Phylogenetic characterization of Newcastle disease virus isolated in themainland of China during 2001–2009

Zhang Rui, Pu Juan, Su Jingliang, Zhao Jixun, Wang Xiaoting, Zhang Shouping,Li Xiaojiao, Zhang Guozhong *

Department of Preventive Veterinary Medicine, College of Veterinary Medicine, China Agricultural University, Beijing 100193, PR China

Veterinary Microbiology 141 (2010) 246–257

A R T I C L E I N F O

Article history:

Received 9 May 2009

Received in revised form 10 September 2009

Accepted 22 September 2009

Keywords:

Newcastle disease virus

Molecular evolution

Genotype

Phylogenetic analysis

Gene recombination

Chicken

A B S T R A C T

Twenty Newcastle disease virus (NDV) isolates from infected chicken flocks during 2001 to

2009 in China were biologically and genetically characterized. All the 20 NDVs were

categorized into velogenic (n = 17) and lentogenic (n = 3) viruses, respectively, according to

the mean death time (MDT) of chicken embryos. Velogenic viruses carry the motif 112R-R-

Q-K-R/F117 (n = 14) or 112G-R-Q-G-R/L117 (n = 3) at the F0 cleavage site, while all the

lentogenic virus had a sequence motif of 112G-R-Q-G-R/L117 (n = 3) at the same site.

Phylogenetic analysis revealed that at least three distinct genotypes (genotypes I, II and

VII) existed in chicken flocks in China and VIId of genotype VII were mainly responsible for

the present ND panzootic. Two natural recombinants (XD/Shandong/08 and QG/Hebei/07)

were supposed and identified by the SimPlot program, and their two parental-like strains

might be from the NDV vaccine lineage and VIId velogenic lineage respectively. The results

indicate that recombination play a role in NDV evolution and the live vaccines have

capacity to boost NDV evolution by homologous recombination with circulating virus.

� 2009 Elsevier B.V. All rights reserved.

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1. Introduction

Newcastle disease (ND) is a highly contagious andwidespread disease which causes severe economic lossesin domestic poultry, especially in chickens (Alexander,2003; Sinkovics and Horvath, 2000). ND is caused by avianparamyxovirus serotype 1 (APMV-1), also known asNewcastle disease virus (NDV), which belongs to thegenus Avulavirus in the family of Paramyxoviridae, orderMononegavirales (Mayo, 2002). NDV strains can beclassified as highly virulent (velogenic), intermediate(mesogenic) or non-virulent (lentogenic) based on theirpathogenicity in embryonating eggs or chickens (Alex-ander, 1998, 2003; Asahara, 1978).

NDV has a single-stranded, negative-sense, nonseg-mented RNA genome of approximately 15,186 nucleotides

* Corresponding author. Tel.: +86 10 62733660; fax: +86 10 62732984.

E-mail address: zhanggz@cau.edu.cn (Z. Guozhong).

0378-1135/$ – see front matter � 2009 Elsevier B.V. All rights reserved.

doi:10.1016/j.vetmic.2009.09.020

in length, which contains six genes encoding the sixstructural proteins (from 30 to the 50 terminus): nucleo-protein (NP), phosphoprotein (P), matrix (M), fusion (F),hemagglutinin–neuraminidase (HN) and RNA-dependentRNA polymerase (L) (Millar et al., 1988). Among these, HNand F are two major spike glycoproteins. The HN protein isresponsible for virus attachment to sialic acid-containingreceptors, and it also has a neuraminidase activity as wellas a yet undefined role in promoting the fusion mediatedby the F protein (Yusoff and Tan, 2001). The F protein canpromote the fusion of host and viral cell membranes,resulting in penetration of the virus particle into the cell,which is an initial step in infection (Rott and Klenk, 1988).The F protein is synthesized as a precursor (F0) which mustbe proteolytically cleaved into F2 and F1 polypeptides toactivate F protein fusion activity (Collins et al., 1993; DeLeeuw et al., 2005; Panda et al., 2004). NDVs are groupedinto genotypes I to IX under one serotype based on theanalysis of the restriction site and nucleotide sequence ofthe F protein gene. Genotypes VI and VII can be further

Z. Rui et al. / Veterinary Microbiology 141 (2010) 246–257 247

divided into seven (VIa–g) and five (VIIa–e) subgenotypes,respectively (Bogoyavlenskiy et al., 2005; Liu et al., 2003,2007a; Wang et al., 2006).

