Akhtar et al. SpringerPlus (2016) 5:1295 DOI 10.1186/s40064-016-2939-1

SHORT REPORT

Genetic characterization and phylogeny of pigeon paramyxovirus isolate (PPMV-1) from PakistanSameera Akhtar1, Muhammad Akram Muneer1, Khushi Muhammad1, Muhammad Yasin Tipu2, Masood Rabbani1, Aziz ul‑Rahman1 and Muhammad Zubair Shabbir3*

Abstract

Background: Knowing the genome characteristics of circulating Newcastle disease viruses [avian paramyxoviruses (APMV‑1) and pigeon paramyxoviruses (PPMV‑1)] is important to devise appropriate diagnostics and control strate‑gies. APMVs originating from chicken and wildlife in Pakistan are well‑elucidated; nevertheless, molecular characteri‑zation for the circulating PPMV‑1 is largely unknown.

Findings: Here, we have performed fusion (F) and hemagglutinin (HN) gene based characterization of PPMV‑1 iso‑lated from an outbreak in a pigeon flock. With F0 proteolytic cleavage site (112RRQKR↓F117), characteristic of velogenic/mesogenic serotype, the complete F and HN gene based sequence analysis of the isolate revealed evolutionary relationship to genotype VI. Further analysis of hyper‑variable region of F‑gene demonstrated clustering of the study isolate with genotype VIb. The deduced residue analysis for both F and HN protein showed a number of substitu‑tion mutations in the functional domains distinct from representative strains of each genotype including the vaccine strains; some of them were found exclusive to the study isolate.

Conclusions: Though limited and preliminary data, the findings enhance our knowledge towards circulating strains of PPMVs in Pakistan. Further studies are needed to ascertain its potential for transmission in the wild birds, commer‑cial and backyard poultry and its subsequent shedding into the environment.

Keywords: Pigeon paramyxovirus type 1 (PPMV‑1), Newcastle disease virus, Pigeon, Pakistan

© 2016 The Author(s). This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

BackgroundPigeon Paramyxovirus type 1 (PPMV-1) is an antigenic and host variant of classical Newcastle Disease virus (NDV) or Avian Paramyxovirus type 1 (APMV-1) that causes Newcastle Disease (ND)-like infection and pathol-ogy in pigeons (Ujvari et al. 2003). Including the earliest known PPMV-1 (Iraq78) originating from the Middle-East (ME), and the strains responsible for third panzootic in 80 s involving a pigeon-adopted APMV-1 are consid-ered likely to be derived from these ME viruses (Kaleta et  al. 1985; Ujvari et  al. 2003) and as yet continue to

circulate around the globe. The virus has an approximate genomic length of 15192 nt and, despite characteristic F protein cleavage site for velogenic strains (112GRQKRF117 or 112RRKKRF117 or 112RRQKRF117), differences in patho-genicity index range from moderate to no virulence for chicken (Collins et al. 1994; Dortmans et al. 2010). How-ever in recent years, some PPMV-1 have been reported to be highly pathogenic for chicken after passage either in chicken or chicken embryo indicating their potential to cause ND outbreaks (Dortmans et  al. 2011). Today, ND caused by either virulent APMV-1 or PPMV-1 is considered endemic to feral and domestic pigeon (Columbiformes) worldwide (Aldous et  al. 2004). Clini-cal symptoms differ depending upon host immune titre and virulence of isolate involved (Dortmans et al. 2011). Most of the times, the observed clinical symptoms relate

Open Access

*Correspondence: shabbirmz@uvas.edu.pk 3 Quality Operations Laboratory, University of Veterinary and Animal Sciences, Lahore 54600, PakistanFull list of author information is available at the end of the article

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to neurotropic form of ND including tremors, torticollis and disturbed equilibrium, however naturally infected pigeon also exhibit respiratory symptoms such as sneez-ing, coughing and tracheal rales (Marlier and Vindevogel 2006).

Since the establishment of well-organized poultry sector in Pakistan to-date, there have been a number of epidemics of ND in commercial poultry, wild birds and domestic pigeons. Although, APMV-1 from com-mercial poultry and wild birds have been isolated and well-characterized as genotype VI and VII (Munir et  al. 2012; Shabbir et al. 2012, 2013a, b), despite a number of clinically suspected outbreaks in pigeons including vac-cinated ones (Lab data, not published), there is absolute paucity of information pertaining to circulating lineage of PPMV-1 and its nucleotide and amino acid profile to reference and vaccinal strains. We analysed complete F and HN genes of an isolate recovered from ND outbreak in a pigeon flock. The obtained sequences were processed for phylogeny and amino acid residue analyses giving an insight towards genetic diversity of the indigenous strain and its comparative evolution to those reported earlier.

