Development and application of a novel triplex protein microarray method for rapid detection of antibodies against avian influenza virus, Newcastle disease virus, and avian infectious bronchitis virus

Archives of Virology volume 166, pages1113–1124(2021)Cite this article

Abstract

Avian influenza virus (AIV), Newcastle disease virus (NDV), and avian infectious bronchitis virus (IBV) inflict immense damage on the global poultry industry annually. Serological diagnostic methods are fundamental for the effective control and prevention of outbreaks caused by these viruses. In this study, a novel triplex protein microarray assay was developed and validated for the rapid and simultaneous visualized detection of antibodies against AIV, NDV, and IBV in chicken sera. The AIV nuclear protein (NP), NDV phosphoprotein (P), and IBV nonstructural protein 5 (nsp5) were produced in a prokaryotic expression system, purified, and immobilized onto an initiator integrated poly(dimethylsiloxane) (iPDMS) film as probes to detect antibodies against these viruses in chicken sera. After optimization of the reaction conditions, no cross-reactivity was detected with infectious bursal disease virus, avian leukosis virus subgroup J and chicken anemia virus antisera. The lowest detectable antibody titers in this assay corresponded to hemagglutination inhibition (HI) titers of 24 and 21 for AIV and NDV, respectively, and to an IDEXX antibody titer of 103 for IBV, using the HI assay and IDEXX commercial ELISA kit as the reference methods. When156 serum samples were tested using the new assay, the HI test and the IBV IDEXX ELISA kit, the assay showed 96.8% (151/156), 97.4% (152/156) and 99.4% (155/156) diagnostic accuracy for detection of AIV, NDV and IBV antibody, respectively. The current study suggests that the newly developed triplex microarray is rapid, sensitive, and specific, providing a viable alternative assay for AIV, NDV, and IBV antibody screening in epidemiological investigations and vaccination evaluations.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

US$ 39.95

Tax calculation will be finalised during checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Availability of data and material

The datasets generated and/or analyzed in the current study are available from the corresponding author on reasonable request.

References

  1. 1.

    Bacarese-Hamilton T, Mezzasoma L, Ardizzoni A, Bistoni F, Crisanti A (2004) Serodiagnosis of infectious diseases with antigen microarrays. J Appl Microbiol 96:10–17

    CAS  Article  Google Scholar 

  2. 2.

    Bande F, Arshad SS, Omar AR, Bejo MH, Abubakar MS, Abba Y (2016) Pathogenesis and diagnostic approaches of avian infectious bronchitis. Adv Virol 2016:1–11

    Google Scholar 

  3. 3.

    Bello MB, Yusoff K, Ideris A, Hair-Bejo M, Peeters BPH, Omar AR (2018) Diagnostic and vaccination approaches for newcastle disease virus in poultry: the current and emerging perspectives. Biomed Res Int 2018:1–18

    Article  Google Scholar 

  4. 4.

    Cianci C, Gerritz SW, Deminie C, Krystal M (2012) Influenza nucleoprotein: promising target for antiviral chemotherapy. Antivir Chem Chemother 23:77–91

    Article  Google Scholar 

  5. 5.

    Das M, Kumar S (2015) Recombinant phosphoprotein based single serum dilution ELISA for rapid serological detection of Newcastle disease virus. J Virol Methods 225:64–69

    CAS  Article  Google Scholar 

  6. 6.

    Delehanty JB, Ligler FS (2002) A microarray immunoassay for simultaneous detection of proteins and bacteria. Anal Chem 74:5681–5687

    CAS  Article  Google Scholar 

  7. 7.

    Engin ED (2019) The use of multiplexing technology in the immunodiagnosis of infectious agents. J Immunoassay Immunochem 40:109–122

    CAS  Article  Google Scholar 

  8. 8.

    Ganar K, Das M, Sinha S, Kumar S (2014) Newcastle disease virus: current status and our understanding. Virus Res 184:71–81

    CAS  Article  Google Scholar 

  9. 9.

    Jackwood MW (2012) Review of infectious bronchitis virus around the world. Avian Dis 56:634–641

    Article  Google Scholar 

  10. 10.

    Jahanshiri F, Eshaghi M, Yusoff K (2005) Identification of phosphoprotein:phosphoprotein and phosphoprotein:nucleocapsid protein interaction domains of the Newcastle disease virus. Arch Virol 150:611–618

    CAS  Article  Google Scholar 

  11. 11.

    Jiang L, Yu Z, Du W, Tang Z, Jiang T, Zhang C, Lu Z (2008) Development of a fluorescent and colorimetric detection methods-based protein microarray for serodiagnosis of TORCH infections. Biosens Bioelectron 24:376–382

    Article  Google Scholar 

  12. 12.

