Development of advanced molecular and biological methods for antigenic characterization of emerging variant infectious bursal disease viruses
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Infectious bursal disease (IBD) is an immunosuppressive disease in chickens which causes economic losses in the poultry industry worldwide. IBD is caused by infectious bursal disease virus (IBDV), a member of the family Birnaviridae. IBDV is a non-enveloped, double-stranded RNA virus targeting proliferating B-lymphocytes causing humoral immunosuppression. Vaccination programs and presence of field viruses probably lead to emergence of antigenically or pathogenically different IBDV due to changes in the viral genome caused by a intrinsic missing proof-reading of the viral replicase. The determination of the antigenicity of IBDV field isolates plays a critical role and is necessary for successful vaccination. To this end, the reverse genetics system (RGS) of IBDV was modified and utilized as a diagnostic tool. In this method, RNA was isolated from bursal samples and amplified with specific primers encompassing the VP2 region responsible for the antigenicity of IBDV. Amplified cDNA was cloned into the segment A of the RGS and nucleotide and amino acid sequences were analyzed. The in vitro transcribed cRNA was transfected into cells, followed by determination of the reactivity of the expressed viral protein with a panel of monoclonal antibodies (MAb) to determine the antigenicity. Using this approach IBDV strains circulating in U.S poultry flocks were identified and resembled a new antigenic subtype of IBDV. Based on this finding, the approach was refined to analyze the antigenicity of IBDV on a global scale. To this end, the RGS was combined with a method where nucleic acids can be transported across borders without importing infectious virus. Using this approach, viruses of the new antigenic subtypes were identified in poultry flocks on different continents. Finally, the relevance of the new variant strains of IBDV were investigated by developing an in vivo experimental model. Using this model for one virus of the new antigenic subtype it was shown that it was indeed different from known IBDV. Taken together, a system was established which enables the identification and antigenic characterization of different IBDVs without transporting infectious virus. This complex system will allow the antigenic analysis of IBDV on a global scale.