MethodsA Newcastle disease outbreak occurred in a flock of racing pigeons (n  =  56) in district Lahore, Pakistan (31.5790°N, 74.3096°E) during December, 2014. Four days post appearance of clinical symptoms, 13 died and 29 were morbid. The clinical symptoms observed in affected pigeons were circling movement and tremors while some birds were also exhibiting mild respiratory symptoms such as sneezing, coughing and nasal discharge. Nec-ropsy was performed and samples (trachea, lungs, and brain) were processed for isolation of NDV in 9 day old embryonated chicken eggs (ECE). Presence of aggluti-nating virus was confirmed in harvested allantoic fluid by spot agglutination assay using 10 % chicken red blood cells (RBCs). Later, identity of the isolate was confirmed as NDV through standard haemagglutination inhibition assay using specific antisera.

Total RNA was extracted from allantoic fluid using commercially available RNA extraction kit as per manu-facturer’s instructions (QIAamp Viral RNA Mini Kit, Qiagen, USA). Quantity (NanoDrop, USA) and quality (QubitFlourometer, USA) of extracted RNA was meas-ured. The extracted RNA was subjected to amplifica-tion of complete F and HN genes spanning the genomic region from 4498 to 6330 nucleotide through reverse transcription polymerase chain reaction (RT-PCR) using the primers and protocols as described previously (Munir et al. 2010). The amplified products were purified by 1.0 % gel electrophoresis, using the Wizard® SV Gel and PCR Clean-Up System (Promega, Co., Madison, WI, USA) as

per manufacturer’s instructions. Using the same prim-ers (as used previously for F and HN gene amplification) and ABI PRISM BigDye Terminator version 3.1 (Applied Biosystems, Foster City, CA, USA), the purified genomic DNAs were processed for sequencing reaction on a 3100 DNA Analyzer (Applied Biosystems, Foster city, CA, USA). Each genomic fragment was sequenced twice in both forward and reverse directions to generate a reliable consensus sequence. The consensus sequences of F and HN genes of the study isolate (Pi/MZS1-UVAS/2014) has been submitted to GenBank under the accession number KU644586 and KU644587, respectively.

The obtained sequences and sequences reported earlier (GenBank) were aligned in BioEdit version 5.0.6 (Hall, 1999) using ClustalW and cut to equal lengths. Phylo-genetic relationships of complete F, HN gene and hyper-variable region of F gene of study isolate were elucidated to the corresponding region of previously characterized viruses around the globe (http://www.ncbi.nlm.nih.gov/nuccore/?term=partial+F+gene+of+Newcastle+disease+virus) at the level of genotype and sub-genotype using the MEGA version 6.0 software (Tamura et al. 2013). The evolutionary distances were inferred and expressed based on the number of nucleotide substitutions per site. The codon positions included in the analysis were the 1st, 2nd, 3rd, and noncoding. All positions containing gaps and missing data were eliminated from the data set (the “complete deletion” option). Furthermore, compara-tive residue analysis of the representative strains of each known genotype was analysed through BioEdit.

Results and discussionAgglutination of harvested virus from allantoic fluid with chicken RBCs (1:128) and subsequent inhibition with specific antisera (1:128) confirmed the presence of NDV. Since a particular emphasis is given to F and HN gene-based molecular characterization of NDV strains in the previous studies (Toyoda et  al. 1988; Yussof and Tan, 2001; Aldous et  al. 2003; Ujvari et  al. 2003; Kim et al. 2008; Munir et al. 2012; Shabbir et al. 2012), we also used complete F and HN gene as well as hyper-variable region of F-gene (47–421 nt, 375 bp) to determine phy-logenic relationship of the study isolate to previously known representative strains of APMV-1 and PPMV-1 around the globe at the level of genotype and sub-gen-otype. Based upon complete F and HN gene phylogeny, the study isolate clustered within genotype VI close to an isolate reported previously from Russia (Pi/Rus/Vladimir/687/05) (Fig.  1a, b). Further analysis of hyper-variable region of F gene revealed clustering of the study isolate to sub-genotype VIb (Fig.  1c). The clustering of study isolate close to the ones from Russia highlights potential ancestor or origin of the virus that may involve

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WF00D WF00G BP01 Muscovy duck/China(Fujian)/FP1/02 ZJ1 SF02 ch/China/JSX1/2010 YZCQ/Liaoning/08 KBNP-C4152R2L GM WF00C SRZ03 ND/03/018 ch/Sukorejo/019/10 cockatoo/Indo/14698/90 ch/Banjarmasin/010/10 goose/Guangdong/2010 GD1003/2010 GD450/2011 ch/Sweden/97 NDV2K35/CH/TN/2003 ch/CP/PK/2010

Genotype VII

ch/U.S.(CA)/1083(Fontana) Ch Sweden 95

Pi/MZS1-UVAS/2014 Pi/Rus/Vladimir/687/05 AV324/96 dove/Italy/2736/00 pi/CH/LJL/100605 pi/CH/LHLJ/110813 pi/CH/LHLJ/110822 pi/CH/LGD/110947 pi/CH/LGD/110945 pi/CH/LJL/120404 pi/CH/LGD/110208 P4 PPMV-1/Belgium/98-321/1998 1.3

