Development and optimization of virus neutralization test in chicken embryonated eggs for indirect identification of avian influenza and Newcastle disease virus
Avian viral problems have been consistently reported in commercial poultry of Pakistan causing heavy economic losses to the poultry farmers. Authentic idenfication and confirmation of the causative agent is always been question mark for the selection of vaccinal strain in this regard. Current study was therefore undertaken to optimize the virus neutralization test for the serological survey of vaccinated poultry particularly for avian influenza virus’s subtypes and Newcastle disease virus. Various physiochemical factors such as concentration of antigen and antibody, Incubation temperature and incubation period for in vitro and in-vivo reaction of antigen and antibody were optimized in chicken embryonated eggs. Serum samples were obtained from vaccinated breeder birds of five commercial poultry breeder companies and subjected for VNT using different concentration of three antigen and their respective homologous antibodies under optimized conditions. AIV H9 (EID50-1×109.0/ml) and NDV (EID50-1×108.2/ml) having biological titer of 10-7 /50ul HA units were neutralize with 10-2/50ul HIU of antibody and incubated at 37°C for 30 minutes was injected subsequently into 10 day old chicken embryo followed by incubation at 37°C for 38 hours showed ≥90% neutralizing specificity. Furthermore, sera obtained from five AIV-H9, AIV-H5 and NDV exposed commercial poultry farms revealed that Big bird broiler, Big bird breeders and A&S chicks are 100% sensitive and specific whereas, Gateway chicks and Waqas poultry breeders showed 100% homology for AIV-H5 virus but do not confers similarity with prevailing AIV-H9 and NDV field strains. Therefore, high sensitivity, reproducibility and specificity VNT, it could be a tool for indirect detection of homology between vaccinal strain and wild virus antigen using known antisera. Particularly, for those organisms possess natural ability to mutate in the adverse climatic conditions.
Keywords: Virus neutralization test, Avian Influenza Virus, Newcastle Disease Virus, Sensitivity, Specificity
2. Amare A, Worku N, Negussie H. Coccidiosis Prevailing in Parent Stocks: A Comparative Study between Growers and Adult Layers in Kombolcha Poultry Breeding and Multiplication Center, Ethiopia. Global Veterinaria. 2012; 8(3):285-291.
3. Muneer MA, Muhammad K, Tahir Y. Pathways to control Avian Influenza in Pakistan. In: Agro Veterinary News. 1995; pp: 2-3.
4. Nauta JJ, Eliminating bias in the estimation of the geometric mean of HA titers. Biology. 2005; 35:149-151.
5. Naeem K, Ullah A, Manvell RJ, Alexander DJ, Avian influenza A subtype H9N2 in poultry in Pakistan. Veterinary Record. 1999; 145(19):560.
6. Bano S, Naeem K, Malik SA. Evaluation of pathogenic potential of avian influenza virus serotype H9N2 in chickens. Avian Distribution. 2003; 47(3):817-822.
7. Barbezange C, Jestin V. Quasi-species nature of an unusual Avian Paramyxovirus type-1 isolated from pigeons. Virus Genes. 2005; 53:224-40.
8. Laoeffen W, Qwak S, Boer-luijtez ED, Hulst M, Poel WVD, Bouwstra R, Mass R. Development of a virus neutralization test to detect antibodies against Schmallenberg virus and serological results in suspect and infected herds. Acta Veterinaria Scandinavica. 2012; 2012:44-54.
9. Stephenson I, Das RG, Wood JM, Katz JM. Comparison of neutralizing antibody assays for detection of antibody to influenza A/H3N2 viruses: an international collaborative study. Vaccine. 2007; 25:4056-4063.
10. Wood JM, Major D, Health A, Newman RW, Stepheson I, Clark T, Katz JM, Zambon MC. Reproducibility of serology assays for pandemic influenza H1N1: collaborative study to evaluate a candidate WHO international standard. Vaccine. 2012; 30:210-217.
11. Burton DR, Poignard P, Stainfield RL, Wilson IA. Broadly neutralizing antibodies present new prospects to counter highly antigenically diverse virses. Science. 2012; 337:183-186.
12. Gerhard W. The role of antibody response in influenza virus infection. Cur tor microbial Immunology. 2001; 260:171-190.
13. Bertram S, Glowacka I, Kuhl A, Pholmann S. Novel insights into proteolytic cleavage of influenza virus heamagglutinin. Rev. Medical Virology. 2010; 20:298-310.
14. Panshin A, Shihmanter E, Weisman Y, Orvell C, Lipkind M. Antigenic heterogeneity among the field isolates of Newcastle disease virus (NDV) in relation to the vaccine strain 1. Studies on viruses isolated from wild birds in Israel Comparative Immunology Microbiology. 2002; 25:95-108.
15. Peter K, Olitsky MD, Casals MD. Neutralization tests for diagnosis of Human virus encephalitides. Journal of the American Medical Association. 1947; 134(15):1224-1228.
16. Tahir MS, Mehmood D, Sultan U, Saeed MH, Khan AR, Ansari F, Salman MM, Majeed KA. A modified strategy of multiplex RT-PCR for simultaneous detection of H5, H7 and H9 subtype of avian influenza virus based on common forward oligo. Journal of Applied Poultry Research. 2016; 25:322-327.
17. Allan WH, Lancaster JE, Toth B. Newcastle disease vaccines, their production and use. FAO animal production and health series. 1978; 57-62.
18. Qihan LI, Alicia GY, Young ML, James H, Marie C. Poliovirus neutralization by antibodies to internal epitopes of VP4 and VP1 results from reversible exposure of these sequences at physiological temperature. Journal of Virology. 1994; 64:3965-3970.
19. Klein M, Schoppel K, Amvrossiadis N, Mach M. Strain specific neutralization of human cytomegalovirus isolates by human sera. Journal of Virology. 1999; 73:878-886.
20. Klasse PJ, Moore JP. Quantitative model of antibody and soluble CD4-mediated neutralization of primary isolates and T-cell line adapted strain of human immunodeficiency virus type 1. Journal of Virology. 1996; 70:3668-3677.
21. Andrewes CH, Elford WJ. Observation on anti-phage sera. I. The percentage law. British Journal of Experimental Pathology. 1993; 14:367-376.
22. Hitchner SB. A virus neutralization screening test: Its limitations in classifying field isolates of infectious bronchitis virus. Avian Pathology. 1973; 2(2):103-109.
23. Olitsky PK, Harford CG. Intraperitoneal and intracerebral routes in serum protection tests with virus of equine encephalomyelitis: Comparison of two routes in protection tests. The Journal of Experimental Medicine. 1938; 68:173-189.
24. Morgan IM. Quantitative study of the neutralization of western equine encephalomyelitis virus by its antiserum and the effect of complement. Journal of Immunology. 1945; 50:359-371.
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