    Jordan B (2017) Vaccination against infectious bronchitis virus: a continuous challenge. Vet Microbiol 206:137–143

    CAS  Article  Google Scholar 

  13. 13.

    Lei J, Shi T, Sun D, Mo K, Yan Y, Jin Y, Liao M, Zhou J (2017) Development and application of nsp5-ELISA for the detection of antibody to infectious bronchitis virus. J Virol Methods 243:182–189

    CAS  Article  Google Scholar 

  14. 14.

    Ma H, Wu Y, Yang X, Liu X, He J, Fu L, Wang J, Xu H, Shi Y, Zhong R (2010) Integrated poly(dimethysiloxane) with an intrinsic nonfouling property approaching “absolute” zero background in immunoassays. Anal Chem 82:6338–6342

    CAS  Article  Google Scholar 

  15. 15.

    Meade P, Latorre-Margalef N, Stallknecht DE, Krammer F (2017) Development of an influenza virus protein microarray to measure the humoral response to influenza virus infection in mallards. Emerg Microbes Infect 6:e110

    Article  Google Scholar 

  16. 16.

    Mezzasoma L, Bacarese-Hamilton T, Di Cristina M, Rossi R, Bistoni F, Crisanti A (2002) Antigen microarrays for serodiagnosis of infectious diseases. Clin Chem 48:121–130

    CAS  Article  Google Scholar 

  17. 17.

    Mostafa A, Abdelwhab EM, Mettenleiter TC, Pleschka S (2018) Zoonotic potential of influenza A viruses: a comprehensive overview. Viruses 10:497

    Article  Google Scholar 

  18. 18.

    Nachbagauer R, Krammer F (2017) Universal influenza virus vaccines and therapeutic antibodies. Clin Microbiol Infect 23:222–228

    CAS  Article  Google Scholar 

  19. 19.

    Okamatsu M, Hiono T, Kida H, Sakoda Y (2016) Recent developments in the diagnosis of avian influenza. Vet J 215:82–86

    CAS  Article  Google Scholar 

  20. 20.

    Pinette MM, Rodriguez-Lecompte JC, Pasick J, Ojkic D, Leith M, Suderman M, Berhane Y (2014) Development of a duplex Fluorescent Microsphere Immunoassay (FMIA) for the detection of antibody responses to influenza A and newcastle disease viruses. J Immunol Methods 405:167–177

    CAS  Article  Google Scholar 

  21. 21.

    Portela A, Digard P (2002) The influenza virus nucleoprotein: a multifunctional RNA-binding protein pivotal to virus replication. J Gen Virol 83:723–734

    CAS  Article  Google Scholar 

  22. 22.

    Qiu X, Fu Q, Meng C, Yu S, Zhan Y, Dong L, Ren T, Sun Y, Tan L, Song C, Han X, Ding C (2016) Kinetic analysis of RNA editing of Newcastle disease virus P gene in the early period of infection. Acta Virol 60:71–77

    CAS  Article  Google Scholar 

  23. 23.

    Starick E, Werner O, Schirrmeier H, Kollner B, Riebe R, Mundt E (2006) Establishment of a competitive ELISA (cELISA) system for the detection of influenza A virus nucleoprotein antibodies and its application to field sera from different species. J Vet Med B Infect Dis Vet Public Health 53:370–375

    CAS  Article  Google Scholar 

  24. 24.

    Sutandy FX, Qian J, Chen CS, Zhu H (2013) Overview of protein microarrays. Curr Protoc Protein Sci 72:27.1.1-27.1.16

    Article  Google Scholar 

  25. 25.

    Villegas P (1998) Viral diseases of the respiratory system. Poult Sci 77:1143–1145

    CAS  Article  Google Scholar 

  26. 26.

    Wang X, Shi L, Tao Q, Bao H, Wu J, Cai D, Wang F, Zhao Y, Tian G, Li Y, Qao C, Chen H (2010) A protein chip designed to differentiate visually antibodies in chickens which were infected by four different viruses. J Virol Methods 167:119–124

    CAS  Article  Google Scholar 

  27. 27.

    Xiao Q, Bi Z, Yao L, Lei J, Yan Y, Zhou J, Yan L (2019) Novel protein microarray for the detection of avian influenza virus antibodies and simultaneous distinction of antibodies against H5 and H7 subtypes. Avian Pathol 48:528–536

    CAS  Article  Google Scholar 

  28. 28.

    Yan L, Hu J, Lei J, Shi Z, Xiao Q, Bi Z, Yao L, Li Y, Chen Y, Fang A, Li H, Song S, Liao M, Zhou J (2018) Novel protein chip for the detection of antibodies against infectious bronchitis virus. BMC Vet Res 14:284

    Article  Google Scholar 

  29. 29.

