Evaluating the Immunogenicity of Avian Influenza Virus Nucleoprotein

Khademi Sayed Bonadaki, Zahra Sadat; Madani, Rasool; Pakzad, Parviz; Golchinfar, Fariba; Emami, Tara

doi:10.30699/ijmm.14.6.643

year 14, Issue 6 (November - December 2020) Iran J Med Microbiol 2020, 14(6): 643-659 | Back to browse issues page

‎ 10.30699/ijmm.14.6.643

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Khademi Sayed Bonadaki Z S, Madani R, Pakzad P, Golchinfar F, Emami T. Evaluating the Immunogenicity of Avian Influenza Virus Nucleoprotein. Iran J Med Microbiol 2020; 14 (6) :643-659
URL: http://ijmm.ir/article-1-1149-en.html

Evaluating the Immunogenicity of Avian Influenza Virus Nucleoprotein

Zahra Sadat Khademi Sayed Bonadaki¹

, Rasool Madani

², Parviz Pakzad¹

, Fariba Golchinfar³

, Tara Emami³

1- Department of Biochemistry, North Tehran Branch, Islamic Azad University, Tehran, Iran
2- Department of Proteomics and Biochemistry, Razi vaccine and serum research institute, Agricultural research education and extension organization (AREEO), Karaj, Iran , madanirasool@gmail.com
3- Department of Proteomics and Biochemistry, Razi vaccine and serum research institute, Agricultural research education and extension organization (AREEO), Karaj, Iran

Abstract: (3171 Views)

Background: Influenza viruses cause Avian Influenza (AI) is a serious infectious disease belonging to type A Orthomyxovirus. A viral RNA synthesis is due to an interaction of the nucleoprotein (NP) with the viral polymerase. In the present study, we have evaluated the immunogenicity of avian influenza virus nucleoprotein.

Materials & Methods: An Influenza Virus N9H2 subtype A/Chicken Iran/259/2014 was selected. In order to perform electrophoresis and purification of nucleoprotein, the protein concentration of the samples was determined. SDS-PAGE and Native-PAGE electrophoresis on polyacrylamide gel were done and differences in gel bands in two methods were compared. Purification of the virus nucleoprotein performed by electroelution method and purified nucleoprotein were assayed by ELISA to obtain the produced antibody titers. Ouchterlony and western blot tests were used for final approval.

Results: By determining the molecular weight of each polypeptides, the molecular weights of H9N2 proteins were ranged from 30 to 140kDa and the molecular weight of the nucleoprotein was 60kDa. The nucleoprotein was purified by electroelution method. Ouchterlony showed that in serum dilution of 1:2 and 1:4, the sediment lines were formed between the serum and the NP antigen. The NP-ELISA enables rapid serological diagnosis in 1:20. Finally, the western blot test confirmed the 60kDa nucleoprotein band.

Conclusion: The nucleoprotein which was purified by electroelution retained its antigenic property and it could be applied in diagnostic kits.

Keywords: Influenza virus, Avian influenza, Nucleoprotein, Western blot

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Type of Study: Original Research Article | Subject: Medical Virology
Received: 2020/05/25 | Accepted: 2020/09/20 | ePublished: 2020/11/10

References

1. Rekstin A, Isakova-Sivak I, Petukhova G, Korenkov D, Losev I, Smolonogina T, et al. Immuno-genicity and cross protection in mice afforded by pandemic H1N1 live attenuated influenza vaccine containing wild-type nucleoprotein. Biomed Res Int 2017; 2017: 1-11. [DOI:10.1155/2017/9359276] [PMID] [PMCID]

2. Isakova-Sivak I, Korenkov D, Smolonogina T, Tretiak T, Donina S, Rekstin A, et al. Comparative studies of infectivity, immunogenicity and cross-protective efficacy of live attenuated influenza vaccines containing nucleoprotein from cold-adapted or wild-type influenza virus in a mouse model. Virology 2017; 500: 209-17. [DOI:10.1016/j.virol.2016.10.027] [PMID] [PMCID]

3. Ghebrehewet S, MacPherson P, Ho A. Influenza. BMJ 2016; 355: 1-8. [DOI:10.1136/bmj.i6258] [PMID] [PMCID]

4. Jin M, Wang G, Zhang R, Zhao S, Li H, Tan Y, Chen H. Development of enzyme-linked immunosorbent assay with nucleoprotein as antigen for detection of antibodies to avian influenza virus. Avian Dis 2004; 48(4): 870-8. [DOI:10.1637/7226-062204r] [PMID]

5. Wahlgren J. Influenza A viruses: an ecology review. Infect Ecol Epidemiol 2011; 1: 1-7. [DOI:10.3402/iee.v1i0.6004] [PMID] [PMCID]

