2020, Scienceline Publication

JWPR

Poultry Research

J. World Poult. Res. 10(2S): 195-202, June 14, 2020

Journal of World’^s

Research Paper, PII: S2322455X2000025-10

License: CC BY 4.0

DOI: https://dx.doi.org/10.36380/jwpr.2020.25

Molecular Identification of a Velogenic Newcastle Disease Virus

Strain Isolated from Egypt

Shakal M.^1*, Mira Maher², Abdulrahman S. Metwally², Mohammed A. AbdelSabour³, Yahia M. Madbbouly³

and Gehan Safwat^2.

¹Department of Poultry Diseases, Faculty of Veterinary Medicine, Cairo University, 12211 Giza, Egypt.

²Faculty of Biotechnology, October University for Modern Sciences and Arts, MSA, 6^thof October City, Egypt.

³Veterinary Serum and Vaccine Research Institute, VSVRI, ARC, Abbassia, Cairo 11381, Egypt.

^*Corresponding author’s Email: shakal2000@gmail.com; ORCID: 0000-0002-1625-7324

Received: 14 Feb. 2020

Accepted: 16 Mar. 2020

ABSTRACT

Newcastle Disease Virus (NDV) is still a major concern for the Egyptian poultry industry in spite of the mass

vaccination programs implemented from a long years ago. The current study aimed to carry out the molecular

identification of surface glycoprotein genes of NDV field strain isolated from the Giza governorate, Egypt.

Tracheae were collected from 10 broilers NDV-vaccinated chicken flocks (at least three samples from each flock)

suffering from mild to moderate respiratory symptoms; with mortalities varying from 10-40% during October 2019.

Only five samples showed HA positive activity after propagation in specific pathogen-free embryonated chicken eggs

and only one sample was positive for Avian avulavirus 1 by real-time reverse transcription-PCR. Sequencing for the

cleavage site of the F protein gene of the positive isolate showed the typical known sequence of velogenic NDV

strains (₁₁₂RRQKRF₁₁₇). Phylogenetic analysis of both F and HN genes showed high similarity and close relation to

Chinese strains of Genotype VII and more specifically subtype VIId, suggesting the role of migratory wild birds in

NDV evolution in Egypt. In conclusion, further epidemiological and surveillance studies are strongly recommended

to define the exact role of migratory wild birds in NDV evolution in Egypt.

Keywords: Broilers, Newcastle Disease, Poultry industry, Velogenic

INTRODUCTION

2015 ). NDV was recorded in Egypt since 1942 (Daubeny

and Mancy, 1947 ) and has been reported ever since.

(Hussein et al., 2000; Mohamed et al., 2011; Selim et al.,

2018 ). Recently in Egypt, NDV outbreaks have been

reported in both vaccinated and non-vaccinated flocks

(Abd El Aziz et al., 2016; Ewies et al., 2017 ). A

subclinical infection manifested by respiratory, intestinal,

and nervous symptoms, with mortalities up to 100% may

be a result of NDV infection according to virus strain

pathogenicity in infected birds. Based on the pathogenicity

of the virus, NDV strains can be categorized into three

main types; lentogenic, mesogenic, and velogenic strains.

NDV can be classified into two classes; class I and class

II. NDV isolates of class I are grouped into one genotype,

whereas NDV isolates of class II are grouped into at least

eighteen genotypes, some with subgenotypes. Genotype

VII viruses are responsible for the fourth panzootic that

has spread from Asia, Africa, Europe, and has even been

isolated in South America, which continues today

(Dimitrov et al., 2016 ). The NDV is an enveloped virus

that has a linear, single-stranded RNA genome of negative

Newcastle Disease (ND) is one of the most important viral

diseases affecting poultry which is caused by Avian

avulavirus 1 (APMV-1) (Abd El Aziz et al., 2016 ). The

natural hosts of ND virus (NDV) include chickens,

turkeys, ducks, geese, pigeons, quail, pheasants, guinea

fowl, ostriches, and several species of wild birds (Wang et

al., 2015).