The genetic change of the virus has been reported as oneof the reasons why NDV virulence changes (Gould et al.,2001). Except for the high mutation rates and largepopulation sizes, recombination is also a potentiallyimportant means of generating more genetic diversity inRNA viruses (Lai, 1992; Worobey and Holmes, 1999). Fornegative strand RNA virus, the recombination, such asambisense arenavirus (Archer and Rico-Hesse, 2002;Charrel et al., 2001), hantavirus (Klempa et al., 2003;Sibold et al., 1999; Sironen et al., 2001), influenza A virus(Gibbs et al., 2001), measles virus (Schierup et al., 2005),respiratory syncytial virus (Spann et al., 2003) andNewcastle disease virus (Chare et al., 2003; Qin et al.,2007) had been reported, but the rates of recombination innegative strand RNA viruses were much lower than thosein positive strand RNA viruses.

Since the 1980s, vaccination has been the widely usedmethod for prevention and control NDV infections inChina, but the disease is still enzootic in some areas andremains a constant threat to domestic poultry. Epidemio-logical studies have revealed that genotype VII virusescirculating predominantly in China in the past decade wereresponsible for disease outbreaks in chicken flocks (Liuet al., 2003). The prevailing NDV strains have significantdifferences from the current vaccine strains in theirbiology, serology and genetics, which is considered asthe main reasons for the outbreaks of this disease invaccinated poultry flocks in recent years (Qin et al., 2008;Tsai et al., 2004). For further characterize and phylogen-etically group clinical NDVs in China, 20 isolates wereobtained from outbreaks in different regions of Chinaduring 2001 to 2009 for sequence comparison, recombi-nant identification and phylogenetic analysis in the study.The study revealed increased changes of circulating NDVsin China, highlighting aspects of NDV evolution acted byhomologous recombination.

2. Materials and methods

2.1. NDV isolates

Twenty isolates in the study were isolated fromclinically diseased chickens in different regions of Chinaduring 2001 to 2009. Viruses were plaque-purified threetimes on primary chicken embryo fibroblasts and thengrown in 10-day-old specific-pathogen-free (SPF) chicken

Table 1

4 pairs of primers used in the study.

Label Sequences (50–30) Targ

F-forward GCTGTCGCAGTGACCG F

F-reverse GGCTCCTCTGACCGTTCTA F

F3943-forward GAAGAGCCCGTTAGTCA F

F3943-reverse GTAATCCACATTGCCACC F

F5215-forward GCCACAAATCACCTCC F

F5215-reverse TCCTCATTCTCCAGCAC F

HN-forward TCACAACATCCGTTCTACC HN

HN-reverse TCAGGATTTGGGATCAGC HN

embryonated eggs. Allantoic fluid samples were harvestedand stored at �80 8C for further use. Allantoic fluids wereused for RNA extraction, hemagglutination inhibition (HI)and mean death time (MDT) (Alexander, 1998; Collinset al., 1994).

2.2. RNA extraction and RT-PCR

Virus genome RNA was extracted from allantoic fluidwith Trizol Reagent (Invitrogen, San Diego, USA) accordingto the manufacturer’s instructions. Reverse transcriptionwas performed using Random Primers (500 mg/ml) (Pro-mega, USA) and 0.5 ml of M-MLV-Reverse Transcriptase(200 U/ml) (Promega). The reverse transcription was thenachieved by incubation at 37 8C for 1 h and reactionstopped by heating at 95 8C for 5 min. In order to obtain thecomplete F and HN gene sequence, the PCR amplificationswere performed with 4 pairs of primers (Table 1) designedaccording to the NDV available nucleotidic sequences(GenBank accession number AY845400 and AF473851).The amplification was performed in PCR Machine (Biome-tra, Germany) with 100 ng previously synthesized cDNA astemplate in a total of 20 ml reaction volume containing20 pmol of each primer and 10 ml of 2� Taq PCR mix(Vigorous Biotechnology Corporation, Beijing, China). TheNDV positive strain cDNA and sterile water were used,respectively, as positive and negative controls. Reactionswere performed according to the following protocol: 95 8Cfor 5 min, followed by 35 cycles (95 8C for 45 s, 53 8C for45 s, 72 8C for 2 min) and a final elongation step of 10 minat 72 8C.

