Alexandria Journal of Veterinary Sciences 2016, July.

50 (1): 145-149
ISSN 1110-2047, www.alexjvs.com
DOI: 10.5455/ajvs.230284

Isolation and Molecular Characterization of Newcastle Disease Virus Genotype VI from

Pigeons in Egypt
Moustafa M. El-Shazly 1, Basem M. Ahmed 2, Ahmed A. El-Sanousi 2, and Youssef I. Youssef 3
1

Veterinary unit, Desouk, province Kafr el-Sheikh, Egypt

Department of Virology, Faculty of Veterinary Medicine, Cairo University, Egypt
3
Department of Poultry and Rabbit Diseases, Faculty of Veterinary Medicine, Cairo University, Egypt
2

ABSTRACT
Key words: This study reports successful isolation and characterization of velogenic genotype VI Newcastle disease
Newcastle
disease virus;
Velogenic;
pigeon;
Genotype IV;
Egypt

virus (NDV) from pigeons in Egypt. A pool of brain sample was collected from three pigeons showing
torticollis and whitish green diarrhea in Desouk, Kafr-elshiekh Province, Egypt, during January, 2014.
Virus Isolation has been pursued by inoculation of brain homogenate into the allantoic sac of 9 day old
specific pathogen free (SPF) embryonated chicken eggs. Identification of the isolate was carried out by
haemagglutination inhibition test using specific polyclonal sera against NDV, RT-PCR and confirmed by
sequencing. Partial deduced amino acid sequence of the fusion protein at the cleavage site revealed that
the isolate possessed the motif 112 K-R-Q-K-R-F117 indicating velogenic entity and phylogenetic analysis
showed clustering of the study sequence with other genotype IV F gene sequences available in GenBank.
The study indicated that pigeons may play epidemiological role in the introduction of a new NDV
genotype VI to commercials chicken, which will increase the epidemiological burden of Newcastle
disease (ND) in Egypt

Corresponding Author: Moustafa M. El-Shazly: elshazlimustafa2015@gmail.com

1.

INTRODUCTION:

clinical signs observed in pigeons were closely

similar to those of the neurotropic NDV included
tremor of the neck and wings, torticollis, paralysis
and disturbed equilibrium (Marlier and Vindevogel,
2006). The respiratory signs were usually absent,
only some naturally infected pigeons showed
respiratory signs, including gasping, coughing,
sneezing and tracheal rales (Guo et al., 2013).
Intracerebral pathogenicity index and F protein
cleavage site motif are the two major parameters of
virulence of NDVs (OIE, 2012). Virulent NDV
strains contain a polybasic F protein cleavage site
with a consensus sequence of 112(R/K)-R-Q-(R/K)R-F117, which can be recognized by ubiquitous host
proteases and thus making it possible to spread
systemically and produce fatal infection (Panda et
al., 2004 and de Leeuw et al., 2005). NDV can be
divided into class I and class II, and each class has
different genotypes and sub-genotypes based on
partial or complete nucleotide sequences of the
fusion protein gene. Up to now, at least 18
genotypes were identified in class II which include
avirulent and virulent viruses (Kim et al., 2007; Liu
et al., 2009; Diel et al., 2012; Courtney et al., 2013
and Snoeck et al., 2013b). The viruses isolated from
pigeons were placed by several studies into different
genotypes, such as genotype II, VI and VII in class

Newcastle disease is highly contagious and

devastating viral disease of poultry worldwide and
has a considerable economic impact on the world
poultry industry (Alexander and Senne, 2008).
ND is caused by virulent NDV or avian
paramyxovirus type 1 (APMV-1) which is an
enveloped negative sense ssRNA virus of the genus
Avulavirus, family Paramyxoviridae (Alexander and
Senne, 2008). The NDV genome is ~15.2 kb in
length and contains genes encoding at least seven
proteins, including the nucleoprotein (NP), the
phosphoprotein (P), the matrix protein (M), the
fusion
protein
(F),
the
haemagglutininneuraminidase (HN), the RNA dependent RNA
polymerase (L), and the V protein (Alexander and
Senne, 2008).
Kaleta and Baldauf (1988) suggested that NDV
infections had been established in at least 241
species of birds representing 27 of the 50 orders of
the class Aves. Pigeon paramyxovirus type 1
(PPMV-1) is an antigenic and host variant of
classical NDV of chickens and some strains could
be distinguished from classical NDV by
hemagglutination inhibition (HI) tests and
monoclonal antibodies (Alexander et al., 1987). The

