Evaluation of chicken egg yolk immunoglobulin Y-based ELISA for serodiagnosis of hepatitis E virus in swine

Abstract

Hepatitis E virus (HEV) is a major zoonotic pathogen, and pigs are an important reservoir. Serology for anti-HEV antibodies poorly reflects active infection, while reverse transcription polymerase chain reaction (RT-PCR) is sensitive but not always feasible for large-scale surveillance. This study aimed to develop an immunoglobulin Y (IgY)-based sandwich enzyme-linked immunosorbent assay (ELISA) for direct detection of HEV antigen (HEV-Ag). An antigenic epitope from the HEV open reading frame 2 capsid protein was identified in silico, synthesized, and used to immunize laying hens; IgY was purified from egg yolk and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An in-house sandwich ELISA was subsequently developed using the anti-HEV IgY antibodies. Its diagnostic performance was evaluated against RT-PCR using 350 field-collected pig serum samples and compared with commercial anti-HEV IgG and IgM ELISA kits. Immunization yielded high-purity IgY antibodies, with antigen-specific antibody kinetics showing a significant increase in specific immunoreactivity from week 4 onward and peak sensitivity at weeks 6-8 post-immunization. The anti-HEV IgY showed high specificity, with no cross-reactivity observed against a heterologous antigen (HBsAg). The optimized sandwich ELISA detected HEV-Ag with a sensitivity of 95.4% and a specificity of 100% relative to RT-PCR. The prevalence of HEV-Ag was 17.7% (62/350), which was not significantly different from RT-PCR (18.6%, 65/350; p=0.250). In contrast, commercial IgG and IgM ELISAs showed high seroprevalence rates (34.9% and 28.3%, respectively) but exhibited significant discrepancies with RT-PCR (p<0.0001), indicating limited utility for identifying active infection. Overall, the developed IgY-based sandwich ELISA demonstrated high diagnostic performance for detecting active HEV infection. This assay represents a viable alternative to RT-PCR for large-scale surveillance, particularly in resource-constrained settings, and may contribute to improved control of zoonotic HEV transmission.

Keywords

Hepatitis E virus / IgY antibodies / Sandwich ELISA / Antigen detection / Pig serum / Diagnostic performance

Acknowledgments

The authors gratefully acknowledge the staff of the Bonaberi-Douala abattoir for their assistance in facilitating blood sample collection at the exsanguination point.

Funding

This research was supported by the International Center for Genetic Engineering and Biotechnology (ICGEB) (grant number: S/CMR15-06), and Ministry of Higher Education of Cameroon under Trimester Research Modernization Allocation.

Conflict of interest

The authors declare that they have no competing interests.

Author contributions

Conceptualization: A.F. Kouam, F.N. Njayou, P.F. Moundipa

Formal analysis: A.F. Kouam, B.F.N. Simo, C.T. Zemnou, K.J. Kongnyuy, A.J. Seukep, E.M. Zeuko’o

Investigation: A.F. Kouam, P.H.N. Njitoyap, B.F.N. Simo, C.T. Zemnou, K.J. Kongnyuy, A.J. Seukep, E.M. Zeuko’o

Methodology: A.F. Kouam, P.H.N. Njitoyap, B.F.N. Simo, C.T. Zemnou, K.J. Kongnyuy, A.J. Seukep, E.M. Zeuko’o

Project administration: A.F. Kouam, F.N. Njayou, P.F. Moundipa

Software: A.F. Kouam, B.F.N. Simo

Resources: A.F. Kouam, P.H.N. Njitoyap, F.N. Njayou, P.F. Moundipa

Supervision: A.F. Kouam, P.H.N. Njitoyap, F.N. Njayou, P.F. Moundipa

Validation: A.F. Kouam, P.H.N. Njitoyap, C.T. Zemnou, A.J. Seukep, E.M. Zeuko’o, F.N. Njayou, P.F. Moundipa

Visualization: A.F. Kouam, P.H.N. Njitoyap, C.T. Zemnou, A.J. Seukep, E.M. Zeuko’o, F.N. Njayou, P.F. Moundipa

Writing-original draft: A.F. Kouam, P.H.N. Njitoyap, B.F.N. Simo

Writing-review & editing: All authors

Ethics approval and consent to participate

This study was approved by the Institutional Animal Care and Use Committee of the University of Buea (UB-IACUC N°59/2023).

Consent for publication

Not applicable.

Availability of data

All data generated or analyzed during this study are included in this published article and its supplementary information files.