Genotype VI

QH1 QH4 AF2240-I

Genotype VIII

NDV-P05 01/10 mixed sp/U.S./Largo/71 APMV-1/ch/Brazil/SJM/75 gamefowl/U.S.(CA)/211472/02 cormorant/US(MN)/92-40140/1992 anhinga/U.S.(Fl)/44083/93 cormorant/US(CA)/92-23071/1997 cormorant/US(CA)/D9704285/1997 cormorant/US(NV)/19529-04(USGS)/2005 cormorant/US(WI)/18719-03(USGS)/2003 cormorant/Canada/98CNN3-V1125/1998 cormorant/Canada/95DC02150/1995 cormorant/Canada/95DC2345/1995

Genotype V

NDV/2K36/peacock/Chennai NDV-2/ch/Namakkal/Tamil Nadu 2K3/Chennai/Tamil Nadu

Genotype IV

MG 1992 MG 725 08 MG MEOLA 08

Genotype XI

F48E8 FJ/1/85/Ch GD09-2 ZJ/1/86/Ch JS/1/97/Ch JS/1/02/Du XBT14 HBNU/LSRC/F3

Genotype IX

mallard/US(MN)/99-376/1999 mallard/US(MN)/99-376/1999 mallard/US(MN)/00-32/2000 mallard/US(MD)/03-632/2003 mallard/US(OH)/04-411/2004 mallard/US(MN)/MN00-39/2000 mallard/US(OH)/86-233/1986 pintail/US(OH)/87-486/1987 mottled duck/US(TX)/01-130/2001

Genotype X

APMV-1/ch/U.S.(TX)/GB/1948 NDV/Ch/Egypt/1/2005 B1 isolate Takaaki LaSota-1 LaSota-2

Genotype II

ch/N.Ireland/Ulster/67 D3 BHG/Sweden/94 I-2progenitor I-2 TS09-C 98-1249 98-1252 98-1154 99-0868hi 99-0868lo 02-1334 99-0655 99-1435 99-1997PR-32 01-1108

Genotype I

Class II

Teal/France/100011/2010 JS10 DE-R49/99 Class I

100

100

100

100

100

100

100

100

100

100100

100

100

100

100

100

100

99

99

99

99

99

97

96100

95100

93

90100

88

87

85

96

81

79

77

66

62

60100

56

55

5470

88

53

4964

100

100

100

63

49

44

42

96

100

84

86

80

40

39

33

50

87

100

100

87

3124

35

61

83

99

99

99

51

99

80

22

27

59

70

95

80

81

83

84

81

90

46

73

100

4642

20

1215

BP01 Muscovy duck/China(Fujian)/FP1/02 WF00D SF02 ZJ1 SRZ03 ch/China/JSX1/2010 YZCQ/Liaoning/08 WF00G GM WF00C KBNP-C4152R2L ND/03/018 cockatoo/Indo/14698/90 ch/Banjarmasin/010/10 ch/Sukorejo/019/10 goose/Guangdong/2010 GD 1003/2010 GD450/2011 ch/Sweden/97 NDV2K35/CH/TN/2003 ch/CP/PK/2010

Genotype VII

ch/U.S.(CA)/1083(Fontana) Ch Sweden 95

Pi/MZS1-UVAS/2014 Pi/Rus/Vladimir/687/05 AV324/96 dove/Italy/2736/00 pi/CH/LLN/110713 pi/CH/LHLJ/110813 pi/CH/LHLJ/110822 pi/CH/LGD/110947 pi/CH/LGD/110945 pi/CH/LJL/100605 pi/CH/LJL/120404 pi/CH/LGD/110208 W4 P4 PPMV-1/Belgium/98-321/1998 PPMV-1/Belgium/98-238/1998 PPMV-1/Belgium/98-248/1998 1.3

Genotype VI

QH1 QH4 AF2240-I

Genotype VIII

NDV-P05 01/10 APMV-1/ch/Brazil/SJM/75 anhinga/U.S.(CA)/211472/02 mixed sp/U.S./Largo/71 cormorant/US(MN)/92-40140/1992 anhinga/U.S.(FI)/44083/93 cormorant/US(CA)/92-23071/1997 anhinga/U.S.(CA)/D9704285/1997 cormorant/US(NV)/19529-04(USGS)/2005 cormorant/US(WI)/18719-03(USGS)/2003 cormorant/Canada/98CNN3-V1125/1998 cormorant/Canada/95DC02150/1995 cormorant/Canada/95DC2345/1995

Genotype V

2K3/Chennai/Tamil Nadu NDV-2/ch/Namakkal/Tamil Nadu NDV/2K36/peacock/Chennai

Genotype IV

MG 1992 MG 75 08 MG MEOLA 08

Genotype XI

HBNU/LSRC/F3 ZJ/1/86/Ch XBT14 FJ/1/85/Ch GD09-2 F48E8 JS/1/02/Du JS/1/97/Ch

Genotype IX

mallard/US(MN)/99-376/1999 mallard/US(MN)/00-32/2000 mallard/US(MN)/MN00-39/2000 mallard/US(MD)/03-632/2003 mallard/US(OH)/04-411/2004 mallard/US(OH)/86-233/1986 pintail/US(OH)/87-486/1987 mottled duck/US(TX)/01-130/2001

