Shabbir et al. Virology Journal 2013, 10:170http://www.virologyj.com/content/10/1/170

RESEARCH Open Access

Genetic diversity of Newcastle disease virus inPakistan: a countrywide perspectiveMuhammad Zubair Shabbir1*, Siamak Zohari2, Tahir Yaqub1, Jawad Nazir3, Muhammad Abu Bakr Shabbir1,Nadia Mukhtar1, Muhammad Shafee4, Muhammad Sajid5, Muhammad Anees6, Muhammad Abbas7,Muhammad Tanveer Khan8, Asad Amanat Ali9, Aamir Ghafoor10, Abdul Ahad11, Aijaz Ali Channa12,Aftab Ahmad Anjum3, Nazeer Hussain13, Arfan Ahmad10, Mohsan Ullah Goraya14, Zahid Iqbal6,Sohail Ahmad Khan6, Hassan bin Aslam3, Kiran Zehra15, Muhammad Umer Sohail16, Waseem Yaqub1,Nisar Ahmad17, Mikael Berg14 and Muhammad Munir18*

Abstract

Background: Newcastle disease (ND) is one of the most deadly diseases of poultry around the globe. The disease isendemic in Pakistan and recurrent outbreaks are being reported regularly in wild captive, rural and commercial poultryflocks. Though, efforts have been made to characterize the causative agent in some of parts of the country, the geneticnature of strains circulating throughout Pakistan is currently lacking.

Material and methods: To ascertain the genetics of NDV, 452 blood samples were collected from 113 flocks,originating from all the provinces of Pakistan, showing high mortality (30–80%). The samples represented domesticatedpoultry (broiler, layer and rural) as well as wild captive birds (pigeons, turkeys, pheasants and peacock). Samples werescreened with real-time PCR for both matrix and fusion genes (1792 bp), positive samples were subjected toamplification of full fusion gene and subsequent sequencing and phylogenetic analysis.

Results: The deduced amino acid sequence of the fusion protein cleavage site indicated the presence of motif(112RK/RQRR↓F117) typical for velogenic strains of NDV. Phylogenetic analysis of hypervariable region of the fusion geneindicated that all the isolates belong to lineage 5 of NDV except isolates collected from Khyber Pakhtunkhwa (KPK)province. A higher resolution of the phylogenetic analysis of lineage 5 showed the distribution of Pakistani NDV strainsto 5b. However, the isolates from KPK belonged to lineage 4c; the first report of such lineage from this province.

Conclusions: Taken together, data indicated the prevalence of multiple lineages of NDV in different poultry populationincluding wild captive birds. Such understanding is crucial to underpin the nature of circulating strains of NDV, theirpotential for interspecies transmission and disease diagnosis and control strategies.

Keywords: Lineages, Newcastle disease virus, Pakistan, Phylogenetic analysis, Poultry

IntroductionNewcastle disease is caused by avian paramyxovirusserotype-1 (APMV-1), which is also known as Newcastledisease virus (NDV). It is a highly contagious viral diseasethat affects domesticated and wild bird species throughoutthe world [1-4]. The disease is considered enzootic in

* Correspondence: vetvains@uvas.edu.pk; drmunir.muhammad@gmail.com1Quality Operations Laboratory, University of Veterinary and Animal Sciences,Lahore, Pakistan18The Pirbright Institute, Compton Laboratory, Compton, Berkshire RG207NN, United KingdomFull list of author information is available at the end of the article

© 2013 Shabbir et al.; licensee BioMed CentraCommons Attribution License (http://creativecreproduction in any medium, provided the or

Pakistan and represents major threat to the economy ofthe country. However, disease severity is variable in differ-ent host species and in different geographical locations.NDV is classified in the genus Avulavirus within subfam-

ily Paramyxovirinae, family Paramyxoviridae and orderMononegavirales [1]. This enveloped virus has a negative-sense, non-segmented, single stranded RNA genome of15186, 15192 or 15198 nucleotides in length [1,5,6]. Thegenome encodes six structural and two non-structural pro-teins [6]. Based on the fusion (F) gene sequence, NDVstrains are classified into lineages or genotypes; however,the discrepancies between the two classification systems are

l Ltd. This is an Open Access article distributed under the terms of the Creativeommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andiginal work is properly cited.

Shabbir et al. Virology Journal 2013, 10:170 Page 2 of 10http://www.virologyj.com/content/10/1/170

nominal [5,7-10]. Clinical manifestation or severity of theND depends largely upon the isolates involved in diseaseoutbreak [1,2]. Based upon pathogenicity, these strains arecommonly categorized into velogenic, mesogenic andlentogenic types [4]. The varying level of pathogenicity is at-tributed to amino acid sequence motif present in the prote-ase cleavage site of the precursor F protein [11]. The aminoacid sequence in more virulent, velogenic and mesogenicstrains is 112R/K-R-Q-R/K-R↓F117. This sequence is cleav-able by a variety of cellular proteases in various organs,resulting in wider systemic infection in respiratory system,gastro-intestinal tract and nervous system. The sequence inless virulent, lentogenic strains of NDV is 112G/E-K/R-Q-G/E-R↓L117. This sequence is cleavable only by trypsin likeproteases, hence limiting the infection only to respiratorysystem and gastro-intestinal tract [1,4,6].Pakistan has an agriculture based economy with livestock

and poultry as an integral part of it. Nearly every family inthe rural areas and every 5th family in the urban areas is as-sociated with poultry in one way or the other [12]. Thepoultry has emerged as the second largest industry inPakistan with an annual increase of 4%, supplying eggs andmeat as the protein sources [13]. Since the development ofan organized poultry sector in Pakistan, the Newcastle dis-ease has caused havocs to the poultry industry several times.Appropriate vaccination and subsequent effective immuneresponse is known to be the only measure to avoid the dis-ease outbreaks, but this approach cannot be extended logis-tically to all domesticated and wild birds. Large number ofdisease outbreaks was recorded during 2010–12 in Pakistan.Reports about the disease in Pakistan have largely

been focused to Punjab province, reporting existence ofvelogenic NDV strains [9,11,14-17]. However, a systematicinvestigation of genetic nature of NDV strains in clinicallydiseased flocks throughout Pakistan has been lacking so far.Failure of previously effective live vaccines in protecting thebirds from current field isolates stimulated us to investigatethe genetic relationship between the past and current viralisolates. This is crucial due to the fact that disease con-tinue to appear in both vaccinated flocks as well as unvac-cinated commercial, wild captive and backyard poultrybirds. This is the first report that encompasses the mo-lecular characterization of prevailing NDV strains fromacross Pakistan.

