A phylogenetic study of South African Newcastle disease virus strains isolated between 1990 and 2002...

Arch Virol (2004) 149: 603–619DOI 10.1007/s00705-003-0218-2

A phylogenetic study of South African Newcastledisease virus strains isolated between 1990 and 2002

suggests epidemiological origins in the Far East

C. Abolnik1, R. F. Horner2, S. P. R. Bisschop3, M. E. Parker2,M. Romito1, and G. J. Viljoen1

1Biotechnology Division, Onderstepoort Veterinary Institute,Pretoria, South Africa

2Allerton Provincial Veterinary Laboratory,Pietermaritzburg, South Africa

3National Poultry Reference Laboratory, University of Pretoria,Pretoria, South Africa

Received March 14, 2003; accepted July 30, 2003Published online November 13, 2003 c© Springer-Verlag 2003

Summary. Genetic comparisons were made of the fusion protein sequences of 155Newcastle disease virus isolates collected in SouthAfrica between 1990 and 2002.Their evolutionary relationships and origins are described. All of the lentogenicfield isolates were shown to be derived from commercial vaccines. No true SouthAfrican lentogenic wild type strain was identified. Furthermore, it was shownthat almost all mesogenic isolates had avirulent F0 cleavage site sequences. Threemajor epizootics occurred in South Africa during the period of this study. The firstoutbreak (1990/1991) was caused by viruses endemic to South Africa since the1960’s (genotypeVIII) but were occasionally also isolated in 2000. GenotypeVIIbviruses, implicated in the severe outbreaks during 1993/1994, persisted until 1999.Genotype VIId viruses, responsible for the most recent outbreak in 1999/2000,had their origins in the Far East like those of the two previous outbreaks.

Introduction

Newcastle disease (ND), classified as a list A disease by the Office Internationaledes Epizooties (OIE) [17], remains one of the most serious poultry diseasesin developing countries. The first recorded entry of ND into South Africa wasthrough the port of Durban during 1944 [11]. After a severe outbreak in the1970’s, outbreaks occurred sporadically until June 1993 when a neuro/respiro-tropic Newcastle disease virus (NDV) was isolated near Pretoria, South Africa

604 C. Abolnik et al.

[D. Verwoerd, personal communication]. The disease spread throughout southernAfrica within six months causing devastating losses in all types of poultry but waseventually brought under control by improved vaccination procedures and biose-curity measures [10, 32, 33]. The most recent SouthAfrican outbreak of ND, whichended in 2000, was limited to village chickens in the Kwa-Zulu/Natal province(KZN) (unpublished data). It has been speculated that village chickens could serveas reservoirs of NDV, although wild birds have also been implicated [33].

NDV is classified in the genus Avulavirus in the family Paramyxoviridae[31]. The negative sense ssRNA genome of 15,586 nucleotides [23] encodes sixgenes: 3′-NP-P-M-F-HN-L-5′ (the nucleoprotein, phosphoprotein, matrix protein,fusion protein, haemagglutinin-neuraminidase protein and the large polymeraseprotein, respectively). The key molecular determinant of NDV pathogenicity is thefusion (F) protein cleavage site sequence. The F0 protein must be cleaved into thedisulphide-linked F1F2 peptides in order to penetrate and induce the fusion ofthe viral envelope with the cell membrane [7, 13, 16]. Velogenic strains containmore basic residues at this site (112R/K-R-Q-R/K-R-F117), which predisposes theF0 protein to undergo cleavage by common cellular proteases found in a widevariety of tissues. Lentogenic strains, however, contain fewer basic residues atthe cleavage site (112G/E-K/R-Q-G/E-R-L117) which limits F0 cleavage only totissues containing trypsin-like proteases [8, 16, 18]. Mesogenic isolates typicallyhave F0 cleavage sites similar to those of velogenic isolates [7]. Molecular typingof this region by RT-PCR is a first line approach to pathotype prediction, followedby DNA sequencing. The F0 cleavage site, as well as other regions in the HN andM protein have been used in phylogenetic studies to determine the evolutionaryrelationships of NDV isolates [38].

Monoclonal antibody (Mab) binding reactions [28] and restriction site anal-yses of RT-PCR amplicons from the F gene [5] have previously been used todefine genetic groupings for Newcastle disease virus from four pandemics. Thefirst outbreak of ND was recorded in 1926 in Southeast Asia [40]. GenotypesII–IV were involved in the first panzootic, each of which was restricted to specificgeographical regions. In particular, strains in genotype V were responsible foroutbreaks in England, California and some other European countries. GenotypeVI isolates caused the second panzootic which began in the Middle East and Greeceduring the late 1960’s and then spread worldwide [1, 15]. The third pandemic wascaused by pigeon PMV-1, which also originated from the Middle East in the late1970’s [1, 6]. Lomniczi et al. [15] described a new genetic group, viz genotypeVII,which has caused many outbreaks of ND around the world. Included are thosecaused by genotypes VIIc and -d in China between 1996 and 2000, genotypesVIIa, -c and -d in Taiwan between 1984 and 2000 [14]; genotype VIIa in westernEurope between 1992 and 1996 [40]; genotype VIIb in southern Africa between1993 and 1995 [9], and genotypes VIIb and -c in the Middle East, northern andeastern Europe, and India between 1993 and 1996 [15].The outbreaks in the UnitedKingdom and northern Europe between 1996 and 1997 [3] were also caused bygenotype VIIb viruses. Genotype VII viruses have been described as causing thefourth ND pandemic, with the most newly emerging NDV strains from China and

Phylogenetic study of NDV origins 605

Taiwan belonging to genotypeVIId [41]. This paper describes the phylogeneticrelationships of South African NDV isolates and the progression of ND in SouthAfrica over the past decade. A secondary objective was to determine whether aSouth African lentogenic wild type strain exists.

