Molecular Characterisation of Infectious Bursal Disease Virus Isolated in Iran for Development of an Enzyme-Linked Immunosorbent Assay Based on Recombinant VPX Protein
Hosseini, Seyed Davood (2005) Molecular Characterisation of Infectious Bursal Disease Virus Isolated in Iran for Development of an Enzyme-Linked Immunosorbent Assay Based on Recombinant VPX Protein. PhD thesis, Universiti Putra Malaysia.
The acute form of infectious bursal disease (IBD) is considered as an economically significant disease among the poultry diseases reported in Iran. It was first reported as being caused by very virulent IBD virus (IBDV) based on conventional methods. Infectious bursal disease outbreaks are still being reported frequently, in spite of vaccination and sanitation measures which are the routine practices for the control of IBD in the country. Since there was no report available on the molecular characteristics of IBDV based on segment A in Iran, which is necessary for the establishment of proper control measures, it was important to characterise the antigenic and virulent properties of the strains prevalent in Iran. Infected bursa of Fabricius were collected from chicken obtained from an unvaccinated farm in Iran. The chickens showed clinical signs of depression, anorexia, ruffled feathers, trembling, whitish or watery diarrhea and mortality. Virus isolation was carried out in embryonated eggs and the isolated virus showed 96% mortality in 4 weeks-old specific pathogen free (SPF) chickens, which was typical of very virulent IBDV. The complete nucleotide sequences of segment A of the isolate, which code for the viral proteins (VPs), VP2, VP4, VP3, and VP5, was amplified by reverse transcriptase-polymerase chain reaction method, sequenced and compared with some published IBDV sequences. A total of 9 common amino acid substitutions, 3 at VP2, (222 Ala, 256 lIe and 294 lIe), 3 at VP4 (685 Asn/Ser, 715 Ser and 751 Asp), 2 at VP3 (990 Val and 1005 Ala) and 1 at VP5 (49 Arg) were found in the isolate as well as in other very virulent (vv) IBDV isolates. However, the Iranian isolate also demonstrated 8 unique amino acid substitutions of which 2 each were in VP2 and VP4, respectively, 3 in VP3 and 1 in VP5. Phylogenetic analysis indicated that the Iran isolate was closely related to vvIBDV isolates from Asian countries, however, it likely shares a common origin as other vv strains isolated from other parts of the world. The characterised IBDV isolate (designated as SDH1) was subjected to expression in prokaryotic system. The VP2 and VPX genes were expressed in Escherichia coli system as a fusion protein with six-histidine tag. Protein bands with the expected molecular weight of 48KD and 51KD were detected by direct protein staining and Western blotting. Since most of the neutralizing epitopes are located on VP2 and VPX, the expressed VPX protein was considered as a suitable candidate antigen for the development of a serological test. However, instead of using whole virion as an antigen, this study focused on the use of recombinant VPX as the antigen for the development of ELISA for the detection of IBDV specific antibody. The results showed that sera obtained from vaccinated broiler chickens reacted specifically using the developed ELISA, suggesting that the recombinant VPX protein is properly folded and expressed the neutralizing epitopes. In addition, when the developed ELISA technique was compared to a commercial ELISA kit from IDEXX, USA, which uses whole virus preparations as test antigen, it showed an excellent correlation value of R2=0.972.
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