Single-cell transcriptomic analyses of HSV-1 reactivation from latently infected tree shrew and mouse trigeminal ganglia reveal differing molecular and cellular processes

Yunshuang Ye; Erlin Wang; Mengqiu Yin; Shiqing Zou; Guijun Chen; Liping Yang; Nigel W. Fraser; Xia Cao; Xinglou Yang; Jumin Zhou

doi:10.1016/j.virs.2026.02.009

February 2026

Citation: Yunshuang Ye, Erlin Wang, Mengqiu Yin, Shiqing Zou, Guijun Chen, Liping Yang, Nigel W. Fraser, Xia Cao, Xinglou Yang, Jumin Zhou. Single-cell transcriptomic analyses of HSV-1 reactivation from latently infected tree shrew and mouse trigeminal ganglia reveal differing molecular and cellular processes .VIROLOGICA SINICA, 2026, 41(1) : 157-171. http://dx.doi.org/10.1016/j.virs.2026.02.009

Single-cell transcriptomic analyses of HSV-1 reactivation from latently infected tree shrew and mouse trigeminal ganglia reveal differing molecular and cellular processes

Yunshuang Ye ^a,b ,
Erlin Wang ^{a,b
,,} ,
Mengqiu Yin ^a,c ,
Shiqing Zou ^a ,
Guijun Chen ^a ,
Liping Yang ^a ,
Nigel W. Fraser ^d ,
Xia Cao ^e ,
Xinglou Yang ^a ,
Jumin Zhou ^{a,f
,,}

a. State Key Laboratory of Genetic Evolution & Animal Models, Yunnan International Joint Laboratory of Zoonotic Viruses, Yunnan Key Laboratory of Biodiversity Information, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650201, China;
b. Department of Neurosurgery, Songjiang Research Institute, Songjiang District Central Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 201615, China;
c. Kunming College of Life Science, University of Chinese Academy of Sciences, Kunming 650201, China;
d. Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia PA 19104, USA;
e. Key Laboratory of Second Affiliated Hospital of Kunming Medical University, Kunming 650201, China;
f. KIZ/CUHK Joint Laboratory of Bioresources and Molecular Research in Common Diseases, Kunming 650201, China

Corresponding author: Erlin Wang, wangerlin@shsmu.edu.cn
Jumin Zhou, zhoujm@mail.kiz.ac.cn
Received Date: 24 September 2025
Accepted Date: 09 February 2026
Available online: 12 February 2026

Fund Project: This study was supported by grants from the Ministry of Science and Technology (China) of China (MOST, 2023YFC2306700) the National Natural Science Foundation of China (NSFC, 81672040) to J. Zhou the National Natural Science Foundation of China-Yunnan Joint Found (NSFC, U2202215) the Ministry of Science and Technology of China Foreign Expert Program to J. Zhou (G2021061008L) and a Thousand Foreign Talent scholarship from Yunnan Province and High-end Foreign Expert Project of Yunnan Revitalization Talent Support Program to J. Zhou. Yunnan Fundamental Research Projects (202401AT070192) and the National Natural Science Foundation of China (NSFC, 82401655) to W.E.

Abstract

Herpes simplex virus type 1 (HSV-1) infects over 70% of the population and establishes lifelong latent infection with periodic reactivation in humans, resulting in various related diseases. However, the molecular and cellular events underlying the transition of HSV-1 from latency to reactivation remain poorly understood. In this study, we used bulk RNA sequencing and single-cell transcriptomic analyses to dissect the cellular and molecular events of HSV-1 latency-reactivation transition in infected trigeminal ganglia (TG) in both mouse and tree shrew infection models. We found that mice exhibited fluctuating host gene responses during the acute phase and relatively quiescent latency, whereas tree shrews displayed a relatively mild acute phase and active latency characteristics. Single-cell analysis revealed that HSV-1 infects TG neuronal subpopulations expressing growth hormone and pituitary hormones. Importantly, we observed that HSV-1 latency in tree shrew TGs exhibited inhibition of cellular autophagy function, while HSV-1 latency in mice was accompanied by the attenuation of monocyte-related immune surveillance. Given that infected cell protein 0 (ICP0) has autophagy inhibitory activity, we further investigated the role of this viral protein in tree shrew models using an ICP0-deficient HSV-1 strain. Notably, the mutant virus could not undergo spontaneous reactivation from latency. These findings support the hypothesis that ICP0 may be essential for spontaneous reactivation by inhibiting autophagy in vivo.
- Herpes simplex virus type 1 (HSV-1)
- , Reactivation
- , Single-cell RNA-seq
- , Autophagy
- , Trigeminal ganglion (TG)
- , Tree shrew

