Research Article

Phylogenetic and Histopathological Characterization of Newcastle Disease Virus (VII.1.1) Recently Isolated from Naturally Infected Quails in Egypt

Mohamed Lebdah1, Sahar Abd El Rahman2*, Ahmed Attia3, Reham Karam2, Naglaa Fathy Saeed Awad1, Mohamed Ibrahim El Bagoury1

1Department of Avian and Rabbit Medicine, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Sharkia, 44511, Egypt; 2Department of Virology; Faculty of Veterinary Medicine, Mansoura University, 35516 El-Gomhoria Street, Mansoura, Egypt; 3Department of Genetics, Faculty of Veterinary Medicine, Mansoura University, 35516 El-Gomhoria Street, Mansoura, Egypt.

Received | January 01, 2022; Accepted | March 14, 2022; Published | June 18, 2022

*Correspondence | Sahar Abd El Rahman, Department of Virology; Faculty of Veterinary Medicine, Mansoura University, 35516 El-Gomhoria Street, Mansoura, Egypt; Email: [email protected]

Citation | Lebdah M, El-Rahman SA, Attia A, Karam R, Awad NFS, El-Bagoury MI (2022). Phylogenetic and histopathological characterization of newcastle disease virus (VII.1.1) recently isolated from naturally infected quails in Egypt. Adv. Anim. Vet. Sci. 10(7):1481-1491.

DOI | https://dx.doi.org/10.17582/journal.aavs/2022/10.7.1481.1491

ISSN (Online) | 2307-8316

Copyright: 2022 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

The Japanese quails have quick growth rate, short life cycle, short hatching period, high fecundity rate and high protein and quality meat (Owen and Dike, 2013; Gecgel et al., 2015; El-Katcha et al., 2015). During the last few decades, the Japanese quails were widely used as table and pet birds as well as they can be used as experimental birds (Ratnamohan, 1985). In Egypt, the introduction and rearing of this table bird have appeared since hundreds of years because they are less expensive, good source of animal-derived protein (Darwish et al., 2018) and more disease resistant than chickens (Yusuf et al., 2016).

Japanese quails are prone to some viral diseases either naturally; avian influenza (Arya et al., 2018) and infectious bronchitis (Fadhilah et al., 2020) or experimentally including Newcastle disease (Sharawi et al., 2015), quail bronchitis (Singh et al., 2016), inclusion body hepatitis (Singh et al., 1995), egg drop syndrome-76 (Mohapatra et al., 2014), Marek’s disease (Adedeji et al., 2019), avian leukosis (Zhang et al., 2019), avian encephalomyelitis (Fan et al., 2017), avian pox (Gülbahar et al., 2005) and reticuloendotheliosis (Carlson et al., 1974). Few of these disease conditions have been investigated extensively.

Newcastle disease is regarded as one of the most important contagious viral diseases affecting the poultry industry (Miller and Koch, 2013). It affects over 240 different avian species (OIE, 2012). It poses threat to both vaccinated and non-vaccinated birds and has challenged all the biosecurity measures. It is the most infectious disease after avian influenza (Shittu et al., 2016). In chickens, it is characterized mainly by respiratory, digestive and nervous manifestations (Mishra et al., 2000). It causes also high morbidity and mortality rates, decrease egg quantity and quality resulting in significant financial economic losses (Orsi et al., 2009; Yan et al., 2011). While, quails are either considered susceptible host for NDV infection producing mild disease (OIE, 2014; Mohamed and Abdel Hafez, 2016) or NDV carriers (Lima et al., 2004).

Newcastle disease is caused by Newcastle disease virus (NDV), a member of the genus Orthoavula viruses, subfamily Avulavirinae, family Paramyxoviridae, and order Mononegavirales (Amarasinghe et al., 2019). Avian paramyxoviruses (APMVs) in wild and domesticated birds have recently been classified into 15 serotypes (APMV-I to APMV-XV) (Thomazelli et al., 2017). The NDV is a single-stranded, enveloped, negative sense RNA virus (Alexander and Senne, 1998). Only virulent APMV-I serotype strains are responsible for the disease. According to pathogenicity indexes, there are five pathotypes of NDV; velogenic viscerotropic viruses, which cause high morbidity, high mortality and hemorrhages in intestinal tract; velogenic neurotropic viruses, which cause nervous signs, respiratory signs and high mortality rates; mesogenic viruses cause low mortality rates, if not complicated with other pathogens, respiratory and nervous signs; lentogenic viruses which are used as a vaccine and asymptomatic enteric NDVs also used as a vaccine since they do not cause any clinical signs (Alexander and Jones, 2001; Mazinga, 2011).

