Abolnik_147-152.indd INTRODUCTION Pigeon paramyxovirus type 1 (PPMV-1) is an anti- genic variant of avian paramyxovirus type 1 (APMV-1; Newcastle disease virus) that was first identified by monoclonal antibody binding studies (Alexander, Wil son, Thain & Lister 1984; Alexander, Russell, Par sons, Abu Elzein, Ballouh, Cernik, Engstrom, Feve reiro, Fleury, Guittet, Kaleta, Kihm, Kosters, Lomniczi, Meister, Meulemans, Nerome, Petek, Po- komunski, Polten, Prip, Richter, Sághy, Samberg, Spanoghe & Tumova 1985; King 1996; Lana, Sny- der, Kink & Marquart 1988). PPMV-1 strains caused outbreaks among racing and show pigeons in Europe in 1981 and re-emerged in 1985 causing a panzootic that continues today (Biancifiori & Fioroni 1983; Alexander et al. 1985; Wilson 1986; Kaleta 1992a, b; Ujvári, Wehman, Kaleta, Werner, Savić, Nagy, Czifra & Lomniczi 2003; Aldous, Fuller, Mynn & Alexander 2004). In pigeons and doves clinical signs of infection include neurological signs such as torticollis and paralysis, and the excretion of large volumes of green, watery diarrhea (Alexander et al. 1984; 1985; Lemahieu, De Vriese & Bijnens 1985). In chickens, intracerebral pathogenicity (ICPI) val- ues for PPMV-1 are typical of mesogenic Newcastle 147 Onderstepoort Journal of Veterinary Research, 75:147–152 (2008) Characterization of pigeon paramyxoviruses (Newcastle disease virus) isolated in South Africa from 2001 to 2006 C. ABOLNIK1*, G.H. GERDES1, J. KITCHING2, S. SWANEPOEL3, M. ROMITO1 and S.P.R. BISSCHOP4 ABSTRACT C. ABOLNIK, G.H. GERDES, J. KITCHING, S. SWANEPOEL, M. ROMITO & S.P.R. BISSCHOP. 2008. Characterization of pigeon paramyxoviruses (Newcastle disease virus) isolated in South Africa from 2001 to 2006. Onderstepoort Journal of Veterinary Research, 75:147–152 Pigeon paramyxovirus type 1 (PPMV-1), a variant of Newcastle disease virus that primarily affects doves and pigeons has been isolated in South Africa since the mid-1980s. Phylogenetic evidence indicates that pigeon paramyxovirus type 1 viruses were introduced into South Africa on multiple oc- casions, based on the presence of two separate lineages, 4bi and 4bii, that have been circulating in Europe and the Far East since the early 1990s. During 2006, a PPMV-1 virus was isolated from an African ground hornbill (Bucorvus leadbeateri) which became acutely infected with PPMV-1 and died, probably after scavenging off infected dove carcasses in the region, since a closely-related PPMV-1 strain was also isolated from doves collected nearby. The hornbill isolate had ICPI and MDT values characteristic of PPMV-1 strains. The threat of PPMV-1 to poultry production and biodiversity in southern Africa highlights the importance of monitoring the spread of this strain. Keywords: African ground hornbill, Newcastle disease virus, paramyxovirus, phylogenetic charac- terization, pigeon * Author to whom correspondence is to be directed. Email: abolnikc@arc.agric.za 1 ARC-Onderstepoort Veterinary Institute, Private Bag X5, Onder stepoort, 0110, South Africa 2 Stellenbosch Provincial Veterinary Laboratory, Private Bag X1, Elsenburg, 7607, South Africa 3 Deltammune Laboratories, P.O. Box 14167, Lyttleton, Cen- turion, 0140, South Africa 4 Poultry Reference Laboratory, University of Pretoria, Private Bag X04, Onderstepoort, 0110 South Africa Accepted for publication 14 April 2008—Editor 148 Pigeon paramyxoviruses (Newcastle disease virus) isolated in South Africa disease viruses but in most cases, PPMV-1 