Materials and methodsHistory and collection of samplesTo ascertain the genetic nature of circulating NDVstrains in Pakistan, blood samples were collected duringan emerging wave of the disease from February to July2012. A total of 113 flocks originating from various dis-tricts in the province of Punjab, Khyber Pakhtunkhwa,Sindh and Baluchistan were examined (Table 1, Figure 1).The under-study flocks consist of commercial (n = 60),

rural (n = 42) and domesticated birds including pigeon,turkeys and peacocks (n = 11). From each diseased flock,whole blood (3–5 mL) was collected aseptically from bra-chial vein of four diseased birds randomly in an anticoagu-lant added vaccutainer (Venoject(R), Belgium). A briefhistory regarding age at infection, clinical symptoms, mor-talities and course of infection was recorded.

Shipment of the samples and extraction of nucleic acidA total 300 μL of whole blood was stored on QIACardFTA Indicator Four Spots (Qiagen, Hilden, Germany).These FTA Indicators have properties to inactivate the virusand preserve the nucleic acid. The samples were shipped atambient temperature from Pakistan to the Department ofBiomedical Sciences and Veterinary Public Health at theSwedish University of Agricultural Sciences (SLU) Uppsala,Sweden, for further processing and analysis.The RNA was extracted from blood-impregnated FTA

Indicator, as we have recently demonstrated [9]. Briefly, onepunch (discs of 2.0 mm diameter) was taken from eachsample using a Harris’ micropunch, according to manufac-turer’s recommendation (BD09; Whatman). These discswere placed individually in a 1.5 mL microcentrifuge tubeand the RNA was eluted with 52 μL of Tris–EDTA elutionbuffer (10 mMTris–HCl with pH 8.0, 0.1 mM EDTA, 50units of RNase Inhibitor and 1 mM DTT) instead ofcompany’s recommended RNA processing buffer. Allreagents used here were purchased from Invitrogen,Carlsbad, CA, USA. These soaked discs were incubated for15 min on ice and were flicked three times after every fiveminutes during the course of incubation). The extractedRNA was stored at –20°C until use for both sample screen-ing and characterization using real-time PCR and sequen-cing, respectively.

Screening of samples using real-time PCRThe detection of nucleic acid for NDV was performedusing real-time PCR for M and F genes, as described previ-ously [9,18]. The reaction was carried out in a Rotor-Gene6000 real-time analyzer (Qiagen). The reporter dye (FAM)signals were measured at the annealing step of each cycle,and the threshold cycle (Ct) for each sample was calculated.The samples that had a Ct value <35 were considered posi-tive in both M and F genes based real-time PCRs.

Amplification and sequencing of F geneAll the samples that appeared positive for real-time PCR(both M and F gene bases) were further processed for theamplification of the complete F gene using degenerateprimers as we described previously [19,20] with one modi-fication. A total 7 μL of eluted RNA (the same as was usedin real-time PCR screen) was added in a 25 μL reactionmix from One-Step RT-PCR Kit (Qiagen). However, thePCR conditions were kept same as reported previously

Table 1 Detail of samples collected from districts and diagnostic efficacy of real-time PCR

Province District Type of birds1Commercial 2Rural 3Wild captive

Flock(n)

Bloodsamples (n)

4Real timePCR

Flock(n)

Bloodsamples (n)

Real TimePCR

Flock(n)

Bloodsamples (n)

Real timePCR

Mgene

Fgene

Mgene

Fgene

Mgene

Fgene

Baluchistan Quetta 09 36 + + 03 12 + + 0.0 0.0 - -

Noshki 0.0 0.0 - - 04 16 - - 0.0 0.0 - -

Zhob 0.0 0.0 - - 01 04 - - 0.0 0.0 - -

Mastoung 0.0 0.0 - - 02 08 - - 0.0 0.0 - -

Loralai 09 03 + + 05 20 + + 0.0 0.0 - -

KPK Abbottabad 11 44 + + 0.0 0.0 - - 0.0 0.0 - -

Mansehra 10 40 + + 0.0 0.0 - - 0.0 0.0 - -

Sindh Karachi 06 24 + + 02 08 + + 0.0 0.0 - -

Hyderabad 03 12 - - 03 12 - - 0.0 0.0 - -

Sukkur 01 04 - - 02 08 - - 0.0 0.0 - -

Punjab Okara 02 08 + + 01 04 + + 04(turkeyand

pigeon

16 + +

Faisalabad 10.0 0.3 + + 07 28 + + 02 (pigeon) 08 +

+

Layyah 12 04 + + 06 24 - - 0.0 0.0 - -

Pakpattan 02 04 + + 0.0 0.0 - - 0.0 0.0 - -

Sheikhupura 09 36 + + 02 08 - - 0.0 0.0 - -

Sialkot 01 04 + + 02 08 - - 0.0 0.0 - -

Sahiwal 01 04 + + 0.0 0.0 - - 0.0 0.0 - -

Lahore 10.0 03 + + 12 05 + + 05 (peafowl andpigeon)

20 +

+

Multan 03 12 + + 0.0 0.0 - - 0.0 0.0 - -

Jhang 02 08 - - 02 08 - - 0.0 0.0 - -1birds kept under full human husbandry conditions and are fed with commercial food and supplements, 2Birds kept under partial human husbandry conditionsand mainly reply on natural source of food. 3Wild birds that are kept under human control and provided commercial food and supplements. 4At least one of thestudied flocks in particular district was positive, + and - indicate that samples were detected positive or negative by real-time PCR, respectively.