Electronic Supplementary Material

10.1016j.virs.2026.02.009-ESM.docx
References
1. Anders, S., Huber, W. 2010. Differential expression analysis for sequence count data. Genome Biol, 11, R106.
2. Aneja, K.K., Yuan, Y. 2017. Reactivation and Lytic Replication of Kaposi's Sarcoma-Associated Herpesvirus: An Update. Front Microbiol, 8, 613.
3. Cairns, D.M., Smiley, B.M., Smiley, J.A., Khorsandian, Y., Kelly, M., Itzhaki, R.F., Kaplan, D.L. 2025. Repetitive injury induces phenotypes associated with Alzheimer’s disease by reactivating HSV-1 in a human brain tissue model. Science Signaling, 18, eado6430.
4. Carter, B., Zhao, K. 2020. The epigenetic basis of cellular heterogeneity. Nature Reviews Genetics, 22, 235-250.
5. D’Aiuto, L., Caldwell, J., Wallace, C., Grams, T., Wesesky, M., Wood, J., Watkins, S., Kinchington, P., Bloom, D., Nimgaonkar, V. 2022. The Impaired Neurodevelopment of Human Neural Rosettes in HSV-1-Infected Early Brain Organoids. Cells, 11, 3539.
6. Ding, J., Adiconis, X., Simmons, S.K., Kowalczyk, M.S., Hession, C.C., Marjanovic, N.D., Hughes, T.K., Wadsworth, M.H., Burks, T., Nguyen, L.T., Kwon, J.Y.H., Barak, B., Ge, W., Kedaigle, A.J., Carroll, S., Li, S., Hacohen, N., Rozenblatt-Rosen, O., Shalek, A.K., Villani, A.C., Regev, A., Levin, J.Z. 2020. Systematic comparison of single-cell and single-nucleus RNA-sequencing methods. Nature Biotechnology, 38, 737-746.
7. Dochnal, S.A., Whitford, A.L., Francois, A.K., Krakowiak, P.A., Cuddy, S., Cliffe, A.R., Goodrum, F. 2024. c-Jun signaling during initial HSV-1 infection modulates latency to enhance later reactivation in addition to directly promoting the progression to full reactivation. Journal of Virology, 98, e0176423.
8. Everett, R.D., Ramakrishna, C., Ferraioli, A., Calle, A., Nguyen, T.K., Openshaw, H., Lundberg, P.S., Lomonte, P., Cantin, E.M. 2015. Establishment of HSV1 Latency in Immunodeficient Mice Facilitates Efficient In Vivo Reactivation. PLOS Pathogens, 11, e1004730.
9. Feldman, L.T., Ellison, A.R., Voytek, C.C., Yang, L., Krause, P., Margolis, T.P. 2002. Spontaneous molecular reactivation of herpes simplex virus type 1 latency in mice. Proceedings of the National Academy of Sciences, 99, 978-983.
10. Halford, W.P., Gebhardt, B.M., Carr, D.J. 1996. Mechanisms of herpes simplex virus type 1 reactivation. Journal of Virology, 70, 5051-5060.
11. Halford, W.P., Schaffer, P.A. 2001. ICP0 Is Required for Efficient Reactivation of Herpes Simplex Virus Type 1 from Neuronal Latency. Journal of Virology, 75, 3240-3249.
12. Heinz, J.L., Hinke, D.M., Maimaitili, M., Wang, J., Sabli, I.K.D., Thomsen, M., Farahani, E., Ren, F., Hu, L., Zillinger, T., Grahn, A., von Hofsten, J., Verjans, G.M.G.M., Paludan, S.R., Viejo-Borbolla, A., Sancho-Shimizu, V., Mogensen, T.H. 2024. Varicella zoster virus-induced autophagy in human neuronal and hematopoietic cells exerts antiviral activity. Journal of Medical Virology, 96, e29690.
13. Hou, F., Sun, Z., Deng, Y., Chen, S., Yang, X., Ji, F., Zhou, M., Ren, K., Pan, D. 2022. Interactome and Ubiquitinome Analyses Identify Functional Targets of Herpes Simplex Virus 1 Infected Cell Protein 0. Front Microbiol, 13, 856471.