isolates have increased their virulence for chickens after passage, and therefore represent a threat to poultry production (Alexander & Parsons 1986; King 1996; Kommers, King, Seal & Brown 2001). Besides pi- geons, doves and chickens, PPMV-1 viruses have also been isolated from kestrels, falcons, cockatoos, budgerigars, pheasants, swans and a robin (Alex- ander et al. 1985; Lister, Alexander & Hogg 1986; Kaleta 1992b; Werner, Römer-Oberdörfer, Köllner, Manvell & Alexander 1999; Monne, Beato, Capua & Mandola 2006; Aldous et al. 2004). Phylogenetic analysis has classified PPMV-1 strains into a dis- crete lineage, VIb (Lomniczi, Wehman, Herczeg, Bal lagi-Pordany, Kaleta, Werner, Meulemans, Jor- gen sen, Manté, Gielkens, Capua & Damoser 1998), recently re-classified as lineage 4b. Lineage 4b was further split into subgroups 4bi and 4bii (Aldous et al. 2004). Large die-offs in doves and pigeons have occasion- ally been reported in various parts of South Africa since the 1980s after the first isolation of PPMV-1 from doves during an outbreak in September 1986 (Pienaar & Cilliers 1987). In the present study, the phylogenetic relationships between 21 South African PPMV-1 viruses isolated from doves, pigeons, chick- ens, a duck and an African ground hornbill (Bucorvus leadbeateri) were investigated, and the pathogenic- ity of the African ground hornbill isolate for chickens was determined. MATERIALS AND METHODS Isolates Virus isolation was performed at the Onderstepoort Veterinary Institute, Stellenbosch Provincial Veter- inary Laboratory, the University of Pretoria’s Poultry Reference Laboratory and Deltammune Laboratory by inoculation into the alantoic cavities of 9 to 10-day-old embryonated specific antibody-negative fowl eggs. Isolates are indicated in Table 1. TABLE 1 South African pigeon paramyxovirus isolates Isolate Year Host Town, Provincea Accession number ZA469/PPMV1/02 PIZA04N230 DOZA05N240 DOZA05N247 PIZA05N277 DOZA05AM68313 DOZA05N417 DOZA05N539 DOZA06N549 DOZA06N589 DOZA06N591 CKZA06N606 DOZA06UP470 PIZA06N642 DOZA06N621 -ZA06N690 PIZA06N699 PIZA06N635 DOZA06N757 BLZA06N779 DKZA06N828 2002 2004 2005 2005 2005 2005 2005 2005 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 2006 28-week-old layers Racing pigeons Doves Laughing dove Pigeon Laughing dove Doves Doves Doves Doves Doves 4-week-old broilers Dove Pigeon Doves Unknown Pigeon Pigeon Laughing doves African ground hornbill Muscovy duck Mooirivier, KZN Oudtshoorn, WC Kimberly, NC Kuilsrivier, WC Pretoria, GP Marikana, NWP Montagu, WC Belville, WC Cape Town, WC Darling, WC Kimberly, NC Sibasa, LP Polokwane, LP Cape Town, WC Cape Town, WC Germiston, GP Middleburg, MPU Stellenbosch, WC Dwaalboom, NWP Dwaalboom, NWP Worchester,WC AY445669 EF030962 EF030953 EF030954 EF030963 EF030952 EF030955 EF030956 EF030957 EF030958 EF030959 EF030951 EF030961 EF030950 EF030960 EF030964 a KZN KwaZulu-Natal WC Western Cape NC Northern Cape LMP Limpopo NWP North West GP Gauteng MPU Mpumahlanga 149 C. ABOLNIK et al. Pathogenicity tests ICPI and MDT tests were performed according to standard procedures (OIE manual of standards: di- agnostic tests and vaccines 2000). Reverse transcription-polymerase chain reaction and nucleotide sequencing Viral RNA was extracted from alantoic fluid using TRIzol® reagent (Gibco, Invitrogen). The fusion (F) protein gene was targeted in a one-step RT-PCR using the oligonucleotide primer pair and thermal cycling parameters described elsewhere (Abolnik, Horner, Maharaj & Viljoen 2004). Cycle sequencing was performed using the ABI PRISM® Big Dye™ Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems) and the reverse primer from the RT-PCR to span the region containing the F0 peptide cleavage site. Reactions were analysed with an ABI3130™ Genetic Analyser (Applied Biosys- tems). Sequence analysis Nucleotide and amino acid sequence analyses and alignment were carried out using Bioedit (Hall 1999) and ClustalW software (http://www.ebi.ac.uk/clustalw/index.html). Phylogenies were reconstructed with MEGA 3.1 soft- ware (Kumar, Tamura & Nei 2004) using the neigh- bour-joining tree inference method with the Kimura 2-parameter substitution model and 1 000 bootstrap replicates to assign confidence levels to branches. RESULTS AND DISCUSSION Phylogenetic analysis of partial F protein genes in- dicated that the South African PPMV-1 isolates do not cluster together as a single geographical entity, but instead are split between the two lineages 4bi and 4bii (Fig. 1). One of the South Africa isolates, PIZA05N277, isolated from a pigeon in Pretoria (Gau teng Province) in May 2005 shared 99 % nucle- otide sequence identity with the other international lineage 4bi viruses, but only 98–99 % and 94–95 % sequence identities with other South African lineage 4bi and lineages 4bii viruses, respectively. Therefore, we suspect that this strain was recently introduced into the country. The muscovy duck (Cairina mos- chata) (DKZA06N828) was a suspected organo- phosphate poisoning case and probably contracted PPMV-1 through contact with faeces from infected doves. CKZA06N606 was isolated from 4-week-old broilers and is only the second reported case of PPMV-1 infection of chickens in South Africa. The African ground hornbill isolate, BLZA06N779 shared 99.4 % sequence identities in the partial fusion pro- tein gene with DOZA06N757 isolated from doves on the same farm during the outbreak. An MDT val- ue of 89.4 h was obtained for BLZA06N779, which falls within the 90h time limit for mesogenic viruses. The ICPI value obtained was 0.33, which indicates that it is not velogenic but does not differentiate be- tween mesogenic and lentogenic viruses. The South African lineage 4bii strains formed a sin- gle clade and these viruses were mainly obtained from the Western Cape Province, apart from single isolates from Gauteng (-ZA06N690) and KwaZulu- Natal Provinces (ZA469/PPMV1/02). Nucleotide se- quence homology within the South African clade varied from 96.7 % to 99.1 %. Relatively longer branch lengths suggest that the lineage 4bii strains may have been circulating in South Africa for an ex- tended period. The sequences of 112RRQKRF117 and 112RRKKRF117 at F0 for lineages 4bi and 4bii, respectively (Fig. 2), are in agreement with other re- ports (Collins, Strong & Alexander 1994; Mase, Imai, Sanada, Sanada, Yuasa, Imada, Tsukamoto & Ya- ma guchi 2002; Meulemans, Van den Berg, De caes- stecker & Boschmanns 2002; Terregino, Cattoli, Gros sele, Bertoli, Tisato & Capua 2003). Although the bootstrap values supporting the phylo- genetic relationships within lineages 4bi and 4bii are low, amino acid sequences (Fig. 2) support the phylogenetic relationships: lineage 4bi clade (c) is distinguished from clades (a) and (b) (Fig. 1) by T3, L10 and R27 substitutions, and lineage 4bii clade (d) differs from clade (e) by a P36→S substitution. We have demonstrated that exotic strains of pigeon paramyxoviruses were introduced