Establishment of an Epstein-Barr virus infection model using human nasal organoids

doi:10.12122/j.issn.1673-4254.2026.02.12

Abstract

Abstract:

Objective To develop an cost-effective and convenient method for culturing human nasal organoids to establish an in vitro Epstein-Barr virus (EBV) infection model. Methods Nasal polyp tissue obtained from surgery was routinely washed, cut, digested and filtered to obtain cell clusters. The cell clusters were then expanded into undifferentiated nasal organoids through dynamic suspension culture under matrix-free conditions, followed by a 14-day differentiation induction treatment to obtain differentiated nasal organoids. The major cellular components of the organoids were identified by immunofluorescence staining and immunohistochemistry. The nasal organoids were infected by EBV in vitro, and viral replication was verified by detecting the expressions of the virus-specific genes EBNA1 and BALF5 using RT-qPCR and immunofluorescence staining. Results Nasal organoids consisting of basal cells, mucous cells, and ciliated cells in a martigel-free system were obtained successfully by dynamic suspension culture. The differentiated nasal organoids expressed high levels of EBV-associated receptors EphA2, NRP1, and NMHCII-A. Both the undifferentiated and differentiated nasal organoids could be infected by EBV. Viral replication in the organoids increased with the viral exposure load, and the differentiated organoids appeared more permissive to viral replication. Conclusion The matrigel-free dynamic suspension culture method is economical and simple for constructing nasal organoids, which can be used as a model of EBV infection for studies of epithelial EBV infection.

Key words: organoids, Epstein-Barr virus, nasal mucosa

Jinyan WEI, Hairui ZHENG, Yunteng ZHAO, Yan YU, Yingying XU, Gang LI. Establishment of an Epstein-Barr virus infection model using human nasal organoids[J]. Journal of Southern Medical University, 2026, 46(2): 345-352.

Figures/Tables 9

Tab.1 Information of the nasal mucosa samples

Sample number	Source	Reason for surgery
ZZH-9580	Nasal polyps	CRSwNP
LHX-4635	Nasal polyps	CRSwNP
XWJ-4024	Nasal polyps	CRSwNP
XCY-8640	Nasal polyps	CRSwNP
LJL-3202	Nasal polyps	CRSwNP
LHB-4635	Nasal polyps	CRSwNP
TRJ-8166	Nasal polyps	CRSwNP

Tab.2 Antibodies for immunofluorescence assay and immunohistochemistry

Antibody	Brand	Cat No.	dilution ratio
Anti-Cytokeratin 5 antibody	Abcam	ab75869	1:100
Anti-Cytokeratin 14 antibody	Abcam	ab119695	1:100
Anti-p63 antibody	Abcam	ab124762	1:100
Anti-MUC5AC antibody	Abcam	ab198294	1:200
Anti-beta Ⅳ tubulin antibody	Abcam	ab179509	1:200
Anti-FoxJ1 antibody	Abcam	ab235445	1:2000
COL4A2 Polyclonal antibody	Proteintech	55131-1-AP	1:300
MYH9 Monoclonal antibody	Proteintech	60233-1-IG	1:200
EPHA2 Monoclonal antibody	Proteintech	66736-1-IG	1:200
Neuropilin 1/CD304 Recombinant antibody	Proteintech	84429-5-RR	1:300
Anti-EBNA1 antibody	Abcam	ab316860	1:2000
Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488	Invitrogen	A11034	1:500
Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488	Invitrogen	A11029	1:500
HRP-conjugated Goat Anti-Rabbit/mouse IgG for IHC (ready to use)	Proteintech	PR30009	No dilution needed

Tab.3 Primer sequence for RT-qPCR

Gene	Foward (5'→3')	Reverse (5'→3')
GAPDH	GGAGCGAGATCCCTCCAAAAT	GGCTGTTGTCATACTTCTCATGG
EPHA2	CCCGATGAGATCACCGTCAG	GGCACCGATATCCTGGAAGG
NMHC-ⅡA	GAGCAAATGGGCCTGCT	TGTTGTCGGGCATGGA
NRP1	ACCCAAGTGAAAAATGCGAATG	CCTCCAAATCGAAGTGAGGGTT
EBNA1	GGTCGTGGACGTGGAGAAAA	GGTGGAGACCCGGATGATG
BALF5	GAGCGATCTTGGCAATCTCT	TGGTCATGGATCTGCTAAACC

Fig.1 Phase-contrast microscopy of nasal organoids in culture. A: Representative images at different time points during the expansion of nasal organoids. B: Representative images of the nasal organoids on days 3, 7, and 14 during differentiation induction.

Fig.2 HE staining of the nasal organoids and immunofluorescence staining for the basal lamina marker collagen IV. A: HE staining of undifferentiated and differentiated nasal organoids. The differentiated nasal organoids exhibit a higher density of cilia on their apical surface. B: Immunofluorescence staining for collagen IV in differentiated nasal organoids.