14. Hovhannisyan, A.H., Son, H., Mecklenburg, J., Barba-Escobedo, P.A., Tram, M., Gomez, R., Shannonhouse, J., Zou, Y., Weldon, K., Ruparel, S., Lai, Z., Tumanov, A.V., Kim, Y.S., Akopian, A.N. 2021. Pituitary hormones are specifically expressed in trigeminal sensory neurons and contribute to pain responses in the trigeminal system. Scientific Reports, 11, 17813.
15. Jones, C., Favoreel, H.W. 2025. Human alpha-herpesvirus 1 (HSV-1) viral replication and reactivation from latency are expedited by the glucocorticoid receptor. Journal of Virology, 99, e0030325.
16. Kalejta, R.F., De Chiara, G., Piacentini, R., Fabiani, M., Mastrodonato, A., Marcocci, M.E., Limongi, D., Napoletani, G., Protto, V., Coluccio, P., Celestino, I., Li Puma, D.D., Grassi, C., Palamara, A.T. 2019. Recurrent herpes simplex virus-1 infection induces hallmarks of neurodegeneration and cognitive deficits in mice. PLOS Pathogens, 15, e1007617.
17. Kim, J.Y., Mandarino, A., Chao, M.V., Mohr, I., Wilson, A.C. 2012. Transient reversal of episome silencing precedes VP16-dependent transcription during reactivation of latent HSV-1 in neurons. PLoS Pathog, 8, e1002540.
18. Krakowiak, P.A., Flores, M.E., Cuddy, S.R., Whitford, A.L., Dochnal, S.A., Babnis, A., Miyake, T., Tigano, M., Engel, D.A., Cliffe, A.R. 2025. Co-option of mitochondrial nucleic acid-sensing pathways by HSV-1 UL12.5 for reactivation from latent infection. Proceedings of the National Academy of Sciences, 122, e2413965122.
19. Li, L., Li, Z., Wang, E., Yang, R., Xiao, Y., Han, H., Lang, F., Li, X., Xia, Y., Gao, F., Li, Q., Fraser, N.W., Zhou, J. 2016. Herpes Simplex Virus 1 Infection of Tree Shrews Differs from That of Mice in the Severity of Acute Infection and Viral Transcription in the Peripheral Nervous System. J Virol, 90, 790-804.
20. Luo, M.-T., Mu, D., Yang, X., Luo, R.H., Zheng, H.-Y., Chen, M., Guo, Y.Q., Zheng, Y.T., Simon, V. 2021. Tree Shrew Cells Transduced with Human CD4 and CCR5 Support Early Steps of HIV-1 Replication, but Viral Infectivity Is Restricted by APOBEC3. Journal of Virology, 95, e0002021.
21. Mostafa, H.H., Thompson, T.W., Kushnir, A.S., Haenchen, S.D., Bayless, A.M., Hilliard, J.G., Link, M.A., Pitcher, L.A., Loveday, E., Schaffer, P.A., Davido, D.J. 2011. Herpes Simplex Virus 1 ICP0 Phosphorylation Site Mutants Are Attenuated for Viral Replication and Impaired for Explant-Induced Reactivation. Journal of Virology, 85, 12631-12637.
22. Perng, G.C., Jones, C. 2010. Towards an Understanding of the Herpes Simplex Virus Type 1 Latency-Reactivation Cycle. Interdisciplinary Perspectives on Infectious Diseases, 2010, 1-18.
23. Preston, C.M. 2007. Reactivation of Expression from Quiescent Herpes Simplex Virus Type 1 Genomes in the Absence of Immediate-Early Protein ICP0. Journal of Virology, 81, 11781-11789.
24. Protto, V., Miteva, M.T., Iannuzzi, F., Marcocci, M.E., Li Puma, D.D., Piacentini, R., Belli, M., Sansone, L., Pietrantoni, A., Grassi, C., Palamara, A.T., De Chiara, G., Cherry, S., Paludan, S.R. 2024. HSV-1 infection induces phosphorylated tau propagation among neurons via extracellular vesicles. mBio, 15, e0152224.