ND as an acute viral infectious disease affects

domestic poultry regardless of gender and age (Saad et al.,

2017) and causes great economic losses, especially in

developing countries (Westbury, 2001 ). Production

inefficiencies are considered as a greater concern

compared to mortality losses in breeders and layers flocks

while mortalities usually reported to be more significant in

broilers (Shahid Mahboob et al., 2020 ). Many NDV

outbreaks were reported in the past years around the

world, as in Japan (Mase et al., 2002 ), in Brazil (Marks et

al., 2014 ), in China (Kang et al., 2014 ), in South America

(Diel et al., 2012 ) and in Malaysia (Jaganathan et al.,

To cite this paper: Shakal M, Maher M, Metwally AS, AbdelSabour MA, Madbbouly YM, Safwat G (2020). Molecular Identification of a velogenic Newcastle Disease Virus Strain

Isolated from Egypt. J. World Poult. Res., 10 (2S): 195-202. DOI: https://dx.doi.org/10.36380/jwpr.2020.25

195

Shakal et al., 2020

polarity; with a genome length of about 15.2ꢀkb (Aldous et

Giza governorate during October of 2019. Tracheae from

the same flock kept together for isolation. Samples history

mentioned in Table 1.

al., 2003; Ashraf et al., 2016 ). The genome of NDV

consists of 15,186, 15,192 nucleotides or 15,198

nucleotides that contains six genes coding six structural

and two non-structural proteins including an RNA-

Table 1. History of flock sampled in the present study

Age of

directed

RNA

polymerase

(L),

hemagglutinin-

Birds

No/Flock

Mortalities

(%)

Sample

birds

(day)

NDV vaccination

neuraminidase protein (HN), fusion protein (F), matrix

protein (M), phosphoprotein (P), and nucleoprotein (N).

Both F with HN proteins play a collective role in NDV

infection prosses. The fusion protein is the most important

key in the NDV virulence determining process (Peeters et

al., 1999). Mutations affecting NDV viral genome which

alter its biological properties and virulence, in addition to

altered immunity, and improper vaccination processes can

increase the incidence of NDV outbreaks in vaccinated

flocks (Kattenbelt et al., 2006). Virulence of ND can be

distinguished on the basis of the cleavage site sequence of

the F protein (Selim et al., 2018 ). HN is one of the

membrane glycoproteins, through its neuraminidase (NA)

activity it mediates attachment to sialic acid-containing

receptors (Wang et al., 2015 ). Recently, the molecular

identification and phylogenetic analysis of any new NDV

isolates become an important and usual approach to find

out which of the applied control measures needs to be

improved (Fringe et al., 2012; Hassan et al., 2016).

Sequence analysis of mainly F and of HN proteins genes -

two surface glycoproteins- is wildly used for molecular

identification of NDV isolates. Brevity, the current applied

NDV vaccination programs consist of live and/or

inactivated genotype I or II NDV or genetically modified

vaccines depending on flock age and type.

10,000

8,000

11,000

4,000

15,000

2,000

12.5 %.

14%.

37.6%.

35.8%.

22.4%.

10.7%.

Twice, live LaSota

1,000

34.5%.

Once, live LaSota

Twice, live LaSota

S10

3,000

4,000

12,000

21.3%.

39.1%.

35.7%.

Isolation

Virus isolation was done from tracheal swabs after

immersion in Phosphate-Buffered Saline (PBS) mixed

with gentamycin antibiotic (50 μg/ml) and mycostatin

(1000 units/mL). Swabs from different birds from the

same flock were immersed in the same PBS solutions.

Samples were named numerically as sample 1 (S1):

sample 10 (S10).

PBS-containing samples were clarified by

centrifugation at 5000 rpm for 15 minutes. A 200 µl of

supernatant fluid from each sample was inoculated into the

allantoic cavity of five 10-day-old Specific Pathogen Free

Embryonated Chicken Eggs (SPF-ECE). Allantoic fluid

from each egg was harvested 3 to 5 days post-inoculation

and was tested for hemagglutination (HA) activity by

rapid slide HA test. HA negative samples were submitted

for two blind passages of SPF-ECE. Collectively, samples

that showed HA positive activity were kept for further

molecular identification (OIE manual, 2018 ).

In the present study, analysis of nucleotides

sequences of F and HN genes were done for a recently

isolated NDV strain obtained from samples collected from

different chicken flocks showing mild to severe respiratory

symptoms with variable mortality rates in Giza

governorate, Egypt.

