J. gen. Virol. (1988), 69, 2115-2122. Printedin Great Britain Key words: NDV/haemagglutinin-neuraminidase/epitope mapping 2115 Location of a Neutralizing Epitope for the Haemagglutinin-Neuraminidase Glycoprotein of Newcastle Disease Virus By P. CHAMBERS,It M. NESBIT, 2 K. YUSOFF, 1 N. S. MILLAR, 1 A. C. R. SAMSON 2. AND P. T. EMMERSON 1 1 Department of Biochemistry and 2 Department of Genetics, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, U.K. (Accepted 26 April 1988) SUMMARY The binding site of a monoclonal antibody to the haemagglutinin-neuraminidase (HN) polypeptide of Newcastle disease virus (NDV) has been located. Complementary DNA or synthetic oligonucleotides corresponding to portions of the HN gene were cloned into the Escherichia coli vector pUC19 and fragments of the HN protein were thereby fused to the ~-peptide of fl-galactosidase. Western blot analysis of E. coli lysates containing expressed fragments of the HN cDNA or synthetic oligonucleotides identified an antibody-binding peptide (Asp-Glu-Gln-Asp-Tyr-Gln-Ile-Arg; amino acid residues 346 to 353). Nucleotide sequence analysis of an antibody-resistant mutant of NDV revealed a Glu (wild-type) to Lys (mutant) substitution within the above sequence. The methods described could be useful for the location of continuous epitopes of other polypeptides. Newcastle disease virus (NDV) is a paramyxovirus that causes a severe and economically important respiratory disease in poultry (Lancaster & Alexander, 1975). The avian antibody response to the virus contains elements directed against both of the viral glycoproteins, the haemagglutinin-neuraminidase (HN) and the fusion (F) protein. Monospecific antisera raised against either purified glycoprotein can neutralize viral infectivity, although anti-F neutral- ization is only effective in the presence of fresh serum (Umino et al., 1984). Anti-HN serum, however, is no more effective than anti-F serum in prolonging the life of chickens after challenge with virulent NDV, but a combination of both is protective (Umino et al., 1987). Protective epitopes on paramyxovirus HN and F proteins may, however, be destroyed during the process of purification (Paterson et al., 1987). At present, therefore, it is not clear whether an antibody response to both glycoproteins is necessary for protection of chickens from challenge with virulent NDV. Studies with murine monoclonal antibodies (MAbs) against NDV suggest that there may be four sites on the H N glycoprotein that stimulate the production of virus-neutralizing antibodies (Iorio & Bratt, 1983; Nishikawa et al., 1986; Le Long et al., 1986). A fifth site is formed by the overlap of two of these sites (Iorio et al., ! 986). This communication describes the location of the epitope for a neutralizing MAb using recombinant plasmids which encode portions of the NDV HN protein and detection of antibody-binding polypeptides by Western blot analysis. Neutralizing MAbs to the NDV HN protein have been obtained from two sources (Russell et al., 1983 ; Le Long et al., 1986). Several of these bind HN in Western blots when the structure of HN has been disrupted by boiling with SDS and 2-mercaptoethanol (Le Long et al., 1986; Samson, 1986; Samson et al., 1988). These antibodies may, therefore, recognize epitopes that are continuous or are readily regenerated by refolding after denaturation. The binding sites of such t Present address: Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, U.K. 0000-8277 © 1988 SGM