Materials and methods

Virus isolates

155 NDV isolates were obtained from the Allerton Provincial Veterinary Laboratory (APVL),the National Poultry Reference Laboratory at the University of Pretoria, Stellenbosch Provin-cial Veterinary Laboratory and Rainbow Farms, as infected allantoic fluid samples (seeTables 1 and 2).

Table 1. List of 85 avirulent NDV isolates collected in South Africa from 1990 to 2002, and reference strains,indicating the cleavage site amino acid sequence and genotype according to phylogenetic analysis

NDV strain District Cleavage site Genotype Accession112GGRQGRL117 number

Hitchner B1 vaccine ------- II U22266LaSota/46 vaccine ------- II AJ249525Miyadera51 -R-K-F III AB070383Avinew61 vaccine --K---- IQueensland66 --K---- I M24693D26/76 --K---- I M24692Ulster2C --K---- I D00243Komarov -RR-R-F IVZA9/L/90∗ Dundee ------- IIZA17/B/91∗ Estcourt ------- IIZA30/GF/92φ Durban/Mayville ------- IIZA138/GF/94 Ixopo ------- IIZA150/B/94∗ Lion’s River/Merrivale ------- IIZA148/B/94φ Camperdown area ------- IIZA50/B/94φ Camperdown area ------- IIZA174/B/95 Bergville ------- IIZA1454/UP/95 South Africa, district unknown ------- II AY210492ZA168/B/95φ Camperdown area ------- IIZA234/B/96∗ Camperdown area ------- IIZA202/B/96∗ KwaZulu/Natal ------- II AF532145ZA204/B/96∗,φ Orange Free State ------- IIZA776/UP/96 Pretoria ------- IIZA216/B/96φ Vryheid ------- IIZA240/B/97 Lower Tugela/Umhlali ------- IIZA719/UP/97 Arnot ------- II AY210489ZA671/UP/97 Phalaborwa ------- II

(continued)


Table 1 (continued)


ZA254/B/97 Mooi river ------- IIZA26/B/97φ Durban/Mayville ------- IIZA249/B/97φ Harding ------- IIZA28/UP/97φ Durban/Mayville ------- IIZA260/B/97∗,φ Port Shepstone area ------- IIZA261/B/97φ Camperdown area ------- IIZA27/UP/97φ Botswana ------- IIZA890/UP/98 Cullinan ------- IIZA281/L/98 Port Shepstone area --K---- IZA271/B/98φ Mooi River ------- IIZA301/B/98∗,φ Lions River/Merrivale ------- IIZA302/B/98∗,φ New Hanover/Wartburg ------- IIZA51/UP/98φ Halfway House ------- IIZA641/UP/98φ Carolina ------- IIZA307/B/99 Dundee ------- II AF532149ZA324/B/99 Camperdown area ------- IIZA312/B/99∗ Port Shepstone area ------- II AF532152ZA1366/UP/99 Hartebeespoort Dam area ------- II AY210490ZA340/P/99 Port Shepstone area --K---- I AF532741ZA336/B/99 Port Shepstone area --K---- IZA340/B/99 Port Shepstone area --K---- IZA345/B/99φ Camperdown area ------- IIZA1453/UP/99φ Lichtenburg ------- IIZA291/B/99φ Camperdown area ------- IIZA339/B/99φ Camperdown area ------- IIZA327/B/99φ Camperdown area ------- IIZA1286/UP/00 Pretoria ------- IIZA11157/Kftn/00 Cape Town ------- IIZA365/B/00 Port Shepstone area ------- IIZA680/UP/00 Pretoria ------- II AY210491ZA375/B/00 Camperdown area ------- II AF532744ZA370/B/00 Estcourt --K---- I AF532742ZA357/B/00φ Camperdown area ------- IIZA362/B/00φ Stanger ------- IIZA361/B/00φ Camperdown area ------- IIZA349/B/00φ Camperdown area ------- IIZA346/B/00φ Camperdown area ------- IIZA6068/Paarl//00φ Paarl ------- IIZA477/UP/00φ Bethal ------- IIZA9240B/Worch/00φ Worchester ------- IIZA368/B/00φ Port Shepstone area ------- IIZA381/B/01 Port Shepstone area ------- II

(continued)


Table 1 (continued)