25. Purushothaman, P., Uppal, T., Verma, S.C. 2015. Molecular biology of KSHV lytic reactivation. Viruses, 7, 116-153.
26. Qiao, H., Chiu, Y., Liang, X., Xia, S., Ayrapetyan, M., Liu, S., He, C., Song, R., Zeng, J., Deng, X., Yuan, W., Zhao, Z. 2023. Microglia innate immune response contributes to the antiviral defense and blood-CSF barrier function in human choroid plexus organoids during HSV-1 infection. Journal of Medical Virology, 95, e28472.
27. Qiao, H., Zhao, W., Guo, M., Zhu, L., Chen, T., Wang, J., Xu, X., Zhang, Z., Wu, Y., Chen, P. 2022. Cerebral Organoids for Modeling of HSV-1-Induced-Amyloid β Associated Neuropathology and Phenotypic Rescue. International Journal of Molecular Sciences, 23, 5981.
28. Rybak-Wolf, A., Wyler, E., Pentimalli, T., Legnini, I., Martinez, A.O., Glazar, P., Loewa, A., Kim, S.J., Kaufer, B., Woehler, A., Landthaler, M., Rajewsky, N. 2023. Modelling viral encephalitis caused by herpes simplex virus 1 infection in cerebral organoids. Nature Microbiology, 8, 1252-1266.
29. Sivasubramanian, M.K., Monteiro, R., Harrison, K.S., Plakkot, B., Subramanian, M., Jones, C., Jung, J.U. 2022. Herpes Simplex Virus Type 1 Preferentially Enhances Neuro-Inflammation and Senescence in Brainstem of Female Mice. Journal of Virology, 96, e0108122.
30. Sodroski, C.N., Knipe, D.M. 2023. Nuclear interferon-stimulated gene product maintains heterochromatin on the herpes simplex viral genome to limit lytic infection. Proceedings of the National Academy of Sciences, 120, e2310996120.
31. Soriano, E., Bakken, T.E., Hodge, R.D., Miller, J.A., Yao, Z., Nguyen, T.N., Aevermann, B., Barkan, E., Bertagnolli, D., Casper, T., Dee, N., Garren, E., Goldy, J., Graybuck, L.T., Kroll, M., Lasken, R.S., Lathia, K., Parry, S., Rimorin, C., Scheuermann, R.H., Schork, N.J., Shehata, S.I., Tieu, M., Phillips, J.W., Bernard, A., Smith, K.A., Zeng, H., Lein, E.S., Tasic, B. 2018. Single-nucleus and single-cell transcriptomes compared in matched cortical cell types. Plos One, 13, e0209648.
32. Stow, N.D., Stow, E.C. 1986. Isolation and Characterization of a Herpes Simplex Virus Type 1 Mutant Containing a Deletion within the Gene Encoding the Immediate Early Polypeptide Vmw110. Journal of General Virology, 67, 2571-2585.
33. Su, X., Yue, P., Kong, J., Xu, X., Zhang, Y., Cao, W., Fan, Y., Liu, M., Chen, J., Liu, A., Bao, F. 2022. Human Brain Organoids as an In Vitro Model System of Viral Infectious Diseases. Frontiers in Immunology, 12, 792316.
34. Sun, B., Yang, X., Hou, F., Yu, X., Wang, Q., Oh, H.S., Raja, P., Pesola, J.M., Vanni, E.A.H., McCarron, S., Morris-Love, J., Ng, A.H.M., Church, G.M., Knipe, D.M., Coen, D.M., Pan, D. 2021. Regulation of host and virus genes by neuronal miR-138 favours herpes simplex virus 1 latency. Nat Microbiol, 6, 682-696.
35. Thompson, R.L., Sawtell, N.M. 2006. Evidence that the Herpes Simplex Virus Type 1 ICP0 Protein Does Not Initiate Reactivation from Latency In Vivo. Journal of Virology, 80, 10919-10930.
