Genomic and pathogenic characterization of a chicken-origin cluster 3.2 Tembusu virus isolated in China

Abstract

Tembusu virus (TMUV) is an emerging flavivirus that causes encephalitis and a decrease in egg production in poultry across East and Southeast Asia. Here, we isolated and characterized a chicken-origin TMUV strain, CHN-WH, from a laying hen farm in China. Whole-genome sequencing and phylogenetic analysis based on the complete open reading frame revealed that CHN-WH belongs to cluster 3.2 and is most closely related to the mosquito-derived strain TMUV-YN2020. Comparative sequence analysis and structural modeling revealed several cluster 3.2-specific signature residues in the E protein, as well as a strain-specific M198L substitution predicted to be located in a surface-exposed region. CHN-WH replicated efficiently in BHK-21 cells, chicken embryo fibroblasts (CEFs), and duck embryo fibroblasts (DEFs). In vivo, CHN-WH caused systemic viral dissemination and neurological lesions in chicks and induced ovarian lesions accompanied by reduced egg production in laying hens. These findings indicate that CHN-WH is a pathogenic chicken-origin cluster 3.2 TMUV strain with distinct genomic and phenotypic characteristics. This study expands the current knowledge of the molecular features and pathogenic potential of the chicken-origin cluster 3.2 TMUV and provides useful information for epidemiological surveillance and vaccine development.

Keywords

Tembusu virus / Phylogenetic analysis / E protein / Pathogenicity / Chicken

References

[1] Cheng Y, Wang R, Wu Q, Chen J, Wang A, Wu Z, Sun F, Zhu S. Advancements in research on duck Tembusu virus infections. Viruses, 2024, 16: 811

[2] Cui Y, Pan Y, Guo J, Wang D, Tong X, Wang Y, Li J, Zhao J, Ji Y, Wu Z. The evolution, genomic epidemiology, and transmission dynamics of Tembusu virus. Viruses, 2022, 14: 1236

[3] Feng C, Jin M, Yang L, Lv J, Qu S, Meng R, Yang B, Wang X, Zhang D. Pathogenicity of a Jinding duck-origin cluster 2.1 isolate of Tembusu virus in 3-week-old Pekin ducklings. Veterinary Microbiology, 2020, 251: 108870

[4] Hamel R, Phanitchat T, Wichit S, Morales Vargas RE, Jaroenpool J, Diagne CT, Pompon J, Missé D. New insights into the biology of the emerging Tembusu virus. Pathogens, 2021, 10: 1010

[5] Hamel R, Vargas REM, Rajonhson DM, Yamanaka A, Jaroenpool J, Wichit S, Missé D, Kritiyakan A, Chaisiri K, Morand S, et al.. Identification of the Tembusu virus in mosquitoes in Northern Thailand. Viruses, 2023, 15: 1447

[6] He D, Zhang X, Chen L, Tang Y, Diao Y. Development of an attenuated live vaccine candidate of duck Tembusu virus strain. Veterinary Microbiology, 2019, 231: 218-225

[7] Hu D, Wu C, Wang R, Yao X, Nie K, Lv Q, Fu S, Yin Q, Su W, Li F. Persistence of Tembusu virus in Culex tritaeniorhynchus in Yunnan Province, China. Pathogens, 2023, 12: 490

[8] Hu T, Wu Z, Wu S, Wang M, Jia R, Zhu D, Liu M, Zhao X, Yang Q, Wu Y. Substitutions at loop regions of TMUV E protein domain III differentially impair viral entry and assembly. Frontiers in Microbiology, 2021, 12: 688172

[9] Huang X, Han K, Zhao D, Liu Y, Zhang J, Niu H, Zhang K, Zhu J, Wu D, Gao L. Identification and molecular characterization of a novel flavivirus isolated from geese in China. Research in Veterinary Science, 2013, 94: 774-780