ZA408/B/01 Camperdown area ------- IIZA392/B/01 Lion’s river/Merrivale ------- IIZA8/UP/01 Potchefstroom ------- IIZA12150/Worc/01 Worchester ------- II AY210493ZA393/B/01 Estcourt ------- II AF532747ZA405/BB/01 Camperdown area ------- II AF532749ZA385/B/01 Camperdown area ------- II AF532746ZA3355/Paarl/01 Paarl --K---- IZA832/UP/01 Potchefstroom --K---- IZA1241/UP/01 Potchefstroom --K---- IZA5157/Paarl/01 Paarl --K---- IZA11127/Malmsb/01 Malmesbury --K---- IZA37401/X/01 Gauteng --K---- I AF532743ZA382/B/01φ New Hanover/Merrivale ------- IIZA388/BB/01φ Pietermaritzburg ------- IIZA397/B/01φ Camperdown area ------- IIZA380/B/01φ Camperdown area ------- IIZA383/B/01φ Camperdown area ------- IIZA395/B/01φ Camperdown area ------- IIZA325/B/99φ Camperdown area ------- IIZA407/B/01φ Camperdown area ------- IIZA383/B/01φ Camperdown area ------- IIZA447/02 KwaZulu/Natal ------- IIZA449/X/02 KwaZulu/Natal --K---- IZA448/X/02 KwaZulu/Natal --K---- I

Mesogenic viruses are marked with ∗. Isolates marked with φ shared 100% sequence similarity tothe LaSota/46 vaccine and were therefore not included in the phylogenetic analysis (Fig. 2)

Table 2. List of 70 virulent NDV isolates collected in South Africa from 1990 to 2002, and reference strains,indicating the cleavage site amino acid sequence and genotype according to phylogenetic analysis

NDV strain District Cleavage site Genotype Accession112GRRQKRF117 number

Kuwait 256 Kuwaitt ------- VIaTW84/C Taiwan ------- VIIc AF083965ZA-18/90 South Africa, district unknown ---R--- VIII AF136766ZA-17/90 South Africa, district unknown ---R--- VIII AF136775ZA16/GF/91 Richmond ---R--- VIII AF532143ZA13/L/91 Richmond ---R--- VIII AF532142

(continued)


Table 2 (continued)


ZA19/B/91 Lion’s River/Merrivale ---R--- VIII AF532752ZA11/B/91 Pietermaritzburg/Thornville ---R--- VIII AF532140ZA-34/94 South Africa, district unknown ---R--- VIII AF136773ZA100/L/94 Durban ------- VIIbZA133/B/94 Camperdown ------- VIIbZA53/BB/94 Camperdown ------- VIIbZA54/BB/94 Camperdown ------- VIIbZA76/L/94 Lion’s river/Merrivale ------- VIIbZA87/X/94 Pretoria ------- VIIbZA108/L/94 Estcourt ------- VIIb AF352139ZA71/B/94 Camperdown area ------- VIIb AF532751ZA60/B/94∗ Camperdown area ----R-- VIIb AF532750ZA110/X/94 Pretoria ----R-- VIIb AF532141ZA52/BB/94 Camperdown area ------- VIIb AF532749ZA360-95 South Africa, district unknown ------- VIIbZA170/B/95 Lower Tugela/Umhlali ------- VIIb AF532144ZA172/B/95 Lower Tugela/Umhlali ------- VIIbZA-35/95 South Africa, district unknown ------- VIIb AF136774ZA983/UP/96 Mpumahlanga ------- VIIbZA903/UP/96 Pretoria ------- VIIbAE232/1/96 United Arab Emirates ------- VIIb AF109884ZA982/UP/97 Kuruman ------- VIIbZA839/UP/97 Kuruman ------- VIIb AY210494ZA837/UP/97 Wakkerstroom ------- VIIbZA928/UP/97 Gabarone, Botswana ------- VIIb AY210495ZA842/UP/97 Pretoria ------- VIIb AY210504ZA825/UP/97 Potgietersrus ------- VIIb AY210499ZA256/X/97 Pretoria ------- VIIb AF532146JS3/98/Go China ------- VIId AF456436ZA751/UP/98 Pretoria ------- VIIb AY210505ZA700/UP/98 Vorna Valley ------- VIIbZA699/UP/98 Johannesburg ------- VIIbZA704/UP/98 Rustenburg ------- VIIb AY210508ZA933/UP/98 Pretoria ------- VIIbZA983/UP/98 Rustenburg ------- VIIb AY210509ZA753/UP/98 Ermelo ------- VIIbZA762/UP/98 Johannesburg ------- VIIbZA949/UP/98 Phalaborwa ------- VIIbZA1003/UP/98 Makapanstad ------- VIIbZA734/UP/98 Rustenburg ------- VIIbZA874/UP/98 Rustenburg ------- VIIb AY210506ZA955/UP/98 Bronkhorstspruit ------- VIIbZA917/UP/98 Witbank ------- VIIb

(continued)


Table 2 (continued)