References

[1] Lagare A, Abdoulkader IK, Fall G, et al. Molecular epidemiology of hepatitis E virus among humans in the Niger Republic, 2017-2023. J Clin Virol. 2025; 176:105761. doi: 10.1016/j.jcv.2025.105761

[2] WHO. News Room Fact Sheets Detail Hepatitis-E. World Health Organization; 2025. Available from: https://www.who.int/news-room/fact-sheets/detail/hepatitis-e [Last accessed on 2025 Jun 11].

[3] Modiyinji AF, Atsama MA, Monamele GC, Nola M, Njouom R. High seroprevalence of hepatitis E among pigs suggests an animal reservoir in Cameroon. J Infect Dev Ctries. 2018; 12(8):676-679. doi: 10.3855/jidc.10310

[4] Purdy MA, Harrison TJ, Jameel S, et al. ICTV virus taxonomy profile: Hepeviridae. J Gen Virol. 2017; 98(11):2645-2646. doi: 10.1099/jgv.0.000940

[5] Cao LC, Ha LNN, Giang TT, et al. Characterization of zoonotic hepatitis E virus in domestic pigs and wild boar in Vietnam: Implications for public health. One Health. 2024; 19:100857. doi: 10.1016/j.onehlt.2024.100857

[6] Meng XJ. From barnyard to food table: The omnipresence of hepatitis E virus and risk for zoonotic infection and food safety. Virus Res. 2011; 161(1):23-30. doi: 10.1016/j.virusres.2011.01.016

[7] Monini M, Di Bartolo I, De Sabato L, Ianiro G, Agostinelli F, Ostanello F. Hepatitis E virus (HEV) in heavy pigs in slaughterhouses of Northern Italy: Investigation of seroprevalence, viraemia, and faecal shedding. Animals. 2023; 13(18):2942. doi: 10.3390/ani13182942

[8] Boxman ILA, Verhoef L, Dop PY, Vennema H, Dirks RAM, Opsteegh M. High prevalence of acute hepatitis E virus infection in pigs in Dutch slaughterhouses. Int J Food Microbiol. 2022; 379:109830. doi: 10.1016/j.ijfoodmicro.2022.109830

[9] He Z, Liu D, Liu B, et al. Prevalence of hepatitis E virus in swine in China: A systematic review with meta-analysis (2004-2023). Front Vet Sci. 2025; 11:1472658. doi: 10.3389/fvets.2024.1472658

[10] Modiyinji AF, Bigna JJ, Kenmoe S, et al. Epidemiology of hepatitis E virus infection in animals in Africa: A systematic review and meta-analysis. BMC Vet Res. 2021; 17:50. doi: 10.1186/s12917-021-02749-5

[11] Yugo DM, Meng XJ. Hepatitis E virus: Foodborne, waterborne and zoonotic transmission. Int J Environ Res Public Health. 2013; 10(10):4507-4533. doi: 10.3390/ijerph10104507

[12] Luo Q, Chen J, Zhang Y, et al. Viral hepatitis E: Clinical manifestations, treatment, and prevention. Liver Res. 2024; 8(1):11-21. doi: 10.1016/j.livres.2024.01.001

[13] Rein DB, Stevens GA, Theaker J, Wittenborn JS, Wiersma ST. The global burden of hepatitis E virus genotypes 1 and 2 in 2005. Hepatology. 2012; 55(4):988-997. doi: 10.1002/hep.25505

[14] Kamar N, Izopet J, Pavio N, et al. Hepatitis E virus infection. Nat Rev Dis Primers. 2017; 3:17086. doi: 10.1038/nrdp.2017.86

[15] Wu C, Wu X, Xia J. Hepatitis E virus infection during pregnancy. Virol J. 2020; 17(1):73. doi: 10.1186/s12985-020-01343-9

[16] Karpe YA. Processing of the hepatitis E virus ORF 1 nonstructural polyprotein. Front Virol. 2024; 3:38. doi: 10.3389/fviro.2023.1327745

[17] Kenney SP, Meng XJ. Hepatitis E virus genome structure and replication strategy. Cold Spring Harb Perspect Med. 2019; 9(1):a031724. doi: 10.1101/cshperspect.a031724

[18] Wang B, Meng XJ. Structural and molecular biology of hepatitis E virus. Comput Struct Biotechnol J. 2021; 19: 1907-1916. doi: 10.1016/j.csbj.2021.03.038

[19] Ankavay M, Montpellier C, Sayed IM, et al. New insights into the ORF₂capsid protein, a key player in the hepatitis E virus lifecycle. Sci Rep. 2019; 9:6243. doi: 10.1038/s41598-019-42737-2

[20] Riddell MA, Li F, Anderson DA. Identification of immunodominant and conformational epitopes in the capsid protein of hepatitis E virus by using monoclonal antibodies. J Virol. 2000; 74(17):8011-8017. doi: 10.1128/jvi.74.17.8011-8017.2000