Genotype X

APMV-1/ch/U.S.(TX)/GB/1948 NDV/Ch/Egypt/1/2005 B1 isolates Takaaki Lasota-1 HX01 Lasota-2

Genotype II

BHG/Sweden/94 ch/N.Ireland/Ulster/67 D3 I-2progenitor I-2 TS09-C 98-1249 98-1252 98-1154 02-1334 01-1108 99-1435 99-0655 99-1997PR-32 99-0868hi 99-0868lo

Genotype I

Class II

Teal/France/100011/2010 JS10 DE-R49/99

Class I

100

100

100

100

100100

100

100

100

100

100100

100

100

100

100

100

100

99

9797

92

91

90

89100

88

87

87

8586

83

82

76

74

72

70

70100

100

100

69

98

100

100

96

6566

62

60

100

100

59

5786

51

51

46

45

44

74

81

100

93

100

94

76

43

93

39

47

38

36

35

100

73

34

3338

4371

98

71

100

96

39

99

98

25

30

44

100

99

100

99

94

96

89

99

100

24

2229

a b

c JS/2/98/Go STP96 NDV05-029 PG/CH/JS/1/05

VIe

YZ-23-07-Pi pi/CH/LJL/120404 pigeon/Guangxi/1015/2013 pi/CH/LGD/110208

VIa

Pi/MZS1-UVAS/2014 HR-155/01 HR-65/95 Pi/Rus/Kemerovo/0267/09 PATPI00323 Pi/Rus/Vladimir/687/05 PPMV-1/PL/H2/10

VIb

HLJ-4/95 TW/99-154 HuN-1/95 NDV-JL-2/97 NDV-BJ-3/97 BHKPI89172 H-310/82 BBGPI95039 S-1/95 CH-1/95 DK-1/95 DK-6/95

VId

BSACK89036 DSACK95121 BBECK97037 BAEHO95114 Pak/Mansehra/2012 BAECK92084 BAECK95088 BAEPI99109 BAEFA99142 BAEOS98034

VIc

100

97

97

94

94

92

8697

7793

7178

100

91

54

50

55

42

99

39

32

32

3072

83

91

63

23

42

28

98

46

18

23

Fig. 1 Phylogenetic consensus tree for the pigeon‑originated NDV isolate for fusion gene (a), hemagglutinin gene (b) and hypervariable region of F gene (c). The nucleotide sequences of study isolate for each gene were compared with corresponding genes of representative strains reported previously to public database, the GenBank. The evolutionary history was inferred using the Neighbor‑Joining method with 1000 bootstrap value in MEGA version 6.0

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migratory route of birds from North to South. Since the first introduction of genotype VI, various strains such as VIa, VIb, VIc and VIdresponsible for pigeon-origin panzootics have been reported from time to time with widespread geographic movement (Kaleta et  al. 1985, Ujvari et al. 2003; Aldous et al. 2004; Lee et al. 2004). The most recent example is the identification of an isolate of genetic clade VIa from China (pi/GX/1015/13) that is supposed to have ancestor similar to isolate (W4/2005) from Europe (Wang et  al. 2015). It is noteworthy that NDVs belonging to genotype VI was identified from chicken in district Karachi back in 2005 (Khan et  al. 2010) and, more recently, genotype VIc was reported from a commercial poultry farm in northern areas of Pakistan (Shabbir et al. 2013a). As the presence of cleav-age motif in mesogenic strains does not necessarily cor-relate with the pathogenicity for chickens (Kommers et  al. 2002; Ujvari et  al. 2003; Kim et  al. 2008), studies ascertaining transmission of pigeon-originated NDVs in vaccinated as well as immunologically naïve flocks and their subsequent shedding are essential in understanding their potential to cause disease in wild, commercial and backyard poultry in Pakistan.

Nucleotide and amino acid residues of the isolate were identified in the complete coding region of the F (4550–6211, 1662  bp, 553  aa) and HN (6418–8133, 1716  bp, 571  aa) genes. Although, differences in amino acid length of HN protein have been reported previ-ously from different strains of NDV (Romer-Oberdorfer et  al. 2006), the study isolates had open reading frame that encoded 571 residues, a feature common to most of the virulent NDVs (Tirumurugaan et al. 2011). While comparing nucleotide and residues of the study isolate to representative strains of different genotypes, the per-cent similarity for F gene was found to be maximum with genotype VI (92.4 and 94.2) while it was observed to vary from 84 to 90 for genotype representing vaccine strains; it was 84.2 and 88.2 for genotype II and 85.8 and 90.2 for genotype III. Similarly for HN gene, the percent similarity for genotype II, III and VI was found to be 82.3 and 87.9, 85.5 and 89.8, and 92.1 and 93.6, respectively. Within hyper-variable region of the F-gene (47–421 nt), we observed predicted multiple basic amino acid resi-dues at proteolytic cleavage site (F0) considered typical of mesogenic or velogenic strains (Aldous et  al. 2003). The corresponding residues at the F2 protein and the N-terminus of F1 protein were found to be 112RRQKR116 and phenylalanine (116↓F117), respectively. This is simi-lar to PPMVs reported after 80  s as the cleavage motif for isolates before 80  s was usually112GRQKR↓F116 (Meulemans et al. 2002). Six potential glycosylation sites [Asp(N)-X-Ser(S)/Thr(T), X could be any residue except proline (P) and aspartic acid (D)] including 85NRT87,