ZA922/UP/98 Johannesburg ------- VIIbZA9357/Moslb/98 Mosselbaai ------- VIIbZA798/UP/98 Cullinan ------- VIIbZA96/UP/98 Tzaneen ------- VIIbZA775/UP/98 Potgietersrus ------- VIIbZA959/UP/98 Potgietersrus ------- VIIbZA756/UP/98 Vereeniging ------- VIIbZA444/B/98 Hammarsdale ------- VIIb AY210511ZA984/UP/98 Gabarone, Botswana ------- VIIb AF109876ZA7351/Rvsdl/98 Riversdale ------- VIIb AY210502ZA296/L/98 Durban ------- VIIb AF532148ZA7381/98 Western Cape ------- VIIb AY210512ZA148/UP/98 Gabarone, Botswana ------- VIIb AY210507ZA328/F/99 Pietermaritzburg/Thornville ------- VIIdZA337/P/99 Durban ------- VIIdZA331/B/99 Stanger ------- VIId AF532739ZA308/B/99 New Hanover/Wartburg ------- VIId AF532150ZA1320/UP/99 Pretoria ------- VIIb AY210510ZA1521/UP/99 Halfway House ------- VIIb AY210501ZA335/B/99 Camperdown area ------- VIIb AF532740ZA549/UP/99 Potchefstroom ------- VIIb AY210500ZA309/B/99 Paul Pietersburg ------- VIIb AF532151ZA8057/99 Western cape ------- VIIbZA3291/Klpmts/99 Klapmuts ------- VIIb AY210503ZA598/UP/00 Pretoria ------- VIII AY210496ZA606/UP/00 Pretoria ------- VIII AY210497ZA377/F/00 Stanger ------- VIIdZA344/B/00 Durban ------- VIIdZA378/F/00 Port Shepstone area ------- VIId AF532745JP/Chiba/2000 Chiba, Japan ------- VIId AB070433TW/2000 Taiwan ------- VIIc AF358786

∗Virus with mesogenic MDT

Virus characterization

NDV strains were isolated in specific pathogen free (SPF) embryonated chicken eggs by stan-dard procedures [2]. APVL obtained pathogenicity indices for all isolates using mean deathtime (MDT) or in some cases intracerebral pathogenicity index (ICPI) tests, according to theprocedures described in the OIE Manual of Standards for diagnostic Tests and Vaccines [17].

Amplification and sequence analysis

Viral RNA was extracted from allantoic fluid using the QIAamp r© Viral RNA Mini Kit(QIAGEN), TriReagentTM (SIGMA) or TRIzol r© reagent (Gibco, Invitrogen), according to


the manufacturer’s instructions. Random hexamers were used to generate first strand cDNAaccording to the method described by Sambrook et al. [29]. The oligonucleotide primers belowwere used to amplify an 1180 base pair fragment spanning the regions between nucleotides581 of the fusion protein and nucleotides 610 of the matrix protein, which includes the F0cleavage site. Reaction mixtures were cycled 35 times at 94 ◦C for 30 sec, 53 ◦C for 30 sec,and 72 ◦C for 1 min on a GeneAmp 2400 PCR System (Perkin Elmer).

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)

DNA was sequenced using the ABI PRISM r© Big DyeTM Terminator Cycle SequencingReady Reaction Kit (Applied Biosystems) according to the manufacturer’s instructions, andwas analysed with an ABI377TM automated sequencer.

Phylogenetic analysis

A 374 nucleotide (nt) fragment of the fusion protein, including the F0 cleavage site wasaligned using GCG Seqlab (Wisconsin package version 10.1-UNIX, Genetics ComputerGroup, Inc). Phylogenetic trees were drawn with PAUP 4.0 Beta version software (DavidL. Swofford, Florida State University). The results are presented as neighbour joining treeswith uncorrected P distances and 100 bootstrap trials to assign confidence values to topology.Maximum parsimony trees were also drawn, however the topology recovered with the differentmethods of analysis was the same.

Results

155 NDV isolates were collected throughout South Africa from 1990 to 2002. RT-PCR and sequencing analyses were performed to determine the F0 cleavage signalsequences and phylogenetic relationships. In an unrooted tree of all South Africanisolates (data not shown), 85 avirulent viruses could clearly be distinguished from70 virulent virus isolates. This data was supported by MDT and ICPI data, whereavailable. Separate trees for the virulent and avirulent viruses were drawn, wherethe virulent virus tree contains the lentogenic La Sota/46 virus sequence as anoutgroup, while the Kuwait/256C virus sequence was used as an outgroup for theavirulent viruses.

Avirulent viruses

Lasota/46 vaccine was isolated almost yearly from 1990 to 2002, Hitchner B1from 1995 onwards, and Avinew61 from 1998 onwards, which corresponds to theavailability of the Avinew61 vaccine in the country from 1997. All the virusesobtained from APVL were pathotyped according to mean death time (MDT) or insome cases by intra cerebral pathogenicity index (ICPI). Eleven viruses showedmesogenic values of between 60 and 90 hours. Ten of the eleven viruses had aviru-lent F0 cleavage site sequences (112GRQGRL117, Fig. 1), and <1% nucleotide dif-ferences in comparison with the genotype II vaccine strains. Isolates ZA373/B/00,ZA303/B/98, ZA137/B/94, ZA130/B/94, ZA260/B/97, ZA206/B/96 are not in-cluded in Fig. 1. Only one strain, ZA60/B/94, had a virulent F0 cleavage sequence


Fig. 1. Phylogenetic tree of avirulent NDV isolates based on a 374 bp region in the F gene.Genotypes are shown on the right. GenBank accession numbers including those of thereference viruses are shown in Table 1. The tree was constructed by the neighbor-joiningalgorithm of PAUP 4.0 Beta, with 100 bootstrap trials to assign confidence to groupings.

Mesogenic viruses are marked with ∗

(112RRQRRF117), with a K115→R substitution and is discussed below (Table 2,Fig. 2).

