Generation of ST6GAL1/ST3GAL4 double-knockout Landrace pigs with reduced respiratory sialylated glycans and decreased influenza A virus susceptibility of derived fibroblasts in vitro

Abstract

Pigs are important hosts in the ecology and cross-species transmission of influenza A viruses because their respiratory tract can contain both human-type α2,6-linked and avian-type α2,3-linked sialylated glycans. These glycan structures may allow pigs to support infection by influenza viruses of different host origins and can facilitate viral reassortment when coinfection occurs. Here, we used CRISPR/Cas9-mediated gene editing to disrupt the porcine ST6GAL1 and ST3GAL4 genes, which encode sialyltransferases involved in the biosynthesis of α2,6-linked and α2,3-linked sialylated glycans, respectively. We generated ST6GAL1/ST3GAL4 double-knockout Landrace pigs and observed markedly reduced Sambucus nigra agglutinin (SNA)- and Maackia amurensis lectin Ⅱ (MAL Ⅱ)-reactive sialylated glycans in the respiratory tract. Fibroblasts derived from the double-knockout pigs showed reduced susceptibility to H1N1, H3N2, and H9N2 influenza A virus infection in vitro. Transcriptomic analysis further revealed distinct gene-expression profiles between knockout and wild-type fibroblasts before and after H1N1 infection. Complementation of ST6GAL1 and ST3GAL4 restored sialylated glycan expression and increased viral susceptibility in cultured cells, supporting a causal contribution of these sialyltransferases to influenza A virus infection. Further in vivo challenge, transmission, safety, and independent-founder studies are required to determine whether this strategy can reduce influenza A virus infection or transmission in live pigs.

Keywords

gene-edited pigs / influenza A virus / ST6GAL1 / ST3GAL4

References

[1] World Health Organization. Influenza (seasonal)[EB/OL]. (2025-02-28) [2025-11-12]. https://www.who.int/news-room/fact-sheets/detail/influenza-(seasonal).

[2] Everest H, Hill SC, Daines R, et al. The evolution, spread and global threat of H6Nx avian influenza viruses[J]. Viruses, 2020, 12(6): 673. doi: 10.3390/v12060673

[3] Petrova VN, Russell CA. Erratum: The evolution of seasonal influenza viruses[J]. Nat Rev Microbiol, 2018, 16(1): 60. doi: 10.1038/nrmicro.2017.146

[4] Böttcher-Friebertshäuser E, Garten W, Matrosovich M, et al. The hemagglutinin: A determinant of pathogenicity[M]// Compans RW, Oldstone MBA. Influenza Pathogenesis and Control-Volume Ⅰ. Cham: Springer, 2014: 3-34.

[5] Park JE, Ryu Y. Transmissibility and severity of influenza virus by subtype[J]. Infect Genet Evol, 2018, 65: 288-292. doi: 10.1016/j.meegid.2018.08.007

[6] Lee CY. Exploring potential intermediates in the cross-species transmission of influenza A virus to humans[J]. Viruses, 2024, 16(7): 1129. doi: 10.3390/v16071129

[7] Kristensen C, Larsen LE, Trebbien R, et al. The avian influenza A virus receptor SA-α2,3-Gal is expressed in the porcine nasal mucosa sustaining the pig as a mixing vessel for new influenza viruses[J]. Virus Res, 2024, 340: 199304. doi: 10.1016/j.virusres.2023.199304

[8] Arruda B, Baker ALV, Buckley A, et al. Divergent pathogenesis and transmission of highly pathogenic avian influenza A(H5N1) in swine[J]. Emerg Infect Dis, 2024, 30(4): 738-751. doi: 10.3201/eid3004.231141

[9] Trombetta C, Piccirella S, Perini D, et al. Emerging influenza strains in the last two decades: A threat of a new pandemic?[J]. Vaccines (Basel), 2015, 3(1): 172-185. doi: 10.3390/vaccines3010172

[10] Świerczyńska M, Mirowska-Guzel DM, Pindelska E. Antiviral drugs in influenza[J]. Int J Environ Res Public Health, 2022, 19(5): 3018. doi: 10.3390/ijerph19053018

[11] Tandel K, Sharma S, Dash PK, et al. Oseltamivir-resistant influenza A(H1N1)pdm09 virus associated with high case fatality, India 2015[J]. J Med Virol, 2018, 90(5): 836-843. doi: 10.1002/jmv.25013

[12] Abed Y, Saim-Mamoun A, Boivin G. Fitness of influenza A and B viruses with reduced susceptibility to baloxavir: A mini-review[J]. Rev Med Virol, 2021, 31(3): e2175. doi: 10.1002/rmv.2175