Shabbir et al. Virology Journal 2013, 10:170 Page 3 of 10http://www.virologyj.com/content/10/1/170

[11,16,20]. The amplified PCR products were visualized in1% (w/v) agarose gel in TBE-containing ethidium bromide.The bands of expected size were cut from the gel and werepurified using the Wizard® SV Gel and PCR Clean-Up Sys-tem (Promega, Co., Madison, WI, USA) according to themanufacturer’s instructions. The purified PCR productswere sequenced with the same primers (used for PCRamplification) by the dideoxy-mediated chain-terminationmethod using ABI PRISM BigDye® Terminator v3.1 CycleSequencing Kit (Applied Biosystems, Foster City, CA) asdescribed by the manufacturer. Sequences were analyzedwith an automated nucleic acid analyzer (ABI PRISM3100; Applied Biosystems). To generate reliable consen-sus, each DNA fragment was sequenced at least twice inboth the directions.

Phylogenetic analysisSequence assembly and editing were performed using theSEQMAN program from DNASTAR Lasergene suite 9(version 9.0.4 39; DNASTAR, Inc., Madison, WI, USA). Todetermine the phylogenetic relationships between APMV-1viruses reported here and characterized previously fromAsia and other parts of the world, a sequence stretch of the373 bases in the F gene were retrieved from GenBank(http://www.ncbi.nlm.nih.gov). Sequences representingeach known lineage were included in the analysis [7,21].Known strains of NDV, representing each lineage/sub-lineage, were included in the trees to determine the distri-bution and clustering pattern of NDV strains studied in thismanuscript. Furthermore, previously characterized stains ofNDV were supported with accession numbers to facilitate

Figure 1 Sampling sites and geographical distribution of Newcastle disease virus lineages in Pakistan.

Shabbir et al. Virology Journal 2013, 10:170 Page 4 of 10http://www.virologyj.com/content/10/1/170

further reading of a specific isolate. All sequences werealigned in BioEdit version 5.0.6 [22] using ClustalW andtrimmed to equal length. A phylogenetic tree was thenconstructed using Bayesian Inference with the programMrBayes version 3.1.2 [23]. Two independent Monte CarloMarkov Chain (MCMC) chains were executed and sampledevery 1000 generations using the default parameters of thepriors’ panel. Once chains reached convergence (standarddeviation values below 0.01), four million additional genera-tions of the MCMC were run. Trees saved in this last stepwere used to construct a majority rule consensus tree. Theanalysis was based on the GTR + I +G model, which allowsignificantly changed posterior probability estimates. Thenomenclature, based on lineages, was used in this study asdescribed by Aldous et al. (2003). To further assess the gen-etic pattern in the tree, same sequences of the F genes wereused for construction of phylogenetic tree using theneighbor-joining method (Kimura 2 parameter) with 2000bootstrap replicates in MEGA4 software (CEMI, Tempe,AZ, USA) [24]. Finally, the labeling of the trees was madein FigTree v3.1.3.

Determination of recombination eventsSix methods (RDP, GeneConv, BootScan, MaxChi,Chimaera and SiScan) integrated in the RDP v3 program[25] were applied on the NDV sequences reported here to

estimate any recombination event and to detect any puta-tive recombination breakpoint. These methods were ap-plied using following parameters: window size = 20, highestacceptable P-value = 0.001 and Bonferroni correction. Forreliable results, any putative recombination events detectedby more than one method were considered.

Sequence submissionAll the sequences (n = 23) used in this study were sub-mitted to GenBank and are available under accessionnumbers from KC191601 to KC191623.

ResultsReal-time PCR screening of clinical samplesOut of all samples analyzed, a total of 23 samples werefound positive in both F and M gene based real-timePCRs. The results indicated that real-time PCR detectedthe samples from both commercial and rural poultry fromall provinces. However, wild captive birds that were col-lected only from Punjab province were appeared positive.A detailed description of the collected, positive and nega-tive samples is provided in Table 1. Majority of positivesamples showed Ct values between 25 and 30. Results in-dicated that the FTA Indicators functioned as an appropri-ate sampling system for shipment at ambient temperature.All the samples with Ct values lower than 35 (threshold

Shabbir et al. Virology Journal 2013, 10:170 Page 5 of 10http://www.virologyj.com/content/10/1/170

for positive samples) were used in conventional PCR forthe amplification of F gene and subsequent sequencing.

Phylogenetic relationshipThe Bayesian phylogenetic tree was constructed on 509sequences, which were used by Aldous et al. (2003) andCattoli et al. (2010) for the classification of NDV strainsinto six different lineages [7,21]. All previously charac-terized NDV isolates from Pakistan were also includedin the phylogenetic analysis to ascertain the genetic di-versity with the isolates characterized here and reportedfrom rest of the world. The topology of the phylogenetictree indicated the division of NDV strains clearly into sixdistinct lineages (Figure 2). Majority of the PakistaniNDV strains were grouped together and clustered withinlineage 5, close to the previously characterized Pakistaniisolates. Interestingly, the NDV isolates from KhyberPakhtunkhwa province clustered with NDV strains oflineage 4.Since both lineage 4 and 5 are further divided into dif-

ferent sub-lineages, a high-resolution phylogenetic analysiswas conducted including the representative isolates ofeach sub-lineages for both lineage (4 and 5). Analysis indi-cated that all isolates, excepting isolates from KhyberPakhtunkhwa province, clustered close to sub-lineage 5b.