Virulent NDV has an intracerebral pathogenicity index (ICPI) of 0.7 or higher or multiple basic amino acids between 112 and 116 residues at the C terminus of the F2 protein and phenylalanine at residue 117 (OIE, 2012). According to the genetic characters, NDV strains are divided into two categories: class I and class II. Non-virulent strains isolated from waterfowl and shorebirds are typically found in Class I, which is made up of a single genotype (Diel et al., 2012). Class II is subdivided into 21 genotypes (І-ХХІ) and multiple subgenotypes (Dimitrov et al., 2019). The most widely distributed NDV strains in chickens and quails all over the world and also in Egypt is genotype VII (Bhuvaneswari et al., 2014; Miller et al., 2015; Ewies et al., 2017; Megahed et al., 2018).

The main source of NDV infection in chickens is avian species that carry and shed the virus (Chukwudi et al., 2012). Quails could be a risk factor for NDV transmission to other poultry species, resulting in great economic losses (Lima et al., 2004; Sharawi et al., 2015). There are few data about the natural infection of quails with NDV in Egypt. Therefore, the present study aimed to isolate the current circulating NDVs from quails in Egypt using serological and molecular characterization in addition to observe the pathological changes induced by these field viruses in naturally infected quails.

MATERIALS AND METHODS

History of the examined flocks and samples collection

Under complete aseptic conditions, 33 quails representing 11 commercial diseased flocks (3 quails/flock) of various ages and breeds were collected. The investigated flocks’ sizes ranged from 2000-10000 birds. They were mainly layers. All the quail flocks were not vaccinated against ND. Before samples collection and viral isolation trials, the quails were subjected for clinical and postmortem examinations. From each quail (liver, spleen, lung, trachea and brain) were collected under aseptic condition. All the collected tissues were expressed as 11 pooled samples (3 quails /flock/1 pool/1sample). The pooled samples were homogenized in 10% tissue suspensions. The homogenates were centrifuged at 1000rpm/10min in cooling centrifuge then the supernatants were collected and aliquoted. The antibiotics (penicillin at 2000 IU/ml and streptomycin at 2 mg/ml) were added to the supernatants to inhibit bacterial contamination (Numan et al., 2008; OIE, 2012).

ECE inoculation

Two hundred specific pathogen free embryonated chicken egg (SPF-ECE supplied by Kom Osheim SPF farm; El-Fayoum Governorate) were used for propagation and isolation of NDV from quails after natural infection. The supernatants of tissue pools homogenates, control negative and control positive (LaSota strain- NDV-CEVAC NEW L, Batch No, 1110A1LN2M) were separately inoculated into SPF-ECEs at 9-11 days via allantoic cavity route (five eggs were used per each sample) for three successive passages (0.2ml/each). The SPF-ECEs were candled for up to 7 days. Deaths within 24 hours were discarded as non-specific deaths. Following that, the eggs containing dead embryos were collected and chilled at 4°C overnight. Then, the allantoic fluids were harvested aseptically and any lesions found in the embryos or chorioallantoic membrane (CAM) was investigated (Alexander, 1989; OIE, 2008).

Hemagglutination and hemagglutination inhibition test

All harvested allantoic fluids, control positive (LaSota strain) and control negative samples were examined for HA activity by slide and microtiter plate HA using 10% and 1% washed chicken red blood cells (RBCs) concentrations, respectively as previously performed (Anon, 1963; OIE, 2012). The positive HA isolates were examined later by HI test using specific-rabbit antisera against NDV obtained from Virology Department, Faculty of Veterinary Medicine, Mansoura University. Hemagglutination inhibition test (HIT) was conducted for determination the titre of NDV in HA positive allantoic fluids as previously described (Alexander, 1989).