[13] Hussain M, Galvin HD, Haw TY, et al. Drug resistance in influenza A virus: The epidemiology and management[J]. Infect Drug Resist, 2017, 10: 121-134. doi: 10.2147/IDR.S105473

[14] Lee N, Hurt AC. Neuraminidase inhibitor resistance in influenza: A clinical perspective[J]. Curr Opin Infect Dis, 2018, 31(6): 520-526. doi: 10.1097/QCO.0000000000000498

[15] World Health Organization. Recommended composition of influenza virus vaccines for use in the 2025-2026 northern hemisphere influenza season[EB/OL]. (2025-02-28) [2025-11-12]. https://www.who.int/publications/m/item/recommended-composition-of-influenza-virus-vaccines-for-use-in-the-2025-2026-nh-influenza-season.

[16] Harrington WN, Kackos CM, Webby RJ. The evolution and future of influenza pandemic preparedness[J]. Exp Mol Med, 2021, 53(5): 737-749. doi: 10.1038/s12276-021-00603-0

[17] Houser K, Subbarao K. Influenza vaccines: Challenges and solutions[J]. Cell Host Microbe, 2015, 17(3): 295-300. doi: 10.1016/j.chom.2015.02.012

[18] Söllner JH, Mettenleiter TC, Petersen B. Genome editing strategies to protect livestock from viral infections[J]. Viruses, 2021, 13(10): 1996. doi: 10.3390/v13101996

[19] Alexander DJ, Brown IH. Recent zoonoses caused by influenza A viruses[J]. Rev Sci Tech, 2000, 19(1): 197-225. doi: 10.20506/rst.19.1.1220

[20] Gao F, Li P, Yin Y, et al. Molecular breeding of livestock for disease resistance[J]. Virology, 2023, 587: 109862. doi: 10.1016/j.virol.2023.109862

[21] Adli M. The CRISPR tool kit for genome editing and beyond[J]. Nat Commun, 2018, 9(1): 1911. doi: 10.1038/s41467-018-04252-2

[22] Qian L, Tang M, Yang J, et al. Targeted mutations in myostatin by zinc-finger nucleases result in double-muscled phenotype in Meishan pigs[J]. Sci Rep, 2015, 5: 14435. doi: 10.1038/srep14435

[23] Ruan J, Xu J, Chen-Tsai RY, et al. Genome editing in livestock: Are we ready for a revolution in animal breeding industry?[J]. Transgenic Res, 2017, 26(6): 715-726. doi: 10.1007/s11248-017-0049-7

[24] Food and Agriculture Organization of the United Nations. Gene editing and food safety: Technical considerations and potential relevance to the work of Codex Alimentarius[EB/OL]. (2023-04-17) [2025-11-12]. https://openknowledge.fao.org/server/api/core/bitstreams/98af0834-aa7b-45ab-9263-65ae0116a0ce/content.

[25] Hallerman E, Bredlau J, Camargo LSA, et al. Enabling regulatory policy globally will promote realization of the potential of animal biotechnology[J]. CABI Agric Biosci, 2024, 5: 25. doi: 10.1186/s43170-024-00221-6

[26] Whitworth KM, Benne JA, Spate LD, et al. Zygote injection of CRISPR/Cas9 RNA successfully modifies the target gene without delaying blastocyst development or altering the sex ratio in pigs[J]. Transgenic Res, 2017, 26(1): 97-107. doi: 10.1007/s11248-016-9989-6

[27] Kwon T, Artiaga BL, McDowell CD, et al. Gene editing of pigs to control influenza A virus infections[J]. Emerg Microbes Infect, 2024, 13(1): 2387449. doi: 10.1080/22221751.2024.2387449

[28] Müller M, Brenig B, Winnacker EL, et al. Transgenic pigs carrying cDNA copies encoding the murine Mx1 protein which confers resistance to influenza virus infection[J]. Gene, 1992, 121(2): 263-270. doi: 10.1016/0378-1119(92)90130-H

[29] Yan Q, Yang H, Yang D, et al. Production of transgenic pigs over-expressing the antiviral gene Mx1[J]. Cell Regen, 2014, 3(1): 11. doi: 10.1186/2045-9769-3-11

[30] European Food Safety Authority, European Centre for Disease Prevention and Control, European Union Reference Laboratory for Avian Influenza, et al. Avian influenza overview December 2022-March 2023[J]. EFSA J, 2023, 21(3): e07917. doi: 10.2903/j.efsa.2023.7917

