作者： 马树杰, mashujie@fafu.edu.cn;
收稿日期： 2026-06-03
录用日期： 2026-09-03

摘要

A型流感病毒（IAV）严重威胁人类和动物健康，开发高效的宿主因子和抗病毒药物筛选方法对于深入研究IAV感染机制及抗病毒干预具有重要意义。报告病毒是开展相关研究的重要工具，但传统报告病毒通常需要插入较大的报告基因，容易对病毒的复制能力和遗传稳定性产生不利影响。本研究以A/WSN/1933（H1N1）病毒为基础，将仅含11个氨基酸的HiBiT标签融合至非结构蛋白1（NS1）中，构建了一株具有复制能力的IAV报告病毒。与亲本病毒相比，该报告病毒具有相似的病毒颗粒形态、复制动力学和体内组织嗜性，同时能够灵敏、快速且定量地反映病毒复制水平。基于该报告病毒，我们进一步建立了稳定、可靠的高通量筛选平台。该平台具有良好的检测性能，并通过宿主因子筛选验证了其在鉴定IAV感染相关宿主因子方面的应用潜力。随后，我们将该平台应用于宿主编码微肽（miPEPs）的筛选，鉴定并命名了细胞因子抑制性微肽53（CIM53），并发现其是一个新的IAV复制调控因子。机制研究表明，CIM53可通过抑制宿主抗病毒先天免疫反应促进IAV复制。值得注意的是，尽管CIM53可促进病毒复制，但它与奥司他韦（OSV）联合治疗条件下能够减轻IAV感染小鼠的肺部损伤，并提高动物存活率。综上，本研究建立了一种实用、高效的IAV报告病毒及高通量筛选平台，为宿主因子和抗病毒药物的筛选提供了新的技术手段；同时发现CIM53具有调控宿主抗病毒免疫反应和减轻炎症损伤的作用，提示其可能作为治疗重症IAV感染的潜在药物。
- 甲型流感病毒
- , HiBiT标签
- , NS1
- , 报告病毒
- , 细胞因子抑制性微肽53

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

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)

References
1. Ayllon, J.,Garcia-Sastre, A., 2015. The NS1 Protein: A Multitasking Virulence Factor. Current Topics in Microbiology and Immunology, 386, 73-107.
2. Basler, C.F., Reid, A.H., Dybing, J.K., Janczewski, T.A., Fanning, T.G., Zheng, H., Salvatore, M., Perdue, M.L., Swayne, D.E., Garcia-Sastre, A., Palese, P.,Taubenberger, J.K., 2001. Sequence of the 1918 pandemic influenza virus nonstructural gene (NS) segment and characterization of recombinant viruses bearing the 1918 NS genes. Proc Natl Acad Sci U S A, 98, 2746-2751.
3. Breen, M., Nogales, A., Baker, S.F.,Martinez-Sobrido, L., 2016. Replication-competent influenza A viruses expressing reporter genes. Viruses, 8, 179.
4. Caceres, C.J., Gay, L.C., Faccin, F.C.,Perez, D.R., 2024. Use of Reverse Genetics for the Generation of Recombinant Influenza Viruses Carrying Nanoluciferase. Methods Mol Biol, 2733, 47-74.
5. Chang, C.C., Tu, H.Y., Peng, S.Y., Hsu, H.J.,Jiang, S.J., 2025. Unveiling the anti-inflammatory potential of RF16, an interleukin 8-derived therapeutic peptide in macrophages. J Inflamm (Lond), 22, 29.
6. Chaudhary, R., Meher, A., Krishnamoorthy, P.,Kumar, H., 2023. Interplay of host and viral factors in inflammatory pathway mediated cytokine storm during RNA virus infection. Curr Res Immunol, 4, 100062.
