1743-422X-10-170

Shabbir et al. Virology Journal 2013, 10:170
http://www.virologyj.com/content/10/1/170
RESEARCH
Open Access
Genetic diversity of Newcastle disease virus in
Pakistan: a countrywide perspective
Muhammad 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 is
endemic in Pakistan and recurrent outbreaks are being reported regularly in wild captive, rural and commercial poultry
flocks. Though, efforts have been made to characterize the causative agent in some of parts of the country, the genetic
nature 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 domesticated
poultry (broiler, layer and rural) as well as wild captive birds (pigeons, turkeys, pheasants and peacock). Samples were
screened with real-time PCR for both matrix and fusion genes (1792 bp), positive samples were subjected to
amplification 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 gene
indicated 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 strains
to 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 population
including wild captive birds. Such understanding is crucial to underpin the nature of circulating strains of NDV, their
potential for interspecies transmission and disease diagnosis and control strategies.
Keywords: Lineages, Newcastle disease virus, Pakistan, Phylogenetic analysis, Poultry
Introduction
Newcastle disease is caused by avian paramyxovirus
serotype-1 (APMV-1), which is also known as Newcastle
disease virus (NDV). It is a highly contagious viral disease
that affects domesticated and wild bird species throughout
the world [1-4]. The disease is considered enzootic in
* Correspondence: vetvains@uvas.edu.pk; drmunir.muhammad@gmail.com
1
Quality Operations Laboratory, University of Veterinary and Animal Sciences,
Lahore, Pakistan
18
The Pirbright Institute, Compton Laboratory, Compton, Berkshire RG20
7NN, United Kingdom
Full list of author information is available at the end of the article
Pakistan and represents major threat to the economy of
the country. However, disease severity is variable in different host species and in different geographical locations.
NDV is classified in the genus Avulavirus within subfamily Paramyxovirinae, family Paramyxoviridae and order
Mononegavirales [1]. This enveloped virus has a negativesense, non-segmented, single stranded RNA genome of
15186, 15192 or 15198 nucleotides in length [1,5,6]. The
genome encodes six structural and two non-structural proteins [6]. Based on the fusion (F) gene sequence, NDV
strains are classified into lineages or genotypes; however,
the discrepancies between the two classification systems are
© 2013 Shabbir et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Shabbir et al. Virology Journal 2013, 10:170
http://www.virologyj.com/content/10/1/170
Page 2 of 10
nominal [5,7-10]. Clinical manifestation or severity of the
ND depends largely upon the isolates involved in disease
outbreak [1,2]. Based upon pathogenicity, these strains are
commonly categorized into velogenic, mesogenic and
lentogenic types [4]. The varying level of pathogenicity is attributed to amino acid sequence motif present in the protease cleavage site of the precursor F protein [11]. The amino
acid sequence in more virulent, velogenic and mesogenic
strains is 112R/K-R-Q-R/K-R↓F117. This sequence is cleavable 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 in
less virulent, lentogenic strains of NDV is 112G/E-K/R-QG/E-R↓L117. This sequence is cleavable only by trypsin like
proteases, hence limiting the infection only to respiratory
system 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 in
the rural areas and every 5th family in the urban areas is associated with poultry in one way or the other [12]. The
poultry has emerged as the second largest industry in
Pakistan with an annual increase of 4%, supplying eggs and
meat as the protein sources [13]. Since the development of
an organized poultry sector in Pakistan, the Newcastle disease has caused havocs to the poultry industry several times.
Appropriate vaccination and subsequent effective immune
response is known to be the only measure to avoid the disease outbreaks, but this approach cannot be extended logistically to all domesticated and wild birds. Large number of
disease outbreaks was recorded during 2010–12 in Pakistan.
Reports about the disease in Pakistan have largely
been focused to Punjab province, reporting existence of
velogenic NDV strains [9,11,14-17]. However, a systematic
investigation of genetic nature of NDV strains in clinically
diseased flocks throughout Pakistan has been lacking so far.