Virulent viruses

The velogenic viruses could be divided into three major groups, viz. genotypesVIII, VIId and VIIb (Tab. 2), which correspond closely to the periods in which theviruses were isolated (Figs. 2 and 3). The earliest viruses sequenced in this study,


ZA11/B/91, ZA19/B/91, ZA13/L/91, ZA16/GF/91, ZA598/UP/00 and ZA606/UP/00, are similar to genotype VIII viruses described by Herczeg et al. [9], viz.ZA-17/90, ZA-18/90 and ZA-34/94. The F0 cleavage site of genotype VIII virusesis unusual, containing an 114R for Q substitution. ZA/11/B/91 was tested byan intravenous pathogenicity test (IVPI) in six week-old chicks, and the IVPIvalue of 2.89 supports the highly virulent nature of genotype VIII viruses, whichHerczeg et al. [9] tentatively attributed to 114R. ZA-18/90 and ZA19/B/91 sharea S124→I substitution. ZA-34/94 was the last genotype VIII virus isolated inSouth Africa until recently, as two more genotype VIII viruses, ZA598/UP/00and ZA606/UP/00, have been isolated. These two viruses as well as ZA-34/94,however contain the conventional 114Q instead of R. Additionally, ZA606/UP/00has a V44→A substitution. ZA598/UP/00 which was isolated east of Pretoria,caused a 2% drop in egg production, with no clinical symptoms apart from nasaldischarges (unpublished laboratory report data).

ZA100/L/94, ZA133/B/94, ZA53/BB/94, ZA54/BB/94, ZA76/L/94 andZA87/X/94, which are all identical in nucleotide sequence (therefore only ZA100/L/94 is shown in Fig. 3), are isolates from the devastating outbreaks in 1993/94(genotype VIIb). Despite a lack of nucleotide sequence data from provinces otherthan KZN, the spread of the outbreak is well documented [10, 32–35]. ZA60/B/94and ZA52/BB/94 share L21→V substitutions and ZA60/B/94 and ZA110/X/94share K115→R substitutions in the F0 cleavage site. The residues responsiblefor the mesogenic characteristics of ZA60/B/94 have not yet been identified. ThegenotypeVIIb viruses isolated in KZN in 1995 (ZA170/B/95 and ZA172/B/95) areidentical at nucleotide level to ZA360-95 [3] and distinct from the viruses of 1994and 1996. A unique S24→C substitution distinguishes these viruses from otherVIIb isolates. The location of ZA360-95, isolated from an ostrich, is not known.

According to nucleotide an amino acid sequences, outbreaks subsequent tothe 1995 outbreaks indicate that certain strains appear to have common origins,for example ZA751/UP/98, ZA839/UP/97 and ZA928/UP/97 (and ZA982/UP/97,ZA837/UP/97, not shown in the alignment) contain unique P10→L, G30→D andL32→F substitutions (Fig. 3).When considered with the dates of isolation, variantscould be traced as they spread from one town to another, along major transportationroutes. This indicates that the genotype VIIb outbreaks which persisted until 1999were perpetuated by the movement of contaminated people and vehicles.

Isolates ZA328/F/99, ZA377/F/00, ZA337/P/99, ZA344/B/00, ZA331/B/99,ZA378/F/00 and ZA308/B/99 were involved in the most recent outbreak from 1March 1999 to 20 December 2000. Only village chickens in an area of about 70square kilometers in KZN were affected. ZA331/B/99 and ZA377/F/00 persisted

�Fig. 2. Phylogenetic tree of virulent NDV isolates based on a 374 bp region in the F gene.Genotypes are shown on the right. GenBank accession numbers including those of thereference viruses are shown in Table 1. The tree was constructed by the neighbor-joiningalgorithm of PAUP 4.0 Beta, with 100 bootstrap trials to assign confidence to groupings. Themesogenic virus is marked with ∗

Fig

.3.

Alig

nmen

tof

the

pred

icte

dam

ino

acid

sequ

ence

sof

Fpr

otei

ns(a

min

oac

ids

1–12

4)fr

omvi

rule

ntN

DV

isol

ates

,with

the

F 0cl

eava

gesi

tebo

xed.

Dif

fere

nces

betw

een

geno

type

san

dis

olat

esar

edi

scus

sed

inth

ere

sults

C. Abolnik et al.: Phylogenetic study of NDV origins 615

in the same flock for at least a year. ZA328/F/99, ZA377/F/00, ZA337/P/99,ZA344/B/00 and ZA331/B/99 are identical in F and M gene sequence, and thesetogether with ZA378/F/00 and ZA308/B/99 all share residues with the orientalstrains JS/3/98/Go (genotype VIId), JP/Chiba/2000 (Genotype VIId), TW/2000(genotype VIIc) and TW84/C (genotype VIIc). 19I, 30S and 101K are common toall the aforementioned viruses, distinguishing them from the genotypes VIIb andVIII. Yu et al. [41] proposed that genotype VIIb evolved from VIb via productionof a VII-specific V121 for I substitution, and then changed to genotypes VIIaand VIIc by producing additional substitutions. Genotype VIIc viruses can bedistinguished from the VIId viruses by residues 4R and 5S. The South Africanisolates share several residues unique to the oriental genotype VIID isolates:27C→R and 71K→R are unique to genotype VIId. The most striking similaritybetween the South African genotype VIId viruses and JS/3/98/Go is the 28S→Psubstitution, whereas the other genotypeVIId isolates (and genotypeVIIc) contain28S→L substitutions.

