Mem Inst Oswaldo Cruz, Rio de Janeiro, VOLUME 121 | 2026
Research Articles
Aedes aegypti salivary gland extract enhances Zika virus replication through immune modulation
1Universidade Federal de Ciências da Saúde de Porto Alegre, Programa de Pós-Graduação em Biociências, Porto Alegre, RS, Brasil
2Universidade Federal de Ciências da Saúde de Porto Alegre, Programa de Pós-Graduação em Ciências da Saúde, Porto Alegre, RS, Brasil
3Universidade Federal do Rio Grande do Sul, Programa de Pós-Graduação em Microbiologia Agrícola e do Ambiente, Porto Alegre, RS, Brasil
4Universidade do Sul de Santa Catarina, Programa de Pós-Graduação em Ciências da Saúde, Tubarão, SC, Brasil
5Universidade de São Paulo, Instituto de Ciências Biomédicas, Laboratório de Virologia Clínica e Molecular, São Paulo, SP, Brasil
6Hospital Israelita Albert Einstein, São Paulo, SP, Brasil
7Universidade Federal de Goiás, Instituto de Patologia Tropical e Saúde Pública, Goiânia, GO, Brasil
8Universidade Federal de Ciências da Saúde de Porto Alegre, Laboratório de Imunovirologia, Porto Alegre, RS, Brasil
DOI: 10.1590/0074-02760250272
ABSTRACT
BACKGROUND Mosquito saliva contains bioactive molecules that modulate host immunity and may influence arboviral infection. The contribution of Aedes aegypti salivary gland extract (SGE) to viral replication and immune regulation during Zika virus (ZIKV) infection remains poorly understood.
OBJECTIVES To investigate the immunomodulatory effects of Ae. aegypti SGE during ZIKV infection.
METHODS Peripheral blood mononuclear cells (PBMCs) and murine antigen-presenting cell lines were exposed to ZIKV with or without SGE. Viral replication was measured by quantitative polymerase chain reaction (qPCR), cell death and immune subsets by flow cytometry, oxidative stress markers by biochemical assays, and cytokine production by enzyme-linked immunosorbent assay (ELISA).
FINDINGS SGE enhanced ZIKV replication, particularly in PBMCs, with increased RNA copies (median Δ = +1,779), reduced late apoptosis of CD4⁺ T cells (p = 0.0055), dendritic cell death (p < 0.01), and impaired T-cell proliferation. SGE attenuated ZIKV-induced oxidative damage by restoring glutathione levels, reducing lipid and protein oxidation (p < 0.001), and increasing nitric oxide (NO) production. Cytokine profiling revealed suppression of interferon-y (IFN-γ) (p < 0.001) and induction of interleukin-4 (IL-4) (p < 0.0001), indicating a Th2-skewed response. Murine cell lines confirmed SGE-driven cytokine modulation.
MAIN CONCLUSIONS Ae. aegypti SGE alters host immune homeostasis, favouring ZIKV infection by weakening antiviral defences and redirecting immune and redox pathways, thereby facilitating viral expansion.
REFERENCES
01. Ryan SJ, Carlson CJ, Mordecai EA, Johnson LR. Global expansion and redistribution of Aedes-borne virus transmission risk with climate change. PLoS Negl Trop Dis. 2019; 13(3): e0007213.
02. Pielnaa P, Al-Saadawe M, Saro A, Dama MF, Zhou M, Huang Y, et al. Zika virus-spread, epidemiology, genome, transmission cycle, clinical manifestation, associated challenges, vaccine and antiviral drug development. Virology. 2020; 543: 34-42.
03. Ribeiro JMC, Arcà B, Lombardo F, Calvo E, Phan VM, Chandra PK, et al. An annotated catalogue of salivary gland transcripts in the adult female mosquito, Aedes ægypti. BMC Genomics. 2007; 8: 6.
04. Ribeiro JMC, Mans BJ, Arcà B. An insight into the sialome of blood-feeding Nematocera. Insect Biochem Mol Biol. 2010; 40(11): 767-84.