Failure of previously effective live vaccines in protecting the
birds from current field isolates stimulated us to investigate
the genetic relationship between the past and current viral
isolates. This is crucial due to the fact that disease continue to appear in both vaccinated flocks as well as unvaccinated commercial, wild captive and backyard poultry
birds. This is the first report that encompasses the molecular characterization of prevailing NDV strains from
across Pakistan.
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 brachial vein of four diseased birds randomly in an anticoagulant added vaccutainer (Venoject(R), Belgium). A brief
history regarding age at infection, clinical symptoms, mortalities and course of infection was recorded.
Materials and methods
Amplification and sequencing of F gene
History and collection of samples
All the samples that appeared positive for real-time PCR
(both M and F gene bases) were further processed for the
amplification of the complete F gene using degenerate
primers as we described previously [19,20] with one modification. A total 7 μL of eluted RNA (the same as was used
in real-time PCR screen) was added in a 25 μL reaction
mix from One-Step RT-PCR Kit (Qiagen). However, the
PCR conditions were kept same as reported previously
To ascertain the genetic nature of circulating NDV
strains in Pakistan, blood samples were collected during
an emerging wave of the disease from February to July
2012. A total of 113 flocks originating from various districts 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),
Shipment of the samples and extraction of nucleic acid
A total 300 μL of whole blood was stored on QIACard
FTA Indicator Four Spots (Qiagen, Hilden, Germany).
These FTA Indicators have properties to inactivate the virus
and preserve the nucleic acid. The samples were shipped at
ambient temperature from Pakistan to the Department of
Biomedical Sciences and Veterinary Public Health at the
Swedish 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, one
punch (discs of 2.0 mm diameter) was taken from each
sample using a Harris’ micropunch, according to manufacturer’s recommendation (BD09; Whatman). These discs
were placed individually in a 1.5 mL microcentrifuge tube
and the RNA was eluted with 52 μL of Tris–EDTA elution
buffer (10 mMTris–HCl with pH 8.0, 0.1 mM EDTA, 50
units of RNase Inhibitor and 1 mM DTT) instead of
company’s recommended RNA processing buffer. All
reagents used here were purchased from Invitrogen,
Carlsbad, CA, USA. These soaked discs were incubated for
15 min on ice and were flicked three times after every five
minutes during the course of incubation). The extracted
RNA was stored at –20°C until use for both sample screening and characterization using real-time PCR and sequencing, respectively.
Screening of samples using real-time PCR
The detection of nucleic acid for NDV was performed
using real-time PCR for M and F genes, as described previously [9,18]. The reaction was carried out in a Rotor-Gene
6000 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 positive in both M and F genes based real-time PCRs.
Shabbir et al. Virology Journal 2013, 10:170
http://www.virologyj.com/content/10/1/170
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Table 1 Detail of samples collected from districts and diagnostic efficacy of real-time PCR
Province
District
Type of birds
1
2
Commercial
Flock
Blood
(n) samples (n)
3
Rural
4
Real time
PCR
M
gene
F
gene
Flock
(n)
Blood
samples (n)
Wild captive
Real Time
PCR
M
gene
F
gene
Flock
(n)
Blood
samples (n)
Real time
PCR
M
gene
F
gene
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
-
-
Abbottabad
11
44
+
+
0.0
0.0
-
-
0.0
0.0
-
-
Mansehra
10
40
+
+
0.0
0.0
-
-
0.0
0.0
-
-
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
-
-
Okara
02
08
+
+
01
04
+
+
04
(turkey
and
pigeon
16
+
+
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 and
pigeon)
20
+
Multan
03
12
+
+
0.0
0.0
-
-
0.0
0.0
-
-
Jhang
02
08
-
-
02
08
-
-
0.0
0.0
-
-
KPK
Sindh
Punjab
Faisalabad
+
+
1
birds kept under full human husbandry conditions and are fed with commercial food and supplements, 2Birds kept under partial human husbandry conditions
and 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 the
studied flocks in particular district was positive, + and - indicate that samples were detected positive or negative by real-time PCR, respectively.