7. Chen, H., Yuan, H., Gao, R., Zhang, J., Wang, D., Xiong, Y., Fan, G., Yang, F., Li, X., Zhou, J., et al., 2014. Clinical and epidemiological characteristics of a fatal case of avian influenza A H10N8 virus infection: a descriptive study. Lancet, 383, 714-721.
8. Chi, X., Huang, G., Wang, L., Zhang, X., Liu, J., Yin, Z., Guo, G., Chen, Y., Wang, S.,Chen, J.L., 2024. A small protein encoded by PCBP1-AS1 is identified as a key regulator of influenza virus replication via enhancing autophagy. PLoS Pathog, 20, e1012461.
9. Chockalingam, A.K., Hamed, S., Goodwin, D.G., Rosenzweig, B.A., Pang, E., Boyne, M.T., 2nd,Patel, V., 2016. The Effect of Oseltamivir on the Disease Progression of Lethal Influenza A Virus Infection: Plasma Cytokine and miRNA Responses in a Mouse Model. Dis Markers, 2016, 9296457.
10. Choudhury, N.R., Trus, I., Heikel, G., Wolczyk, M., Szymanski, J., Bolembach, A., Dos Santos Pinto, R.M., Smith, N., Trubitsyna, M., Gaunt, E., Digard, P.,Michlewski, G., 2022. TRIM25 inhibits influenza A virus infection, destabilizes viral mRNA, but is redundant for activating the RIG-I pathway. Nucleic Acids Res, 50, 7097-7114.
11. Diamond, M.S.,Farzan, M., 2013. The broad-spectrum antiviral functions of IFIT and IFITM proteins. Nat Rev Immunol, 13, 46-57.
12. Dixon, A.S., Schwinn, M.K., Hall, M.P., Zimmerman, K., Otto, P., Lubben, T.H., Butler, B.L., Binkowski, B.F., Machleidt, T., Kirkland, T.A., Wood, M.G., Eggers, C.T., Encell, L.P.,Wood, K.V., 2016. NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells. ACS Chem Biol, 11, 400-408.
13. Elton, D., Simpson-Holley, M., Archer, K., Medcalf, L., Hallam, R., Mccauley, J.,Digard, P., 2001. Interaction of the influenza virus nucleoprotein with the cellular CRM1-mediated nuclear export pathway. J Virol, 75, 408-419.
14. Fajgenbaum, D.C.,June, C.H., 2020. Cytokine storm. N Engl J Med, 383, 2255-2273.
15. Fukuyama, S., Katsura, H., Zhao, D., Ozawa, M., Ando, T., Shoemaker, J.E., Ishikawa, I., Yamada, S., Neumann, G., Watanabe, S., Kitano, H.,Kawaoka, Y., 2015. Multi-spectral fluorescent reporter influenza viruses (Color-flu) as powerful tools for in vivo studies. Nat Commun, 6, 6600.
16. Gu, C., Maemura, T., Guan, L., Eisfeld, A.J., Biswas, A., Kiso, M., Uraki, R., Ito, M., Trifkovic, S., Wang, T., Babujee, L., Presler, R., Jr., Dahn, R., Suzuki, Y., Halfmann, P.J., Yamayoshi, S., Neumann, G.,Kawaoka, Y., 2024. A human isolate of bovine H5N1 is transmissible and lethal in animal models. Nature, 636, 711-718.
17. Gu, Y., Zuo, X., Zhang, S., Ouyang, Z., Jiang, S., Wang, F.,Wang, G., 2021. The Mechanism behind Influenza Virus Cytokine Storm. Viruses, 13, 1362.
18. Hao, Y., Zhang, L., Niu, Y., Cai, T., Luo, J., He, S., Zhang, B., Zhang, D., Qin, Y., Yang, F.,Chen, R., 2018. SmProt: a database of small proteins encoded by annotated coding and non-coding RNA loci. Brief Bioinform, 19, 636-643.
