Abstract
BackgroundNewcastle disease is a devastating disease in poultry caused by virulent Newcastle disease virus (NDV), a paramyxovirus endemic in many regions of the world despite intensive vaccination. Phylogenetic analyses reveal ongoing evolution of the predominant circulating genotype 2.VII, and the relevance of potential antigenic drift is under discussion. To investigate variation within neutralization-sensitive epitopes within the protein responsible for receptor binding, i.e. the Hemagglutinin-Neuraminidase (HN) spike protein, we were interested in establishing genotype-specific monoclonal antibodies (MAbs).MethodsAn HN-enriched fraction of a gradient-purified NDV genotype 2.VII was prepared and successfully employed to induce antibodies in BalbC mice that recognize conformationally intact sites reactive by haemagglutination inhibition (HI). For subsequent screening of mouse hybridoma cultures, an NDV-ELISA was established that utilizes Concanavalin A (ConA-ELISA) coupled glycoproteins proven to present conformation-dependent epitopes.ResultsSix out of nine selected MAbs were able to block receptor binding as demonstrated by HI activity. One MAb recognized an epitope only present in the homologue virus, while four other MAbs showed weak reactivity to selected other genotypes. On the other hand, one broadly cross-reacting MAb reacted with all genotypes tested and resembled the reactivity profile of genotype-specific polyclonal antibody preparations that point to minor antigenic differences between tested NDV genotpyes.ConclusionsThese results point to the concurrent presence of variable and conserved epitopes within the HN molecule of NDV. The described protocol should help to generate MAbs against a variety of NDV strains and to enable in depth analysis of the antigenic profiles of different genotypes.
Highlights
Newcastle disease virus (syn. avian orthoavulavirus-1; avian paramyxovirus-1 (APMV-1), NDV) is a member of the family of Paramyxoviridae within the genusMoharam et al Virol J (2021) 18:86Orthoavulavirus, which together with the genera Metaand Paraavulavirus form the subfamily Avulavirinae [1]
Purified virus preparations were dominated in sodium dodecyl sulphate (SDS)-Polyacrylamide gel electrophorese (PAGE) and subsequent Coomassie staining by a band at 55 kD, likely representing comigrating proteins of NP (53 kD), P (53–56 kD) and F1 (55 kD) (Fig. 1a)
This protein fraction is recognized as a major immunogenic fraction in the virion by antibodies of NDV vaccinated chicken (Fig. 1b)
Summary
Newcastle disease virus (syn. avian orthoavulavirus-1; avian paramyxovirus-1 (APMV-1), NDV) is a member of the family of Paramyxoviridae within the genusMoharam et al Virol J (2021) 18:86Orthoavulavirus, which together with the genera Metaand Paraavulavirus form the subfamily Avulavirinae [1]. The genome size of either 15,186, 15,192 or 15,198 nucleotides [2] encodes for six structural proteins [3, 4] with two outer spike glycoproteins, the hemagglutinin-neuraminidase- (HN) and fusion protein (F), that facilitate attachment to and subsequent entry into the host cell. The latter protein is translated as a precursor molecule (F0) and cleaved by cellular proteases into disulfide bond-linked subunits F1 and F2 [5, 6]. To investigate variation within neutralization-sensitive epitopes within the protein responsible for receptor binding, i.e. the Hemagglutinin-Neuraminidase (HN) spike protein, we were interested in establishing genotype-specific monoclonal antibodies (MAbs)
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