Lineage

Lineage 4

Lineage 5

Isolates studiedin this work

Figure 2 A phylogenetic analysis of the partial sequence of F gene restudy are colored red in both lineage 5 and lineage 4.

However, all these Pakistani isolates present a separatebranch (Figure 3). The isolates from Khyber Pakhtunkhwaprovince (n = 4) were clustered with isolates belonging tosub-lineage 4c (Figure 4).

Analysis of the amino-acid sequences of F proteinProteolytic cleavage site motifs (amino acids 112–117) forthe F0 protein of the all the isolates, putatively indicatingthe level of pathogenicity, were analyzed. Based onthe cleavage site of the F protein, it was possible to dem-onstrate that all the isolates carry velogenic motif112RRQKRF117 regardless of their phylogenetic distribu-tion. However, three isolates collected from Faisalabad(MM26), Lahore (MM35), and Gujranwala (MM41) carry112RKQKRF117motif at their cleavage sites. Both these mo-tifs are generally identified in the strains of NDV that arehighly virulent in chickens.Comparison of the predicted amino acid sequences of

the complete F gene showed that the seven neutralizingepitopes (D72, E74,A75, K78, A79, L343), believed to becritical for structure and function of F protein, wereconserved and identical in all studied isolates. The po-tential N-glycosylation sites in F-glycoprotein (Asn-X-Ser/Thr or N-X-S/T, where X present any amino acidexcept aspartic acid or proline) were found at

Lineage 6

Lineage 2

Lineage 1

3

presenting all the lineage of NDV. The sequences reported in this

AY

1357

54 1

SA

CK

0018

4 Li

neag

e5d

AY

1357

54 1

SA

CK

0018

4 Li

neag

e5d(

2)92

AY

1756

32 1

AE

OS

9910

1 Li

neag

e5d

AY

1756

84 -

CN

BR

--30

4 Li

neag

e5d

88

SH

X 2

/99

(AF3

7824

5)

AY17

5685

-CN

OS-

-303

Lin

eage

5d

SHD 1/9

9 (A

F378

260)

91

XJ 2/97

(AF37

8253

)

90

GX 1/97 (AF378254)

GX 2/98 (AF378255)

97

GX 3/98 (AF378256)

TW 156/99 (AF234031)

TW 154b/99 (AF326521)TW 157/99 (AF234032)TW 1/98 (AF083963)

TW 4/98 (AF083964)

TW 2/98 (AF083973)

TW 165/99 (AF326523)

TW 156a/99 (AF326522)

TW 174/99 (AF326524)

757279

86

AY135743 1CZCK96048 Lineage5c

TW 9/95 (AF083968)

TW 7/95 (AF083968)

99

TW 159/99 (AF234034)

TW P/96 (AF083971)

TW C

/84 (AF083965)

TW

P/84 (A

F083967)

96

QG

B445/97 (A

F109886)

TW P

/94 (AF083961)

TW

4/95 (AF

083969)T

W 1/95 (A

F083960)

9288

98

AY

1756

82 C

IDC

K96

271

Line

age5

a

D 8

2/94

(A

F00

1117

) )811100F

A(59/ 38

DA

Y17

5746

NC

HH

E94

139

Line

age5

aA

Y17

5749

NE

SC

K94

143

Line

age5

a

NL

2/93

(AF0

0112

5)

AY

1357

51 N

BE

CK

8115

8 Li

neag

e5a

AY17

5747

ND

ECK9

4147

Lin

eage

5a

NL 1/

93 (A

F001

124)

D 85/96

(AF00

1119

)

AY175748 -DECK9623 Lineage5a

AY175687 -DEFO95092 Lineage5a

DE 143/95 (AF109881)

8396

98

7994

AY135752 APECK92173 Lineage5b

AY175677 BMYBU87078 Lineage5b

99AY175678 BMYCK82175 Lineage5b

AY175675 BINCK94170 Lineage5b

80

AY175635 1NIPH97097 Lineage5b

AY175634 1NIPH97041 Lineage5b

97AY175633 1NICK97068 Lineage5b

AY135746 1NICK97069 Lineage5b(2)

AY135746 1NICK97069 Lineage5b

AY175627 1IRPO96043 Lineage5b

AY175690 DINCK96042 Lineage5b75 AY175683 1ITCK00179 Lineage5b87

AY175636 1Q-HO99272 Lineage5b96AY175650 ASEPA90046 Lineage5bAY175652 AESCK90174 Lineage5b

AY175640 1ZAOS95044 Lineage5b

AY175761 -PTTY91146 Lineage5b

70

AY175641 1ZAOS99141 Lineage5b

AY175740 JPTCK94045 Lineage5b

AY175711 FBG--93149 Lineage5b

AY175741 JPTCK96082 Lineage5b

AY175739 JPTBR95104 Lineage5b

79

80

89

88

9798

99

Pak/Balochistan/Quetta/MM38/2012

Pak/Balochistan/Quetta2/MM44/2012

Pak/Punjab/Sheikhupura/MM31/2012

Pak/Punjab/Kasour/MM

37/2012

Pak/Punjab/Sialkot/MM

43/2012

Pak/P

unjab/Pakpattan2/M

M30/2012

Pak/P

unjab/Okara1/M

M25/2012

Pak/P

unjab/Layyah/MM

32/2012

Pak/P

unjab/Sahiw

al/MM

33/2012P

ak/Sindh/K

arachi/MM

39/2012P

ak/Punjab/O

kara2/MM

26/2012

Pak/P

unjab/Okara3/M

M27/2012

Pak/Punjab/Lahore1/MM

34/2012

Pak/Sindh/Karachi2/MM

40/2012

Pak/Punjab/Lahore2/MM

35/2012

Pak/Punjab/Gugranwala/MM41/2012

Pak/Punjab/Faisalabad/MM28/2012

Pak/Punjab/Pakpattan1/MM29/2012

Pak/Punjab/Lahore3/MM36/2012

Pak/Punjab/Multan/MM42/2012

78

99

0.01

Sublineage 5a

Sublineage 5b

Sublineage 5d

Sublineage 5c

Sublineage 5d

Figure 3 A high-resolution phylogenetic tree for lineage 5. Sequences presenting all the sub-lineages within lineage 5 are shown. Isolatesshown in this study formed a separate cluster within sub-lineage 5b.

