Characterization of a pigeon paramyxovirus ( PPMV-1 ) isolated from chickens in South Africa

ABOLNIK, C., HORNER, R.F., MAHARAJ, R. & VILJOEN, G.J. 2004. Characterization of a pigeon paramyxovirus (PPMV-1) isolated from chickens in South Africa. Onderstepoort Journal of Veterinary Research, 71:157–160 A paramyxovirus with a thermostability of 60 min (typical of velogenic viruses) and a mean death time of > 90 h (typical of lentogenic viruses) was isolated from layers near Mooi River, South Africa. Our results, based on comparative nucleotide sequence data indicated that the virus is pigeon paramyxovirus 1 (PPMV-1), a variant of Newcastle disease virus. The F0 cleavage site contains a 112RRKKRF117 motif, and the virus had 98 % sequence identity with PPMV-1 strains from the Far East. PPMV-1 was last reported in South Africa during the 1980s, with this being the first report of PPMV-1 isolated from chickens in South Africa.


INTRODUCTION
Newcastle disease virus (NDV), or avian paramyxovirus-1, is a member of the Avulavirus genus in the family Paramyxoviridae (Van Regenmortel, Fauquet, Bishop, Carsten & Maniloff 2000).It is classified as a list A disease by the Office Internationale des Epizooties (OIE) because it is highly contagious and causes severe disease and high mortalities in susceptible birds.A pandemic caused by the pigeon variant of avian paramyxovirus-1 (PPMV-1) arose in the late 1970s, and reached Europe by 1981 before spreading worldwide (Collins, Strong & Alexander 1994;Alexander 1998), including South Africa in 1986 (Pienaar & Cilliers 1987).

Virus characterization
South African isolate ZA469/PPMV1/02 was isolated from 28-week-old layers with symptoms of moderate mucoid tracheitis from the Mooi River area in KwaZulu-Natal Province, South Africa in December 2002.Pooled tissues of trachea and caecal tonsils were prepared and inoculated into the allantoic cavity of 9-11 day-old embryonated chicken eggs.Mean death time (MDT) tests were performed as described in the OIE Manual of Standards for Diagnostic Tests and Vaccines (2000).

RNA extraction and RT-PCR
Viral RNA was extracted from allantoic fluid using TRIzol reagent (Gibco, Invitrogen), according to the manufacturer's instructions.Random hexamers were used to generate first strand cDNA according to the method described by Sambrook, Fritsch & Maniatis (1989).The oligonucleotide primers mentioned below were used to amplify an 1 180 base pair fragment spanning the regions between nucleotides 581 of the fusion protein and nucleotides 610 of the matrix protein, which includes the F 0 cleavage site.Reaction mixtures were subjected to 35 cycles of 94 °C for 30 s, 53 °C for 30 s, and 72 °C for 1 min.M610 5'-CTG TAC AAT CTT GCG CTC AAT GTC -3' (forward primer) NDVF581 5'-CTG CCA CTG CTA GTT GTG ATA ATC C -3' (reverse primer) Sequencing and phylogenetic analysis DNA was sequenced using the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems) according to the manufacturer's instructions, in an ABI377 automated sequencer.
A 374 nucleotide (nt) fragment of the fusion protein gene, including the F 0 cleavage site was aligned using GCG Seqlab (Wisconsin package version 10.1-UNIX, Genetics Computer Group, Inc).Phylogenetic trees were drawn with the DNAML program from the PHYLIP software package (version 3.4) (Feselstein 1991).

RESULTS AND DISCUSSION
The thermostability assay of the virus that was isolated gave a value of 60 min, which is typical for velogenic ND viruses.This was in contrast to the value obtained (> 90 h which is typical for lentogenic viruses).RT-PCR was performed on the viral 158 Pigeon paramyxovirus (PPMV-1) isolated from chickens in South Africa Residues 112-117 form the F 0 cleavage site; the unusual 114K for Q substitution is printed in bold RNA using NDV-specific oligonucleotides, and the F 0 cleavage site was sequenced in order to determine the pathotype.The partial nucleotide sequence of the F gene and amino acid sequence at F 0 cleavage site was typical of PPMV-1.Phylogenetic analysis indicated that, although similar to recent European PPMV-1 isolates, the South African isolate is most closely related to Japanese strains of pigeon paramyxoviruses isolated in the late 1990s (at 98 % nucleotide sequence homology).The phylogenetic relationships furthermore suggest that currently circulating Italian strains 117/01 and 2736/00 could share a common origin with the strains from the Far East.In a recent epidemiological study of 155 NDV isolates isolated in South Africa over the past 12 years, no evidence of PPMV-1 was found.PPMV-1, however, was present in South Africa during the 1980s (Pienaar & Cilliers 1987) Siberia, which mingle there with others migrating from the Far East).The isolation of a pigeon paramyxovirus from chickens with the potential of causing disease is nevertheless a further motivation for highlighting the importance of vaccinating poultry and domestic racing pigeons against ND.

TABLE 1
Viruses used in the phylogentic analysis and comparison of F 0 cleavage site sequences Phylogenetic relationships of PPMV-1 isolates in Table1based on (A) nucleotides 269 to 374 (106 bp) of the F protein gene and (B) nucleotides 1 to 374 of the F protein gene, with insert showing higher resolution of the phylogenetic relationship of some of these viruses