Reverse transcriptase polymerase chain reaction

The manufacturer’s instructions were followed to extract viral RNA from the positive HI allantoic fluids, control positive (LaSota strain) and control negative using the QIAamp-MinElut virus spin kit. The RT-PCR was used to detect velogenic and lentogenic NDV strains by amplification of a 362 bp fragment of the NDV F gene using primers (A+B) at annealing temperature 52°C and to differentiate velogenic and lentogenic NDV strains by amplification of a 254 bp fragment of the NDV F gene for velogenic NDV strains only using primers (A+C) at annealing temperature 45°C. The primers’ sequences were A: 5’-TTGATGGCAGGCCTCTTGC-3’, B: 5’-GGAGG ATGTTGGC AGC ATT-3’ and C: 5’-AGCGT(C/T)TCTGTCTCCT-3) (Kant et al., 1997). According to the manufacturer’s instructions, a one-step RT-PCR kit (enzymotics, South Korea) was used. 10µl 2x reaction buffer, 4µl extracted RNA, 0.8µl enzyme, 2µl of forward and reverse primers were mixed then nuclease free water was finally added up to 20µl. Cycling condition began with reverse transcription at 45°C/10 minutes by one cycle then inactivation and initial denaturation at 95°C/10 minutes for one cycle. Later, denaturation at 95°C/30 seconds, annealing at 52°C/30 seconds for A+B primer pair and 45°C/30 seconds for A+C primer pair and elongation at 72°C/1 minutes by 40 cycles. Final elongation at 72°C /10minutes by one cycle. The RT-PCR products were electrophoresed on agarose gel according to (Vögtlin et al., 1999). The specific bands (362 bp F gene fragment) for five selected PCR positive samples (sharp bands) and control positive (LaSota strain) were used for DNA sequencing. The DNAs were extracted from the agarose gel according to Lee et al. (2012). The extracted DNA targeting F gene (362 pb) was submitted to MACROGEN clinical laboratory, Korea. The nucleotide sequences of 362bp DNA of NDV F gene were used for phylogenetic analysis using alignment by MEGA X and bioedit software.

Histopathological examination

Tissue specimens from the lung, brain, liver, and trachea were fixed in 10% buffer formalin for 24 hours, transferred to a 70% ethanol solution, fixed in paraffin and processed using traditional methods as described previously (Kim, 2018). Tissue sections of fixed lesions were stained with hematoxylin and eosin and then examined microscopically for any changes in quails’ organs.

RESULTS and Discussion

Clinical and postmortem findings

In most of the investigated quails, neither the clinical signs nor the gross lesions are diagnostic for ND. Notably, in eleven non-vaccinated quail flocks, most of the clinically examined birds were apparently healthy with no clinical signs. Meanwhile, few diseased quails showed signs in the form of depression, anorexia, sneezing, torticollis (Figure 1A), opisthotonos (Figure 1B), paralysis of one or two legs and whitish green diarrhea (Figure 1C). Additionally, to our knowledge, this is the first study to show reproductive problems in naturally infected quails, including a 25% decrease in egg production, thin shell or shell-less eggs, loss of pigmentation and easily broken eggs (Figure 1D). The morbidity and mortality rates in the investigated flocks were ranged from 1-90%. The main postmortem lesions were congested liver, spleen, brain and muscles besides the greenish contents of the gizzard (Figure 2). Also, pneumonia, consolidation of lung and emaciation were observed in some flocks (Table 1).

Virus isolation, hemagglutination and hemagglutination inhibition test

All collected samples prepared from naturally infected quails were inoculated into SPF- ECEs for three passages with homogenate tissue suspensions samples. The dead embryos showed hemorrhages, congestion and dwarfism (Figure 3A). The positive HA allantoic fluids were used for HI test using known antirabbit hyperimmune serum. The HI results expressed 5 positive samples for NDV (Table 2).

 

Table 1: Clinical and Postmortem Examination of quails collected from infected flocks.