2.3. Gel extraction of PCR products and nucleotide sequencing

5 ml of PCR reaction mixture was loaded onto 1%agarose gel and electrophoresed for 40 min in TAE buffer,containing 0.5 mg/ml ethidium bromide. The PCR productswere purified with AxyPrep DNA Gel Extraction Kit(AxyGEN, USA) according to the manufacturer’s instruc-tions. Sequencing reactions were performed by SunbioBiotech Company (Beijing, China).

2.4. Analysis of nucleotide and deduced amino acid sequences

Nucleotide and deduced amino acid sequences of the Fand HN genes of NDV obtained in this study were alignedusing a J. Hein method with PAM250 residue weight tableof DNASTAR software (DNASTAR Inc., Madison, WI, USA).The nucleotide and amino acid sequences analysis of the

et gene Position Expected size (bp)

4295–4310 1895

6172–6190

3943–3959 1351

5277–5293

5215–5240 1244

6446–6458

6367–6386 2037

8383–8400

Table 2

NDV isolates and their accession numbers used in phylogenetic analysis.

Virus strains Year Country Genotype Accession number

V4* Australia I AF217084

JL01* 2001 China I EF464163

NDV981634 China I EU258651

NDV016540 China I EU258668

I-2progenitor Australia I AY935500

99-0655 1999 I AY935494

Texas GB/48 1948 USA II M24698

Beaudette C/45 1945 USA II X04719

B1* 1947 USA II NC_002617

Clone-30* II Y18898

LaSota* 1946 USA II AY845400

HB9* China II AY225110

B1 isolate USA II AF375823

Miyadera/51 1951 Japan III M18456

AUS/Victoria/32 1932 Australia III M21881

Mukteswar* China III EF201805

Herts/33 Netherands IV AY741404

Italien* China IV EU293914

Mixed species/USA(FL)/Largo/71 1971 USA V AY288987

Chicken/Mexico/37821/96 1996 Mexico V AY288999

Gamefowl/U.S(CA)/211472/02 2002 USA V AY562987

Anhinga/U.S(FL)/44083/93 1993 USA V AY562986

Chicken/U.S(CA)/1083(Fonta na)/72 1972 USA VI AY562988

Warwick* VI Z12111

Dove/Italy/2736/00 2000 Italy VI AY562989

IT-227/82 Italy VI AJ880277

Cockatoo/Indonesia/14698/90 1990 Indonesia VIIc AY562985

Sterna/Astr/2755/2001 2001 Australia VIIb AY865652

NA-1* 2006 China VIId DQ659677

ZJI* 2000 China VIId AF431744

GM China VIId DQ486859

TW/2000 2000 Taiwan VIId AF358786

NDV06-064 2006 China VIId EF221726

Ch-501/02 2002 China VIId AY630438

JS/5/01 2001 China VIId AF456442

Kr-188/02 2002 South Korea VIId AY630436

NDV05-085 2005 China VIId DQ439937

TW/84C 1984 Taiwan VIIc AF083965

TW/96P 1996 Taiwan VIIc AF083971

RI-1/88 1988 Hungary VIIa AF001134

NL-2/93 1993 Hungary VIIa AF001125

DE143/95 1995 UK VIIa AF109881

TW/94P 1994 Taiwan VIIe AF083961

TW/95-2 1995 Taiwan VIIe AF083972

Q-GB 445/97 1997 UK VIIe AF109886

AE 232/1/96 1996 UK VIIb AF109884

ZA 360/95 1995 UK VIIb AF109876

ZW 3422/95 1995 UK VIIb AF109877

AF2240 1998 Malaysia VIII AF048763

Trenque Lauquen/Argentina/70 1970 Argentina VIII AY734534* The strains used as query in the analysis of putative recombinant sequence with the SimPlot program.