05. Melo B, Silva N, Gomes R, Navegantes K, Oliveira A, Almeida L, et al. Bioactive compounds of the salivary glands from Aedes aegypti with anti-hemostatic action. Annu Res Rev Biol. 2015; 8(2): 1-17.
06. Pompon J, Manuel M, Ng GK, Wong B, Shan C, Manokaran G, et al. Dengue subgenomic flaviviral RNA disrupts immunity in mosquito salivary glands to increase virus transmission. PLoS Pathog. 2017; 13(7): e1006535.
07. Cardenas JC, Drame PM, Luque-Burgos KA, Berrio JD, Entrena- Mutis E, González MU, et al. IgG1 and IgG4 antibodies against Aedes aegypti salivary proteins and risk for dengue infections. PLoS One. 2019; 14(1): e0208455.
08. Howell MM, Olajiga OM, Cardenas JC, Parada-Higuera CA, Gonzales-Pabon MU, Gutierrez-Silva LY, et al. Mosquito salivary antigens and their relationship to dengue and P. vivax malaria. Pathogens. 2024; 13(1): 52.
09. Wasserman HA, Singh S, Champagne DE. Saliva of the yellow fever mosquito, Aedes aegypti, modulates murine lymphocyte function. Parasite Immunol. 2004; 26(6-7): 295-306.
10. Sri-in C, Thontiravong A, Bartholomay LC, Tiawsirisup S. Effects of Aedes aegypti salivary protein on duck Tembusu virus replication and transmission in salivary glands. Acta Trop. 2022; 228: 106310.
11. Valenzuela-Leon PC, Shrivastava G, Martin-Martin I, Cardenas JC, Londono-Renteria B, Calvo E. Multiple salivary proteins from Aedes aegypti mosquito bind to the Zika virus envelope protein. Viruses. 2022; 14(2): 221.
12. Chisenhall DM, Christofferson RC, McCracken MK, Johnson AMF, Londono-Renteria B, Mores CN. Infection with dengue-2 virus alters proteins in naturally expectorated saliva of Aedes aegypti mosquitoes. Parasit Vectors. 2014; 7(1): 252.
13. Cerqueira ARA, Rodrigues L, Coavoy-Sánchez SA, Teixeira SA, Feitosa KB, Taniguchi EY, et al. Aedes aegypti salivary gland extract alleviates acute itching by blocking TRPA1 channels. Front Physiol. 2023; 14: 1055706.
14. Gavor E, Choong YK, Liu Y, Pompon J, Ooi EE, Mok YK, et al. Identification of Aedes aegypti salivary gland proteins interacting with human immune receptor proteins. PLoS Negl Trop Dis. 2022; 16(9): e0010743.
15. Sales-Campos H, de Souza PR, Basso PJ, Ramos AD, Nardini V, Chica JEL, et al. Aedes aegypti salivary gland extract ameliorates experimental inflammatory bowel disease. Int Immunopharmacol. 2015; 26(1): 13-22.
16. Gomes RS, Navegantes-Lima K, Monteiro V, Oliveira AB, Rodrigues D, Reis J, et al. Salivary gland extract from Aedes aegypti improves survival in murine polymicrobial sepsis through oxidative mechanisms. Cells. 2018; 7(11): 182.
17. Guerrero D, Cantaert T, Missé D. Aedes mosquito salivary components and their effect on the immune response to arboviruses. Front Cell Infect Microbiol. 2020; 10: 407.
18. Cox J, Mota J, Sukupolvi-Petty S, Diamond MS, Rico-Hesse R. Mosquito bite delivery of Dengue virus enhances immunogenicity and pathogenesis in humanized mice. J Virol. 2012; 86(14): 7637-49.
19. Garcia M, Alout H, Diop F, Damour A, Bengue M, Weill M, et al. Innate immune response of primary human keratinocytes to West Nile virus infection and its modulation by mosquito saliva. Front Cell Infect Microbiol. 2018; 8: 387.