[11,16,20]. The amplified PCR products were visualized in
1% (w/v) agarose gel in TBE-containing ethidium bromide.
The bands of expected size were cut from the gel and were
purified using the Wizard® SV Gel and PCR Clean-Up System (Promega, Co., Madison, WI, USA) according to the
manufacturer’s instructions. The purified PCR products
were sequenced with the same primers (used for PCR
amplification) by the dideoxy-mediated chain-termination
method using ABI PRISM BigDye® Terminator v3.1 Cycle
Sequencing Kit (Applied Biosystems, Foster City, CA) as
described by the manufacturer. Sequences were analyzed
with an automated nucleic acid analyzer (ABI PRISM
3100; Applied Biosystems). To generate reliable consensus, each DNA fragment was sequenced at least twice in
both the directions.
Phylogenetic analysis
Sequence assembly and editing were performed using the
SEQMAN program from DNASTAR Lasergene suite 9
(version 9.0.4 39; DNASTAR, Inc., Madison, WI, USA). To
determine the phylogenetic relationships between APMV-1
viruses reported here and characterized previously from
Asia and other parts of the world, a sequence stretch of the
373 bases in the F gene were retrieved from GenBank
(http://www.ncbi.nlm.nih.gov). Sequences representing
each known lineage were included in the analysis [7,21].
Known strains of NDV, representing each lineage/sublineage, were included in the trees to determine the distribution and clustering pattern of NDV strains studied in this
manuscript. Furthermore, previously characterized stains of
NDV were supported with accession numbers to facilitate
Shabbir et al. Virology Journal 2013, 10:170
http://www.virologyj.com/content/10/1/170
Page 4 of 10
Figure 1 Sampling sites and geographical distribution of Newcastle disease virus lineages in Pakistan.
further reading of a specific isolate. All sequences were
aligned in BioEdit version 5.0.6 [22] using ClustalW and
trimmed to equal length. A phylogenetic tree was then
constructed using Bayesian Inference with the program
MrBayes version 3.1.2 [23]. Two independent Monte Carlo
Markov Chain (MCMC) chains were executed and sampled
every 1000 generations using the default parameters of the
priors’ panel. Once chains reached convergence (standard
deviation values below 0.01), four million additional generations of the MCMC were run. Trees saved in this last step
were used to construct a majority rule consensus tree. The
analysis was based on the GTR + I + G model, which allow
significantly changed posterior probability estimates. The
nomenclature, based on lineages, was used in this study as
described by Aldous et al. (2003). To further assess the genetic pattern in the tree, same sequences of the F genes were
used for construction of phylogenetic tree using the
neighbor-joining method (Kimura 2 parameter) with 2000
bootstrap replicates in MEGA4 software (CEMI, Tempe,
AZ, USA) [24]. Finally, the labeling of the trees was made
in FigTree v3.1.3.
Determination of recombination events
Six 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 putative recombination breakpoint. These methods were applied using following parameters: window size = 20, highest
acceptable P-value = 0.001 and Bonferroni correction. For
reliable results, any putative recombination events detected
by more than one method were considered.
Sequence submission
All the sequences (n = 23) used in this study were submitted to GenBank and are available under accession
numbers from KC191601 to KC191623.
Results
Real-time PCR screening of clinical samples
Out of all samples analyzed, a total of 23 samples were
found positive in both F and M gene based real-time
PCRs. The results indicated that real-time PCR detected
the samples from both commercial and rural poultry from
all provinces. However, wild captive birds that were collected only from Punjab province were appeared positive.