36. van Weperen, V.Y.H., Littman, R.J., Arneson, D.V., Contreras, J., Yang, X., Ajijola, O.A. 2021. Single-cell transcriptomic profiling of satellite glial cells in stellate ganglia reveals developmental and functional axial dynamics. Glia, 69, 1281-1291.
37. Vilaboa, N., Bloom, D.C., Canty, W., Voellmy, R. 2024. A Broad Influenza Vaccine Based on a Heat-Activated, Tissue-Restricted Replication-Competent Herpesvirus. Vaccines, 12, 703.
38. Viret, C., Duclaux-Loras, R., Nancey, S., Rozieres, A., Faure, M. 2021. Selective Autophagy Receptors in Antiviral Defense. Trends in Microbiology, 29, 798-810.
39. Waisner, H., Kalamvoki, M., Sandri-Goldin, R.M. 2019. The ICP0 Protein of Herpes Simplex Virus 1 (HSV-1) Downregulates Major Autophagy Adaptor Proteins Sequestosome 1 and Optineurin during the Early Stages of HSV-1 Infection. Journal of Virology, 93, e01258-01219.
40. Waisner, H., Lasnier, S., Suma, S.M., Kalamvoki, M., Cliffe, A.R. 2023. Effects on exocytosis by two HSV-1 mutants unable to block autophagy. Journal of Virology, 97, e0075723.
41. Wang, E., Huang, X., Ye, Y., Zou, S., Chen, G., Yang, L., Fraser, N.W., Bao, F., Zhou, J., Cao, X. 2023. Persistent inflammation and neuronal loss in the mouse brain induced by a modified form of attenuated herpes simplex virus type I. Virol Sin, 38, 108-118.
42. Wang, E., Ye, Y., Zhang, K., Yang, J., Gong, D., Zhang, J., Hong, R., Zhang, H., Li, L., Chen, G., Yang, L., Liu, J., Cao, H., Du, T., Fraser, N.W., Cheng, L., Cao, X., Zhou, J. 2020. Longitudinal transcriptomic characterization of viral genes in HSV-1 infected tree shrew trigeminal ganglia. Virology Journal, 17, 95.
43. Wang, Z., Liu, J., Han, J., Zhang, T., Li, S., Hou, Y., Su, H., Han, F., Zhang, C. 2024. Herpes simplex virus 1 accelerates the progression of Alzheimer’s disease by modulating microglial phagocytosis and activating NLRP3 pathway. Journal of Neuroinflammation, 21, 176.
44. Yang, W., Tang, C.Y., Fan, D.Y., Wang, Y.S., Wang, P.G., An, J., Luan, G.M. 2024. Mice with type I interferon signaling deficiency are prone to epilepsy upon HSV-1 infection. Virol Sin, 39, 251-263.
45. Yordy, B., Iijima, N., Huttner, A., Leib, D., Iwasaki, A. 2012. A Neuron-Specific Role for Autophagy in Antiviral Defense against Herpes Simplex Virus. Cell Host & Microbe, 12, 334-345.
46. Yu, W., Geng, S., Suo, Y., Wei, X., Cai, Q., Wu, B., Zhou, X., Shi, Y., Wang, B. 2018. Critical Role of Regulatory T Cells in the Latency and Stress-Induced Reactivation of HSV-1. Cell Reports, 25, 2379-2389.e2373.
47. Zanini, F., Pu, S.Y., Bekerman, E., Einav, S., Quake, S.R. 2018. Single-cell transcriptional dynamics of flavivirus infection. eLife, 7, 111-123.
48. Zhang, J., Zhao, J., Xu, S., Li, J., He, S., Zeng, Y., Xie, L., Xie, N., Liu, T., Lee, K., Seo, G.J., Chen, L., Stabell, A.C., Xia, Z., Sawyer, S.L., Jung, J., Huang, C., Feng, P. 2018. Species-Specific Deamidation of cGAS by Herpes Simplex Virus UL37 Protein Facilitates Viral Replication. Cell Host & Microbe, 24, 234-248.e235.
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