[10] Huang Y, Chu X, Zhang Y, Yang S, Shi Y, Wu J, Chen Q. Duck Tembusu virus infection causes testicular atrophy. Theriogenology, 2022, 188: 52-62

[11] Huang Y, Hu Z, Dong J, Li L, Zhang J, Kuang R, Gao S, Sun M, Liao M. Chicken-origin cluster 3.2 Tembusu virus exhibits higher infectivity than duck-origin cluster 2 Tembusu virus in chicks. Frontiers in Veterinary Science, 2023, 10: 1152802

[12] Kono Y, Tsukamoto K, Abd Hamid M, Darus A, Lian TC, Sam LS, Yok CN, Di KB, Lim KT, Yamaguchi S. Encephalitis and retarded growth of chicks caused by Sitiawan virus, a new isolate belonging to the genus Flavivirus. American Journal of Tropical Medicine and Hygiene, 2000, 63: 94-101

[13] Li Q, Feng C, Yang B, Meng R, Wang X, Zhang D. Antibody prophylaxis against Tembusu virus-associated disease. Archives of Virology, 2022, 167: 1687-1691

[14] Liao JY, Tang H, Xiong WJ, Liu TN, Li JY, Xia JY, Xiao CT. Isolation and characterization of a novel goose ovaritis-associated cluster 3 Tembusu virus. Poultry Science, 2023, 102: 102867

[15] Lin J, Liu Y, Wang X, Yang B, He P, Yang Z, Duan H, Xie J, Zou L, Zhao J. Efficacy evaluation of an inactivated duck Tembusu virus vaccine. Avian Diseases, 2015, 59: 244-248

[16] Liu M, Chen Shaoying, Chen Y, Liu C, Chen Shilong, Yin X, Li G, Zhang Y. Adapted Tembusu-like virus in chickens and geese in China. Journal of Clinical Microbiology, 2012, 50: 2807-2809

[17] Liu P, Lu H, Li S, Moureau G, Deng YQ, Wang Y, Zhang L, Jiang T, De Lamballerie X, Qin CF, et al.. Genomic and antigenic characterization of the newly emerging Chinese duck egg-drop syndrome flavivirus: Genomic comparison with Tembusu and Sitiawan viruses. Journal of General Virology, 2012, 93: 2158-2170

[18] Liu X, Yan D, Peng S, Zhang Y, Xu B, Li L, Shi X, Ma T, Li X, Teng Q. 326K in the E protein is critical for mammalian adaptation to TMUV. Viruses, 2023, 15: 2376

[19] Luo, X., Y. Liu, R. Jia, H. Shen, X. Wang, M. Wang, D. Zhu, S. Chen, M. Liu, and X. Zhao. 2018. Ultrastructure of duck Tembusu virus observed by electron microscopy with negative staining. Acta Virol 62. https://doi.org/10.4149/av_2018_227.

[20] Mackenzie JS, Williams DT. Zoonotic flaviviruses of Southern, South-Eastern and Eastern Asia and Australasia: The potential for emergent viruses. Zoonoses and Public Health, 2009, 56: 338-356

[21] Piewbang C, Yi L, Zahro AN, Poonsin P, Panyathi P, Kasantikul T, Kosoltanapiwat N, He B, Techangamsuwan S. Natural fatal infection of Tembusu virus in bottlenose dolphins in Thailand. Science and Reports, 2025, 15: 9917

[22] Qiu G, Cui Y, Li Yeqiu, Li Yongdong, Wang Y. The spread of Tembusu virus in China from 2010 to 2019. Virus Research, 2021, 300: 198374

[23] Tang Y, Gao X, Diao Y, Feng Q, Chen H, Liu X, Ge P, Yu C. Tembusu virus in human, China. Transboundary and Emerging Diseases, 2013, 60: 193-196

[24] Wang HJ, Li XF, Liu L, Xu YP, Ye Q, Deng YQ, Huang XY, Zhao H, Qin ED, Shi PY, et al.. The emerging duck flavivirus is not pathogenic for primates and is highly sensitive to mammalian interferon antiviral signaling. Journal of Virology, 2016, 90: 6538-6548