Z. Rui et al. / Veterinary Microbiology 141 (2010) 246–257248

NDV isolates, based on a variable portion (nt1-389)covering the F cleavage site, was processed by Clustal Wmultiple alignment method in MegAlign program ofDNASTAR software.

2.5. Identification of putative recombinant sequence

Putative recombinant sequence and its parents wereidentified with the SimPlot program (version 3.5) (Loleet al., 1999). We used the complete sequence of F gene andHN gene of the current vaccine strains or the typicalprevailing isolates in China collected from the GenBank(Table 2) as a query to determine the potential recombi-

nant sequence. Bootscanning (Salminen et al., 1995) wasalso carried out employing subprogram from the SimPlotprogram, using the putative recombinant sequence as aquery, to monitor the parental-like strains. A clusteranalysis maximizing the value of x2 was then used to selectbreakpoints among the clusters, combined with smallsample Akaike Information Criterion (AIC) (implementingGARD online) (Kosakovsky Pond et al., 2006). P values forthe resultant divisions of sites were calculated by usingFisher’s exact test (Lole et al., 1999). The phylogenetic treesbased on the different fragments of F gene wereconstructed as a powerful evidence for recombination(Worobey et al., 2002).

Table 3

The data of 18 Chinese NDV isolates analyzed in the present study.

NDV strains Origin Genotype MDT* Virulence Accession numbers

(F/HN gene)

Separatum Host Year of isolation Age (day)

GM/Shandong/01 Shandong Breeder 2001 13 VII 35 V FJ608343/FJ608361

WN/Tianjin/03 Tianjin Broiler 2003 6 VII 35 V FJ608334/FJ608352

MQ/Liaoning/05 Liaoning Broiler 2005 33 II 40 V FJ608340/FJ608358

KQ/Liaoning/06 Liaoning Layer 2006 13 II 48 V FJ608342/FJ608360

SH/Zhejiang/06 Zhejiang Native 2006 42 VII 23 V FJ608338/FJ608356

ZSM/Hebei/07 Hebei Broiler 2007 26 VII 46 V FJ608336/FJ608354

CJG/Xinjiang/07 Xinjiang Breeder 2007 22 VII 35 V FJ608335/FJ608353

QG/Hebei/07 Hebei Broiler 2007 26 VII 35 V FJ608337/FJ608355

AG/Tianjin/07 Tianjin Broiler 2007 20 VII 39 V FJ608339/FJ608357

TYQ/Shanxi/07 Shanxi Broiler 2007 9 II >90 L FJ608341/FJ608359

DF3Q/Beijing/08 Beijing Breeder 2008 40 II >90 L FJ608344/FJ608362

DFC3Q/Liaoning/08 Liaoning Broiler 2008 25 VII 50 V FJ608346/FJ608364

XD/Shandong/08 Shandong Broiler 2008 21 VII >90 L FJ608347/FJ608365

DFZD/Jilin/08 Jilin Breeder 2008 18 VII 46 V FJ608345/FJ608363

TCQQ/Tianjin/08 Tianjin Broiler 2008 32 VII 49 V FJ608348/FJ608366

LGQQ/Liaoning/08 Liaoning Broiler 2008 16 VII 48 V FJ608349/FJ608367

DFQS/Beijing/08 Beijing Broiler 2008 18 VII 38 V FJ608350/FJ608368

YZCQ/Liaoning/08 Liaoning Broiler 2008 16 VII 30 V FJ608351/FJ608369

SG/Liaoning/2009 Liaoning Broiler 2009 20 VII 45 V FJ882014/FJ882012

HG/Beijing/2009 Beijing Broiler 2009 22 VII 40 V FJ882015/FJ882013* Mean death time in embryonating eggs (hours) (<60: velogenic; 60–90: mesogenic; >90: lentogenic) (Alexander, 1998).