Flock No.	Age	Mortality	Flock/ capacity	Clinical signs					Postmortem lesions
				Resp. signs	Anorexia	Diarrhea	Nervous signs	Decrease in egg production	Intestinal ulcer	Congested liver, muscles and brain
1	2M	90%	3000	+	+	+	-	+	-	+
2	3M	↓1%	12000	-	-	-	+	+	-	+
3	4M	↓1%	4000	mild+	-	+	-	+	-	+
4	1M	↑90%	5000	+	+	+	-	-	-	+
5	2M	5%	4000	+	-	-	-	-	-	+
6	3M	↓1%	5000	+	-	+	-	+up to 25%	-	+
7	2M	↓1%	4000	-	-	-	+	+up to25%	-	+
8	5M	↓1%	8000	-	-	+	-	+up to 25%	-	+
9	4M	↓1%	6000	-	-	+	-	-	-	-
10	2M	↑90%	2000	-	+	+	-	+	-	+
11	3M	↑90%	4000	-	+	+	-	+	-	+

 

Table 2: Descriptive data and NDV detection steps of the examined quail flocks.

Sample No	Mortalities in inoculated SPF-ECEs in the third passage	Serological identification by			Molecular characterization by RT-PCR
		HA	HA Titrelog 2	HI	362 Bp fragment	254 Bp fragment
1	1 after 24 h 4 after 48 h	+	5	_	_	_
2	2 after 72 h 3 after 96 h	+	4	+	+	_
3	3 after 48 h 2 after 72 h	+	5	+	+	+
4	5 after 24 h	+	6	_	_	_
5	5 after 72 h	+	4	_	_	_
6	2 after 48 h 3 after 96 h	+	5	+	+	+
7	3 after 48 h 2 after 96 h	+	4	+	+	+
8	4 after 72 h 1 after 96 h	+	5	+	+	+
9	5 after 48 h	_	Nd	Nd	_	_
10	5 after 24 h	+	5	_	_	_
11	5 after 72 h	+	6	_	_	_
Total	55	10		5	5	4
C+ve		+	10		+	+
C-ve		_	_	_	_	_

 

Nd, means not done

 

Detection of NDV by RT-PCR

Five positive HI allantoic fluids were confirmed by conventional RT-PCR and showed clear bands at 362 and 254 bp of the F gene fragment using primers A and B for all NDV pathotypes and using primers A and C for virulent NDV strains, respectively. The results revealed that 5 samples out of 11 examined samples (5/11; 45.45%) were virulent NDV field isolates (Table 2).

Sequencing and phylogenetic analysis

On gel electrophoresis, five samples were chosen for DNA sequencing from RT-PCR positive samples (clear bands). The obtained sequence targeting the F gene at 362 bp fragment was designated as quail/Egypt/Zagazig/2021 and was deposited in GenBank as accession number OK310497 (Table 4). In addition, the identified sequence was compared to reference strains from GenBank that belonged to class II sub-genotypes VII.1.1, VII.1.2, and VII.2 of NDV (F gene sequences) (Table 3). The isolated field strain was found in sub-genotype VII.1.1 with other Egyptian strains. The sequencing of the 362-bp F gene revealed that one isolate had the 112 RRQKRF 117 motif, which is characteristic of virulent strains.

 

Table 3: Orthoavula viruses reference strains used in the phylogenetic tree.

Accession number	Strain	Year	% of nucleotides identity	Sub-genotype
OK310497	Quail/Egypt/Zagazig	2021	100%	VII 1.1
MW566177	Chicken/Egypt/QALUIBIA/NRC	2019	97.54%	VII 1.1
MG717683	quail/Egypt/SDU-2	2016	96.89%	VII 1.1
MN481243	Chicken/Egypt/ME4	2017	96.89%	VII 1.1
MH377309	turkey/isolate PHL255913	2016	96.89%	VII 1.1
MH377297	turkey/isolate PHL257758	2016	96.89%	VII 1.1
MH371065	turkey/isolate PHL245632	2016	96.89%	VII 1.1
KY075889	chicken/Egypt/Qualyobia26	2016	96.89%	VII 1.1
MW603771	Chicken/Egypt/KSH/VVTNRC	2021	96.89%	VII 1.1
MN481246	Chicken/Egypt/ME7	2016	96.58%	VII 1.1
MN481241	Chicken/Egypt/ME2	2017	96.58%	VII 1.1
MN481240	Chicken/Egypt/ME1	2016	96.58%	VII 1.1
MH377314.1	chicken/isolate PHL266018	2016	96.58%	VII 1.1
MH377313.1	chicken/isolate PHL265559	2016	96.58%	VII 1.1
MH377294.1	chicken/isolate PHL253817	2016	96.58%	VII 1.1
MG966505.1	chicken/Egypt/48Ob/NRC-CU	2014	96.58%	VII 1.1
KX686725.1	Egypt -GZ-339F	2015	96.58%	VII 1.1
KY075881.1	chicken/Egypt/Ismailia8	2016	96.58%	VII 1.1
MN481251.1	Chicken/Egypt/ME12	2014	96.27%	VII 1.1
MW580389.1	CHICKEN-EGY-ALEX-NRC	2020	96.27%	VII 1.1
MK495908.1	Chicken/Egypt/Qena/4	2012	98.7	VII 1.1
AY028995.1	China/CH-A7/96	1996	90.15%	VII 1.2
AF458010.1	chicken/China/JS-3/00	2000	90.15%	VII 1.2
AF140343	Chicken/China/LZ	1998	90.15%	VII 1.2
GQ338309	Pigeon/China/03/018	2003	90.15%	VII 1.2
KY747481	Chicken/Namibia/6196	2016	88%	VII.2
KY747482	Chicken/Namibia/6403	2016	87.69%	VII.2