6. Wang X, Zeng Z, Zhang Z, Zheng Y, Li B, Su G, et al. The appropriate combination of hemagglutinin and neuraminidase prompts the predominant H5N6 highly pathogenic avian influenza virus in birds. Front Microbiol 2018; 9: 1-11. [DOI:10.3389/fmicb.2018.01088] [PMID] [PMCID]

7. Blijleven JS, Boonstra S, Onck PR, van der Giessen E, van Oijen AM. Mechanisms of influenza viral membrane fusion. Semin Cell Dev Biol 2016; 60: 78-88. [DOI:10.1016/j.semcdb.2016.07.007] [PMID]

8. Chlanda P, Schraidt O, Kummer S, Riches J, Oberwinkler H, Prinz S, et al. Structural analysis of the roles of influenza a virus membrane-associated proteins in assembly and morphology. J Virol 2015; 89(17): 8957-66. [DOI:10.1128/JVI.00592-15] [PMID] [PMCID]

9. Noda T, Kawaoka Y. Structure of influenza virus ribonucleoprotein complexes and their packaging into virions. Rev Med Virol 2010; 20(6): 380-91. [DOI:10.1002/rmv.666] [PMID] [PMCID]

10. Biswas SK, Boutz PL, Nayak DP. Influenza virus nucleoprotein interacts with influenza virus polymerase proteins. J Virol 1998; 72(7): 5493-501. [DOI:10.1128/JVI.72.7.5493-5501.1998] [PMID] [PMCID]

11. Chang C, Chen C, Wu C, Chen S, Shih S, Kuo R. Cellular hnRNP A2/B1 interacts with the NP of influenza A virus and impacts viral replication. PLoS One 2017; 12(11): 1-6. [DOI:10.1371/journal.pone.0188214] [PMID] [PMCID]

12. Vreede FT, Ng AK, Shaw PC, Fodor E. Stabilization of influenza virus replication intermediates is dependent on the RNA-binding but not the homo-oligomerization activity of the viral nucleoprotein. J Virol 2011; 85(22): 12073-8. [DOI:10.1128/JVI.00695-11] [PMID] [PMCID]

13. Tseng YF, Weng TC, Lai CC, Chen PL, Lee MS, Hu AY. A fast and efficient purification platform for cell-based influenza viruses by flow-through chromatography. Vaccine 2018; 36(22): 3146-52. [DOI:10.1016/j.vaccine.2017.03.016] [PMID]

14. Kon TC, Onu A, Berbecila L, Lupulescu E, Ghiorgisor A, Kersten GF, et al. Influenza vaccine manufacturing: effect of inactivation, splitting and site of manufacturing. Comparison of influenza vaccine production processes. PLoS One 2016; 11(3): 1-6. [DOI:10.1371/journal.pone.0150700] [PMID] [PMCID]

15. Iwasaki A, Pillai PS. Innate immunity to influenza virus infection. Nat Rev Immunol 2014; 14(5): 315-28. [DOI:10.1038/nri3665] [PMID] [PMCID]

16. Altstein AD, Gitelman AK, Smirnov YA, Piskareva LM, Zakharova LG, Pashvykina GV, et al. Immunization with influenza A NP-expressing vaccinia virus recombinant protects mice against experimental infection with human and avian influenza viruses. Arch Virol 2006; 151(5): 921-31. [DOI:10.1007/s00705-005-0676-9] [PMID]

17. Wang W, Li R, Deng Y, Lu N, Chen H, Meng X, et al. Protective efficacy of the conserved NP, PB1, and M1 proteins as immunogens in DNA- and vaccinia virus-based universal influenza A virus vaccines in mice. Clin Vaccine Immunol 2015; 22(6): 618-30. [DOI:10.1128/CVI.00091-15] [PMID] [PMCID]

18. Chen X, Liu S, Goraya M, Maarouf M, Huang S, Chen JL. Host immune response to influenza A virus infection. Front Immunol 2018; 9: 320-8. [DOI:10.3389/fimmu.2018.00320] [PMID] [PMCID]

19. Laurie KL, Horman W, Carolan LA, Chan KF, Layton D, Bean A. Evidence for viral interference and cross-reactive protective immunity between influenza B virus lineages. J Infect Dis 2018; 217(4): 548-59. [DOI:10.1093/infdis/jix509] [PMID] [PMCID]

20. Grant E, Wu C, Chan KF, Eckle S, Bharadwaj M, Zou QM, et al. Nucleoprotein of influenza A virus is a major target of immunodominant CD8+ T-cell responses. Immunol Cell Biol 2013; 91(2): 184-94. [DOI:10.1038/icb.2012.78] [PMID]

21. Babaie M, Zolfagharian H, Jamili S, Zolfaghari M. Biochemical, Hematological effects and complications of Pseudosynanceia melanostigma envenoming. J Pharmacopuncture 2019; 22(3): 7-11.