[31] Wu D, Huang W, Wang Y, et al. Gene silencing of β-galactosamide α-2,6-sialyltransferase 1 inhibits human influenza virus infection of airway epithelial cells[J]. BMC Microbiol, 2014, 14: 78. doi: 10.1186/1471-2180-14-78

[32] Zhao Y, Zou J, Gao Q, et al. CMAS and ST3GAL4 play an important role in the adsorption of influenza virus by affecting the synthesis of sialic acid receptors[J]. Int J Mol Sci, 2021, 22(11): 6081. doi: 10.3390/ijms22116081

[33] Trebbien R, Larsen LE, Viuff BM. Distribution of sialic acid receptors and influenza A virus of avian and swine origin in experimentally infected pigs[J]. Virol J, 2011, 8: 434. doi: 10.1186/1743-422X-8-434

[34] Nelli RK, Kuchipudi SV, White GA, et al. Comparative distribution of human and avian type sialic acid influenza receptors in the pig[J]. BMC Vet Res, 2010, 6: 4. doi: 10.1186/1746-6148-6-4

[35] Costa T, Chaves AJ, Valle R, et al. Distribution patterns of influenza virus receptors and viral attachment patterns in the respiratory and intestinal tracts of seven avian species[J]. Vet Res, 2012, 43(1): 28. doi: 10.1186/1297-9716-43-28

[36] Kimble B, Nieto GR, Perez DR. Characterization of influenza virus sialic acid receptors in minor poultry species[J]. Virol J, 2010, 7: 365. doi: 10.1186/1743-422X-7-365

[37] Byrd-Leotis L, Jia N, Dutta S, et al. Influenza binds phosphorylated glycans from human lung[J]. Sci Adv, 2019, 5(2): eaav2554. doi: 10.1126/sciadv.aav2554

[38] Chung C, Yin B, Wang Q, et al. Assessment of the coordinated role of ST3GAL3, ST3GAL4 and ST3GAL6 on the α2, 3 sialylation linkage of mammalian glycoproteins[J]. Biochem Biophys Res Commun, 2015, 463(3): 211-215. doi: 10.1016/j.bbrc.2015.05.023

[39] Ito T, Suzuki Y, Suzuki T, et al. Recognition of N-glycolylneuraminic acid linked to galactose by the α2,3 linkage is associated with intestinal replication of influenza A virus in ducks[J]. J Virol, 2000, 74(19): 9300-9305. doi: 10.1128/JVI.74.19.9300-9305.2000

[40] Couceiro JNSS, Paulson JC, Baum LG. Influenza virus strains selectively recognize sialyloligosaccharides on human respiratory epithelium; the role of the host cell in selection of hemagglutinin receptor specificity[J]. Virus Res, 1993, 29(2): 155-165. doi: 10.1016/0168-1702(93)90056-S

[41] Monteerarat Y, Suptawiwat O, Boonarkart C, et al. Inhibition of H5N1 highly pathogenic influenza virus by suppressing a specific sialyltransferase[J]. Arch Virol, 2010, 155(6): 889-893. doi: 10.1007/s00705-010-0658-4

[42] Byrd-Leotis L, Liu R, Bradley KC, et al. Shotgun glycomics of pig lung identifies natural endogenous receptors for influenza viruses[J]. Proc Natl Acad Sci U S A, 2014, 111(22): E2241-E2250. doi: 10.1073/pnas.1323162111

[43] Ni Z, Wang J, Yu X, et al. Influenza virus uses mGluR2 as an endocytic receptor to enter cells[J]. Nat Microbiol, 2024, 9(7): 1764-1777. doi: 10.1038/s41564-024-01713-x

[44] Hedlund M, Ng E, Varki A, et al. α 2-6-Linked sialic acids on N-glycans modulate carcinoma differentiation in vivo[J]. Cancer Res, 2008, 68(2): 388-394. doi: 10.1158/0008-5472.CAN-07-1340

[45] Cazet A, Julien S, Bobowski M, et al. Tumour-associated carbohydrate antigens in breast cancer[J]. Breast Cancer Res, 2010, 12(3): 204. doi: 10.1186/bcr2577

[46] Ednie AR, Horton KK, Wu J, et al. Expression of the sialyltransferase, ST3Gal4, impacts cardiac voltage-gated sodium channel activity, refractory period and ventricular conduction[J]. J Mol Cell Cardiol, 2013, 59: 117-127. doi: 10.1016/j.yjmcc.2013.02.013