Discussion

In this study, 155 NDV isolates collected in South Africa over the past decadewere genotypically characterized. Previously, Herczeg et al. [9] found that strainsisolated in Southern Africa between 1990 and 1995 were caused by group VIIIand group VIIb viruses. That study, however, could not determine the fluctuationor dominance of a particular epidemiological strain. This was due to the presenceof group VIII viruses alone between 1990 and 1993, and group VIIb strains onlybetween 1993 and 1995. We have been able to describe NDV epidemics between1990 and 2002, and establish the origin of the most recent outbreaks in villageflocks. Neither wild type virus nor signs of disease could be detected between2000 and 2002 and there were no signs that the disease was seen in South Africain that period either.

The avirulent viruses could be classified into one of three vaccine types viz.LaSota/46, Hitchner B1 or the more recently-introduced Avinew61, or their closederivatives. These vaccine strains could be isolated regularly over several years.The lack of sufficient changes at the nucleotide level in vaccine variants alsosuggests that vaccine strains do not circulate for long periods of time before furtherinflux from administered vaccine strains occurs, as ND vaccination is performedroutinely and frequently in all commercial poultry houses. Furthermore, based onthe isolates investigated, we found no evidence that a true SouthAfrican lentogenicwild type exists. We are particularly interested in those viruses having mesogenicMDT test results. Only isolate ZA60/B/94 had a virulent F0 cleavage sequence,the remainder were avirulent in contrast to the accepted sequence pattern indicatedfor mesogenic strains [6]. The molecular determinants for the increased virulenceof these viruses presumably occur elsewhere in the genomes and are currentlyunder investigation.

Herczeg et al. [9] first suggested that genotype VIII viruses were endemicin South Africa, as their branching order and inter-group heterogeneity indicated


that they developed locally over time. Genotype VIII virus was already present inSingapore in 1965 and in the Chinese Qinghai province in 1979, 1984 and 1985.This suggests that these genotypic strains may have a common ancestor in the FarEast [14]. Chinese genotype VIII viruses, however, all contain the 114Q insteadof 114R that is present in South African isolates. Like the South African genotypeVIII viruses, the former do show higher ICPI values than genotype VIIb virusesEast [14]. Recently (twice in 2000), genotype VIII viruses containing 114Q wereisolated in South Africa, but high mortality rates were not recorded. This raisesthe question whether genotype VIII is endemic but inactive for long periods inSouth Africa, or whether the newer viruses are recent introductions. If the formeris the case, the maintenance host has yet to be identified.

Genotype VII, responsible for the fourth pandemic and the progenitor to VIIa,VIIb, VIIc and VIId viruses, appeared in Taiwan and Indonesia in the 1980’s[41]. The first recorded entry of genotype VIIb into South Africa was in June1993 at Hartebeespoort Dam just west of Pretoria. Despite a lack of nucleotidesequence data from provinces other than KZN, the spread of the outbreak is welldocumented [10, 32–35]. Although VIIb peaked in 1993/1994, we have shownthat the virus persisted until 1999 in most provinces, even spreading across thewestern border into Botswana on several occasions. In almost all cases, towns orcities infected with specific variants lie along main transport routes. Horizontaltransfer via workers or traders from rural settlements into commercial poultryhouses is the most likely route of transmission [33], rather than dissemination bywild birds.

The most recent outbreak in 1999/2000 affected only village chickens in KZN.The isolates lack diversity which suggests a recent introductory event. The closestrelative to the SouthAfrican strains was isolated from a goose in China during 1998(JS/3/98/Go). This directly links the South African outbreak to the Far East, wheregenotypes VIIc and VIId were recently described as emerging viruses. VelogenicNDV certainly occurs periodically in village chickens, and small numbers ofsurvivors may well serve as a link in the dissemination of virus. Their role asmaintenance hosts or NDV reservoirs is, however, questionable considering theirown susceptibility to highly velogenic NDV and epidemiological patterns of NDoutbreaks over the past decade, especially since a link with the Far East has beenestablished.

ND viruses exist as a single serotype (avian paramyxovirus serotype 1) basedon the neutralising antibody test and cross-protective analysis [3]. In practice,however, vaccination of poultry against NDV does not necessarily prevent periodicoutbreaks of disease [12, 20, 39, 40, 27] and especially in young immunologicallyimmature birds (pers. obs). Emerging NDV strains are causing concerns that theycan overcome vaccination barriers [21]. Results from cross-protective experimentsputatively indicated that LaSota/46-vaccinated chickens were only partially pro-tected against challenge with Ch-A7/96, an immune response-escaping antigenicvariant belonging to genotype VIIc [41]. Since we have determined that in allmajor epidemics in South Africa, the causative genotypes had caused outbreaksin the Far East in prior months or years, it remains crucial to monitor such


emerging strains, particularly in the Far East. This would be necessary for makingpredictions of the expected genotype responsible for the next outbreak, and forplanning suitable vaccination strategies accordingly. Although the exact sourceof transmission of these viruses to South Africa remains unknown, migratorybirds appear to be possible candidates. Several species of birds, mostly waders,are known to migrate between Southern Africa and their breeding grounds inSiberia, via communal stopover grounds in the African Rift Valley, the MiddleEast, and central Asia, where they are likely to come into contact with birds fromthe Far East [24–26, 30]. Pfitzer et al. [22] were able to show a high serologicallypositive incidence amongst wild aquatic birds in South Africa. Verwoerd [33]speculated that migratory waterfowl played a role in the dissemination of NDVduring previous epidemics in South Africa. The latter, however, is likely to bedifficult to prove as has been stated elsewhere [4]. There is evidence that genotypeVIIa, which circulated in Western European countries in the 1990’s was alsosomehow transmitted from the Far East [15, 37, 40]. If the Siberian breedinggrounds of migratory waterbirds are important as an incubator for future strainsof pandemic ND viruses, as has been reported for avian influenza virus [19], thenthe emerging NDV strains in the Far East may well have importance for Eurasia,Australasia and the rest of Africa too.