Shabbir et al. Virology Journal 2013, 10:170 Page 6 of 10http://www.virologyj.com/content/10/1/170

87

100

95

77

94

99

9898

99

AY175756 PIEPI00242 Lineage4b

AY175754 PDEPI99063 Lineage4b

AY175770 PUKPI99064 Lineage4b

AY175772 PUKPI99130 Lineage4b

AY1757

73 P

UKPI9913

1 Line

age4

b

AY17

5779

PU

KPI9

9065

Lin

eage

4b

AY

1757

71 P

UK

PI9

9128

Lin

eage

4b

AY

1757

52 P

DE

CT9

5204

Lin

eage

4b

AY

1757

59 P

ITTD

0017

7 Li

neag

e4b

AY

1757

53 P

DE

PI9

4216

Lin

eage

4b

b4egaeni L11159

KC

RT-

677571Y

A

AY

1757

60 P

SA

PI9

8201

Lin

eage

4b

AY

1757

51 P

AE

PI9

6210

Lin

eage

4b

AY

1357

49 P

DE

PI9

4102

Lin

eage

4b

AY

1757

66 P

UK

PI8

4125

Lin

eage

4b

AY

1757

55 P

DK

PI9

5103

Lin

eage

4b

AY17

5769

PU

KPI9

0126

Lin

eage

4b

AY1757

67 P

UKPI882

84 L

inea

ge4b

AY1757

58 P

ITPH95

294

Linea

ge4b

AY1757

78 -U

KPI891

16 L

ineag

e4b

AY175768 PUKPI89124 Lineage4b

AY175765 PUKPI83299 Lineage4b

AY175764 PUKPI83279 Lineage4bAY175757 PIEPI96302 Lineage4b

97

86

AY175763 PTRPI95107 Lineage4a

100

AY175738 JAEFA96038 Lineage4a

AY135748 JAESA90099 Lineage4a

AY135748 JAESA90099 Lineage4a

91 100

HLJ 4/95 (AF378259)

AY175655 GTZDK95058 Lineage4a

JL 2/97 (AF400615)

BJ-3/97 (A

F400616)

99

AY175673 BHKPI89172 Lineage4d

93

TW 1

54/9

9 (A

F2340

30)

HUN 1/9

5 (A

F378

258)

78

H 3

10/8

2 (A

F001

112)

CH

1/9

5 (A

F001

132)

AY

1357

42 B

BG

PI9

5039

Lin

eage

4d

S 1

/95

(AF0

0113

1)

DK

1/9

5 (A

F00

1129

)D

K 6

/95

(AF

0011

30)

7296

99

AY175667 BAEPE92085 Lineage4c

AY175680 BSACK89036 Lineage4c

AY175668 BAEPI99109 Lineage4c

AY175665 BAEFA99142 Lineage4c

c4egaeniL43089SOEAB666571YAAY175663 BAECK95088 Lineage4c

AY175664 BAEHO95114 Lineage4c

AY175662 BAECK92084 Lineage4c

Pak/KPK/Abbottabad1/MM21/2012

Pak/KPK/Abbottabad2/MM22/2012

Pak/KPK/Mansehra1/MM23/2012

Pak/KPK/Mansehra2/M

M24/2012

100

9990

0.01

Sublineage 4c

Sublineage 4b

Sublineage 4a

Sublineage 4d

Figure 4 A high-resolution phylogenetic tree for lineage 4. Sequences presenting all the sub-lineages within lineage 4 are shown. Isolatesshown in this study clustered within sub-lineage 4c.

Shabbir et al. Virology Journal 2013, 10:170 Page 7 of 10http://www.virologyj.com/content/10/1/170

position85NRT87, 191NNT193, 366NTS368, 447NIS449,471NNS473 and 541NNT543. Comparison of thecomplete F gene sequences among all the lineages showedthat the studied strains of NDV shared highest nucleotideand predicted amino acid (95.7% and 91.3%) identity withthe lineage5 whereas lowest (86.5% and 90.9%) withlineage 1 except for four isolates belonging to lineage 4.

Recombination among F genes of NDV isolatesEvidence of recombination among poultry and ostrichNDV has been reported [26-28]. Recombination analysesperformed on the F genes of the NDV isolates belonging

to all lineages reported before and isolates characterizedhere appear to lack any recombination events.

DiscussionGeographically, Pakistan is located (33°40′N and 73°10′E) at the crossroads of the strategically important re-gions of South, Central and Western Asia (Figure 1).From 2009 to mid 2012, a number of outbreaks ofNewcastle disease have been reported to WorldOrganization for Animal Health (OIE-WAHID inter-face, available at: http://www.oie.int/wahis/public.php)from Pakistan as well as neighboring countries. Most ofthe outbreaks have been reported from Iran and India