19. Haviernik, J., Eyer, L., Yoshii, K., Kobayashi, S., Cerny, J., Nougairede, A., Driouich, J.S., Volf, J., Palus, M., De Lamballerie, X., Gould, E.A.,Ruzek, D., 2021. Development and characterization of recombinant tick-borne encephalitis virus expressing mCherry reporter protein: A new tool for high-throughput screening of antiviral compounds, and neutralizing antibody assays. Antiviral Res, 185, 104968.
20. Hou, Y., Deng, G., Cui, P., Zeng, X., Li, B., Wang, D., He, X., Yan, C., Zhang, Y., Li, J., Ma, J., Li, Y., Wang, X., Tian, G., Kong, H., Tang, L., Suzuki, Y., Shi, J.,Chen, H., 2024. Evolution of H7N9 highly pathogenic avian influenza virus in the context of vaccination. Emerg Microbes Infect, 13, 2343912.
21. Iversen;, P.W., Beck;, B., Chen;, Y.-F., Dere;, W., Devanarayan;, V., Eastwood;, B.J., Farmen;, M.W., Iturria;, S.J., Montrose;, C., Moore;, R.A., Weidner;, J.R.,Sittampalam, G.S., 2012. HTS Assay Validation. Eli Lilly & Company and the National Center for Advancing Translational Sciences.
22. Iwasaki, A.,Pillai, P.S., 2014. Innate immunity to influenza virus infection. Nat Rev Immunol, 14, 315-328.
23. Jiao, P.R., Tian, G.B., Li, Y.B., Deng, G.H., Jiang, Y.P., Liu, C., Liu, W.L., Bu, Z.G., Kawaoka, Y.,Chen, H.L., 2008. A single-amino-acid substitution in the NS1 protein changes the pathogenicity of H5N1 avian influenza viruses in mice. Journal of Virology, 82, 1146-1154.
24. Killip, M.J., Smith, M., Jackson, D.,Randall, R.E., 2014. Activation of the interferon induction cascade by influenza a viruses requires viral RNA synthesis and nuclear export. J Virol, 88, 3942-3952.
25. Kim, J.H., Lee, S.R., Li, L.H., Park, H.J., Park, J.H., Lee, K.Y., Kim, M.K., Shin, B.A.,Choi, S.Y., 2011. High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. PLoS One, 6, e18556.
26. Kong, H., Zhang, Q., Gu, C., Shi, J., Deng, G., Ma, S., Liu, J., Chen, P., Guan, Y., Jiang, Y.,Chen, H., 2015. A live attenuated vaccine prevents replication and transmission of H7N9 virus in mammals. Sci Rep, 5, 11233.
27. Lee, S., Yoon, S.S., Jo, M., Kang, M., Lee, S., Seo, Y.J., Park, S., Paik, Y.K.,Jo, D., 2024. Intracellular delivery of nuclear localization sequence peptide mitigates COVID-19 by inhibiting nuclear transport of inflammation-associated transcription factors. Mol Ther, 32, 227-240.
28. Li, X., Zhang, H., Zhang, Y., Li, J., Wang, Z., Deng, C., Jardim, A.C.G., Terzian, A.C.B., Nogueira, M.L.,Zhang, B., 2019. Development of a rapid antiviral screening assay based on eGFP reporter virus of Mayaro virus. Antiviral Res, 168, 82-90.
29. Li, Y., Zhou, H., Chen, X., Zheng, Y., Kang, Q., Hao, D., Zhang, L., Song, T., Luo, H., Hao, Y., Chen, R., Zhang, P.,He, S., 2021. SmProt: A Reliable Repository with Comprehensive Annotation of Small Proteins Identified from Ribosome Profiling. Genomics Proteomics Bioinformatics, 19, 602-610.
30. Liang, X.Y., Zhu, Q.C., Liang, J.Q., Liu, S.Y., Liu, D.X.,Fung, T.S., 2020. Development of HiBiT-Tagged Recombinant Infectious Bronchitis Coronavirus for Efficient in vitro and in vivo Viral Quantification. Front Microbiol, 11, 2100.