Acknowledgements

The authors thank the following for their invaluable technical assistance or discussion: RainaMaharaj, Madre Nortje, Hanlie Moolman, Dirk Verwoerd, Arrie Klopper, Don Thomson andRetha Brandt.

References1. Alexander DJ (1988) Historical aspect. In: Alexander DJ, Newcastle disease. Kluwer,

Boston, pp 1–102. Alexander DJ (1989) Newcastle disease. In: Purchase HG, Arp LH, Domermuth CH,

Perason JE (eds) A laboratory manual for the isolation and identification of avianpathogens, 3rd edn. American Association of Avian Pathologists, Kenner Square, PA,pp 114–120

3. Alexander DJ, Banks J, Collins MS, Manvell RJ, Frost KN, Speidel EC, Aldous EW(1999) Antigenic and genetic characterisation of Newcastle disease viruses isolated inoutbreaks in domestic fowl and turkeys in Great Britain during 1997. Vet Rec 145:417–421

4. Alexander DJ (2001) Newcastle disease. Br Poultry Sci 42: 5–225. Ballagi-Pordany A, Wehmann E, Herczeg J, Belak S, Lomniczi B (1996) Identification

and grouping of Newcastle disease virus strains by restriction site analysis of a regionfrom the F gene. Arch Virol 14: 243–261

6. Collins MS, Bashiruddin JB, Alexander DJ (1993) Deduced amino acid sequences at thefusion protein cleavage site of Newcastle disease virus showing variation in antigenicityand pathogenicity (Brief report). Arch Virol 128: 363–370

7. Glickman RL, Syddall RJ, Iorio RM, Sheehan JP, Bratt MA (1988) Quantitative basicresidue requirements in the cleavage activation site of the fusion glycoprotein as adeterminant of virulence for Newcastle disease virus. J Virol 62: 354–356


8. Gotoh B, Ohnishi N, Inocentio M, Esaki E, Nakayama K, Barr PJ, Thomas G,Nagai Y (1992) Mammalian subtilisin-related proteinases in cleavage activation of theparamyxovirus fusion glycoprotein: superiority of furin/PACE to PC2 or PC1/PC3.J Virol 66: 6391–6397

9. Herczeg J, Wehmann E, Bragg RR, Travassos Dias PM, Hadjiev G, Werner O, LomnicziB (1999) Two novel genetic groups (VIIb and VIII) responsible for recent Newcastledisease outbreaks in Southern Africa, one (VIIb) of which reached Southern Europe.Arch Virol 144: 2087–2099

10. Huchzermeyer FW, Gerdes GH (1993) Newcastle disease virus isolated from ostrichesin South Africa. J S Afr Vet Assoc 64: 14

11. Kaschula VR, Canham AS, Diesel AM, Coles JDWA (1945) Newcastle disease in Natal.J S Afr Vet Med Assoc 17: 1–14

12. Khalafalla AI, Fadol MA, Hameid OA, Hussein YA, El Nur M (1992) Pathogenicproperties of Newcastle disease virus isolates isolated in the Sudan. Acta Vet Hung 40:329–333

13. Le Long L, Brasseur C,Wemers C, Meulemans G, BurnyA (1988) Fusion (F) protein geneof Newcastle disease virus: sequence and hydrophobicity comparative analysis betweenvirulent and avirulent strains. Virus Genes 1: 333–350

14. Liang R, Cao DJ, Li JQ, Chen J, Guo X, Zhuang FF, Duan MX (2002) Newcastle diseaseoutbreaks in western China were caused by the genotypes VIIa and VIII. Vet Microbiol87: 193–203

15. Lomniczi B, Wehman E, Herczeg J, Ballagi-PordanyA, Kaleta EF, Werner O, MeulemansG, Jorgensen PH, Mante AP, Gielkens ALJ, Capua I, Damoser J (1998) Newcastledisease outbreaks in recent years in Western Europe were caused by old (VI) and a novelgenotype (VII). Arch Virol 143: 49–64

16. Nagai Y, Klenk HD, Rott R (1976) Proteolytic cleavage of the viral glycoproteinsand its significance for the virulence of Newcastle disease virus. Virology 72:494–508

17. OIE (2000) Office International des Epizooties, Manual of standards for diagnostic testsand vaccines 2000. pp 221–232

18. Ogasawara T, Gotoh B, Suzuki H, Asaka J, Shimokata K, Rott R, Nagai Y (1992)Expression of factor X and its significance for the determination of paramyxovirustropism in the chick embryo. EMBO J 11: 467–472