[21] Li Y, Yang HS, Klasse PJ, Zhao Z. The significance of antigen-antibody-binding avidity in clinical diagnosis. Crit Rev Clin Lab Sci. 2025; 62(1):9-23. doi: 10.1080/10408363.2024.2379286

[22] Adams NM, Leelawong M, Benton A, Quinn C, Haselton FR, Schmitz JE. COVID‐19 diagnostics for resource‐limited settings: Evaluation of “unextracted” qRT‐PCR. J Med Virol. 2021; 93(1):559-563. doi: 10.1002/jmv.26328

[23] Lee L, Samardzic K, Wallach M, Frumkin LR, Mochly- Rosen D. Immunoglobulin Y for potential diagnostic and therapeutic applications in infectious diseases. Front Immunol. 2021; 12:696003. doi: 10.3389/fimmu.2021.696003

[24] Pereira EPV, Van Tilburg MF, Florean EOPT, Guedes MIF. Egg yolk antibodies (IgY) and their applications in human and veterinary health: A review. Int Immunopharmacol. 2019; 73:293-303. doi: 10.1016/j.intimp.2019.05.015

[25] Modiyinji AF, Tankeu LTA, Monamele CG, et al. Hepatitis E virus infections among patients with acute febrile jaundice in two regions of Cameroon: First molecular characterization of hepatitis E virus genotype 4. PLoS One. 2024; 19(2):e0298723. doi: 10.1371/journal.pone.0298723

[26] Locarnini SA, Yuen L. Molecular genesis of drug-resistant and vaccine-escape HBV mutants. Antivir Ther. 2010; 15(3 Pt B):451-461. doi: 10.3851/IMP1499

[27] Can H, Köseoğlu AE, Erkunt Alak S, et al. In silico discovery of antigenic proteins and epitopes of SARS-CoV-2 for the development of a vaccine or a diagnostic approach for COVID-19. Sci Rep. 2020; 10:22387. doi: 10.1038/s41598-020-79645-9

[28] Chaudhuri D, Datta J, Majumder S, Giri K. In silico designing of peptide based vaccine for hepatitis viruses using reverse vaccinology approach. Infect Genet Evol. 2020; 84:104388. doi: 10.1016/j.meegid.2020.104388

[29] Shams M, Nourmohammadi H, Asghari A, et al. Construction of a multi-epitope protein for human Toxocara canis detection: Immunoinformatics approach multi-epitope construct for T. Canis serodiagnosis. Inform Med Unlocked. 2021; 26:100732. doi: 10.1016/j.imu.2021.100732

[30] Shey RA, Ghogomu SM, Esoh KK, et al. In-silico design of a multi-epitope vaccine candidate against onchocerciasis and related filarial diseases. Sci Rep. 2019; 9:4409. doi: 10.1038/s41598-019-40833-x

[31] Hodek P, Trefil P, Simunek J, Hudecek J, Stiborova M. Optimized protocol of chicken antibody (IgY) purification providing electrophoretically homogenous preparations. Int J Electrochem Sci. 2013;8(1):113-124. doi: 10.1016/S1452-3981(23)14006-5

[32] Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951; 193(1):265-275.

[33] Bagulo H, Majekodunmi AO, Welburn SC, Bimi L. Hepatitis E seroprevalence and risk factors in humans and pig in Ghana. BMC Infect Dis. 2022; 22:132. doi: 10.1186/s12879-022-07127-6

[34] Mbencho MN, Hafza N, Cao LC, et al. Prevalence, genotype distribution, and risk factors of hepatitis E virus in blood donors, HIV patients, and pregnant women in Southwest Cameroon. Diagn Microbiol Infect Dis. 2025; 111(4):116748. doi: 10.1016/j.diagmicrobio.2025.116748

[35] Meta-Djomsi D, Atsama-Amougou M, Ngamaleu MR, et al. Molecular surveillance of hepatitis E virus in wastewater in Yaoundé Cameroon. PLoS One. 2025; 20(8):e0322765. doi: 10.1371/journal.pone.0322765

[36] Modiyinji AF, Sanding GMAM, Atsama MA, Monamele CG, Nola M, Njouom R. Serological and molecular investigation of hepatitis E virus in pigs reservoirs from Cameroon reveals elevated seroprevalence and presence of genotype 3. PLoS One. 2020; 15(2):e0229073. doi: 10.1371/journal.pone.0229073

[37] Landry ML. Immunoglobulin M for acute infection: True or False? Clin Vaccine Immunol. 2016; 23(7):540-545. doi: 10.1128/CVI.00211-16