A detailed description of the collected, positive and negative samples is provided in Table 1. Majority of positive
samples showed Ct values between 25 and 30. Results indicated that the FTA Indicators functioned as an appropriate 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
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Page 5 of 10
for positive samples) were used in conventional PCR for
the amplification of F gene and subsequent sequencing.
Phylogenetic relationship
The Bayesian phylogenetic tree was constructed on 509
sequences, which were used by Aldous et al. (2003) and
Cattoli et al. (2010) for the classification of NDV strains
into six different lineages [7,21]. All previously characterized NDV isolates from Pakistan were also included
in the phylogenetic analysis to ascertain the genetic diversity with the isolates characterized here and reported
from rest of the world. The topology of the phylogenetic
tree indicated the division of NDV strains clearly into six
distinct lineages (Figure 2). Majority of the Pakistani
NDV strains were grouped together and clustered within
lineage 5, close to the previously characterized Pakistani
isolates. Interestingly, the NDV isolates from Khyber
Pakhtunkhwa province clustered with NDV strains of
lineage 4.
Since both lineage 4 and 5 are further divided into different sub-lineages, a high-resolution phylogenetic analysis
was conducted including the representative isolates of
each sub-lineages for both lineage (4 and 5). Analysis indicated that all isolates, excepting isolates from Khyber
Pakhtunkhwa province, clustered close to sub-lineage 5b.
However, all these Pakistani isolates present a separate
branch (Figure 3). The isolates from Khyber Pakhtunkhwa
province (n = 4) were clustered with isolates belonging to
sub-lineage 4c (Figure 4).
Analysis of the amino-acid sequences of F protein
Proteolytic cleavage site motifs (amino acids 112–117) for
the F0 protein of the all the isolates, putatively indicating
the level of pathogenicity, were analyzed. Based on
the cleavage site of the F protein, it was possible to demonstrate that all the isolates carry velogenic motif
112
RRQKRF117 regardless of their phylogenetic distribution. However, three isolates collected from Faisalabad
(MM26), Lahore (MM35), and Gujranwala (MM41) carry
112
RKQKRF117motif at their cleavage sites. Both these motifs are generally identified in the strains of NDV that are
highly virulent in chickens.
Comparison of the predicted amino acid sequences of
the complete F gene showed that the seven neutralizing
epitopes (D72, E74,A75, K78, A79, L343), believed to be
critical for structure and function of F protein, were
conserved and identical in all studied isolates. The potential N-glycosylation sites in F-glycoprotein (Asn-XSer/Thr or N-X-S/T, where X present any amino acid
except aspartic acid or proline) were found at
Lineage 3
Lineage 5
Lineage 1
Lineage 2
Lineage 6
Isolates studied
in this work
Lineage 4
Figure 2 A phylogenetic analysis of the partial sequence of F gene representing all the lineage of NDV. The sequences reported in this
study are colored red in both lineage 5 and lineage 4.