20. Le Coupanec A, Babin D, Fiette L, Jouvion G, Ave P, Misse D, et al. Aedes mosquito saliva modulates Rift Valley Fever virus pathogenicity. PLoS Negl Trop Dis. 2013; 7(6): e2237.
21. Pingen M, Bryden SR, Pondeville E, Schnettler E, Kohl A, Merits A, et al. Host Inflammatory response to mosquito bites enhances the severity of arbovirus infection. Immunity. 2016; 44(6): 1455-69.
22. Surasombatpattana P, Hamel R, Patramool S, Luplertlop N, Thomas F, Desprès P, et al. Dengue virus replication in infected human keratinocytes leads to activation of antiviral innate immune responses. Infect Genet Evol. 2011; 11(7): 1664-73.
23. Wichit S, Diop F, Hamel R, Talignani L, Ferraris P, Cornelie S, et al. Aedes Aegypti saliva enhances chikungunya virus replication in human skin fibroblasts via inhibition of the type I interferon signaling pathway. Infect Genet Evol. 2017; 55: 68-70.
24. Salazar MI, Richardson JH, Sánchez-Vargas I, Olson KE, Beaty BJ. Dengue virus type 2: replication and tropisms in orally infected Aedes aegypti mosquitoes. BMC Microbiol. 2007; 7(1): 9.
25. Styer LM, Lim PY, Louie KL, Albright RG, Kramer LD, Bernard KA. Mosquito saliva causes enhancement of West Nile virus infection in mice. J Virol. 2011; 85(4): 1517-27.
26. Jin L, Guo X, Shen C, Hao X, Sun P, Li P, et al. Salivary factor LTRIN from Aedes aegypti facilitates the transmission of Zika virus by interfering with the lymphotoxin-β receptor. Nat Immunol. 2018; 19(4): 342-53.
27. Sun P, Nie K, Zhu Y, Liu Y, Wu P, Liu Z, et al. A mosquito salivary protein promotes flavivirus transmission by activation of autophagy. Nat Commun. 2020; 11(1): 260.
28. Surasombatpattana P, Ekchariyawat P, Hamel R, Patramool S, Thongrungkiat S, Denizot M, et al. Aedes aegypti saliva contains a prominent 34-kDa protein that strongly enhances Dengue virus replication in human keratinocytes. J Invest Dermatol. 2014; 134(1): 281-4.
29. Bizzarro B, Barros MS, Maciel C, Gueroni DI, Lino CN, Campopiano J, et al. Effects of Aedes aegypti salivary components on dendritic cell and lymphocyte biology. Parasit Vectors . 2013; 6(1): 329.
30. Briant L, Desprès P, Choumet V, Missé D. Role of skin immune cells on the host susceptibility to mosquito-borne viruses. Virology. 2014; 464-5: 26-32.
31. Donald CL, Brennan B, Cumberworth SL, Rezelj VV, Clark JJ, Cordeiro MT, et al. Full genome sequence and sfRNA interferon antagonist activity of Zika virus from Recife, Brazil. PLoS Negl Trop Dis. 2016; 10(10): e0005048.
32. Cavalcante RR, Pereira MH, Gontijo NF. Anti-complement activity in the saliva of phlebotomine sand flies and other haematophagous insects. Parasitology. 2003; 127(1): S0031182003003329.
33. Slámová M, Skallová A, Páleníková J, Kopecký J. Effect of tick saliva on immune interactions between Borrelia afzelii and murine dendritic cells. Parasite Immunol. 2011; 33(12): 654-60.
34. Schneider BS, Soong L, Coffey LL, Stevenson HL, McGee CE, Higgs S. Aedes aegypti saliva alters leukocyte recruitment and cytokine signaling by antigen-presenting cells during West Nile virus infection. PLoS One. 2010; 5(7): e11704.