191NNT193, 366NTS368, 447NVS449, 471NNS473, 541NNT543 were identified in the F protein. Although there were variations in residue composition of glycosylation sites for different genotypes, the 3rd glycosylation site was found to be unique in a way that all the representative strains of each genotype had residues (447NIS449) in com-parison to the study isolate; Val (V) was present at posi-tion 448. Moreover, 13 cysteine (C) residues located at position 25, 27, 76, 199, 338, 347, 362, 370, 394, 399, 401, 424 and 523 were observed in the study isolate. The sites for glycosylation and cysteine residues are thought to be conserved (Umali et al. 2013), however, we found differences in residues composition for a given glyco-sylation site and variations in both number and site of cysteine residues. For example, there were differences in composition of residues in glycosylation sites located at position 191–193, 447–449 and 471–473. Likewise, compared to the study isolate, we found 12 cysteine resi-dues in Ulster-67 (genotype-I) with replacement of two residues [76(C → F) and 401 (C → S)] and addition of one cysteine residue at position 514 than the study iso-late (G). While characterizing pigeon isolate (PPMV-1) from China, Wang et al. (2015) reported 13 cysteine residues in one of six isolates in a pattern similar to the study isolate with additional residue (13th) at position 27. Compared to consensus sequence of vaccine strains used in Pakistan and other representative strains, we observed a number of substitution mutations in residue sequences for fusion peptide (117–142  aa) at position 121 (V → I), 124 (S → G) and 132 (A → S), hydrophilic region-a (143–185  aa) at position 179 (V →  I), hydro-philic region-b (268–299  aa) at position 272 (N →  H), 288 (T → N) and hydrophilic region-c (471–500 aa) at position 482 (E → A), 487 (K → R), 492 (N → D). Muta-tion at position 506 (V →  I) and 516 (I/M → S) of the major transmembrane region (501–521  aa) further indicates lack of conserveness of this particular part of genome (Fig. 2).

On the other hand, for HN gene, analysis of predicted amino acid sequence revealed conserveness of cysteine residues at positions 123, 172, 186, 196, 238, 247, 251, 344, 455, 461, 465, 531 and 542; key residues for recep-tor (sialic acid) binding at positions R174, E401, R416, Y526; residues for the hydrophobic core of the stalk 4HB at positions Y85, V88, S92, L96, T99, I103, I107, L110 and I114; and stalk residues involved in direct interaction with F protein at positions R83, A89, L90, L94 and L97 (Ke et al. 2010; Yuan et al. 2011). Six N-glycosylation sites at positions 119NNS121, 341NNT343, 433NKT435, 481NHT483, 508NIS510, and 538NKT540 were identified. When com-pared to most common vaccine strain used in Pakistan (genotype II), beside substitutions at several point in total length of HN gene, we found substitutions in the

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10 20 30 40 50 60 70 80 90 100. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

Pi/MZS1-UVAS/2014 MGSKSSTRIPTPLMLITRIMLILSCICLTNSLDGRPLAAAGIVVTGDKAVNIYTSSQTGSIIVKLLPNMPKDKEACAKAPLEAYNRTLTTLLTPLGDSIRUlster/67 (I) ...R......V....TV.VA.E...V.P.S....................T.S...............L......F.......F......W.........LaSota (II) ...RP..KN.A.M..TI.VA.V.....PA..I....................................L............D..................Mukteswar (III) ..PR......V....TI..T.A..YVR..S......................................................................Herts/33 (IV) ...R......V.P...I..V.T....R..S......................................................................Largo/71 (V) ....P...L.V........T........AS......................................................................Pi/Rus/687/05 (VI) .S........A....T..........R..S................................I..............R...........A..N.......14698/90 (VII) ....P.....V..................S......................................................................QH4 (VIII) ........FL..S..............P.G...............................................RT.....................MG_1992 (XI) .......W..ISP.PTIL.A.A.G.VR..S..................................................V...................