31. Liu, Q., Zhou, Y.H.,Yang, Z.Q., 2016. The cytokine storm of severe influenza and development of immunomodulatory therapy. Cell Mol Immunol, 13, 3-10.
32. Liu, S., Yan, R., Chen, B., Pan, Q., Chen, Y., Hong, J., Zhang, L., Liu, W., Wang, S.,Chen, J.L., 2019. Influenza Virus-Induced Robust Expression of SOCS3 Contributes to Excessive Production of IL-6. Front Immunol, 10, 1843.
33. Ma, S., Zhang, B., Shi, J., Yin, X., Wang, G., Cui, P., Liu, L., Deng, G., Jiang, Y., Li, C.,Chen, H., 2020. Amino acid mutations A286V and T437M in the nucleoprotein attenuate H7N9 viruses in mice. J Virol, 94, e01530-01519.
34. Miyakawa, K., Jeremiah, S.S., Ohtake, N., Matsunaga, S., Yamaoka, Y., Nishi, M., Morita, T., Saji, R., Nishii, M., Kimura, H., Hasegawa, H., Takeuchi, I.,Ryo, A., 2020. Rapid quantitative screening assay for SARS-CoV-2 neutralizing antibodies using HiBiT-tagged virus-like particles. J Mol Cell Biol, 12, 987-990.
35. Nagashima, S., Primadharsini, P.P., Nishiyama, T., Takahashi, M., Murata, K.,Okamoto, H., 2023. Development of a HiBiT-tagged reporter hepatitis E virus and its utility as an antiviral drug screening platform. J Virol, 97, e0050823.
36. Nakaya, Y., Onomura, D., Hoshi, Y., Yamagata, T., Morita, H., Okamoto, H.,Murata, K., 2024. Establishment of a hepatitis B virus reporter system harboring a HiBiT-tag in the PreS2 region. J Infect Dis, 231, 204-213.
37. Neumann, G., Watanabe, T., Ito, H., Watanabe, S., Goto, H., Gao, P., Hughes, M., Perez, D.R., Donis, R., Hoffmann, E., Hobom, G.,Kawaoka, Y., 1999. Generation of influenza A viruses entirely from cloned cDNAs. Proceedings of the National Academy of Sciences of the United States of America, 96, 9345-9350.
38. Niu, L., Lou, F., Sun, Y., Sun, L., Cai, X., Liu, Z., Zhou, H., Wang, H., Wang, Z., Bai, J., Yin, Q., Zhang, J., Chen, L., Peng, D., Xu, Z., Gao, Y., Tang, S., Fan, L.,Wang, H., 2020. A micropeptide encoded by lncRNA MIR155HG suppresses autoimmune inflammation via modulating antigen presentation. Sci Adv, 6, eaaz2059.
39. Okura, T., Miyakawa, K., Tahara, M., Someya, K., Seki, F., Nishi, M., Otsuki, N.,Ryo, A., 2024. Rapid quantitative detection system for measles virus-neutralizing antibodies using HiBiT-tagged virus-like particles. Microbiol Immunol, 68, 160-164.
40. Peacock, T., Moncla, L., Dudas, G., Vaninsberghe, D., Sukhova, K., Lloyd-Smith, J.O., Worobey, M., Lowen, A.C.,Nelson, M.I., 2025. The global H5N1 influenza panzootic in mammals. Nature, 637, 304-313.
41. Primadharsini, P.P., Nagashima, S., Nishiyama, T.,Okamoto, H., 2023. Three distinct reporter systems of hepatitis E virus and their utility as drug screening platforms. Viruses, 15, 1989.
42. Puckette, M., Burrage, T., Neilan, J.G.,Rasmussen, M., 2017. Evaluation of Gaussia luciferase and foot-and-mouth disease virus 2A translational interrupter chimeras as polycistronic reporters for transgene expression. BMC Biotechnol, 17, 52.