Shabbir et al. Virology Journal 2013, 10:170
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Page 6 of 10
Sublineage 5d
P
TW
1C
TW
K9
48
9/9
ea
68
)
/9
99
9
(A
5/9 F32
TW 9 (AF652
2/9
2
8 (A 3265)
TW 4
/98 (A F083293)
F0839 73 757
64))
279
TW 1/98 (AF083
963
)
(AF234032))
TW 157/99 F32652311)
)
97
40
256
9 (A
54b/9 (AF23
378 )
F
9
TW 1
A
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8 ( 2552)54
156
3/9
8 8
TW
GX
37 7
AF AF3 )
(
8
(
3
9
2/ /97
25
1
GX
78
X
3
G
F
(A
97
/
2
XJ
94
79
b
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-C
85
99
56
AY
17
2
01
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37
12
M
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2
r/M
43
201
30/
ou MM
as ot/
/MM
/K ialk tan2
2
201
/
ab /S pat
nj b ak
a
M25
P
j
/
1/M
Puun jab
ara
2
kk///PPun
/Ok
32/201
Paa k
ah/MM
P
njab
/Pu jab/Layy
97 98 Pa
akk/Pun
MM33/2012
unjab/Sahiwal/
k/Pind
Pa
P
/Pun h/Karachi/MM39/2
PPPaaaakk/S
012
kkk/P jab/O
/
S/Pi uunnjab kara2/M
nd ja /Ok
M26/2
012
h/ b/L ara
3
K a
/
M
h
a
M
ra ore
1/M 27/20
c
12
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M
12
Sublineage 5d
ag
ne
Li
82
b
60
e5
K9 eag
TC Lin
JP 04
41 51
57 R9
17PTB
AY9 J
3
57
17
AY
D
1/
99
NO (A
S- F3
AY1
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756
SH
84 CNB
X 2 3 Lin260
R--3
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AY175
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04 L
632 1A
(AF age
in
5
EOS9
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AY135754 1S
91
5)
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Line
0184
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92
88
90
86
a
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ag
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5a
n
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ge
ea
1
3L
01 962 92 Lin
0
F CK 950
Sublineage 5b
(A
b
5b
1)
6 DE FO
e5
ge
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ea
5
ag
n
e
8 5768 5 (AF
i
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AYE 1
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AY AY1
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99
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Y13 1NNICKK97069
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043 Lin
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A1Y
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AYY1175652
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PTT 950
17Y1
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SH
79
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(A
16
98
6a
99
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39
17
15
08
5c
TW
TW
ge
92 88
AF
Lin
TW
5(
8)
96
83
F0
(A
60
95
7/
ZC
TW
43
98
57
age5a
271 Line
CIDCK96
)
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82
D8
3 /9 5 (A F 0 0 1 1 1 8 )
D
AY
ANYLA
17
57
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NL1A23/51
93
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NE
1Y/ 17751(AF001 SCK94143 L
93 5 NB 125
ineage
5a
(A747 ECK )
F0 ND 81
01 EC 158
12 K9 Lin
4)
41 eag
47
e
Lin 5a
ea
ge
5a
13
Sublineage 5a
AY175682
AY
96
9)
F083960)
TW 1/95 (AAF08396
1)
)
/95 (
97
TW 4
886
83 4)
9
3
F0
7)
)
F10
396 5) 6 (A 340
961 /97 (A
F08 96
083
9
A
3
(
8
F2
P/
(AF
445
P/84 (AF0
(A
/94
GB
W
W
Q
T
T
4
99
C/8
9/
TW
15
Sublineage 5c
2
01
9/2
M2
/M /2012
n1
6
tta M3
12
pa /M
ak re3
2/20
/P ho MM4
jaabb/La ultan/
uunnjja
b/M
k///PP
Pun
Pa k
hi
2
01
/2
40
2
99
2
01
/2
2
41
M /201
M
a/ M28
al
nw d/M
ra ba
ug ala
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Shabbir et al. Virology Journal 2013, 10:170
http://www.virologyj.com/content/10/1/170
Page 7 of 10
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position85NRT87, 191NNT193, 366NTS368, 447NIS449,
471NNS473 and 541NNT543. Comparison of the
complete F gene sequences among all the lineages showed
that the studied strains of NDV shared highest nucleotide
and predicted amino acid (95.7% and 91.3%) identity with
the lineage5 whereas lowest (86.5% and 90.9%) with
lineage 1 except for four isolates belonging to lineage 4.
Recombination among F genes of NDV isolates
Evidence of recombination among poultry and ostrich
NDV has been reported [26-28]. Recombination analyses
performed on the F genes of the NDV isolates belonging
to all lineages reported before and isolates characterized
here appear to lack any recombination events.
Discussion
Geographically, Pakistan is located (33°40′N and 73°10′
E) at the crossroads of the strategically important regions of South, Central and Western Asia (Figure 1).