110 120 130 140 150 160 170 180 190 200. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

Pi/MZS1-UVAS/2014 RIQGSVSTSGGRRQKRFIGAIIGGVALGVATSAQITAAAALIQANQNAANILRLKESIAATNEAVHEVTDGLSQLAVAIGKMQQFVNDQFNNTARELDCIUlster/67 (I) ...E..T....GK.G.L.......A......A......S.......................................V............K..Q.....LaSota (II) ...E..T....G..G.L..............A............K.................................V............K..Q.....Mukteswar (III) ...E..T.......R........S.......A......S..............................G........V...............Q.....Herts/33 (IV) ...E..T.......R........S.......A......S.......................................V...............Q.....Largo/71 (V) .....AT..........V.....S.......A...................V..........................V.....................Pi/Rus/687/05 (VI) ...........K...........S......................................................V.....................14698/90 (VII) K...................V..S.......A...........................................S..V.....................QH4 (VIII) ......T.............V..S.......A..............................................V.....................MG_1992 (XI) ...E.AT......RR..V..V..S.......A............K.................................V............K..Q....V

210 220 230 240 250 260 270 280 290 300. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

Pi/MZS1-UVAS/2014 KITQQVGIELNLYLTELTTVFGPQITSPALTQLTIQALYNLAGGNIDYLLTKLGIGNNQLSSLIGSGLITGHPILYDSQTQLLGIQVNLPSVGNLNNMRAUlster/67 (I) .......V.....................................M........V................N...............T............LaSota (II) ..A....V......................NK.............M........V................N...............T............Mukteswar (III) ..A....V..........................V..........V........V................N..F............T............Herts/33 (IV) .......V.....................................M........V................N.........I.....T............Largo/71 (V) .......V.........S.......................V...M........V..............S.NL...........................Pi/Rus/687/05 (VI) .V..................................................................................................14698/90 (VII) .......V.....................................M........V................Y............................QH4 (VIII) ..A....V..........................V..........M........V................N...............T............MG_1992 (XI) ..A....V..S................................S.M........V................S.........I.....TM....S......

310 320 330 340 350 360 370 380 390 400. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

Pi/MZS1-UVAS/2014 TYLGTLSVSTTKGFASALVPRVVTQVGSVIEELDTSHCIESDLDLYCTRIVTFPMSPGIYSCLSGNTSACMYSKTEGALTTPYMTLKGSVIANCKITTCRUlster/67 (I) ...E................K...............Y...T......................................................M....LaSota (II) ...E.......R........K...............Y...T............................................I.........M....Mukteswar (III) ...E................K...............Y...A.................................................V...QM....Herts/33 (IV) ...E................K...............Y..GT...........................................A..........M....Largo/71 (V) ...E................K.G.............Y............V.............................S....A..........M....Pi/Rus/687/05 (VI) ....................K...............................................................A...............14698/90 (VII) ...E................K...............Y...............................................A.....V.........QH4 (VIII) ...E................K...............Y...............................................A..........M....MG_1992 (XI) ...E......S.........K...............Y..GT..N.....V.............R......T............L...........MI...

410 420 430 440 450 460 470 480 490 500. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

Pi/MZS1-UVAS/2014 CVDPPGIISQHYGEAVSLIDRHSCNILSLDGITLRLSGEFDATYQKNVSILDSQVIVTGNLDISTELGNVNNSISNALDKLAESNSRLDKVDVKLTSTSAUlster/67 (I) SA........N..........Q...V.....................I..Q..............................E....K....N........LaSota (II) ..N.......N.........KQ...V...G...........V.....I..Q.....I.....................N..E...RK....N........Mukteswar (III) .A........N.........K....VV....................I.......L...............H.........E....K....N.R......Herts/33 (IV) .A........N..............V.....................I..............................N..E....K....N.R......Largo/71 (V) .A........N.........K....V................I....I...........................S.........NK.N..N.N......Pi/Rus/687/05 (VI) ..........N..............V...............V.....I...............................R.V....K....N........14698/90 (VII) .A........N..............V...............T.....I......................................K....N........QH4 (VIII) .A........N.........K....V.....K...............I.................................S....K....K........MG_1992 (XI) .A......T................................S.....I...........................K..N..E....K....N...A....

510 520 530 540 550. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . .

Pi/MZS1-UVAS/2014 LITYIILTVISLVFGSLSLVLACYLMYKQKAQQKTLMWLGNNTLDQMRATTRA*Ulster/67 (I) .....V.......C.I....................L..............KM*LaSota (II) .....V..I......I...I................L..............KM*Mukteswar (III) .....V.......L.M................R...L..............KM*Herts/33 (IV) .....V.........V....................L..............KI*Largo/71 (V) .....V.A.......VI...................I.F.............T*Pi/Rus/687/05 (VI) .....V.........A....................I...............T*14698/90 (VII) .....V.........A....................L................*QH4 (VIII) ..I..V.I.....S.V.....T..............L...............T*MG_1992 (XI) .....V...V.....V.....I..............L...............I*

HRa (143-185)

Fusion peptide (117-142) HRb (268-299)

HRc (471-500)

Major transmembrane domain (501-521)

Fig. 2 Alignment of deduced amino acid sequence of complete F gene of pigeon isolate. The residue profile of study isolate is compared with strains of NDVs representing different genotypes including the vaccine strains. Structurally and functionally important residues are boxed and highlighted

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10 20 30 40 50 60 70 80 90 100. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