Shabbir et al. Virology Journal 2013, 10:170 Page 8 of 10http://www.virologyj.com/content/10/1/170

that shares border with Pakistan. Despite some of the re-ports from selective regions only and emergence of novelNDV (5i) from Pakistan [9], it is of the essence to screenand characterize the NDV throughout the country.The F gene sequence data of all the analyzed samples

clustered virulent NDVs among the lineage 5 (sub-lineage5b) except KPK province, where the ND strains were clus-tered together in lineage 4 (sub-lineage 4c). Throughoutthe world, the NDVs belonging to lineage 5 are consideredto be the one involved in outbreaks in Far East [29-31]South Africa [32] and Europe [8]. Among many Asiancountries and particularly those that shares border withPakistan, there have been found and characterized numberof velogenic NDVs within class II that belongs to variouslineage and sub-lineages [7,33-35]; however, the dominantone is found to be lineage 5. Likewise, NDVs belonging tovarious sub-lineage like 5a, 5b, and novel 5i has beenreported from Pakistan from different type of birds re-cently [9,11,16]. Contrary to recent study analysis of gen-etic nature of circulating NDVs except those of KPKprovince, where sub-lineage 5b was found circulatingamong rural, commercial and wild birds, we have seen aclear distinction between the viruses isolated from varioustypes of birds, wild and commercial in the recent past.The isolates from wild birds were clustered within sub-lineage 5a and closely related to Indonesian isolate(AY562985) [15,17] while the ones from commercial andrural poultry were found within sub-lineage 5b, novel 5iand closely related to Swedish (GU585905) and Russianisolates (AY865652) [17,19,20].Since 2005, lineage 4 (sub-lineage 4c) has been found

now and from a different geographical region,Abbottabad and Mansehra districts of KPK province.Previously, the lineage 4 was isolated and characterizedfrom areas in and around coastal border of Pakistan[14]. The re-emergence of lineage 4 (sub-lineage 4c)from KPK province might be attributed either to the factthat viruses have not been characterized from this par-ticular region before or either to the movement of mi-gratory/caged birds across the country or from north tosouth involving Europe, Asia and Middle East. Historic-ally, lineage 4 has been isolated and characterized amongdomestic fowls, ostrich, falcons and pigeons from middleeast countries (United Arab Emirates and Saudi Arabia),Asian (Japan and China) and European countries(UK, Italy, Peru and Belgium) [1,34]; however, most ofNDVs belonging to sub-lineage 4c have been reportedfrom wild captive and domestic fowls from United ArabEmirates [7]. Isolation and characterization of NDV (JP/Chiba-pa/97) from parakeets exported to Japan in 1997[29] gives an evidence that lineage 4 is present inPakistan since 1997. Since it is the only report from past,it could be hypothesized that lineage 4 is present evenbefore and is still circulating in the environment.

Further, it is imperative to describe the fact that sampleswere collected during a designated period of six monththroughout the country and all of the clinical outbreaksare not necessarily included in the study. This meansthat there may exist more diversity in NDV strainsin Pakistan and therefore, presence of lineage 4 (sub-lineage 4c) in already reported area (Karachi) or otherscannot be ignored. The isolation and subsequentcharacterization of isolates from turkey and domesticfowls in UK (Q-GB506/97) [1], exported parakeets fromPakistan to Japan (JP/Chiba-pa/97) [29], and from pigeonsin china (C/98-1) [34] which clustered together with Ital-ian exotic isolate (IT-148/94) [8] in lineage 4 provides apossible epidemiological transmission through migratorybirds from north to south. Considering migratory/cagedbirds as reservoir/carrier of NDVs in an inapparent formand the potential of viruses to infect multiple avian specieswithout prior adaptation [1,2,26,36], raises the concerns inworldwide distribution of velogenic pathotypes throughtrading and migration of birds across regional and inter-national boundaries.Recent outbreaks of ND in Pakistan are supposed to be

due to a breach in the biosecurity measures. However, therole of vaccines in providing protection against the fieldchallenge also needs to be evaluated. Inability of live vac-cines to elicit protective immune response might be due toseveral reasons like improper cold chain supply system, in-appropriate route of vaccination, or uneven vaccinationschedules. Administration of both live and inactivated NDvaccine could be practiced to protect the birds from viru-lent NDVs [37]. Presently, lentogenic (LaSota) or mesogenicstrains (Muktesewar) of NDVs are being used to vaccinatethe birds in Pakistan. However, it is still a matter of ques-tion whether these vaccinal strains are able to elicit a pro-tective immune response against the prevailing field strainswith high genetic gap in relation to vaccinal strains asevidenced and reported previously [9]. It has been reportedthat the currently practiced NDV vaccines give better pro-tection against the velogenic NDVs isolated in 1930 to 70s(Herts33/56, California 71) than the ones, which have beenisolated in past few years [5,9]. Hence parameters for selec-tion of a vaccinal strain are needed to be reconsidered. Fur-thermore, monitoring the immune response of birds toNDV vaccines along with strict biosecurity measures shouldbe employed to keep the flocks free of vNDVs infection.

ConclusionsSimultaneous detection of multiple velogenic strains ofNDV (lineage 4 and 5) from various regions of the countrywarrants continuous isolation and molecular epidemio-logical investigations involving wild/caged birds. Vaccinestrategies that can elicit high antibody response along withcontinuous monitoring from the laboratory coupled withimproved biosecurity measures in the form is suggested.

Shabbir et al. Virology Journal 2013, 10:170 Page 9 of 10http://www.virologyj.com/content/10/1/170

AbbreviationsNDV: Newcastle disease virus; F: Fusion protein; M: Matrix protein; APMV-1: Avianparamyxoviruses serotype 1; KPK: Khyber Pakhtunkhwa.

Competing interestsThe authors declare that they have no competing interests.

Authors’ contributionsMZS, TY, JN, MABS, MS, MS, MA, MA, MTK, AAA, AG, AA, AAC, AAA, MZK, AA,MUG, ZI, HA, KZ, NM, SAK, NA, WY and MUS collected samples from variousgeographical areas of Pakistan. MZS, MM wrote the manuscript. MB, SZdiscussed and reviewed the manuscript. MM and MZS designed the manuscriptand analyzed the data. All authors read and approved the final manuscript.