43. Rajan, A., Nair, A.S., Pillai, V.S., Kumar, B., Pai, A.R., Benny, B.,Veettil, M.V., 2024. Highly sensitive and quantitative HiBiT-tagged Nipah virus-like particles: A platform for rapid antibody neutralization studies. Heliyon, 10, e31905.
44. Saito, M., Itoh, Y., Yasui, F., Munakata, T., Yamane, D., Ozawa, M., Ito, R., Katoh, T., Ishigaki, H., Nakayama, M., et al., 2021. Macrocyclic peptides exhibit antiviral effects against influenza virus HA and prevent pneumonia in animal models. Nat Commun, 12, 2654.
45. Schwinn, M.K., Machleidt, T., Zimmerman, K., Eggers, C.T., Dixon, A.S., Hurst, R., Hall, M.P., Encell, L.P., Binkowski, B.F.,Wood, K.V., 2018. CRISPR-Mediated Tagging of Endogenous Proteins with a Luminescent Peptide. ACS Chem Biol, 13, 467-474.
46. Shi, J., Deng, G., Kong, H., Gu, C., Ma, S., Yin, X., Zeng, X., Cui, P., Chen, Y., Yang, H., et al., 2017. H7N9 virulent mutants detected in chickens in China pose an increased threat to humans. Cell Res, 27, 1409-1421.
47. Shi, Y.J., Li, J.Q., Zhang, H.Q., Deng, C.L., Zhu, Q.X., Zhang, B.,Li, X.D., 2023. A high throughput antiviral screening platform for alphaviruses based on Semliki Forest virus expressing eGFP reporter gene. Virol Sin, 38, 585-594.
48. Sun, H., Gu, R., Tang, T., Rai, K.R.,Chen, J.L., 2025a. Current Perspectives on Functional Involvement of Micropeptides in Virus-Host Interactions. Int J Mol Sci, 26, 3651.
49. Sun, H., Li, H., Tong, Q., Han, Q., Liu, J., Yu, H., Song, H., Qi, J., Li, J., Yang, J., et al., 2023. Airborne transmission of human-isolated avian H3N8 influenza virus between ferrets. Cell, 186, 4074-4084.e4011.
50. Sun, J., Yang, J., Tao, J., Yang, Y., Wang, R., Zhang, H., Liu, W., Zhao, S., Shao, R., He, Y., et al., 2025b. Delaying pyroptosis with an AI-screened gasdermin D pore blocker mitigates inflammatory response. Nat Immunol, 26, 1660-1672.
51. Te Velthuis, A.J.W., Long, J.C., Bauer, D.L.V., Fan, R.L.Y., Yen, H.L., Sharps, J., Siegers, J.Y., Killip, M.J., French, H., Oliva-Martin, M.J., Randall, R.E., De Wit, E., Van Riel, D., Poon, L.L.M.,Fodor, E., 2018. Mini viral RNAs act as innate immune agonists during influenza virus infection. Nat Microbiol, 3, 1234-1242.
52. Thangavel, R.R.,Bouvier, N.M., 2014. Animal models for influenza virus pathogenesis, transmission, and immunology. J Immunol Methods, 410, 60-79.
53. Wang, G., Jiang, L., Wang, J., Zhang, J., Kong, F., Li, Q., Yan, Y., Huang, S., Zhao, Y., Liang, L., et al., 2020. The G protein-coupled receptor FFAR2 promotes internalization during influenza A virus entry. J Virol, 94, e01707-e01719.
54. Wang, G., Zhang, J., Kong, F., Li, Q., Wang, J., Ma, S., Zhao, Y., Liang, L., Li, J., Sun, N., Guan, L., Zhou, Y., Zhou, C., Huang, S., Bu, Z., Jiang, L., Chen, H.,Li, C., 2018a. Generation and application of replication-competent Venus-expressing H5N1, H7N9, and H9N2 influenza A viruses. Sci Bull (Beijing), 63, 176-186.