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II (Liu et al., 2006; Qin et al., 2008; Snoeck et al.,

2013a and Wang et al., 2015), however most
recently detected NDVs circulating in Egypt were
isolated from chickens belonged to class II genotype
VII (subgenotype VIId) (Radwan et al., 2013;
Abdel-Glil et al., 2014 and Hussein et al., 2014).

positive HA-allantoic fluid samples as well as from

passaged LaSota vaccinal strain as positive control
and non-infected allantoic fluid as negative control
using QIAamp Viral RNA Mini Kit (Qiagen,
Germany) and the extract was subjected to one step
RT-PCR using VersoTM 1-step RT-PCR Kit
(Thremo Scientific, USA) for partial amplification
of F gene containing cleavage site. The RT-PCR
reaction mixture was adjusted to 25 l as
recommended by the kit manual instruction as
follows: Nuclease free water 3.75 l, Verso 1-step
Master Mix12.5 l, Verso enzyme mix 0.5 l,
Primers 2 l (F-NDV3: 5'-GGA GGA TGT TGG
CAG CATT-3' and R-NDV4: 5'-GTC AAC ATA
TAC ACC TCA TC-3) (Pang et al., 2002), RT
Enhancer 1.25 l and Extracted RNA 5 l. Each
tube was spined for 10-20 seconds to remove any
drops in the tubes lids and then transferred to
thermal cycler adjusted as showed in table (1). 5 l
from each amplified PCR products were visualized
in ethidium bromide stained 1.5 % agarose gel and
documented using ultraviolet trans-illuminator.
1.1. Virus characterization:
Purified PCR products were sequenced in both
orientations by the di-deoxy chain-termination
method using the same primers of amplification.
Gene sequencing was carried out using a BigDye
Terminator v3.1 cycle sequencing kit (Applied
Biosystems, Foster City, CA) at MACROGEN,
Korea using 3730XL DNA sequencer. The obtained
sequences were subjected to BLAST analysis then
sequences were imported into BioEdit version
7.0.4.1. Multiple nucleotide and amino acid
sequence alignment were conducted using Clustal
W application embedded in the BioEdit to detect the
pathotype of isolated NDV. The phylogenetic tree
was generated by the neighbor-joining method with
the MEGA program version (7.0.18).

2. MATERIAL AND METHODS

2.1. Samples
Aseptically brain pool sample was collected from
pigeons (Columba livia domestica) showing
torticollis and whitish green diarrhea in Desouk,
Kafr-elshiekh Province, during investigation of free
living birds in the vicinity of broiler chicken farms
for prevalence of avian influenza (AI) and ND in
2014.
2.2. Virus isolation
supernatant fluid of brain tissue homogenize was
inoculated into the allantoic cavity of 9 to 11 dayold SPF embryonated chicken eggs, 0.2 ml/egg.
Eggs were incubated at 37 oC daily for 4 days. Eggs
showed embryonic deaths before 24 hours of
inoculation were discarded and considered nonspecific. Eggs showed embryonic death after 24
hours and/or remained alive up to 4 days were
chilled. Allantoic fluid was harvested and tested for
hemagglutination (HA) using 10 and 1 % (V/V)
chicken RBCs (OIE, 2012).
2.3. Virus identification
The allantoic fluid harvest was tested by
haemagglutination inhibition (HI) assay using
specific polyclonal antisera against NDV and AIV (
subtype H5 and H9), 96 well V-bottomed HA
microtiter plate, 1 % (V/V) chicken RBCs, PBS,
control HA-positive (egg passaged LaSota vaccine)
and control negative (non-infected allantoic fluid)
(OIE, 2012). Viral RNA was extracted from 140 l

Table (1): Thermo cycler protocol for amplification of partial F gene of NDV
Step
Temperature
Time

