Identification and subgrouping of pigeon type Newcastle disease virus strains by restriction enzyme cleavage site analysis
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).
Section snippets
Virus strains
Origin, propagation and handling of PPMV1 viruses were as described previously (Ballagi-Pordány et al., 1996). The same viruses were used in the present study whose phylogenetic analysis was already published (Ujvári et al., 2003, Ujvári et al., 2004). Therefore, to keep data within reasonable limits the list, which contains isolation data and phylogenetic characterization of strains, is not included.
Viral RNA extraction
This procedure was carried out as described previously (Ballagi-Pordány et al., 1996) with
Results
Pigeon type NDV strains were subjected routinely to analysis by RE cleavage as they arrived in the laboratory. The results of these are described. The majority of strains included in the current study were isolated from domesticated pigeons (83 strains) but some isolates (23) from wild pigeons, poultry, ornamental and zoo birds were also included. Viruses were obtained during the period of 1978–2002 from 16 countries in the Middle East, Europe and North America. A detailed description of the
Discussion
RE analysis of pigeon type NDV strains has resulted in several important findings relevant to diagnosis and epidemiology of these viruses.
Firstly, the salient finding of the current work is that all PPMV1 isolates lacked the BstOI site at nucleotide 1601 whose occurrence is a shared property of NDV strains of diverse origin. In addition to this marker, with the exception of sublineage IQ, all other currently circulating subgroups are characterised by the additional loss of a BstOI site at 1260
Acknowledgement
Parts of this work were supported by grant from the National Science Fund (OTKA, No. T 038254), Hungary.
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