Z. Rui et al. / Veterinary Microbiology 141 (2010) 246–257 249

2.6. Phylogenetic analysis

The sequences obtained in the study were comparedwith other NDV sequences available in GenBank (Table 2),which are either the reference strains, the strains used forthe vaccine or the isolates from China reported in recentyears in eight genotypes. The alignment, based on thenucleotide sequences of F gene (from 1 to 1662) and HNgene (from 1 to 1731 or 1713), was processed with ClustalW multiple alignment method. Phylogenetic tree wasconstructed with the maximum composition likelihood(MCL) in the program MEGA4 (Molecular EvolutionaryGenetics Analysis, version 4.0) using the Kimura two-parameter model and the neighbor-joining algorithm with1000 bootstraps (Tamura et al., 2007). And all branchessupported by >50% bootstrap value are considered as thesame group in the trees.

2.7. Nucleotide sequence accession numbers

The generated sequence data are available fromGenBank accession numbers FJ608334 to FJ608369 andFJ882012 to FJ882015.

3. Results

3.1. Virus isolates

Totally 20 NDV isolates, from outbreaks in differentchicken flocks, dates, and regions in China between 2001and 2009, were collected and summarized in Table 3.

3.2. Nucleotide sequencing and sequences alignment

The amplified F gene coding sequence for each isolatewas 1662 nucleotides, directing the synthesis of a proteinpredicted to be 554 amino acids in length. The HN protein

of 4 NDV isolates had 577 amino acids possessing a singlearginine at position 572 and 16 isolates had 571 aminoacids, which encoded by 1731 nucleotides and 1713nucleotides, respectively (Fig. 1).

The analysis and comparison of nucleotide and deducedamino acid sequences of the 20 NDV isolates with theLaSota, the frequently used vaccine strain in China, wereperformed. The results revealed that the nucleotide andamino acid identities of HN gene sequences (1731 bp or1713 bp) among the 20 NDV isolates ranged from 79.93% to100%. There were 12.82–19.03% amino acid differencesbetween the 16 isolates and LaSota strain, whereas therewere only 1.04–1.73% amino acid differences betweenother 4 isolates and LaSota strain (data not shown). As for Fgenes, the nucleotide and amino acid identities among the20 NDV isolates ranged from 82.97% to 100%. 14 out of 16isolates had 11.57–16.61% amino acid differences withLaSota strain, two isolates (XD/Shandong/08 and QG/Hebei/07) had 7.41–10.31% amino acid differences withLaSota strain. Another four isolates had only 0.72–2.35%amino acid differences with LaSota strain (data notshown).

3.3. Sequence analysis of the F protein cleavage site

Genetic analyses of NDVs have demonstrated arelationship between the amino acid sequence at the Fprotein cleavage site and virulence (Nagai et al., 1976a). Sowe analyzed the proteolytic cleavage site motifs for the F0

protein of the 20 NDV isolates. The results demonstratedthat 14 virulent isolates had K101 and V121, a characteristicof genotype VII viruses (Lomniczi et al., 1998), andpossessed the motif of 112RRQKR/F117 (Table 3 andFig. 2) (Collins et al., 1993; Toyoda et al., 1987). Threeavirulent isolates have a cleavage site sequence of typicalavirulent NDVs, 112G-R-Q-G-R/L117, and amino acid resi-dues R101, I121. Unexpectedly, another three virulent

Fig. 2. Alignment partial predicted amino acid sequences of fusion protein (F) of Newcastle disease virus from residues 101 to 121 (NDV F protein

numbering) to illustrate discrepancies among reported F protein. The line between residues 116 and 117 indicates the F protein cleavage site.

Fig. 1. The relationship between the length of HN protein and the NDV genotype. The length of HN protein of 16 NDV isolates is 571 aa which belong to the

genotype VII. The 4 NDV isolates of genotype II has 577 aa in the HN protein. The single arginine at position 572 (in the box) seems to be proteolytic cleavage

site and is present in all genotype II strains.