35. Dorneles GP, Boeira MCR, Schipper LL, Silva IRV, Elsner VR, Dal Lago P, et al. Acute strenuous exercise induces an imbalance on histone H4 acetylation/histone deacetylase 2 and increases the proinflammatory profile of PBMC of obese individuals. Oxid Med Cell Longev. 2017; 2017: 1530230.
36. Ohki CMY, Benazzato C, van der Linden V, França JV, Toledo CM, Machado RRG, et al. Zika virus infection impairs synaptogenesis, induces neuroinflammation, and could be an environmental risk factor for autism spectrum disorder outcome. Biochim Biophys Acta Mol Basis Dis. 2024; 1870(5): 167097.
37. Lanciotti RS, Kosoy OL, Laven JJ, Velez JO, Lambert AJ, Johnson AJ, et al. Genetic and serologic properties of Zika virus associated with an epidemic, Yap State, Micronesia, 2007. Emerg Infect Dis. 2008; 14(8): 1232-9.
38. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem. 1979; 95(2): 351-8.
39. Griffith O. Determination of glutatione reductase and glutathione disulfide using glutathione reductase and 2-vinylpyridine. Anal Biochem. 1980; 106(1): 207-12.
40. Miranda KM, Espey MG, Wink DA. A rapid, simple spectrophotometric method for simultaneous detection of nitrate and nitrite. Nitric Oxide. 2001; 5(1): 62-71.
41. Henke E, Oliveira VS, Silva IM, Schipper L, Dorneles G, Elsner VR, et al. Acute and chronic effects of high intensity interval training on inflammatory and oxidative stress markers of postmenopausal obese women. Transl Sports Med. 2018; 1(6): 257-64.
42. Kodani A, Knopp KA, Di Lullo E, Retallack H, Kriegstein AR, De- Risi JL, et al. Zika virus alters centrosome organization to suppress the innate immune response. EMBO Rep. 2022; 23(9): e52211.
43. Zeng Q, Liu J, Hao C, Zhang B, Zhang H. Making sense of flavivirus non-strctural protein 1 in innate immune evasion and inducing tissue-specific damage. Virus Res. 2023; 336: 199222.
44. Yu J, Zheng YM, Sheridan MA, Ezashi T, Roberts RM, Liu SL. Autophagy-mediated downregulation of AXL and TIM-1 promotes sustained Zika virus infection. Proc Natl Acad Sci USA. 2025; 122(21): e2427241122.
45. Hu Y, Dong X, He Z, Wu Y, Zhang S, Lin J, et al. Zika virus antagonizes interferon response in patients and disrupts RIG-I– MAVS interaction through its CARD-TM domains. Cell Biosci. 2019; 9(1): 46.
46. Gim E, Shim DW, Hwang I, Shin OS, Yu JW. Zika virus impairs host NLRP3-mediated inflammasome activation in an NS3-dependent manner. Immune Netw. 2019; 19(6): e40.
47. Sarratea MB, Alberti AS, Redolfi DM, Truant SN, Lopez LVI, Bivona AE, et al. Zika virus NS4B protein targets TANK-binding kinase 1 and inhibits type I interferon production. Biochim Biophys Acta Gen Subj. 2023; 1867(12): 130483.
48. Serman TM, Gack MU. Evasion of innate and intrinsic antiviral pathways by the Zika virus. Viruses. 2019; 11(10): 970.
49. Cheang KW, Chen WY, Wu-Hsieh BA, Shiao SH. Infecting mosquitoes alters DENV-2 characteristics and enhances hemorrhageinduction potential in Stat1-/- mice. Murray KO, editor. PLoS Negl Trop Dis. 2021; 15(8): e0009728.
50. Conway MJ, Londono-Renteria B, Troupin A, Watson AM, Klimstra WB, Fikrig E, et al. Aedes aegypti D7 saliva protein inhibits Dengue virus infection. PLoS Negl Trop Dis. 2016; 10(9): e0004941.
51. McCracken MK, Gromowski GD, Garver LS, Goupil BA, Walker KD, Friberg H, et al. Route of inoculation and mosquito vector exposure modulate Dengue virus replication kinetics and immune responses in rhesus macaques. PLoS Negl Trop Dis. 2020; 14(4): e0008191.
