title: Dual function of the hemagglutinin H5 fused to chicken CD154 in a potential strategy of DIVA against avian influenza disease: preliminary study.
abstract: 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.
authors.affiliation: Animal Biotechnology Department, Center for Genetic Engineering and Biotechnology (CIGB), P. O. Box 6162, Havana 10600, Cuba.
authors.forename: A G
authors.initials: AG
authors.lastname: Pose
authors.affiliation: Animal Biotechnology Department, Center for Genetic Engineering and Biotechnology (CIGB), P. O. Box 6162, Havana 10600, Cuba.
authors.forename: E S
authors.initials: ES
authors.lastname: Rodríguez
authors.affiliation: Molecular Biology Department, Faculty of Medicine, Ibero-american University (UNIBE), Ave. Francia, No. 129. Gazcue, Santo Domingo, Dominican Republic.
authors.forename: A C
authors.initials: AC
authors.lastname: Méndez
authors.affiliation: Virology Department, National Center for Animal and Plant Health (CENSA), Apdo 10, San José de Las Lajas, Mayabeque 32700, Cuba.
authors.forename: J N
authors.initials: JN
authors.lastname: Gómez
authors.affiliation: Virology Department, National Center for Animal and Plant Health (CENSA), Apdo 10, San José de Las Lajas, Mayabeque 32700, Cuba.
authors.forename: A V
authors.initials: AV
authors.lastname: Redondo
authors.affiliation: Animal Biotechnology Department, Center for Genetic Engineering and Biotechnology (CIGB), P. O. Box 6162, Havana 10600, Cuba.
authors.forename: E R
authors.initials: ER
authors.lastname: Rodríguez
authors.affiliation: Animal Biotechnology Department, Center for Genetic Engineering and Biotechnology (CIGB), P. O. Box 6162, Havana 10600, Cuba.
authors.forename: E M G
authors.initials: EM
authors.lastname: Ramos
authors.affiliation: Animal Biotechnology Department, Center for Genetic Engineering and Biotechnology (CIGB), P. O. Box 6162, Havana 10600, Cuba.
authors.forename: A Á
authors.initials: AÁ
authors.lastname: Gutiérrez
authors.affiliation: Animal Biotechnology Department, Center for Genetic Engineering and Biotechnology (CIGB), P. O. Box 6162, Havana 10600, Cuba.
authors.forename: M P R
authors.initials: MP
authors.lastname: Moltó
authors.affiliation: Animal Biotechnology Department, Center for Genetic Engineering and Biotechnology (CIGB), P. O. Box 6162, Havana 10600, Cuba.
authors.forename: D G
authors.initials: DG
authors.lastname: Roche
authors.affiliation: Virology Department, Laboratory for Avian Investigations and Diagnostic (LIDA), Boyeros, Havana 10800, Cuba.
authors.forename: Y S
authors.initials: YS
authors.lastname: Ugalde
authors.affiliation: Virology Department, Laboratory for Avian Investigations and Diagnostic (LIDA), Boyeros, Havana 10800, Cuba.
authors.forename: A M
authors.initials: AM
authors.lastname: López
keywords: Avian influenza virus; CD154; Competition ELISA; DIVA; Hemagglutinin