Pi/MZS1-UVAS/2014 MDRAVRKIVLENEEREAKNTWRLVFRIAVLFLIVMTLAISAAVLVHSMRASTPHDIAGISAAISRTEEKVTSLLNSNQDVVDRIYKQVALESPLALLNTEUlster/67 (I) .....SQVA...D...............I.L.T.V.......A.AY..E....S.LI..PT....A...I..A.G.........................LaSota (II) .....SQVA...D..........I....I...T.V.....V.S.LY..G....S.LV..PTR...A...I..T.G......................K..Mukteswar (III) ..S..SQVA...DR....D.........A.L.M.I...V..VA.AY..E....G.LVS.PT..Y.A..RI..A.G.........................Herts/33 (IV) .....SRVA.............F.....I.L...I.......A..Y..E....G.LV..PTV...A...I..A.S.........................Largo/71 (V) .....SRV....D.................S.........V.A..YG.G.R..S.L....TV..KA.D......S.........................Pi/Rus/687/05 (VI) ..Q...RV......................L.V....................R..VS..TV..K....I.A..G.........................14698/90 (VII) .....NRV...................M..L.T.........A.AY.TG....R.L....TV..K..D......S.S...I...................QH4 (VIII) .....NRV..........................T.......A.IY..G....SNL...PT...KA.DRI....S.........................MG_1992 (XI) ...V.SRVA..............A....I.L..TII..L.T.T.IY..E....G.LV..PTE...A..RI..A.S..R..M..V.............S..

110 120 130 140 150 160 170 180 190 200. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

Pi/MZS1-UVAS/2014 SMIMNAITSLSHQINGAANNSGCGAPVHDPDYIGGIGKELIVDDTSDVTSFYPSAYQEHLNFIPAPTTGSGCTRIPSFDMSATHYCYTHNVILSGCRDHSUlster/67 (I) .T.........Y.......S......I.................A..........F............................................LaSota (II) TT.........Y..........W...I.................A..........F............................................Mukteswar (III) .I.........Y.....T.....................................F............................................Herts/33 (IV) .V.........Y................................A..........F.......................I....................Largo/71 (V) .I.........Y................................A..........F............................................Pi/Rus/687/05 (VI) .I..........................................I.......................................................14698/90 (VII) .I.........Y.....................................................................T..................QH4 (VIII) .I.........Y................................V.......................................................MG_1992 (XI) .V.........Y..................................E........F.......................I....................

210 220 230 240 250 260 270 280 290 300. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

Pi/MZS1-UVAS/2014 HSHQYLALGVLRTSATGRVFFSTLRSINLDDTQNRKSCSVSATPLGCDILCSKVTETEEEDYRSVTPTPMVHGRLGFDGQYHEKDLDIAVLFRDWVANYPUlster/67 (I) ........................H.......................M.............N.AV..S..................VTT..E.......LaSota (II) ..Y.............................................M.............N.AV..R..................VTT..G.......Mukteswar (III) ...............................A................M....I........K..I..S..................VTT..........Herts/33 (IV) ...........................I...N................M....I........S.....S..................VIT..K.......Largo/71 (V) ................................................M.....I.......K.....SV.................VT...K.......Pi/Rus/687/05 (VI) ..............................................................K.I...S..........R........T...K.......14698/90 (VII) ................................................M.............K.....S..............R...TT...K.G.....QH4 (VIII) ..Y.............................................M..F..........K.....S..............E....T...K.......MG_1992 (XI) .......I...........L...........N.....................I..............S..................VIT..K.......

310 320 330 340 350 360 370 380 390 400. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

Pi/MZS1-UVAS/2014 GVGSGSFIDDRVWFPVYGGLKPSSPSDTAQEGNYVIYKRYNNTCPDEQDYQIRMAKSSYKPGRFGGKRVQQAILSIKVSTSLGEDPVLTVPPNTVTLMGAUlster/67 (I) ...G.....N............N.........K........D..........................................................LaSota (II) ...G.....S....S.......N.....V...K........D..........................I...............................Mukteswar (III) ...G....NN......................R........D.....................................................A....Herts/33 (IV) ...G.....N............N.....V...R........D...............................................I..........Largo/71 (V) ...G..L.............R.N.........R..................V................................................Pi/Rus/687/05 (VI) ......................N.........................S.......A.....................................I.....14698/90 (VII) ...G.........V........N.........K..............................IR...............F..K.....I.L.PI.....QH4 (VIII) ...G................D.N.........R............................R......................................MG_1992 (XI) ...G.....G............N....I....R........D............................................M.............

410 420 430 440 450 460 470 480 490 500. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

Pi/MZS1-UVAS/2014 EGRVLTVGTSHFLYQRGSSYFSPALLYPMTVHNKTATLHSPYTFNAFTRPGSVPCQASARCPNSCITGVYTDPYPLVFHRNHTLRGVFGTMLDDVQARLNUlster/67 (I) ...............................S............D....................V............Y...............K.....LaSota (II) ...I...........................S....................I..........P.V..........I.Y..............G......Mukteswar (III) ...............................N.......N.........................V............................K.....Herts/33 (IV) ........................I......N.................................V..........I.................G.....Largo/71 (V) ...........................................................................V.................NE.....Pi/Rus/687/05 (VI) ...I.........................I.....................I........................I.................I.....14698/90 (VII) ...............................YT.........................S.................I.................E.....QH4 (VIII) ...............................N.......................H....................I.................E.....MG_1992 (XI) .............................I.N...................II..........P.V............................E.....