52. Schneider BS, Higgs S. The enhancement of arbovirus transmission and disease by mosquito saliva is associated with modulation of the host immune response. Trans R Soc Trop Med Hyg. 2008; 102(5): 400-8.
53. Turpin J, Frumence E, Desprès P, Viranaicken W, Krejbich-Trotot P. The Zika virus delays cell death through the anti-apoptotic Bcl- 2 family proteins. Cells. 2019; 8(11): 1338.
54. Morris G, Gevezova M, Sarafian V, Maes M. Redox regulation of the immune response. Cell Mol Immunol. 2022; 19(10): 1079-101.
55. Geng S, Lin R, Wu Y, Wang J, Li L. Modulation of innate immune memory dynamics by subcellular reactive oxygen species. Antioxid Redox Signal. 2023; 39(16-18): 1027-38.
56. Manoharan RR, Prasad A, Pospíšil P, Kzhyshkowska J. ROS signaling in innate immunity via oxidative protein modifications. Front Immunol. 2024; 15: 1359600.
57. Mukherjee A, Ghosh KK, Chakrabortty S, Gulyás B, Padmanabhan P, Ball WB. Mitochondrial reactive oxygen species in infection and immunity. Biomolecules. 2024; 14(6): 670.
58. Bode K, Hauri-Hohl M, Jaquet V, Weyd H. Unlocking the power of NOX2: a comprehensive review on its role in immune regulation. Redox Biol. 2023; 64: 102795.
59. Hillyer JF, Christensen BM. Characterization of hemocytes from the yellow fever mosquito, Aedes aegypti. Histochem Cell Biol. 2002; 117(5): 431-40.
60. Gomes RS, Navegantes-Lima KC, Monteiro VVS, Oliveira ALB, Rodrigues DVS, Reis JF, et al. Salivary gland extract from Aedes aegypti improves survival in murine polymicrobial sepsis through oxidative mechanisms. Cells. 2018; 7(11): 182.
61. Carpenter A, Santos SR, Clem RJ. Expressing the pro-apoptotic reaper protein via insertion into the structural open reading frame of Sindbis virus reduces the ability to infect Aedes aegypti mosquitoes. Viruses. 2022; 14(9): 2035.
62. Ayers JB, Coatsworth HG, Kang S, Dinglasan RR, Zhou L. Clustered rapid induction of apoptosis limits ZIKV and DENV-2 proliferation in the midguts of Aedes aegypti. Commun Biol. 2021; 4(1): 69.
63. Limonta D, Jovel J, Kumar A, Airo AM, Hou S, Saito L, et al. Human fetal astrocytes infected with Zika virus exhibit delayed apoptosis and resistance to interferon: implications for persistence. Viruses. 2018; 10(11): 646.
64. Silveira GF, Wowk PF, Cataneo AHD, dos Santos PF, Delgobo M, Stimamiglio MA, et al. Human T lymphocytes are permissive for Dengue virus replication. J Virol. 2018; 92(10): e02181-17.
65. Boppana VD, Thangamani S, Adler AJ, Wikel SK. SAAG‐4 is a novel mosquito salivary protein that programmes host CD4 + T cells to express IL‐4. Parasite Immunol. 2009; 31(6): 287-95.
66. Souissi C, Marzouki S, Elbini-Dhouib I, Jebali J, Oliveira F, Valenzuela JG, et al. PpSP32, the Phlebotomus papatasi immunodominant salivary protein, exerts immunomodulatory effects on human monocytes, macrophages, and lymphocytes. Parasit Vectors. 2023; 16(1): 1.
67. Sun X, Hua S, Gao C, Blackmer JE, Ouyang Z, Ard K, et al. Immune- profiling of ZIKV- infected patients identifies a distinct function of plasmacytoid dendritic cells for immune cross-regulation. Nat Commun. 2020; 11(1): 2421.