[25] Wang X, Chen X, Ding Y, Xu P, Wang C, Wei X, Peng H, Cui C, Kang O, Chen Y. Isolation, identification, and pathogenicity evaluation of a novel cluster 3 Tembusu virus isolated from geese in China. Poultry Science, 2025, 104: 104684

[26] Yan D, Li X, Wang Z, Liu X, Dong X, Fu R, Su X, Xu B, Teng Q, Yuan C. The emergence of a disease caused by a mosquito origin cluster 3.2 Tembusu virus in chickens in China. Veterinary Microbiology, 2022, 272: 109500

[27] Yan P, Zhao Y, Zhang X, Xu D, Dai X, Teng Q, Yan L, Zhou J, Ji X, Zhang S. An infectious disease of ducks caused by a newly emerged Tembusu virus strain in mainland China. Virology, 2011, 417: 1-8

[28] Yang, C. C., W. H. Tan, R. J. Zou, Y. Liang, X. L. Zhang, and J. W. Shao. 2026. Expansion of cluster 3 Tembusu virus in Guangdong geese with hepatitis-splenomegaly syndrome and evidence of recombination. Comp Immunol Microbiol Infect Dis 126:102439. https://doi.org/10.1016/j.cimid.2026.102439.

[29] Yang Q, Ding Y, Yao W, Chen S, Jiang Y, Yang L, Bao G, Yang K, Fan S, Du Q. Pathogenicity and interspecies transmission of cluster 3 Tembusu virus strain TMUV HQ-22 isolated from geese. Viruses, 2023, 15: 2449

[30] Yu Z, Ren H, Sun M, Xie W, Sun S, Liang N, Wang H, Ying X, Sun Y, Wang Y. Tembusu virus infection in laying chickens: Evidence for a distinct genetic cluster with significant antigenic variation. Transboundary and Emerging Diseases, 2022, 69: e1130-e1141

[31] Zhang J, An D, Fan Y, Tang Y, Diao Y. Effect of TMUV on immune organs of TMUV infected ducklings. Veterinary Microbiology, 2021, 255: 109033

[32] Zhang W, Chen S, Mahalingam S, Wang M, Cheng A. An updated review of avian-origin Tembusu virus: A newly emerging avian Flavivirus. Journal of General Virology, 2017, 98: 2413-2420

[33] Zhou P, Li Y, Liu A, Zhang Q, Wu W, Jin H, Jongkaewwattana A, He Q, Luo R. Tembusu virus nonstructural protein 2B antagonizes type I interferon production by targeting MAVS for degradation. Journal of Virology, 2022, 96: e00816-22

[34] Zhou P, Liu D, Zhang Q, Wu W, Chen D, Luo R. Antiviral effects of duck type I and type III interferons against duck Tembusu virus in vitro and in vivo. Veterinary Microbiology, 2023, 287: 109889

[35] Zhou P, Ma B, Gao Y, Xu Y, Li Z, Jin H, Luo R. Epidemiology, genetic diversity, and evolutionary dynamics of Tembusu virus. Archives of Virology, 2023, 168: 262

[36] Zhu Y, Hu Z, Lv X, Huang R, Gu X, Zhang C, Zhang M, Wei J, Wu Q, Li J. A novel Tembusu virus isolated from goslings in China form a new subgenotype 2.1. 1. Transboundary and Emerging Diseases, 2022, 69: 1782-1793

Funding

National Natural Science Foundation of China(32573344)

Natural Science Foundation of Hubei Province(2025AFB600)

Earmarked Fund for Modern Agro-industry Technology Research System(CARS-41)

Natural Science Foundation of Wuhan(2026040301020043)

Fundamental Research Funds for the Central Universities(2662026DKQD005)

Rights & permissions

The Author(s)