Z. Rui et al. / Veterinary Microbiology 141 (2010) 246–257250

isolates also had a motif of 112G-R-Q-G-R/L117 at thecorresponding positions, as shown in Fig. 2. A Gln-to-Argchange at position 114 in WN/Tianjin/03, and an Arg-to-Lys change at position 113 in QG/Hebei/07, becameapparent (Table 3 and Fig. 2).

3.4. Identification of putative recombinant sequence

We performed the identification of potential mosaicamong the 20 NDV isolates with the SimPlot program

(version 3.5). The results demonstrated that two naturalrecombinants, XD/Shandong/08 and QG/Hebei/07, weredescended from two putative parents in the gene F, and onecrossover was found respectively (Supplementary Fig. S1).

The potential breakpoints analyses based on the gene Fof XD/Shandong/08 revealed that the single breakpointwas located in a parsimonious region from 705 to 716 withthe maximization of x2 using the program SimPlot. Themost likely breakpoint was found exactly at site 706according to Single Breakpoint Analysis in GARD (Fig. 3A

Fig. 3. The evidences for recombination in F gene of strain XD/Shandong/08 and QG/Hebei/07. (A) and (C) F gene similarity from SimPlot analysis of XD/

Shandong/08 and QG/Hebei/07 are shown respectively. Standard similarity plot (constructed using all sites) of the complete F gene, with a window size of

200 bp and a step size of 20 bp. Vertical lines indicate breakpoints identified by maximization of x2 and small sample Akaike Information Criterion as

described in Section 2. The x2 value and P value of each breakpoint are shown near (or on) the vertical lines. Informative sites and their bifurcating trees are

shown in each crossover block. Herts/33 and LaSota are used as the outgroup to determine the breakpoints respectively. (B) and (D) The results of

Bootscanning of XD/Shandong/08 and QG/Hebei/07. The y-axis gives the percentage of permutated trees using a sliding window of 200 bp wide centered on

the position plotted, with a step size between plots of 20 bp. The rest is the same as panels (A) and (C).

Z. Rui et al. / Veterinary Microbiology 141 (2010) 246–257 251

and B). The phylogenetic tree based on the N-terminal(nucleotide position 1–705) of F gene showed that XD/Shandong/08 fell into the cluster of genotype II NDVstrains, while the tree based on the C-terminal (nucleotideposition 706–1662) of F gene showed that XD/Shandong/08 fell into the cluster of genotype VII NDV strains (Fig. 4).A standard similarity plot, constructed using all sites of Fgene of XD/Shandong/08, revealed that this strain exhib-ited the greatest affinity with one putative parent lineageof LaSota (genotype II) in the region from 1 to 705, andshared the highest sequence similarity with anotherlineage of NA-I (genotype VII) of China from 706 to1662 (Fig. 3A and B).

Another probable F gene mosaic (QG/Hebei/07) wasfound when we conducted a similarity analysis. The resultsshowed that the most likely breakpoint was located at site693. The F sequence exhibited greatest affinity with V4(genotype I) from position 1 to 692, and shared highest

sequence similarity with ZJ1 (genotype VII) of China fromposition 693 to 1662 (Fig. 3C and D). A significantdiscrepancy between phylogenetic trees inferred fornucleotide sequences of each recombination region inthe gene F of QG/Hebei/07 is also an important evidence forrecombination (Fig. 4).

3.5. Phylogenetic analysis

According to the phylogenetic tree of F gene (nt1–1662)and HN gene (nt1–1731 or 1713), all 20 NDV isolates wereassigned to two genotypes, II and VII, respectively. Most ofthe NDV isolates (80%), including the two naturalrecombinants, were demonstrated to be of genotype VII,and four isolates were of genotype II (Fig. 5A and B). Theresults of further VII subtype analyses based on thevariable region (nt1-389) of F gene demonstrated that 14of 16 Genotype VII isolates belonged to the genotype VIId,

Fig. 4. Neighbor-joining phylogenies inferred for the 2 regions delimited by the breakpoints. The percentage of replicate trees in which the associated taxa

clustered together in the bootstrap test (1000 replicates) is shown next to the branches (only >50% is shown). The evolutionary distances were computed

using the Maximum Composite Likelihood model as described in Section 2, and are in the units of the number of base substitutions per site. Phylogenetic

analyses were conducted in MEGA4. (A) The phylogenetic tree of the region from position 1 to 705. (B) The phylogenetic tree of the region from position 706

to 1662. The putative mosaic is indicated in bold.

