Passaging of a Newcastle disease virus pigeon variant in chickens results in selection of viruses with mutations in the polymerase complex enhancing virus replication and virulence

Abstract

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 ...

Occurrence, characteristics and control of pigeon paramyxovirus type 1 in pigeons

“…Sequencing of the whole genome revealed that the increase in virulence of the virus passaged in chickens was not associated with changes in the F protein, but with mutations which occurred in genes encoding L and P proteins. These proteins are responsible for replication of the genome, including the synthesis of RNA strands of positive polarity, which serves as a template for the RNA strand of negative polarity (Dortmans et al 2011). Therefore, the pathogenicity of PPMV-1, and thus the ICPI ratio, is determined not only by the amino acid composition of the F protein, but also by the viral replication complex (proteins NP, P and L) (Dortmans et al 2011).…”

“…The complex of proteins NP, P and L is responsible for the transcription and replication of the viral genome. NP protein forms a nucleocapsid enclosing the viral RNA, protein P enables the synthesis of RNA and protein L functions as an RNA-dependent RNA polymerase and is responsible for the post-transcriptional modifications of mRNA (Dortmans et al 2011). HN and F glycoproteins, forming two types of viral surface projection, play a special role in the course of infection and are the major antigens, which cause an immune response.…”

“…However, despite this fact, some PPMV-1 strains have low virulence. Determination of the virulence can be done using biological assays such as ICPI (intracerebral pathogenicity index) for one-day-old specific pathogens free (SPF) chickens (Dortmans et al 2011). As defi-973ned by the OIE (2008), Newcastle disease, among other factors, occurs when the avian paramyxovirus serotype 1 has ICPI equal to 0.7 or higher.…”

An active site mutation increases the polymerase activity of the guinea pig-lethal Marburg virus

“…Rhabdoviridae (Kim et al, 2014), Filoviridae (Ebihara et al, 2006) and Bornaviridae (Ackermann et al, 2007). This Cterminal component of L proteins of NNS RNA viruses, especially the region between domains V and VI, is highly variable, and considered as a domain of L which interacts with unique host cell transacting transcriptional cofactors (Dortmans et al, 2011;Poch et al, 1990;Sidhu et al, 1993). The mutations in guinea pig-lethal MARV L protein, however, affect the domain III close to the GDNQ/E motif, presumably directly modulating the processivity of viral RNA-dependent RNA polymerase (Liang et al, 2015).…”

The molecular basis of the pathogenicity of Newcastle disease virus in chickens

“…This was associated with 3 amino acid mutations, 2 in the L protein and 1 in the P. 148 A further study examining all 6 NDV genes showed that whilst the F gene is the main determinant of pathogenicity, the polymerase L gene is the second most important contributor. 120 …”