From 2009 to mid 2012, a number of outbreaks of
Newcastle disease have been reported to World
Organization for Animal Health (OIE-WAHID interface, available at: http://www.oie.int/wahis/public.php)
from Pakistan as well as neighboring countries. Most of
the outbreaks have been reported from Iran and India
Shabbir et al. Virology Journal 2013, 10:170
http://www.virologyj.com/content/10/1/170
that shares border with Pakistan. Despite some of the reports from selective regions only and emergence of novel
NDV (5i) from Pakistan [9], it is of the essence to screen
and characterize the NDV throughout the country.
The F gene sequence data of all the analyzed samples
clustered virulent NDVs among the lineage 5 (sub-lineage
5b) except KPK province, where the ND strains were clustered together in lineage 4 (sub-lineage 4c). Throughout
the world, the NDVs belonging to lineage 5 are considered
to be the one involved in outbreaks in Far East [29-31]
South Africa [32] and Europe [8]. Among many Asian
countries and particularly those that shares border with
Pakistan, there have been found and characterized number
of velogenic NDVs within class II that belongs to various
lineage and sub-lineages [7,33-35]; however, the dominant
one is found to be lineage 5. Likewise, NDVs belonging to
various sub-lineage like 5a, 5b, and novel 5i has been
reported from Pakistan from different type of birds recently [9,11,16]. Contrary to recent study analysis of genetic nature of circulating NDVs except those of KPK
province, where sub-lineage 5b was found circulating
among rural, commercial and wild birds, we have seen a
clear distinction between the viruses isolated from various
types of birds, wild and commercial in the recent past.
The isolates from wild birds were clustered within sublineage 5a and closely related to Indonesian isolate
(AY562985) [15,17] while the ones from commercial and
rural poultry were found within sub-lineage 5b, novel 5i
and closely related to Swedish (GU585905) and Russian
isolates (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 characterized
from 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 fact
that viruses have not been characterized from this particular region before or either to the movement of migratory/caged birds across the country or from north to
south involving Europe, Asia and Middle East. Historically, lineage 4 has been isolated and characterized among
domestic fowls, ostrich, falcons and pigeons from middle
east countries (United Arab Emirates and Saudi Arabia),
Asian (Japan and China) and European countries
(UK, Italy, Peru and Belgium) [1,34]; however, most of
NDVs belonging to sub-lineage 4c have been reported
from wild captive and domestic fowls from United Arab
Emirates [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 in
Pakistan since 1997. Since it is the only report from past,
it could be hypothesized that lineage 4 is present even
before and is still circulating in the environment.
Page 8 of 10
Further, it is imperative to describe the fact that samples
were collected during a designated period of six month
throughout the country and all of the clinical outbreaks
are not necessarily included in the study. This means
that there may exist more diversity in NDV strains
in Pakistan and therefore, presence of lineage 4 (sublineage 4c) in already reported area (Karachi) or others
cannot be ignored. The isolation and subsequent
characterization of isolates from turkey and domestic
fowls in UK (Q-GB506/97) [1], exported parakeets from
Pakistan to Japan (JP/Chiba-pa/97) [29], and from pigeons
in china (C/98-1) [34] which clustered together with Italian exotic isolate (IT-148/94) [8] in lineage 4 provides a
possible epidemiological transmission through migratory
birds from north to south. Considering migratory/caged
birds as reservoir/carrier of NDVs in an inapparent form
and the potential of viruses to infect multiple avian species
without prior adaptation [1,2,26,36], raises the concerns in
worldwide distribution of velogenic pathotypes through
trading and migration of birds across regional and international boundaries.
