Identification and subgrouping of pigeon type Newcastle disease virus strains by restriction enzyme cleavage site analysis

Abstract

A host variant of Newcastle disease virus (NDV, genus Avulavirus, family Paramyxoviridae) is responsible for an autonomous disease in pigeons. It emerged in the late 1970s in the Mediterranean region. Despite great genetic diversity the vast majority of strains belong to a monophyletic group (sublineage VIb) within genotype VI of NDV strains that were indigenous in the region at that time. To date only a monoclonal antibody assay is available for the specific identification of pigeon type strains. A specific genetic assay is described suitable for the identification of pigeon isolates. Cleavage site analysis of a 1349 bp amplicon of the fusion protein gene was carried out using restriction enzymes (RE) HinfI, BstOI and RsaI. RE analysis of over 100 strains isolated between 1978 and 2002 deriving from 16 countries has revealed nine RE-patterns, which were progressive site variants of the parental (group VI) genotype. In spite of substantial site variation, extant pigeon viruses lacked a BstOI cleavage site at nucleotide 1601 shared by other NDV strains of chicken origin. RE analysis is a simple and reliable method both for the identification and subgrouping of pigeon type viruses.

Introduction

Newcastle disease (ND) is highly contagious and economically one of the most important diseases of poultry. The symptoms of NDV infection in domestic avian species vary from inapparent to severe, the latter is associated with respiratory, enteric and neurological signs (Alexander et al., 2003). ND is spread widely in developing countries and strains display a great genetic diversity. Using phylogenetic analysis at least eight genotypes (with several sub-types) of NDV were described whose distribution shows host, temporal and/or geographical restrictions (Aldous et al., 2003, Czeglédi et al., 2002, Herczeg et al., 1999, Herczeg et al., 2001, Huang et al., 2004, Lee et al., 2004, Liu et al., 2003, Lomniczi et al., 1998, Mase et al., 2002, Tsai et al., 2004, Wehmann et al., 2003a, Wehmann et al., 2003b, Wise et al., 2004, Yu et al., 2001). In 1981 in Italy and the Sudan, an epizootic form of an ND-like disease was diagnosed among racing, show and food pigeons (Biancifiori and Fioroni, 1983, Eisa and Omer, 1984) and within a few years it spread not only through Europe but appeared in North America and the Far East (Alexander et al., 1985, Kaleta, 1992a, Kaleta, 1992b, Vindevogel and Duchatel, 1988, Wilson, 1986). Retrospective analysis revealed that the disease was already present in the Middle East in the late 1970s (Kaleta et al., 1985). The causative agent, a host variant of ND-virus (NDV), was called variably pigeon paramyxovirus type 1 (PPMV1) or pigeon type NDV. Despite vaccination, pigeon type ND is still enzootic in many countries and affects not only domesticated but feral and wild pigeons and zoo birds as well (Abolnik et al., 2004, Aldous et al., 2004, Alexander et al., 1985, Alexander et al., 1997, Kommers et al., 2001, Meulemans et al., 2002, Terregino et al., 2003, Ujvári et al., 2003, Ujvári et al., 2004, Werner et al., 1999, Zanetti et al., 2001).

In a previous study based on sequence analysis of the F gene, the vast majority of PPMV1 strains clustered in a monophyletic group (VIb) of the genotype VI of NDV within which four subgroups (Iraqi, early European, North American and recent European) could be recognized (Aldous et al., 2004, Ujvári et al., 2003, Ujvári et al., 2004) (Fig. 1). Most pigeon NDV strains had reduced virulence for chickens, nevertheless based on the presence of ‘virulent type’ proteolytic cleavage site of the fusion (F) protein, these are also classified as pathogens (Aldous et al., 2004, Alexander and Parsons, 1986, Kissi, 1988, Meulemans et al., 2002, Ujvári et al., 2003, Werner et al., 1999). In this way they pose a constant threat to susceptible chicken populations. Up to now PPMV1 strains were differentiated from chicken NDV isolates by monoclonal antibody (mAb) (Alexander et al., 1985, Alexander et al., 1997).

The aim of the current study was to develop a simple genetic method for the differentiation of PPMV1 from ‘more classical’ (that is chicken and wild waterbird) NDV strains. We describe here the application of restriction enzyme (RE) cleavage site analysis for not only diagnostic identification but also for the subgrouping of pigeon type strains. Previously, the technique proved useful for the grouping of NDV strains and the identification of vaccine viruses (Ballagi-Pordány et al., 1996).