Viral RNA extraction

MATERIALS AND METHODS

Viral RNA from HA positive allantoic fluid was

extracted using Pure Link® (Invitrogen, USA) RNA Mini

Kit following the manual’s instruction.

Ethical approval

Institutional, national, and international animal care

guidelines were followed.

Real-time reverse transcription-polymerase chain

reaction

Sampling and samples history

Tracheae (at least three samples from each flock)

from 10 freshly dead broilers, NDV vaccinated chicken

flocks suffering from mild to moderate respiratory

symptoms; with mortalities varying from 10 to 40 % as

well as a range of NDV indicative postmortem lesions at

Real-time Reverse Transcription PCR (RT-qPCR)

was performed in one step. Using TOPreal™ One-step -

SYBR Green with low ROX - RT qPCR Kit (Enzynomics,

Korea) according to the manufacturer’s instructions and

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J. World Poult. Res., 10(2S): 195-202, 2020

using the CFX96 Touch real-time PCR detection system

Table 3. Primers used for F and HN genes amplification

and sequencing.

(Bio-Rad Laboratories, USA). Primers used were designed

according to Wise et al. (2004 ) which are specific for the

matrix protein gene of APMV-1 viruses selected from a

conserved region of the M gene (Table 2).

Target

gene

Primer

Sequence

Name

Fus-F

Fus-R

5’-ATGGGCTCCAAACTTTCT-3’

F protein

gene

5’-CATGCTCTTGTAGTGGCTCTC-3’

The thermal conditions were as follows; reverse

transcription at 50°C for 30 mins followed by 10 mins at

95 °C for reverse transcriptase inactivation and initial

denaturation. Then, followed by 40 amplification cycles of

95 °C denaturation for 5 s, 52°C annealing for 10 s, and 60

°C extension for 30 s.

protein

gene

Hae-F

Hae-R

5’-CATGGACCGCGCGGTTAAC -3’

5’-CTAAACTCTATCATCCTTG-3’

Sequencing

Sequencing of the purified RT-PCR products was

done by the Bigdye Terminator V3.1 cycle sequencing kit

(Perkin- Elmer, Foster City, CA) and Applied Biosystems

3130 genetic analyzer machine (ABI, USA).

Melting curve analysis was performed to determine

the specificity of amplification as follows: 95°C

denaturation for 10 s, 65°C annealing for 5 s, and heating

to 95 °C with an increment 0.5 °C for 0.05 s.

Genetic alignment

The quality of obtained F and HN genes sequences

The melting temperature (Tm) of melting curves and

Cp values were calculated using the Bio-Rad CFX

manager 3.1 software (Figure 1).

were checked, assembled, edited using Bioedit software

version 7.0.4.1 (Hall, 1999), and submitted to GenBank

using

BankIt

tool

the

GenBank

Table 2. Primers used for Newcastle disease virus

(http://www.ncbi.nlm.nih.gov/WebSub/?tool=genbank ),

with accession numbers MN905162 and MN905163,

respectively.

detection using RT-qPCR.

Primer

Sequence

Name

F Primer

R primer

5’-AGTGATGTGCTCGGACCTTC-3’

5’-CCTGAGGAGAGGCATTTGCTA-3’

Phylogenetic analysis

The tree was constructed using the neighbor-joining

method; bootstrapping at 500 repeats using Mega 6

software version 7.0.26 (Tamura et al., 2013).

F and HN genes amplification

Positive NDV RNA samples (by RT-qPCR) samples

were subjected to one-step RT-PCR using SuperScript™

III One-Step RT-PCR System with Platinum™ Taq DNA

Polymerase according to the manufacturer’s instructions to

amplify full-length F protein gene and HN protein gene

using two sets of primes kindly provided by Dr.

Mohammed Rohaim, Virology Department, Cairo

University, Egypt (Table 3) and using the ProFlex

PCR thermal cycler (Applied biosystem, USA).

RESULTS

Hemagglutination activity

After three blind passages only S3, S4, S7, S9, and

S10 samples were positive for hemagglutination activity.

S7 and S10 were positive for HA after the 1^stegg passage,

S3, and S4 were positive for HA after the 2^ndegg passage,

and S9 was positive for HA after the 3^rdegg passage. RNA

from 5 positive HA samples were sent for one-step RT-

qPCR.