 

 

 

The isolated strain was closely related to strains of NDV sub-genotype VII 1.1 including the QALUIBIA/NRC/2019, ME4/2017, turkey/isolate PHL257758/2016, turkey/isolate PHL245632/2016 and EG-SH2/NRC (Table 3). The nucleotide sequence of the present strain quail/Egypt/Zagazig/2021 showed high identity percent as 97.54% with QALUIBIA/NRC/2019, then 96.89% identity with the Egypt/ ME4/2017, turkey/ isolate PHL257758/2016, turkey/ isolate PHL245632/2016, EG-SH2/NRC, Qualyobia26/ 2016 and EGY-KSH-VVTNRC-2021 (Tables 3, 4, Figures 4, 5).

 

 

 

 

Table 4: OK310497 Quail/Egypt/Zagazig and sub genotype type VII 1.1 - specific residue substitutions in the deduced F nucleotide sequence.

Strain / Position	35	43	47	94	104	153	196
Majority	T	A	T	A	G	T	C
MW566177.1/Chicken/Egypt/QALUIBIA/NRC	.	.	.	.	.	.	.
OK310497 Quail/Egypt/Zagazig	G	G	G	C	C	C	A
MG717683.1/quail/Egypt/SDU-2	.	.	.	.	.	.	.
MN481243.1/Chicken/Egypt/ME4	.	.	.	.	.	.	.
MH377309.1/turkey/isolate PHL2	.	.	.	.	.	.

 

The mark (.) means the same nucleotide.

 

Table 5: The first comparison of histopathological features between naturally infected quails with NDV VII and healthy quails.

Affected organ	Lesions	Healthy quail	Naturally infected quails
Brain	Non suppurative encephalitis Homorrhage and hyperemia in meninges Degenerated neuron Encephalomalacia and gliosis	_ _ _ _	++ +++ ++ ++
Liver	congestion of hepatic sinusoids coagulative necrosis of the hepatic parenchyma portal cholestasis degenerative changes in the hepatic cells interstitial lymphocytic aggregation	_ _ _ _	+++ + ++ ++
Lung	focal pneumonic areas organization hemorrhage and hemosiderosis	_ _ _	++ ++ +
Trachea	Tracheitis epithelial destruction	_ _	++ ++

 

Histopathological changes

The trachea of the naturally infected quails showed mucous exudate in the tracheal lumen admixed with a few leukocytes and erythrocytes (Figure 5B). Additionally, thickened tracheal wall (tracheitis), infiltration of leukocytes mainly lymphocytes, proliferative glands and partial epithelial destruction (Figure 5C). The lungs of the examined quails exhibited inflammatory exudate varied from sero-fibrinous to caseous with or without heterophils and erythrocytes in the bronchial lumen and peribronchial air vesicles together with hypertrophy and necrosis of the bronchial smooth muscles (Figure 5E). Focal pneumonic areas consisted of necrotic debris, living and degenerated heterophils were scattered within the lung tissues besides necrotic tissues invaded by fibroblasts together with macrophages and giant cells (organization) (Figure 5F).