510 520 530 540 550 560 570. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . .

Pi/MZS1-UVAS/2014 PVSAVFDNISRSRVTRVSSSSTKAAYTTSTCFKVVKTNKTYCLSIAEISNTLFGEFRIVPLLVEILKGDRV*Ulster/67 (I) .......S.....I.....................................................D.G.RLaSota (II) .A.....ST....I.....................................................D.G.RMukteswar (III) .............I........R............................................DGG.*Herts/33 (IV) .............I.....R...............................................N.G.*Largo/71 (V) .......YT....IA........................V...........................DN..*Pi/Rus/687/05 (VI) ......................................................................I*14698/90 (VII) .I.....................................A...........................D...*QH4 (VIII) .............M.........................V...........................D...*MG_1992 (XI) .............I........................R............................DG.I*

HRa (74-88)

Site 1 and 14 (345-355)

Site 12 (494)

Site 2 (569)Site 2 and 12 (513-521)

HRb (96-110)

Site 23 (193-211)

Fig. 3 Alignment of deduced amino acid sequence of complete HN gene of pigeon isolate. The residue profile of study isolate is compared with strains of NDVs representing different genotypes including the vaccine strains. Structurally and functionally important residues are boxed and highlighted

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transmembrane domain (I29V, T33I, V35M, S43V, L45V, Y46H, G49R), antigenic sites (K98N, G494D, I514V) and neutralization epitope (N263R) (Fig. 3). It is interesting to note that some substitutions were found to be exclusive to the study isolate notably at positions V179I, K321R, I448V, K487R, K/N492D, V506I, L537M for F protein and Q/R7K, V8I, T61A, S/G75N, S/R269P, I/T289A for HN protein (Figs.  2, 3). This is important as some geo-graphically conserved mutation have been reported recently; K4I is conserved for strains reported from Japan (Umali et al. 2013) while I52V, K78R, R101K are consid-ered conserved for isolates originating from the Far East Asia (Japan, China and Taiwan). Taken together, varia-tion in nucleotide and subsequent substitutions/alterna-tions in amino acid profile such as observed in this study is consistent with previously described theories of evo-lution of RNA viruses particularly NDV (Yu et al. 2001; Umali et al. 2013).

As the pigeon flock had a history of vaccination with lentogenic strain (LaSota), identification of cleavage motif similar to velogenic raises concerns for the type of vaccine used to vaccinate the flock and the need for post-vaccine evaluation. Substitution and subsequent muta-tions at fusion peptide, HR regions, trans-membrane domain and antigen neutralization sites could affect the fusion activity of NDVand, alteration in antigenic epitopes particularly those that are involved in virus attachment, could result in escape variants and subse-quent vaccine failure (Cho et al. 2007; Umali et al. 2014; Wang et  al. 2015). Beside potential compromise in pro-cedures used in vaccine storage and administration, the expected genetic distance between vaccine strains and the study isolate seems to be well-explained by Wang et al. (2015) through cross-HI assay. While evaluating the antigenic diversity of different strains through cross-HI assay, they reported a lower R-value (0.13–0.18) for inter-action of PPMVs to LaSota than between PPMVs (VIa and VIb, 0.7) indicating an obvious antigenic difference with vaccine strain.

ConclusionWe characterized the circulating genotype of pigeon (PPMV-1) closely related to previously known clades of VIb. Since outbreaks of NDV in commercial poul-try has been reported in one of the province of Pakistan from a closely related genotype (VIc) to the study isolate (VIb), future studies relating to disease surveillance cou-pled with experiments involving potential to transmit and shedding from either vaccinates or non-vaccinates are essential. Moreover, immunity barrier provided by LaSota and other vaccinal strains needs to be evaluated in the face of newly emerging NDV strains.

Authors’ contributionsConceived and designed the experiment: SA, MAM, MR and MZS. Sampling and laboratory work: SA, MYT, AR and MZS. Analyzed the data: SA, KM, AR and MZS. Wrote the manuscript: SA, AR and MZS. All authors read and approved the final manuscript.

Author details1 Department of Microbiology, University of Veterinary and Animal Sciences, Lahore 54600, Pakistan. 2 Department of Pathology, University of Veterinary and Animal Sciences, Lahore 54600, Pakistan. 3 Quality Operations Laboratory, University of Veterinary and Animal Sciences, Lahore 54600, Pakistan.

AcknowledgementsWe thank Defense Threat Reduction Agency of USA for capacity building of the faculty as well as strengthening of existing laboratory facilities utilized in this work at the University of Veterinary and Animal Sciences, Lahore Pakistan through collaborative research projects with The Pennsylvania State University, USA. Further, thanks are due to Dr. Irshad Hussain for reading and editing the revised version of the manuscript.

Competing interestsThe authors declare that they have no competing interests.

Ethical approvalThis article does not contain any experiment or research with animals per‑formed by any of the authors.

Received: 7 April 2016 Accepted: 28 July 2016

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