68. Sun X, Hua S, Chen HR, Ouyang Z, Einkauf K, Tse S, et al. Transcriptional changes during naturally acquired Zika virus infection render dendritic cells highly conducive to viral replication. Cell Rep. 2017; 21(12): 3471-82.
69. Bowen JR, Quicke KM, Maddur MS, O’Neal JT, McDonald CE, Fedorova NB, et al. Zika virus antagonizes type I interferon responses during infection of human dendritic cells. PLoS Pathog. 2017; 13(2): e1006164.
70. Michlmayr D, Andrade P, Gonzalez K, Balmaseda A, Harris E. CD14+CD16+ monocytes are the main target of Zika virus infection in peripheral blood mononuclear cells in a paediatric study in Nicaragua. Nat Microbiol. 2017; 2(11): 1462-70.
71. Clinton JLS, Vogt MB, Kneubehl AR, Hibl BM, Paust S, Rico- Hesse R. Sialokinin in mosquito saliva shifts human immune responses towards intracellular pathogens. PLoS Negl Trop Dis. 2023; 17(2): e0011095.
72. Shrivastava G, Valenzuela-Leon PC, Botello K, Calvo E. Aedes aegypti saliva modulates inflammasome activation and facilitates flavivirus infection in vitro. iScience. 2024; 27(1): 108620.
73. Mu X, Lin Z, Sun Y, Chen L, Lv Q, Ji C, et al. Aedes albopictus salivary adenosine deaminase is an immunomodulatory factor facilitating dengue virus replication. Sci Rep. 2023; 13(1): 16660.
74. Suzuki T, Miyata Y, Haga S, Itoh Y, Tanaka T, Hishinuma T, et al. Selective TLR ligand stimulation enhances in vivo mosquitoborne flavivirus pathogenicity. Cell Rep. 2025; 44(9): 116210.
75. Barros MS, Lara PG, Fonseca MT, Moretti EH, Filgueiras LR, Martins JO, et al. Aedes aegypti saliva impairs M1-associated proinflammatory phenotype without promoting or affecting M2 polarization of murine macrophages. Parasit Vectors. 2019; 12(1): 239.
76. Wanasen N, Nussenzveig RH, Champagne DE, Soong L, Higgs S. Differential modulation of murine host immune response by salivary gland extracts from the mosquitoes Aedes aegypti and Culex quinquefasciatus. Med Vet Entomol. 2004; 18(2): 191-9.
77. Lara PG, Esteves E, Sales-Campos H, Assis JB, Henrique MO, Barros MS, et al. AeMOPE- 1, a novel salivary peptide from Aedes aegypti, selectively modulates activation of murine macrophages and ameliorates experimental colitis. Front Immunol. 2021; 12: 681671.
78. Cloherty APM, Rader AG, Patel KS, Eisden TJTHD, van Piggelen S, Schreurs RRCE, et al. Dengue virus exploits autophagy vesicles and secretory pathways to promote transmission by human dendritic cells. Front Immunol. 2024; 15: 1260439.
79. Loh SN, Anthony IR, Gavor E, Lim XS, Kini RM, Mok YK, et al. Recognition of Aedes aegypti mosquito saliva protein LTRIN by the human receptor LTβR for controlling the immune response. Biology (Basel). 2024; 13(1): 42.
Financial support: FAPERGS, CAPES, INCT PROBIAM.
+ Corresponding authors: luizcrj@ufcspa.edu.br | 
https://orcid.org/0000-0002-5380-8930 / pedror@ufcspa.edu.br | https://orcid.org/0000-0002-1039-2509
Received 06 October 2025
Accepted 14 January 2026
HOW TO CITE
Hilario G, Dorneles GP, de Freitas DN, da Silva OS, Prophiro JS, Fazolo T, et al. Aedes aegypti salivary gland extract enhances Zika virus replication through immune modulation. Mem Inst Oswaldo Cruz. 2026; 121: e250272.
HANDLING EDITOR
Ademir de Jesus Martins Jr | https://orcid.org/0000-0001-5739-1215