Recent outbreaks of ND in Pakistan are supposed to be
due to a breach in the biosecurity measures. However, the
role of vaccines in providing protection against the field
challenge also needs to be evaluated. Inability of live vaccines to elicit protective immune response might be due to
several reasons like improper cold chain supply system, inappropriate route of vaccination, or uneven vaccination
schedules. Administration of both live and inactivated ND
vaccine could be practiced to protect the birds from virulent NDVs [37]. Presently, lentogenic (LaSota) or mesogenic
strains (Muktesewar) of NDVs are being used to vaccinate
the birds in Pakistan. However, it is still a matter of question whether these vaccinal strains are able to elicit a protective immune response against the prevailing field strains
with high genetic gap in relation to vaccinal strains as
evidenced and reported previously [9]. It has been reported
that the currently practiced NDV vaccines give better protection against the velogenic NDVs isolated in 1930 to 70s
(Herts33/56, California 71) than the ones, which have been
isolated in past few years [5,9]. Hence parameters for selection of a vaccinal strain are needed to be reconsidered. Furthermore, monitoring the immune response of birds to
NDV vaccines along with strict biosecurity measures should
be employed to keep the flocks free of vNDVs infection.
Conclusions
Simultaneous detection of multiple velogenic strains of
NDV (lineage 4 and 5) from various regions of the country
warrants continuous isolation and molecular epidemiological investigations involving wild/caged birds. Vaccine
strategies that can elicit high antibody response along with
continuous monitoring from the laboratory coupled with
improved biosecurity measures in the form is suggested.
Shabbir et al. Virology Journal 2013, 10:170
http://www.virologyj.com/content/10/1/170
Abbreviations
NDV: Newcastle disease virus; F: Fusion protein; M: Matrix protein; APMV-1: Avian
paramyxoviruses serotype 1; KPK: Khyber Pakhtunkhwa.
Page 9 of 10
9.
Competing interests
The authors declare that they have no competing interests.
10.
Authors’ contributions
MZS, 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 various
geographical areas of Pakistan. MZS, MM wrote the manuscript. MB, SZ
discussed and reviewed the manuscript. MM and MZS designed the manuscript
and analyzed the data. All authors read and approved the final manuscript.
11.
Acknowledgments
The authors would like to thank the field veterinarians and poultry farmers
who helped sample collection from the mentioned districts of each province
of Pakistan.
12.
Author details
1
Quality Operations Laboratory, University of Veterinary and Animal Sciences,
Lahore, Pakistan. 2National Veterinary Institute, Uppsala, Sweden.
3
Department of Microbiology, University of Veterinary and Animal Sciences,
Lahore, Pakistan. 4University of Baluchistan, Quetta, Pakistan. 5Veterinary
Research and Disease Investigation Center, Abbottabad, Pakistan. 6Livestock
and Dairy Development Department, Punjab, Pakistan. 7Veterinary Research
Institute, Lahore, Pakistan. 8Department of Biology, University of Bergen,
Bergen, Norway. 9Poultry Research Institute, Rawalpindi, Pakistan. 10University
Diagnostic Laboratory, University of Veterinary and Animal Sciences, Lahore,
Pakistan. 11Department of Microbiology, Chittagong Veterinary and Animal
Sciences University, Chittagong, Bangladesh. 12Department of
Theriogenology, University of Veterinary and Animal Sciences, Lahore,
Pakistan. 13Sindh Poultry Vaccine Center, Karachi, Pakistan. 14Swedish
University of Agricultural Sciences, Uppsala, Sweden. 15Department of
Geography, Government College University, Lahore, Pakistan. 16Government
Collage University Faisalabad, Faisalabad, Pakistan. 17Department of
Parasitology, University of Veterinary and Animal Sciences, Lahore, Pakistan.
18
The Pirbright Institute, Compton Laboratory, Compton, Berkshire RG20
7NN, United Kingdom.
14.
13.
15.
16.
17.
18.
19.
20.
Received: 11 March 2013 Accepted: 29 May 2013
Published: 30 May 2013
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Cite this article as: Shabbir et al.: Genetic diversity of Newcastle disease
virus in Pakistan: a countrywide perspective. Virology Journal 2013 10:170.
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