Fig.3 Immunofluorescence analysis of cellular composition of undifferentiated and differentiated nasal organoids. A: Representative images of undifferentiated organoids showing positive expressions of epithelial basal stem cell markers CK5, CK14, and p63. B: Representative images of differentiated organoids consisting of basal cells (CK5), goblet cells (MUC5AC), and ciliated cells (β-tubulin IV).

Fig.4 Immunohistochemical analysis of cellular composition of nasal mucosal tissue and organoids. Representative images of nasal mucosal tissue are shown in the upper panel and differentiated nasal organoids in the lower panel. The organoids exhibit comparable expression patterns to nasal mucosal tissue for P63 (basal cells), MUC5AC (goblet cells), and FOXJ-1 (ciliated cells).

Fig.5 Differentiated nasal organoids exhibit high expressions of Epstein-Barr virus (EBV) receptors. A: Relative mRNA expressions of EBV receptors NMHC-IIA, EPHA2 and NRP1 in undifferentiated and differentiated nasal organoids. ***P<0.001 vs undifferentiated group. B: Immunofluorescence staining for EBV receptors NMHC-IIA, EPHA2 and NRP1 in differentiated nasal organoids (n=3).

Fig.6 Establishment of EBV-infected nasal organoids. A, B: Relative mRNA expression levels of EBV-specific genes EBNA1 and BALF5 in differentiated organoids harvested at 72 h post-EBV infection at MOI=250 and 2500 (A; *P<0.05, ***P<0.001, ****P<0.0001 vs Mock group) and in both undifferentiated and differentiated organoids harvested 72 h after EBV infection at MOI=2500 (B; ***P<0.001 vs undifferentiated group). C, D: Immunofluorescence staining for EBNA1 undifferentiated (C) and differentiated (D) organoids with EBV infection at MOI=2500 (n=3).