22. Babaie M, Ghaempanah A, Mehrabi Z, Mollaei A, Khalilifard Brojeni S. Partial purification and characterization of antimicrobial effect from snake (Echis carinatus), scorpion (Mesosobuthus epues) and bee (Apis mellifera) venoms. Iran J Med Microbiol. 2020; 14 (4): 108-124. [DOI:10.30699/ijmm.14.5.460]

23. Nowakowski AB, Wobig WJ, Petering DH. Native SDS-PAGE: High resolution electrophoretic separation of proteins with retention of native properties including bound metal ions. Metallomics 2014; 6(5): 1068-78. [DOI:10.1039/C4MT00033A] [PMID] [PMCID]

24. Vázquez-Iglesias L, Estefanell-Ucha B, Barcia-Castro L, Páez de la Cadena M, Álvarez-Chaver P, Ayude-Vázquez D, et al. A simple electroelution method for rapid protein purification: isolation and antibody production of alpha toxin from Clostridium septicum. Peer J 2017; 5: 1-16. [DOI:10.7717/peerj.3407] [PMID] [PMCID]

25. Hornbeck P. Double-immunodiffusion assay for detecting specific antibodies (ouchterlony). Curr Protoc Immunol. 2017; 116: 1-8. [DOI:10.1002/cpim.18] [PMID]

26. Alizadeh H, Madani R, Babaie M, Kavid N, Golchinfar F, Emami T. Preparation and purification of polyclonal antibodies against Mycobacterium avium paratuberculosis antigens in rabbit. J Fasa Univ Med Sci 2012; 2(3): 168-73.

27. Wolkerstorfer A, Katinger D, Romanova J. Factors affecting the immunogenicity of the live attenuated influenza vaccine produced in continuous cell line. Microbiol Ind Resh J 2016; 3(1): 13-24. [DOI:10.18527/2500-2236-2016-3-1-13-24]

28. Lang PO, Samaras D, Samaras N, Govind S, Aspinall R. Influenza vaccination in the face of immune exhaustion: Is herd immunity effective for protecting the elderly? Influenza Res Treat 2011; 419216: 1-6. [DOI:10.1155/2011/419216] [PMID] [PMCID]

29. Nazari A, Samianifard M, Khalili I. Immunogenicity of concentrated and purified inactivated avian influenza vaccine formulation. Arch Razi Inst 2018; 73(4): 319-24.

30. Du L, Zhou Y, Jiang S. Research and development of universal influenza vaccines. Microb Infec. 2010; 12:280-6. [DOI:10.1016/j.micinf.2010.01.001] [PMID]

31. Ellebedy AH, Webby RJ. Influenza vaccines. Vaccine. 2009; 27: 65-68. [DOI:10.1016/j.vaccine.2009.08.038] [PMID] [PMCID]

32. Comin A, Toft N, Stegeman A, Klinkenberg D, Marangon S. Serological diagnosis of avian influenza in poultry: is the haemagglutination inhibition test really the gold standard? Influenza Other Respir Viruses 2013; 7(3): 257-64. [DOI:10.1111/j.1750-2659.2012.00391.x] [PMID] [PMCID]

33. Sapachova M. Comparative analysis of methods of molecular detection of avian influenza virus. Online J Public Health Inform 2017; 9(1): 1-8. [DOI:10.5210/ojphi.v9i1.7739] [PMCID]

34. Jensen TH, Ajjouri G, Handberg KJ, Slomka MJ, Coward VJ, Cherbonnel M, et al. An enzyme-linked immunosorbent assay for detection of avian influenza virus subtypes H5 and H7 antibodies. Acta Vet Scand 2013; 55(1): 84-91. [DOI:10.1186/1751-0147-55-84] [PMID] [PMCID]

35. Lee YT, Kim KH, Ko EJ, Lee YN, Kim MC, Kwon YM. New vaccines against influenza virus. Clin Exp Vaccine Res 2014; 3(1): 12-28. [DOI:10.7774/cevr.2014.3.1.12] [PMID] [PMCID]

36. Londo DR, Davis AR, Nayak DP. Complete nucleotide sequence of the nucleoprotein gene of influenza B virus. J Virol 1983; 47(3): 642-8. [DOI:10.1128/JVI.47.3.642-648.1983] [PMID] [PMCID]

37. Panahi Y, Farahmand B, Soleimani-Stiar R, Saghiri R, Fattahi SH, Tabatabaeian M, et al. Characterization of nucleoprotein extracted from human influenza a virus cultured in two different cell lines. Iranian J Virol 2013; 7(1&2): 51-6. [DOI:10.21859/isv.7.1.2.51]

38. Qiu D, Tannock GA, Barry RD, Jackson DC. Western blot analysis of antibody responses to influenza virion proteins. Immunol Cell Biol 1992; 70(3): 181-91. [DOI:10.1038/icb.1992.23] [PMID]