Verification of H5 gene expression from H5-recombinant fowlpox viruses co-expressing host cytokine using indirect immunofluorescence antibody test (IFAT)

Fowlpox virus (FWPV) has been used in vaccine development against avian influenza virus (AIV) by the expression of the AIV haemagglutinin (HA) gene. Recombinant FWPV (rFWPV) vaccines can be improved by co-expression of interleukins (IL) that can act as immunostimulatory molecule. Prior to any field applications, rFWPV ability to stably express HA and IL genes in cells and its efficacy to induce immune response needs to be verified. Thus, the objective of this study is to verify the H5 gene expression from rFWPV-H5 and rFWPV-H5-IL-15 via indirect immunofluorescence antibody test (IFAT) method. Firstly, freshly prepared chicken embryonic fibroblast (CEF) cells were infected with recombinant viruses and wild type fowlpox virus served as a negative control. After overnight incubation, the cells were overlaid with H5 primary antibodies raised in rabbits for 2 hours. Counter-staining was done using fluorescein (FITC)- conjugated anti-rabbit secondary antibody raised in goats. Phase-contrast fluorescence microscopy showed faint green fluorescence signals in rFWPV-H5 and false positive in rFWPV-H5-IL-15 when ab21292 H5 primary antibody was used. Bright green fluorescence signals were exhibited in both recombinants, but not negative controls, when ab62587 H5 primary antibody was used, indicative of successful H5 recombinant protein expression.

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