55. Wang, J., Wang, Z., Liu, R., Shuai, L., Wang, X., Luo, J., Wang, C., Chen, W., Wang, X., Ge, J., He, X., Wen, Z.,Bu, Z., 2018b. Metabotropic glutamate receptor subtype 2 is a cellular receptor for rabies virus. 14, e1007189.
56. Yang, W., Yin, X., Guan, L., Li, M., Ma, S., Shi, J., Deng, G., Suzuki, Y.,Chen, H., 2018. A live attenuated vaccine prevents replication and transmission of H7N9 highly pathogenic influenza viruses in mammals. Emerg Microbes Infect, 7, 153.
57. Zhang, C., Zhang, G., Zhang, Y., Lin, X., Zhao, X., Cui, Q., Rong, L.,Du, R., 2023. Development of an HiBiT-tagged reporter H3N2 influenza A virus and its utility as an antiviral screening platform. J Med Virol, 95, e28345.
58. Zhang, C., Zhao, C., Huang, J., Wang, Y., Jiang, B., Zheng, H., Zhuang, M., Peng, Y., Zhang, X., Liu, S., Qiang, H., Wang, H., Zeng, X., Guo, G., Chen, J.L.,Ma, S., 2025a. Emergence of a novel reassortant H3N3 avian influenza virus with enhanced pathogenicity and transmissibility in chickens in China. Vet Res, 56, 56.
59. Zhang, J.H., Chung, T.D.,Oldenburg, K.R., 1999. A smple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen, 4, 67-73.
60. Zhang, J.W., Wang, H., Liu, J., Ma, L., Hua, R.H.,Bu, Z.G., 2021. Generation of a stable GFP-reporter zika virus system for high-throughput screening of zika virus inhibitors. Virol Sin, 36, 476-489.
61. Zhang, Q., Shi, J., Deng, G., Guo, J., Zeng, X., He, X., Kong, H., Gu, C.Y., Li, X.Y., Liu, J.X., et al., 2013a. H7N9 influenza viruses are transmissible in ferrets by respiratory droplet. Science, 341, 410-414.
62. Zhang, T., Li, Z., Li, J.,Peng, Y., 2025b. Small open reading frame-encoded microproteins in cancer: identification, biological functions and clinical significance. Mol Cancer, 24, 105.
63. Zhang, Y., Zhang, Q., Kong, H., Jiang, Y., Gao, Y., Deng, G., Shi, J., Tian, G., Liu, L., Liu, J., Guan, Y., Bu, Z.,Chen, H., 2013b. H5N1 hybrid viruses bearing 2009/H1N1 virus genes transmit in guinea pigs by respiratory droplet. Science, 340, 1459-1463.
64. Zhao, C., Huang, J., Zhang, C., Wang, Y., Zhang, X., Liu, S., Qiang, H., Wang, H., Zheng, H., Zhuang, M., Peng, Y., Chen, F., Zeng, X., Chen, J.-L.,Ma, S., 2025a. Characteristics of the first domestic duck-origin H12N8 avian influenza virus in China. Int J Mol Sci, 26, 2740.
65. Zhao, C., Zhang, X., Wang, H., Qiang, H., Liu, S., Zhang, C., Huang, J., Wang, Y., Li, P., Chen, X., Zhang, Z.,Ma, S., 2025b. Proteomic analysis of differentially expressed proteins in A549 cells infected with H9N2 avian influenza virus. Int J Mol Sci, 26, 657.
66. Zumla, A., Rao, M., Wallis, R.S., Kaufmann, S.H., Rustomjee, R., Mwaba, P., Vilaplana, C., Yeboah-Manu, D., Chakaya, J., Ippolito, G., Azhar, E., Hoelscher, M.,Maeurer, M., 2016. Host-directed therapies for infectious diseases: current status, recent progress, and future prospects. Lancet Infect Dis, 16, e47-63.
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