In this study we demonstrated that the
vaccine candidate against
avian influenza virus H5N1 based on the
hemagglutinin H5 (HA) fused to the chicken CD154 (HACD) can also be used for differentiating infected from vaccinated animals (DIVA). As the strategy of DIVA requires at least two
proteins, we obtained a variant of the
nucleoprotein (NP49-375) in E. coli. After its purification by
IMAC, the competence of the
proteins NP49-375 and HACD as coating
antigens in indirect ELISA assays were tested by using the sera of chickens immunized with the
proteins HA and HACD and the reference sera from several
avian influenza subtypes. Together with these sera, the sera from different species of birds and the sera of chickens infected with other avian
viral diseases were analyzed by competition ELISA assays coated with the
proteins NP49-375 and HACD. The results showed that the segment CD154 in the chimeric
protein HACD did not interfere with the recognition of the molecule HA by its specific
antibodies. Also, we observed variable detection levels when the reference sera were analyzed in the ELISA plates coated with the
protein NP49-375. Moreover, only the
antibodies of the reference serum subtype H5 were detected in the ELISA plates coated with the
protein HACD. The competition ELISA assays showed percentages of inhibition of 88-91% for the positives sera and less than 20% for the negative sera. We fixed the cut-off value of these assays at 25%. No antibody detection was observed in the sera from different species of birds or the sera of chickens infected with other avian
viral diseases. This study supported the fact that the ELISA assays using the
proteins NP49-375 and HACD could be valuable tools for
avian influenza surveillance and as a strategy of DIVA for counteracting the highly pathogenic
avian influenza virus H5N1 outbreaks.