Z. Rui et al. / Veterinary Microbiology 141 (2010) 246–257252

and the two natural recombinants changed into thegenotype I and II, respectively (Fig. 5C).

4. Discussion

Newcastle disease is a most important disease ofpoultry throughout the world because of its high morbidityand mortality (Alexander, 2003). In China, although anintensive vaccination policy has been implemented,virulent NDV can still be frequently isolated in well-vaccinated flocks. Genetic analysis of NDV strains haveproved its utility in determining pathotype, recent and pasthistory of NDV strains and epidemiological relationships ingeographically widely separated areas. In this study, 20isolates of NDV isolated from infected commercial flocks indifferent regions of China in recent years have beencharacterized genotypically in laboratory.

The length of HN gene of NDV can be regarded as one ofthe phylogenetic feature. The sizes of HN protein were

different and could be classified into six length types (571 aa,577 aa, 578 aa, 580 aa, 585 aa, and 616 aa) (Ujvari, 2006). Weobserved that the length of HN gene was related with thegenotype, but there was no special relationship withvirulence or year of isolation. The length of HN protein of16 NDV isolates is 571 aa which belong to the genotype VII.While the NDV isolates of genotype II have 577 aa in the HNprotein and possess a single arginine at position 572 (Fig. 1).The ability to bind a sialic acid-containing receptor is one offunction of the HN protein and plays a key role in the initialsteps of the NDV life cycle (Huang et al., 2004a,b). Severalamino acids residues, R174, Y526, E401, and R416 in HN proteinwere reported to be involved in the activity of sialic acidbinding (Connaris et al., 2002). In our study, these aminoacids were completely conserved in all 20 strains. Theresults may facilitate the site-directed mutations to destroysialic acid binding, which would be in conjunction with theadditional of targeting sequences, to get a targeted virus infurther study.

Z. Rui et al. / Veterinary Microbiology 141 (2010) 246–257 253

It is widely accepted that sequence analysis of the Fprotein cleavage site can be used to predict potentialpathogenicity of NDV instead of conventional methodssuch as mean death time (MDT) and intracerebralpathogenic index tests (ICPI) (Collins et al., 1993; Pandaet al., 2004; Toyoda et al., 1987). However, in our

Fig. 5. Phylogenetic tree of NDV isolates. (A) Phylogenetic tree of NDV isolates b

1662). (B) Phylogenetic tree of NDV isolates based on complete HN gene sequenc

isolates, except the two natural recombinants (XD/Shandong/08 and QG/Hebei/

389). The percentage of replicate trees in which the associated taxa clustered tog

(only>50% is shown). The tree is drawn to scale, with branch lengths in the same

tree. The evolutionary distances were computed using the Maximum Composit

number of base substitutions per site. All positions containing gaps and miss

Phylogenetic analyses were conducted in MEGA4. The putative mosaic is indic

experiments, even when the three NDV strains presenteda typical cleavage site sequence of avirulent NDV (112G-R-Q-G-R/L117), the virus performance in the MDT assay wassimilar to virulent virus, and this could be due to thepresence of other influencing factors. Nevertheless, someprevious reports also supplied the experimental evidences

ased on complete F gene sequence (1662 bp, between nt positions 1 and

e (1731 bp, between nt positions 1 and 1731). (C) Phylogenetic tree of NDV

07) based on partial F gene sequence (389 bp, between nt positions 1 and

ether in the bootstrap test (1000 replicates) is shown next to the branches

units as those of the evolutionary distances used to infer the phylogenetic

e Likelihood model as described in Section 2, and are in the units of the

ing data were eliminated from the data set (complete deletion option).

ated in bold.