Thermal amplification conditions were as follows;

reverse transcription at 50 °C for 30 min followed by

initial denaturation for 2 min at 94 °C. Then followed by

40 cycles of denaturation at 94 °C for 15 s, annealing at 65

°C for 30 s for F gene while 51 °C for 30 s for HN gene,

and extension at 68 °C for 120 s followed by one cycle of

final extension at 68 °C for 5 min.

NDV detection by RT-qPCR

Only S4 was positive for Avian avulavirus 1 by RT-

qPCR with a threshold cycle (CT) of 29.34 with a starting

quantity of 3.033 log10 in comparison with a standard

curve (Figure 2) with melting peak at 79 °C (Figure 3 and

4).

PCR products were analyzed by agarose gel

electrophoresis (1%) and then purified using a QIAquick

Gel Extraction Kit (Qiagen) following the manufacturer’s

instructions.

197

Shakal et al., 2020

protein gene showed that S4 isolate is closely related to

genotype VII subtype D (Figure 5).

Amplification of full F and full HN proteins genes

by RT-PCR

RT PCR products gel electrophoresis revealed the

expected and correct size bands for full-length F and HN

proteins genes.

HN protein gene

Blasting of sequence results obtained for the full HN

protein gene showed similarities with Chinese genotype

VII strains with similarities varying from 95.69 % to

98.72% and with some Egyptian isolates varying from

94.9% to 95.45 %The phylogenetic tree of the full HN

protein gene showed that S4 isolate is closely related to

the Chinese genotype VII (Figure 6). Three-dimensional

structure of F and HN monomer for S4 isolate was created

by SWISS-Model modeling online server and visualized

by PyMOL program version 2.3.4 (Figure 7 and 8).

Genetic and phylogenetic analysis

F protein gene

Blasting of sequence results obtained for the full F

protein gene showed similarities with Chinese genotype

VII strains with similarities varying from 95.5 % to

97.28% and with many Egyptian isolates varying from

94.5% to 95.5 %. The phylogenetic tree of the full F

Figure 1. Thermal conditions applied at RT-qPCR and for the melting curve.

Figure 2. Threshold cycles of tested samples, green lines represent positive control samples (standard curve samples), the red line

represents positive for Avian avulavirus sample (S4) appeared after 29.34 CT, and pink lines represent the negative for Avian

avulavirus samples.

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J. World Poult. Res., 10(2S): 195-202, 2020

Figure 3. Melting curve of tested samples, green lines represent positive control samples (standard curve samples), the red line

represents positive for Avian avulavirus sample (S4), and pink lines represent the negative for Avian avulavirus samples.

Figure 4. Melting peak of tested samples, green lines represent positive control samples (standard curve samples), the red line

represents positive for Avian avulavirus sample (S4) showed a different melting peak, and pink lines represent the negative for

Avian avulavirus samples.

199

Shakal et al., 2020

Figure 6. Neighbor-joining phylogenetic tree of the full-

length HN gene of Egyptian isolate of Newcastle disease

virus (NDV) (S4) in comparison to other NDV strains from

GenBank. Bootstrap values are shown above the branches. S4

isolate is indicated by a solid green circle.

Figure 7. 3D structure for F protein of Newcastle disease

virus (S4 isolate) created by SWISS-Model modeling online

server and visualized by PyMOL program version 2.3.4, red

color represents the cleavage site.

Figure 5. Neighbor-joining phylogenetic tree of the full-

length F gene of Egyptian isolate of Newcastle disease virus

(NDV) (S4) in comparison to other NDV strains from

GenBank. Bootstrap values are shown above the branches. S4

isolate is indicated by a solid green circle.

Figure 8. 3D structure for HN protein of Newcastle disease

virus (S4 isolate) created by SWISS-Model modeling online

server and visualized by PyMOL program version 2.3.4.

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J. World Poult. Res., 10(2S): 195-202, 2020

migratory wild birds in NDV evolution in Egypt with

DISCUSSION

defining the main causes of the inability of currently used

vaccines to protect chickens against infection with

Newcastle disease virus.

In the current study, only five samples (50% samples)

showed HA positive activity indicating infection with a

hemagglutinating virus. To confirm NDV infection, RT-

qPCR was performed using the HA positive samples.