    Yoo G, Bong JH, Kim S, Jose J, Pyun JC (2014) Microarray based on autodisplayed Ro proteins for medical diagnosis of systemic lupus erythematosus (SLE). Biosens Bioelectron 57:213–218

    CAS  Article  Google Scholar 

  30. 30.

    Zhao Y, Wang X, Chen P, Zeng X, Bao H, Wang Y, Xu X, Jiang Y, Chen H, Li G (2015) Detection of antibodies against avian influenza virus by protein microarray using nucleoprotein expressed in insect cells. J Vet Med Sci 77:413–419

    CAS  Article  Google Scholar 

  31. 31.

    Zhu H, Snyder M (2003) Protein chip technology. Curr Opin Chem Biol 7:55–63

    CAS  Article  Google Scholar 

Download references

Funding

This study was supported by the National Key Research and Development Program of China (2016YFD0500800), the Forestry Science and Technology Innovation and Promotion Project of Jiangsu Province (LYKJ[2018]22), the China Agriculture Research System (CARS-40-K13), and Priority Academic Program Development of Jiangsu Higher Education Institutions.

Author information

Affiliations

  1. MOE Joint International Research Laboratory of Animal Health and Food Safety, Jiangsu Engineering Laboratory of Animal Immunology, Jiangsu Detection Center of Terrestrial Wildlife Disease, Institute of Immunology and College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, 210095, People’s Republic of China

    Yuan Li, Jing Lei, Wentao Fan, Zhenwei Bi, Suquan Song & Liping Yan

  2. Comprehensive Enforcement Brigade of Agriculture, Forestry and Water Conservancy, Suzhou New District, Suzhou, 215000, People’s Republic of China

    Jianhua Hu

Authors
  1. Yuan Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jianhua Hu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jing Lei
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Wentao Fan
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Zhenwei Bi
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Suquan Song
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Liping Yan
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

LPY carefully conceived and designed the study. YL and JHH collected clinical serum samples, performed the experiments and analyzed data. YL and LPY wrote the manuscript. LPY, SQS, JL, ZWB and WTF revised the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Liping Yan.

Ethics declarations

Conflict of interest

No potential financial or non-financial conflict of interest was declared by the authors.

Ethical approval

The animals in this study were treated in compliance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Ministry of Health, China. All sample collection procedures in the current study were scrutinized and authorized by the Institutional Animal Care and Use Committee of Nanjing Agricultural University (approval. no. SYXK (Su) 2017-0007).

Consent to participate

Written informed consent to use 104 clinical serum samples, which were collected from breeding poultry farms in Jiangsu province of China, was obtained from the owner of the animals. All efforts were made to minimize animal suffering during sample collection.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Handling Editor: William G Dundon.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Fig. S1 SDS-PAGE analysis of the expression and purification of recombinant pET-32a-AIV-NP (a), pCold-І-NDV-P (b), and pGEX-4T-1-IBV-nsp5 (c) after transformation of E. coli BL21. M, standard protein marker; lane 1, uninduced E. coli transformed with pET-32a, pCold І, or pGEX-4T-1; lane 2, induced E. coli transformed with pET-32a, pCold І, or pGEX-4T-1; lane 3, uninduced E. coli transformed with pET-32a-AIV-NP, pCold-І-NDV-P, or pGEX-4T-1-IBV-nsp5; lane 4, induced E. coli transformed with pET-32a-AIV-NP, pCold-І-NDV-P, or pGEX-4T-1-IBV-nsp5; lane 5, purified AIV-NP, NDV-P, or IBV-nsp5 protein. Fig. S2 Western blot verification of the immunoreactivity of an AIV-positive serum with AIV NP (a), NDV-positive serum with NDV-P (b), and IBV-positive serum with IBV nsp5 (c). M, standard protein marker; lane 1, purified AIV NP, NDV P or IBV nsp5 fusion protein; lane 2, induced E. coli transformed with pET-32a, pCold І, or pGEX-4T-1. Fig. S3 The limit of detection for AIV NP, NDV P, and IBV nsp5 protein determined by testing 50 standard negative chicken sera. Each dot represents one negative chicken serum sample. Fig. S4 Stability of the triplex protein microarray slides. The signal intensities of AIV NP, NDV P and IBV nsp5, positive control, and negative control spots were compared between freshly prepared slides and slides stored at 4 °C for six months (PDF 295 KB)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Li, Y., Hu, J., Lei, J. et al. Development and application of a novel triplex protein microarray method for rapid detection of antibodies against avian influenza virus, Newcastle disease virus, and avian infectious bronchitis virus. Arch Virol 166, 1113–1124 (2021). https://doi.org/10.1007/s00705-021-04962-x

Download citation