Fig. 5. (Continued )

Z. Rui et al. / Veterinary Microbiology 141 (2010) 246–257254

considering that multiple determinants, apart from theamino acids sequence at F0 cleavage site, could beresponsible for the virulence displayed by NDV strains(Mebatsion et al., 2001; Romer-Oberdo rfer et al., 2003;Peeters et al., 1999). We conjectured it could be possiblethat when a NDV strain bears basic amino acids atpositions 112, 113, 115, 116 and phenylalanine at position117, the manifestation of its virulence is independent ofother factors, and when a NDV strain possess less basicamino acids at the F protein cleavage site, it may need thecontribution of other determinants to show its virulence.

Yang et al. (1999) reported that K101 and V121 were theunique features for genotype VII, which was in agreementwith our findings in 16 Chinese isolates (VII) collected

between 2001 and 2009 (Fig. 1), but we also found thatexceptions do exist, such as QG/Hebei/07 and XD/Shandong/08, which had R101 and I121. Although the twostrains were descended from two different parentsrespectively, the results of the phylogenetic analysisplaced the 2 NDV isolates into genotype VII, as shown inFig. 5. Whether this exception has any functionalsignificance in pathogenicity was not clear, which canultimately be determined using reverse genetics techni-ques. It will be of interest to determine whether thisdifference is consistent in other recombination of genotypeVII NDV or it is only special in the two strains we found.

For NDV, the recombinations had been found in the HNgene, F gene and complete genome in previous study

Fig. 5. (Continued ).

Z. Rui et al. / Veterinary Microbiology 141 (2010) 246–257 255

(Chare et al., 2003; Qin et al., 2007; Han et al., 2008), butthe natural homologous recombination involving vaccinestrains was not reported on the level of F gene. The result ofthe identification of potential mosaic in our study providedthe evidence that homologous recombination can occurnaturally not only between circulating NDV viruses, butalso between circulation NDV and vaccine strain, andsuggested that NDV vaccines might play an important rolein the evolution of the virus and the frequency of naturalhomologous recombination might not be low at least inNDV. In China, commercial live vaccines, such as LaSota, B1and Clone-30, are still widely used for protecting chickenfrom Newcastle disease. However, the role of vaccine is notclear in NDV evolution. Because the negative-sense RNAviruses, especially nonsegmented negative-sense RNAviruses, have low rates of recombination according someprevious reports, the natural recombination between NDVvaccines and circulating viruses is often neglected, whichmight contribute to the circumstances that allowed thiscourse of evolution to occur under natural conditions.According to the epidemiological investigation on the

infected chicken flocks, we found that such naturalrecombinants have the ability to spread widely and causeepidemics in chickens (data not shown). Further char-acterization of such viruses is important for the develop-ment of effective NDV vaccination strategies.

The genotype VII of NDV has become the mostprevalent in Asia since 1980s. The results of thephylogenetic analysis showed that genotype VIId virushad become the predominant pathogen in poultry in China,and the percentage is increasing with year (Table 3), whichwas same with some previous reports (Liu et al., 2007b). Inaddition, we also found that the genotype VIId virusescirculating predominantly in China had an increaseddiscrepancy in recent years and could be further categor-ized into two groups (Fig. 5C). The Group I belonged to thetypical ‘Asia newly described isolates’, and were close toKr-188/02 from Korea, JS/5/02, NDV05-085, ZJ1, Ch-501/02from China, and Japan/Chiba/2000, Japan/Gunma/2001from Japan (Lien et al., 2007). While the Group II familyemerged the latest (2006–2009) and exhibited the highestaffinity with a previously Chinese isolate, GM (Fig. 5C).

Z. Rui et al. / Veterinary Microbiology 141 (2010) 246–257256

Therefore, it can be postulated that part of Chinese isolatescollected between 2006 and 2009 originated from localstains which had its own endemic character and hadbecame the dominant strains responsible for recentoutbreaks in China.

Appendix A. Supplementary data

Supplementary data associated with this article can be

found, in the online version, at doi:10.1016/j.vet-

mic.2009.09.020.

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