References 33

[1]	Alsaadawe M, Radman BA, Long JY, et al. Epstein Barr virus: a cellular hijacker in cancer[J]. Biochim Biophys Acta BBA Rev Cancer, 2024, 1879(6): 189218. doi：10.1016/j.bbcan.2024.189218
[2]	Damania B, Kenney SC, Raab-Traub N. Epstein-Barr virus: Biology and clinical disease[J]. Cell, 2022, 185(20): 3652-70. doi：10.1016/j.cell.2022.08.026
[3]	Schindele A, Holm A, Kraft S, et al. Cross-evaluating Epstein-Barr virus, human Papilloma virus, human cytomegalovirus and human adenovirus in nasal polyps and turbinate mucosa[J]. Acta Otolaryngol, 2025, 145(2): 164-7. doi：10.1080/00016489.2024.2445025
[4]	Zaravinos A, Bizakis J, Spandidos DA. Prevalence of human Papilloma virus and human herpes virus types 1-7 in human nasal polyposis[J]. J Med Virol, 2009, 81(9): 1613-9. doi：10.1002/jmv.21534
[5]	谭学君, 姚文昊, 陈晓平, 等.三峡库区重庆万州段鼻息肉与HPV及EBV阳性表达的相关性研究[J].中国中西医结合耳鼻咽喉科杂志,2015, 23(1): 7-11.
[6]	Jia Z, Zhang D, Zhu L, et al. Animal models of human herpesvirus infection[J]. Animal Model Exp Med, 2025, 8(4): 615-28. doi：10.1002/ame2.12575
[7]	Huang PY, Zeng TT, Li MQ, et al. Proteomic analysis of a nasopharyngeal carcinoma cell line and a nasopharyngeal epithelial cell line[J]. Tumori, 2015, 101(6): 676-83. doi：10.5301/tj.5000345
[8]	Li HM, Man C, Jin Y, et al. Molecular and cytogenetic changes involved in the immortalization of nasopharyngeal epithelial cells by telomerase[J]. Int J Cancer, 2006, 119(7): 1567-76. doi：10.1002/ijc.22032
[9]	Song LB, Zeng MS, Liao WT, et al. Bmi-1 is a novel molecular marker of nasopharyngeal carcinoma progression and immortalizes primary human nasopharyngeal epithelial cells[J]. Cancer Res, 2006, 66(12): 6225-32. doi：10.1158/0008-5472.can-06-0094
[10]	Collett S, Torresi J, Silveira LE, et al. Investigating virus–host cell interactions: Comparative binding forces between hepatitis C virus-like particles and host cell receptors in 2D and 3D cell culture models[J]. J Colloid Interface Sci, 2021, 592: 371-84. doi：10.1016/j.jcis.2021.02.067
[11]	Yan HHN, Chan AS, Lai FP, et al. Organoid cultures for cancer modeling[J]. Cell Stem Cell, 2023, 30(7): 917-37. doi：10.1016/j.stem.2023.05.012
[12]	Zhu S, Chen D, Yang X, et al. Organoid models to study human infectious diseases[J]. Cell Prolif, 2025: e70004. doi：10.1111/cpr.70004
[13]	汪珂,于言,韩日,等.分化可控的人类鼻粘膜类器官模型的建立[J].南方医科大学学报,2022,42(06):868-77.
[14]	于言,曹浚垣, 刘蓉, 等.人鼻粘膜类器官冠状病毒感染模型可用于抗病毒药物的筛选和评价[J].南方医科大学学报,2024,44(11):2227-34.
[15]	Kaur S, Kaur I, Rawal P, et al. Non-matrigel scaffolds for organoid cultures[J]. Cancer Lett, 2021, 504: 58-66. doi：10.1016/j.canlet.2021.01.025
[16]	Co JY, Margalef-Català M, Monack DM, et al. Controlling the polarity of human gastrointestinal organoids to investigate epithelial biology and infectious diseases[J]. Nat Protoc, 2021, 16(11): 5171-92. doi：10.1038/s41596-021-00607-0
[17]	Chiu MC, Li C, Liu X, et al. Human nasal organoids model SARS-CoV-2 upper respiratory infection and recapitulate the differential infectivity of emerging variants[J]. mBio, 2022, 13(4): e0194422. doi：10.1128/mbio.01944-22
[18]	Xia TL, Li XY, Wang XP, et al. N(6)‑methyladenosine-binding protein YTHDF1 suppresses EBV replication and promotes EBV RNA decay[J]. EMBO Rep, 2021, 22(4): e50128. doi：10.15252/embr.202050128
[19]	Zhong LY, Xie C, Zhang LL, et al. Research landmarks on the 60th anniversary of Epstein-Barr virus[J]. Sci China Life Sci, 2025, 68(2): 354-80. doi：10.1007/s11427-024-2766-0
[20]	谭德重, 冯源, 胡月, 等. 病毒感染与鼻息肉关系的Meta分析[J].临床耳鼻咽喉头颈外科杂志,2018, 32(12): 910-6.
[21]	Wong KCW, Hui EP, Lo KW, et al. Nasopharyngeal carcinoma: an evolving paradigm[J]. Nat Rev Clin Oncol, 2021, 18(11): 679-95. doi：10.1038/s41571-021-00524-x
[22]	Tsang CM, Zhang G, Seto E, et al. Epstein-Barr virus infection in immortalized nasopharyngeal epithelial cells: regulation of infection and phenotypic characterization[J]. Int J Cancer, 2010, 127(7): 1570-83. doi：10.1002/ijc.25173
[23]	Wang HB, Zhang H, Zhang JP, et al. Neuropilin 1 is an entry factor that promotes EBV infection of nasopharyngeal epithelial cells[J]. Nat Commun, 2015, 6: 6240. doi：10.3410/f.725353089.793504525
[24]	Kozlowski MT, Crook CJ, Ku HT. Towards organoid culture without Matrigel [J]. Commun Biol, 2021, 4(1): 1387. doi：10.1038/s42003-021-02910-8
[25]	Marchini A, Gelain F. Synthetic scaffolds for 3D cell cultures and organoids: applications in regenerative medicine[J]. Crit Rev Biotechnol, 2022, 42(3): 468-86. doi：10.1080/07388551.2021.1932716
[26]	Capeling MM, Huang S, Childs CJ, et al. Suspension culture promotes serosal mesothelial development in human intestinal organoids[J]. Cell Rep, 2022, 38(7): 110379. doi：10.1016/j.celrep.2022.110379
[27]	Kumar SV, Er PX, Lawlor KT, et al. Kidney micro-organoids in suspension culture as a scalable source of human pluripotent stem cell-derived kidney cells[J]. Development, 2019, 146(5): dev172361. doi：10.1242/dev.172361
[28]	Wu H, Wang J, Liu S, et al. Large-scale production of expandable hepatoblast organoids and polarised hepatocyte organoids from hESCs under 3D static and dynamic suspension conditions[J]. Cell Prolif, 2025, 58(7): e70001. doi：10.1111/cpr.70001
[29]	Stroulios G, Brown T, Moreni G, et al. Apical-out airway organoids as a platform for studying viral infections and screening for antiviral drugs[J]. Sci Rep, 2022, 12(1): 7673. doi：10.1038/s41598-022-11700-z
[30]	Zhao KY, Du YX, Cao HM, et al. The biological macromolecules constructed Matrigel for cultured organoids in biomedical and tissue engineering[J]. Colloids Surf B Biointerfaces, 2025, 247: 114435. doi：10.1016/j.colsurfb.2024.114435
[31]	Bu GL, Xie C, Kang YF, et al. How EBV infects: the tropism and underlying molecular mechanism for viral infection[J]. Viruses, 2022, 14(11): 2372. doi：10.3390/v14112372
[32]	Hayman IR, Temple RM, Burgess CK, et al. New insight into Epstein-Barr virus infection using models of stratified epithelium[J]. PLoS Pathog, 2023, 19(1): e1011040. doi：10.1371/journal.ppat.1011040
[33]	Nawandar DM, Wang A, Makielski K, et al. Differentiation-dependent KLF4 expression promotes lytic Epstein-Barr virus infection in epithelial cells[J]. PLoS Pathog, 2015, 11(10): e1005195. doi：10.1371/journal.ppat.1005195