Only S4 isolate was positive for NDV using

universal primers for APMV-1. Negative RT-qPCR results

for S3, S7, S9, and S10 may indicate an infection with

another hemagglutinating virus-like avian influenza H9 or

H5; however, history of mortalities and symptoms severity

indicated H9 infection mixed with other respiratory

pathogens other than H5 (Hussein et al., 2014; Sedeik et

al., 2018 ). The most important pathogenicity indicator for

NDV is the F protein gene sequence analysis mainly for

cleavage site in which velogenic strains have polybasic

amino acid sequences; therefore, molecular identification

and phylogenetic analysis of the F gene is a major

determinant

of NDV virulence instead of conventional methods

(Mohamed et al., 2011; Damena et al., 2016). Also, it can

be considered as a reliable way for NDV virulence

evaluation when compared to traditional ways of

evaluation (Ganar et al., 2014 ).

Results of F protein gene sequencing revealed that

the cleavage site motif of S4 isolate has the sequence of

velogenic NDV strains ₁₁₂RRQKRF₁₁₇in agreement with

(Sedeik et al., 2019). Also, the neurological effects of

NDV infections by is thought to be due to the presence of

the phenylalanine (F) residue at position 117 (Collins et

al., 1993). The full sequence of both F and HN protein

genes were submitted to the GenBank database with

accession number MN905162 for the full F protein gene

sequence and MN905163 for the full HN protein gene

sequence.

DECLARATION

Authors' contributions

All authors reviewed the final manuscript. This work

is a part of Mira Maher, and Abdulrahman S. Metwally

thesis under the supervision of Shakal M, and Gehan

Safwat. Shakal M. designed, supervised the experiments,

and co-wrote the paper. Gehan Safwat co-designed the

experiment and co-wrote the paper. Mohammed A. Abdel

Sabour conducted samples pooling, virus isolation, and co-

wrote the paper. Mira Maher and Abdulrahman S.

Metwally conducted RNA extraction, genes amplification

by PCR, and conducted genetic alignment. Yahia M.

Madbouly conducted RNA extraction, real-time reverse

transcription PCR, GenBank submission, phylogenetic

analysis, and co-wrote the paper.

Competing interests

The authors declare that they have no competing

interests.

Acknowledgments

Authors gratefully acknowledge Dr. Mohammed

Rohaim, Virology Department, Cairo University, Egypt

for his kind support and comments.

REFERENCES

Abd El Aziz M, Abd El-Hamid H, Ellkany H, Nasef S, Nasr S, and El

Bestawy A (2016). Biological and molecular characterization of

Newcastle disease virus circulating in chicken flocks, Egypt, during

2014-2015. Zagazig Veterinary Journal, 44(1): 9–20. DOI:

https://dx.doi.org/10.21608/zvjz.2016.7827.

F and HN proteins genes genetic and phylogenetic

analysis in the present study revealed high similarity of S4

isolate with Chinese isolates and relatively fewer

similarities with the Egyptian isolates which may strongly

refer to the role of migratory wild birds in NDV evolution

in Egypt.

Aldous EW, Mynn JK, Banks J, and Alexander DJ (2003). A molecular

epidemiological study of avian paramyxovirus type 1 (Newcastle

disease virus) isolates by phylogenetic analysis of a partial

nucleotide sequence of the fusion protein gene. Avian Pathology,

32: 239–256. DOI: https://doi.org/10.1080/030794503100009783.

Ashraf A, Shah MSU, Habib M, Hussain M, Mahboob S, and Al-Ghanim

K (2016). Isolation, identification and molecular characterization of

highly pathogenic Newcastle disease virus from field outbreaks.

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DOI: https://doi.org/10.1590/1678-4324-2016160301.

CONCLUSION

Collins MS, Bashiruddin JB, and Alexander DJ (1993). Deduced amino

acid sequences at the fusion protein cleavage site of Newcastle

disease viruses showing variation in antigenicity and pathogenicity.

Newcastle disease still occurs in sporadic cases despite

massive vaccination programs implemented in the

Egyptian poultry field. Migratory wild birds are supposed

to have a big role in the continuous evolution of NDV in

Egypt. Further epidemiological and surveillance work is

strongly recommended to define the exact role of