Mapping of three antigenic sites on the haemagglutinin-neuraminidase protein of Newcastle disease virus

Abstract

Nine neutralizing monoclonal antibodies (MAbs), each of which react with the haemagglutinin-neuraminidase (HN) glycoprotein of the Beaudette C strain of Newcastle disease virus (NDV), have been used in competitive binding assays to delineate three non-overlapping antigenic sites A, B and C. Epitopes within these sites have been identified on the basis of cross-reactivity of MAb-resistant mutants against the panel of MAbs, determined by plaque assays and Western blotting. Site A contains three non-overlapping epitopes (A1, A2 and A3). A1 is the only linear epitope; all remaining epitopes are conformational. MAbs which react with epitopes A2 and A3 inhibit neuraminidase activity (NA) when assayed with neuraminlactose. Site B contains three partially overlapping epitopes (B1, B2 and B3) and site C is represented by a single epitope (C1). HN gene sequence analysis of MAb-resistant mutants showed that they each had only single amino acid substitutions which range from amino acid residues 347-460 for site A, 284-325 for site B, and at 481 for the C1 epitope. The apparent molecular mass of the HN glycoprotein of one mutant was increased from 72 to 75 kDa. This correlates well with the creation of an additional potential glycosylation site in this mutant from Asn-Ser-Pro(325) to Asn-Ser-Ser(325).

Sequence of the haemagglutinin‐neuraminidase gene of the Newcastle disease virus oral vaccine strain V4(UPM)

“…Glycosylation has been found to be essential for the correct folding of the HN protein of Sendai virus and subsequent recognition by some monoclonal antibodies (Vidal et al, 1989). Glycosylation has also been shown to directly influence epitope recognition of the HN protein of NDV (Nishikawa et al, 1986;Gotoh et al, 1988;Yusoff et al, 1988). However, no significant changes in the structure, hydrophilicity and charge profiles were observed in this domain when compared to the parental strain.…”

“…NDV genomic RNA was extracted from purified virions and sequenced directly according to Yusoff et al (1988). Fifteen oligonucleotide antisense primers were constructed based on consensus sequences from Sakaguchi et al (1989) and used for sequencing the genomic RNA.…”

Heterologous Expression of Hemagglutinin-Neuraminidase Protein from Newcastle Disease Virus Strain AF2240 in Centella Asiatica

“…The NDV was cultivated in 9-10-day-old embryonated chicken eggs and purified using 30-60% (w/v) sucrose gradient centrifugation at 275,000 g for 4 h at 4°C. The resulting virus pellet was resuspended in NTE buffer (10 mM Tris, 100 mM NaCl, 1mM EDTA, pH 8.0) and used for viral RNA extraction as described previously (Yusoff et al, 1988).…”

Location of Neutralizing Epitopes on the Fusion Protein of Newcastle Disease Virus Strain Beaudette C

“…same antigenic site (site A). In contrast, the NDV HN protein has at least three antigenic sites (Yusoff et al, 1988).…”

“…The MAbs were purified from ascites fluids, labelled with peroxidase as described (Yusoff et al, 1988) and used in reciprocal competitive binding assays using wild-type Beaudette C virions to determine whether or not these antibodies recognize distinct sites on the F protein. As indicated in Table I (Nishikawa et al, 1983).…”