AcknowledgmentsThe authors would like to thank the field veterinarians and poultry farmerswho helped sample collection from the mentioned districts of each provinceof Pakistan.

Author details1Quality Operations Laboratory, University of Veterinary and Animal Sciences,Lahore, Pakistan. 2National Veterinary Institute, Uppsala, Sweden.3Department of Microbiology, University of Veterinary and Animal Sciences,Lahore, Pakistan. 4University of Baluchistan, Quetta, Pakistan. 5VeterinaryResearch and Disease Investigation Center, Abbottabad, Pakistan. 6Livestockand Dairy Development Department, Punjab, Pakistan. 7Veterinary ResearchInstitute, Lahore, Pakistan. 8Department of Biology, University of Bergen,Bergen, Norway. 9Poultry Research Institute, Rawalpindi, Pakistan. 10UniversityDiagnostic Laboratory, University of Veterinary and Animal Sciences, Lahore,Pakistan. 11Department of Microbiology, Chittagong Veterinary and AnimalSciences University, Chittagong, Bangladesh. 12Department ofTheriogenology, University of Veterinary and Animal Sciences, Lahore,Pakistan. 13Sindh Poultry Vaccine Center, Karachi, Pakistan. 14SwedishUniversity of Agricultural Sciences, Uppsala, Sweden. 15Department ofGeography, Government College University, Lahore, Pakistan. 16GovernmentCollage University Faisalabad, Faisalabad, Pakistan. 17Department ofParasitology, University of Veterinary and Animal Sciences, Lahore, Pakistan.18The Pirbright Institute, Compton Laboratory, Compton, Berkshire RG207NN, United Kingdom.

Received: 11 March 2013 Accepted: 29 May 2013Published: 30 May 2013

References1. Alexander D: Newcastle disease, other avian paramyxoviruses, and

pneumovirus infections. In Diseases of Poultry. 4th edition. Edited by SaifYM, Barnes HJ, Glisson JR, Fadly AM LRM, Swayne D. Ames: Iowa StateUniversity Press; 2003:63–99.

2. Miller PJ, Decanini EL, Afonso CL: Newcastle disease: evolution ofgenotypes and the related diagnostic challenges. Infect Genet Evol 2010,10:26–35.

3. Seal BS, King DJ, Meinersmann RJ: Molecular evolution of the Newcastledisease virus matrix protein gene and phylogenetic relationships amongthe paramyxoviridae. Virus Res 2000, 66:1–11.

4. OIE: Newcastle disease. In Chapter 2115 OIE Manual of Standards forDiagnostic Tests and Vaccines, in Manual of Diagnostic Tests and Vaccines forTerrestrial Animals: Mammals, Birds and Bees. Paris: Office International desEpizooties; 2004:270–282.

5. Czegledi A, Ujvari D, Somogyi E, Wehmann E, Werner O, Lomniczi B: Thirdgenome size category of avian paramyxovirus serotype 1 (Newcastledisease virus) and evolutionary implications. Virus Res 2006, 120:36–48.

6. Kolakofsky D, Roux L, Garcin D, Ruigrok RW: Paramyxovirus mRNA editing,the “rule of six” and error catastrophe: a hypothesis. J Gen Virol 2005,86:1869–1877.

7. Aldous EW, Mynn JK, Banks J, Alexander DJ: A molecular epidemiologicalstudy of avian paramyxovirus type 1 (Newcastle disease virus) isolatesby phylogenetic analysis of a partial nucleotide sequence of the fusionprotein gene. Avian Pathol 2003, 32:239–256.

8. Herczeg J, Wehmann E, Bragg RR, Travassos Dias PM, Hadjiev G, Werner O,Lomniczi B: Two novel genetic groups (VIIb and VIII) responsible for

recent Newcastle disease outbreaks in Southern Africa, one (VIIb) ofwhich reached Southern Europe. Arch Virol 1999, 144:2087–2099.

9. Munir M, Cortey M, Abbas M, Qureshi ZU, Afzal F, Shabbir MZ, Khan MT,Ahmed S, Ahmad S, Baule C, et al: Biological characterization andphylogenetic analysis of a novel genetic group of Newcastle diseasevirus isolated from outbreaks in commercial poultry and from backyardpoultry flocks in Pakistan. Infect Genet Evol 2012, 12:1010–1019.

10. Diel DG, Susta L, Cardenas Garcia S, Killian ML, Brown CC, Miller PJ, AfonsoCL: Complete genome and clinicopathological characterization of avirulent Newcastle disease virus isolate from South America. J ClinMicrobiol 2012, 50:378–387.

11. Munir M, Shabbir MZ, Yaqub T, Shabbir MA, Mukhtar N, Khan MR, Berg M:Complete genome sequence of a velogenic neurotropic avianparamyxovirus 1 isolated from peacocks (Pavocristatus) in a wildlife parkin Pakistan. J Virol 2012, 86:13113–13114.

12. Sadiq M: Pakistan poultry sector still on an upward swing. World Poult2004, 20:10–11.

13. Numan M, Zahoor MA, Khan HA, Siddique M: Serological status ofNewcastle disease in broilers and layers in Faisalabad and surroundingdistricts. Pak Vet J 2005, 25:55–58.

14. Khan TA, Rue CA, Rehmani SF, Ahmed A, Wasilenko JL, Miller PJ, Afonso CL:Phylogenetic and biological characterization of Newcastle disease virusisolates from Pakistan. J Clin Microbiol 2010, 48:1892–1894.

15. Munir M, Abbas M, Khan MT, Zohari S, Berg M: Genomic and biologicalcharacterization of a velogenic Newcastle disease virus isolated from ahealthy backyard poultry flock in 2010. Virol J 2012, 9:46.

16. Munir M, Zohari S, Abbas M, Berg M: Sequencing and analysis of thecomplete genome of Newcastle disease virus isolated from acommercial poultry farm in 2010. Arch Virol 2012, 157:765–768.

