Some Newcastle disease virus (NDV) variants isolated from pigeons (pigeon paramyxovirus type 1; PPMV-1) do not show their full virulence potential for domestic chickens but may become virulent upon spread in these animals. In this study we examined the molecular changes responsible for this gain of virulence by passaging a low-pathogenic PPMV-1 isolate in chickens. Complete genome sequencing of virus obtained after 1, 3 and 5 passages showed the increase in virulence was not accompanied by changes in the fusion protein -a well known virulence determinant of NDV -but by mutations in the L and P replication proteins. The effect of these mutations on virulence was confirmed by means of reverse genetics using an infectious cDNA clone. Acquisition of three amino acid mutations, two in the L protein and one in the P protein, significantly increased virulence as determined by intracerebral pathogenicity index tests in dayold chickens. The mutations enhanced virus replication in vitro and in vivo and increased the plaque size in infected cell culture monolayers. Furthermore, they increased the activity of the viral replication complex as determined by an in vitro minigenome replication assay. Our data demonstrate that PPMV-1 replication in chickens results in mutations in the polymerase complex rather than the viral fusion protein, and that the virulence level of pigeon paramyxoviruses is directly related to the activity of the viral replication complex.
INTRODUCTIONNewcastle disease caused by Newcastle disease virus (NDV), or avian paramyxovirus type 1, is an economically important disease of birds. Periodic outbreaks of Newcastle disease severely affect the poultry industry and, therefore, many countries rely on compulsory vaccination. NDV is classified in the genus Avulavirus of the family Paramyxoviridae (Mayo, 2002) and has a non-segmented negative-strand RNA genome consisting of six transcriptional units (Lamb & Parks, 2007). These encode at least six proteins: the nucleocapsid protein (NP), the phosphoprotein (P), the matrix protein (M), the fusion protein (F), the haemagglutinin-neuraminidase (HN) and the polymerase (L) protein. During P gene transcription an additional, non-structural protein (V) is produced by means of mRNA editing (Steward et al., 1993) and functions as an interferon antagonist (Park et al., 2003). The M, F and HN proteins are associated with the viral envelope, in which the M protein is involved in budding and morphogenesis, whereas F and HN mediate the entry and release of NDV. The virulence of NDV is mainly determined by the amino acid sequence of the protease cleavage site of the F protein. Virulent NDV strains can be discriminated from low-or non-virulent strains by the presence of multiple basic amino acids at the proteolytic cleavage site of the F protein (Nagai et al., 1976; Ogasawara et al., 1992). The NP protein encapsidates the RNA genome to form the nucleocapsid and associates with the P and L proteins. The P protein is essential for viral RNA synthesis and is involved in all of ...