19. Okazaki K, Takada A, Ito T, Imai M, Takakuwa H, Hatta M, Ozaki H, Tanizaki T, NaganoT, Ninomiya A, Demenev VA, Tyaptirganov MM, Karatayeva TD, Yamnikova SS, LvovDK, Kida H (2000) Precursor genes of future pandemic influenza viruses are perpetuatedin ducks nesting in Siberia. Arch Virol 145: 885–893

20. Oncel T, Alexander DJ, Manvell RJ, Ture O (1997) Characterisation of Newcastle diseaseviruses isolated from chickens and pigeons in the South Marmara region of Turkey. AvianPathol 26: 129–137

21. Panshin A, Shihmanter E, Weisman Y, Orvell C, Lipkind M (2002) Antigenicheterogeneity among the field isolates of Newcastle disease virus (NDV) in relationto the vaccine strain. 1. Studies on viruses isolated from wild birds in Israel. ComparativeImmununology. Microbiol Infect Dis 25: 95–108

22. Pfitzer S, Verwoerd DJ, Gerdes GH, Labuschagne AE, Erasmus A, Manvell RJ, Grund C(2000) Newcastle disease and avian influenza A virus in wild waterfowl in South Africa.Avian Dis 44: 655–660

23. Phillips RJ, Samson ACR, Emmerson PT (1998) Nucleotide sequence of the 5′ terminusof Newcastle disease virus and assembly of the complete genomic sequence: agreementwith the “rule of six”. Arch Virol 143: 1993–2002


24. Piersma T, Davidson NC (1992a) The migration of knots. Wader Study Group Bull64(Suppl): 1–209

25. Piersma T, Davidson NC (1992b) The migrations and annual cycles of five subspecies ofknots in perspective. Wader Study Group Bull 64(Suppl): 187–197

26. Rogacheva EV (1992) The birds of Central Siberia. Husum Druck- undVerlagsgesellschaft, Husum, Germany

27. Roy P, Venugopalan AT, Manvell R (2000) Characterization of Newcastle diseaseviruses isolated from chickens and ducks in Tamilnadu. Indian Vet Res Commun 24:135–142

28. Russel PH, Alexander DJ (1983) Antigenic variation of Newcastle disease virus strainsdetected by monoclonal antibodies. Arch Virol 75: 243–253

29. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: A Laboratory Manual.2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

30. Underhill LG, Tree AJ, Oschadleus HD, Parker V (1999) In: Review of ringrecoveries of waterbirds in southern Africa. Avian Demography Unit, University ofCape Town

31. Van Regenmortel MH, Fauquet CM, Bishop DHL et al. (eds) (2000) Seventh reportin Virus Taxonomy. Reports of the International Committee on Taxonomy of Viruses.Academic Press, New York, pp 1024

32. Verwoerd DJ (1995a) Newcastle disease and poultry production in Africa. In: MeissnerHH (ed) Proc Second All Africa Conference on Animal Agriculture: Food security inAfrica

33. Verwoerd DJ (1995b)Velogenic Newcastle disease epidemic in SouthAfrica II. Ostriches,waterfowl, exotic bird collections and wild birds. S Afr Vet Med 8: 44–49

34. Verwoerd DJ, Gerdes GH, Olivier AJ, Williams R (1997) Experimental infections ofvaccinated slaughter ostriches with virulent Newcastle disease virus. Onderstepoort J VetRes 64: 213–216

35. Verwoerd DJ, Olivier B, Gummow B, Gerdes GH, Williams R (1999) Experimentalinfections of vaccinated slaughter ostriches in a natural, open-air feedlot facility, withvirulent Newcastle disease virus. Avian Dis 43: 93–103

36. Wan H, Wu Y, Liu X, Zhang R (2002) Sequence analysis of the fusion (F) protein genesof four Newcastle disease virus strains causing clinical disease in geese. Wei Sheng WuHsueh Pao (in press)

37. Wehmann E, Czegledi A, Werner O, Kaleta EF, Lomniczi B (2003) Occurrence ofgenotypes IV, V, VI and VIIa in Newcastle disease outbreaks in Germany between 1939and 1995. Avian Pathol 32: 157–163

38. Westover KM, Hughes AL (2001) Molecular evolution of viral fusion and matrix proteingenes and phylogenetic relationships among the Paramyxoviridae. Mol Phylogen Evol21(1): 128–134

39. Yang CY, Chang PC, Hwang JM, Shieh HK (1997) Nucleotide sequence and phylogeneticanalysis of Newcastle disease virus isolates from recent outbreaks in Taiwan. Avian Dis41: 365–373

40. Yang CY, Shieh HK, Lin YL, Chang PC (1999) Newcastle disease virus isolated fromrecent outbreaks in Taiwan phylogenetically related to viruses (genotype VII) from recentoutbreaks in Western Europe. Avian Dis 43: 125–130

41. Yu I, Wang Z, Jiang Y, Chang L, Kwang J (2001) Characterization of newly emergingNewcastle disease virus isolates from the People’s Republic of China and Taiwan. J ClinMicrobiol (Oct): 351

Author’s address: Gerrit Viljoen, Biotechnology Division, Onderstepoort Veterinary In-stitute, Private Bag X5, Onderstepoort, 0110, South Africa; e-mail: [email protected]

A phylogenetic study of South African Newcastle disease virus strains isolated between 1990 and 2002...

Documents

Transcript of A phylogenetic study of South African Newcastle disease virus strains isolated between 1990 and 2002...