17. Shabbir MZ, Abbas M, Yaqub T, Mukhtar N, Subhani A, Habib H, Sohail MU,Munir M: Genetic analysis of Newcastle disease virus from Punjab,Pakistan. Virus Genes 2012, 46:309–315.

18. Wise MG, Suarez DL, Seal BS, Pedersen JC, Senne DA, King DJ, KapczynskiDR, Spackman E: Development of a real-time reverse-transcription PCRfor detection of newcastle disease virus RNA in clinical samples. J ClinMicrobiol 2004, 42:329–338.

19. Munir M, Linde AM, Zohari S, Stahl K, Baule C, Engstrom B, LH MR, Berg M:Whole genome sequencing and characterization of a virulent Newcastledisease virus isolated from an outbreak in Sweden. Virus Genes 2011,43:261–271.

20. Munir M, Linde AM, Zohari S, Stahl K, Baule C, Holm K, Engstrom B, Berg M:Complete genome analysis of an avian paramyxovirus type 1 strainisolated in 1994 from an asymptomatic black-headed gull (Larusridibundus) in southern Sweden. Avian Dis 2010, 54:923–930.

21. Cattoli G, Fusaro A, Monne I, Molia S, Le Menach A, Maregeya B, Nchare A,Bangana I, Maina AG, Koffi JN, et al: Emergence of a new genetic lineageof Newcastle disease virus in West and Central Africa–implications fordiagnosis and control. Vet Microbiol 2010, 142:168–176.

22. Hall T: BioEdit: a user-friendly biological sequence alignment editor andanalysis program for Windows 95/98/NT. Nucleic Acids Sym Ser 1999,41:95–98.

23. Ronquist F, Huelsenbeck JP: MrBayes 3: Bayesian phylogenetic inferenceunder mixed models. Bioinformatics 2003, 19:1572–1574.

24. Tamura K, Dudley J, Nei M, Kumar S: MEGA4: Molecular EvolutionaryGenetics Analysis (MEGA) software version 4.0. Mol Biol Evol 2007,24:1596–1599.

25. Martin DP, Lemey P, Lott M, Moulton V, Posada D, Lefeuvre P: RDP3: aflexible and fast computer program for analyzing recombination.Bioinformatics 2010, 26:2462–2463.

26. Miller PJ, Kim LM, Ip HS, Afonso CL: Evolutionary dynamics of Newcastledisease virus. Virology 2009, 391:64–72.

27. Qin Z, Sun L, Ma B, Cui Z, Zhu Y, Kitamura Y, Liu W: F gene recombinationbetween genotype II and VII Newcastle disease virus. Virus Res 2008,131:299–303.

28. Yin Y, Cortey M, Zhang Y, Cui S, Dolz R, Wang J, Gong Z: Molecularcharacterization of Newcastle disease viruses in Ostriches(Struthiocamelus L.): further evidences of recombination within avianparamyxovirus type 1. Vet Microbiol 2011, 149:324–329.

29. Mase M, Imai K, Sanada Y, Sanada N, Yuasa N, Imada T, Tsukamoto K,Yamaguchi S: Phylogenetic analysis of Newcastle disease virus genotypesisolated in Japan. J Clin Microbiol 2002, 40:3826–3830.

Shabbir et al. Virology Journal 2013, 10:170 Page 10 of 10http://www.virologyj.com/content/10/1/170

30. Qin ZM, Tan LT, Xu HY, Ma BC, Wang YL, Yuan XY, Liu WJ: Pathotypicalcharacterization and molecular epidemiology of Newcastle disease virusisolates from different hosts in China from 1996 to 2005. J Clin Microbiol2008, 46:601–611.

31. Tan LT, Xu HY, Wang YL, Qin ZM, Sun L, Liu WJ, Cui ZZ: Molecularcharacterization of three new virulent Newcastle disease virus variantsisolated in China. J Clin Microbiol 2008, 46:750–753.

32. Abolnik C, Horner RF, Bisschop SP, Parker ME, Romito M, Viljoen GJ: Aphylogenetic study of South African Newcastle disease virus strainsisolated between 1990 and 2002 suggests epidemiological origins in theFar East. Arch Virol 2004, 149:603–619.

33. Tirumurugaan KG, Kapgate S, Vinupriya MK, Vijayarani K, Kumanan K,Elankumaran S: Genotypic and pathotypic characterization of Newcastledisease viruses from India. PLoS One 2011, 6:e28414.

34. Yu L, Wang Z, Jiang Y, Chang L, Kwang J: Characterization of newlyemerging Newcastle disease virus isolates from the People’s Republic ofChina and Taiwan. J Clin Microbiol 2001, 39:3512–3519.

35. Zhang S, Wang X, Zhao C, Liu D, Hu Y, Zhao J, Zhang G: Phylogenetic andpathotypical analysis of two virulent Newcastle disease viruses isolatedfrom domestic ducks in China. PLoS One 2011, 6:e25000.

36. Clavijo A, Robinson Y, Booth T, Munroe F: Velogenic Newcastle disease inimported caged birds. Canadian Vet J 2000, 41:404–406.

37. Senne DA, King DJ, Kapczynski DR: Control of Newcastle disease byvaccination. Dev Biol 2004, 119:165–170.

doi:10.1186/1743-422X-10-170Cite this article as: Shabbir et al.: Genetic diversity of Newcastle diseasevirus in Pakistan: a countrywide perspective. Virology Journal 2013 10:170.

Submit your next manuscript to BioMed Centraland take full advantage of:

• Convenient online submission

• Thorough peer review

• No space constraints or color figure charges

• Immediate publication on acceptance

• Inclusion in PubMed, CAS, Scopus and Google Scholar

• Research which is freely available for redistribution

Submit your manuscript at www.biomedcentral.com/submit