Number of cycles

Initial denaturation
RT

50 C
94 C

15 minutes
2 minutes

1cycle
1cycle

Denaturation

94 C

30 seconds

40 cycles

Primer annealing

50 C

30 seconds

Extension

72 C

45 seconds

Final extension

72 C

10 minutes

1cycle

Cooling

4 C

---------

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3. RESULTS AND DISCUSSION

Bangladesh that had a virulent-like fusion protein

cleavage site 112 R-R-Q-K-R-F 117 and Wang et al.
2015 reported genetic characterization of PPMV-1
isolates in China during 2011 to 2013 belonged to
genotype VI. It is worth noting that in contrary to
concurrently isolated genotype VII viruses from
other free living birds that killed embryos of the
inoculated eggs within 48 hours (Data not shown),
the study virus didnt kill the embryos of inoculated
eggs over 2 successive passages thus more in depth
molecular and antigenic analysis are required in
addition to in vivo characterization to understand the
nature of the study virus.

Isolation of HA virus that only inhibited by the

polyclonal antiserum of NDV with HI titer reached
(211) and didnt kill the embryos up to 4 days of
incubation and after 2 successive passages.
RT-PCR product revealed positive target fragment
of F gene with correct size 320-bp on agarose gel
electrophoresis (Pang et al., 2002). Deduced amino
acid sequence alignment revealed high identity
95.7% to genotype VI of Ukraine (KJ914671.1
NDV Pigeon/Dnipropetrovsk/1-18-11) and showed
that the pigeon isolate had possessed the motif of 112
k-R-Q-K-R-F117 which is a characteristic motif for
the velogenic NDVs (Kim et al., 2008 and OIE,
2012). Phylogenetic analysis proved that pigeon
strain sequence was clustered with velogenic
genotype
VI
(KJ914671.11
NDV
Pigeon/Dnipropetrovsk/1-18-11) and did not cluster
with any of the representative Egyptian isolates
(Fig. 2), also these finding increase the role of
migratory birds (Mediterranean/Black Sea Flyway)
that connect Europe and central and western Asia to
Africa. Isolation of NDV possessing velogenic
motif entity from pigeons are consistent with Liu et
al. 2006 who characterized NDV isolates from sick
pigeons in China with amino acids sequences motifs
112
R/K-R-Q-K-R-F 117 at fusion protein cleavage
site. However isolation of NDV genotype VI from
pigeons is a new record in Egypt, Nooruzzaman et
al. 2015 isolated genotype VI from sick pigeons in

4. CONCLUSION
Isolation of velogenic NDV genotype VI from
pigeons will increase the epidemiological burden of
NDV in Egypt and asses the role of semidomesticated or feral wild birds in prevalence of
new genotypes of NDV however, no recent study
reported the detection of the same genotype from
commercial chicken in Egypt till now therefore,
extensive surveillance of wild birds in addition to
routine surveillance of domestic poultry is a
fundamental requirement.
Acknowledgments
The work team is grateful to all members of
Virology Department, Faculty of Veterinary
Medicine, Cairo University for their excellent
technical assistance.

Fig. 1: Ethidium bromide stained agarose gel electrophoresis containing the RT-PCR products of internal part
of F gene of NDV (320bp), L= DNA ladder (100-3000 bp), CP= Positive control, CN = Negative control,
Lane 2 and 3 = Positive NDV, Lane 1,4 and 5 negative NDV.

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El-Shazly et al. /Alexandria Journal of Veterinary Sciences 2016, Apr. 49 (1): 145-149

Fig. 2: Neighbor-Joining phylogenetic tree for selected NDV sequences from Genotypes (I, II, VI, VII),
rooted to Genotype I and showing the clustering of Study Isolate (black square) with genotype VI NDV
sequences. Tree was generated using MEGA version (7.0.18) with 500 bootstrap replicates.
Submission to GenBank:
Sequence of the isolate (NDV
pigeon/Desouk/Egypt/MS3I/2014) has been
submitted to the GenBank with Accession No.
KR082484
URL:http://www.ncbi.nlm.nih.gov/gquery/?term=
KR082484.

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