into South Africa on multiple occasions. Routes of introduction into South Africa are speculative, although they are most likely via the importation of infected pigeons for rac- ing and ornamental purposes as reported in other countries (Aldous et al. 2004). A second case of PPMV-1 infection of chickens in South Africa is re- ported here, highlighting the importance of the threat of PPMV-1 to poultry, but PPMV-1 also potentially threatens biodiversity of wild birds, particularly rare indigenous dove and pigeon species. We report the first isolation of PPMV-1 from an African ground hornbill (Bucorvus leadbeateri), an increasingly rare species, which became acutely infected with PPMV-1 and died, although the virus did not display increased pathogenicity for chickens. It is likely that the ground hornbill became infected by scavenging off the dead doves in the area. Our findings suggest that PPMV-1 150 Pigeon paramyxoviruses (Newcastle disease virus) isolated in South Africa FIG.1 Phylogenetic tree of a 374-bp region of the fusion protein gene of PPMV-1 viruses. South African strains are framed (bold type) PUKPI99065 PUKPI99128 PEBPI98328 PDEPI99063 PAEPI00377 PBEPI98327 99299 PIEPI00246 PIEPI00242 -IEPI00242 PUKPI00248 PCAPI91374 PAEPI00318 PUKPI02440 PUKPI02440 PUKPI99131 PIEPI01387 PIEPI00339 PUKPI02422 PUKPI02423 PUKPI99064 PIZA05N277 PUKPI01421 SE-8/00 PAEPI00366 DE-1266/01 DE-690/00 SE-9/00 SE-7/00 PDKPI00316 PDKPI01322 PITDO01321 PUKPI01426 PUKPI01383 PIEPI01369 PUKPI02435 PUKPI02434 PUKPI02431 PUKPI02429 BLZA06N779 DOZA06N757 PIZA06N699 DOZA06UP470 DOZA05AM68313 DKZA06N828 DOZA05N240 DOZA06N591 CKZA06N606 99106 PUKPI02428 Pigeon/Italy/1166/00 YU Vo-595/01 PTRBU95211 DE-2663/98 PATP97205 DOZA06N549 PIZA06N635 PIZA06N642 DOZA05N539 DOZA06N621 DOZA05N417 PIZA04N230 DOZA06N589 DOZA05N247 ZA469/PPMV1/02 -ZA06N690 JP/Gunma-pg/2000 PDECK95331 PITTD00177 PITPI99232 Dove/Italy/2736/00 PFRPI98372 PUKPI98355 JP/Shiga-pg/96 PIEPI96349 DE-1383/99 PDEPI94217 PDKPI3202 DE-51/93 JP-Utsunomiya-pg/95 JP/Tochigi-pg/95 DE-60/93 DE-61/93 PDEPI94216 DE-48/92 ES-3/92 DE-55/94 PATPI95295 PDEPI9593 PUKPI90213 88 67 64 1 2 55 27 41 7 5 55 8 14 31 36 2 71 53 41 36 46 38 34 57 60 41 9 72 41 62 93 61 5174 72 54 54 66 42 72 58 64 38 41 23 79 35 57 21 17 45 97 8 7 8 12 15 0 0 6 9 14 14 GB Western Cape, KwaZulu-Natal and Gauteng Provinces (e) Western Cape Province (d) Lineage 4bii Europe, South Africa and Japan Northern Cape, Western Cape, North-West and Limpopo Province (c) North-West, Limpopo and Mpumalanga Provinces (b) Gauteng Province (a) Lineage 4bi Europe, United Arab Emirates and South Africa 0.005 40 61 46 49 151 C. 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V . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . . K . . . . . . . . . . - Z A 0 6 N 6 9 0 . . . T . F . . . L . . . . . . . . . . . I . . . . . . . . . . . . . . . . . . . . . . . . . . V . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S . . . . . . . . . . . . . . . . . . . . V . . K . . . . . . . . . . F IG . 2 M u lti p le a m in o a ci d s e q u e n ce a lig n m e n t o f p a rt ia l S o u th A fr ic a n p ig e o n p a ra m yx o vi ru s fu si o n p ro te in s e q u e n ce s. T h e f u si o n p ro te in c le a va g e s ite ( F 0 ) is in d ic a te d in b o ld 152 Pigeon paramyxoviruses (Newcastle disease virus) isolated in South Africa infection may be lethal to species other than doves and pigeons. 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