The liver of the investigated quails showed intense congestion of hepatic sinusoids together with degenerative changes (micro steatosis), apoptosis and minute coagulative necrosis of the hepatic parenchyma (Figure 6B). Other birds had interstitial lymphocytic aggregation with various degenerative changes in the hepatic cells (Figure 6C). The quails with nervous signs had a picture of non-suppurative encephalitis, hemorrhages in meninges and Virchow robin spaces (perivascular) and perivascular edema (Figure 6E). Additionally, the mid brain mainly cerebellum had degenerated Purkinjean neurons along with meningeal blood vessels usually hyperemic with a few extravasated erythrocytes, lymphocyte and edema in sub meningeal tissues (Figure 6F).

DISCUSSION

In the current work, few naturally infected quails revealed whitish green diarrhea, neurological signs. This result was compatible with Moustafa et al. (1983). Respiratory signs were mild in some farms and absent in others. This was in agreement with Abd-el-Latif et al. (2009) and to our knowledge, it is the first report to show reproductive disorders in the form of drop in egg production reached 25%, thin shell or shell-less eggs, loss of pigmentation and easily broken eggs in naturally infected quails with low mortality and morbidity not exceed 1%. Postmortem examinations revealed congested brain, liver, lung and spleen in addition to greenish content of the gizzard. There was absence of the pathognomonic lesions which are present in the chicken such as hemorrhage of proventriculus, ulcer of intestine and hemorrhage in cecal tonsils. These results were in accordance with Lima et al. (2004). Based on these findings, it could be concluded that APMV-I pathogenicity in quails differs from that in domestic chickens. These findings come in agreement with Abd-el-Latif et al. (2009) who found, nervous manifestations and mild respiratory signs with 1.6% mortalities in a 3 week-old quail flock containing 5000 birds. Similarly, Momayez et al. (2007) found diarrhoea, nervous signs, morbidity of more than 10% and mortality of about 5% in a flock of 1500 quails aged 1 day, 3 weeks, 6 weeks, and 3-6 months in Tehran province, Iran, without NDV vaccination. Czirjak et al. (2007), on the other hand, reported that in a natural outbreak of ND, the morbidity and mortality rates were 100%. Also, Lima et al. (2004) found no clinical signs or mortality in experimentally infected quails.

The birds, submitted for histopathological examination in this study, showed microscopic changes in the brain including non-suppurative encephalitis, perivascular edema, degenerated neurons, and neuronophagia with early encephalomalacia besides gliosis. These results were in accordance with those of Susta et al. (2018). In addition to, thickened tracheal wall (tracheitis) by infiltrating leukocytes mainly lymphocytes and epithelial discontinuation were also recorded. This was in agreement with Ayoob et al. (2019).

Regarding to viral isolation, all collected samples were inoculated via allantoic cavity route in SPF-ECEs, NDV suspected samples caused congestion, hemorrhagic lesions and death of the embryo within 96 hours of inoculation (OIE, 2012). All of the samples came from flocks that had not been vaccinated against Newcastle. The HA titers for the tested allantoic fluids were low, indicating that the quails could be carriers for NDV and spread the virus to other poultry species, particularly domestic chickens, which are highly susceptible to NDV. This was consistent with the findings of Sa’idu et al. (2004), who found low HA titers in quails.

The results of the HI test for NDV detection were confirmed by the RT-PCR test and agreed with those of Munir et al. (2012). To identify, portray and pathotype the isolated NDVs, researchers used a molecular diagnostic technique called RT-PCR, which uses Taq polymerase enzyme and forward and reverse specific primers (Tan et al., 2008). Five isolates from extracted viral RNA from the allantoic fluid of 11 samples representing 11 quail flocks revealed five clear bands at the correct expected size (362 bp) for NDV on electrophoresis (El-Bagoury et al., 2015). The 362-bp fragment of the F gene, which is conserved in all NDNs pathotypes, was amplified using the A+B primer set for preliminary NDV identification (Kant et al., 1997). The NDV characterization and surveillance are currently carried out using reverse transcriptase polymerase chain reaction (RT-PCR) amplification of the partial coding region of the F gene, followed by sequencing and phylogenetic analysis of the fusion protein gene cleavage site (Khan et al., 2010). The pathogenicity of NDV is initially determined by the amino acid sequence of the fusion protein cleavage site and the cellular protease that cleaves the fusion proteins (Nagai et al., 1976).

It was critical to pathotype and genotype the circulating virus in these flocks. The OIE defines Newcastle disease as an infection of birds caused by a virus (NDV) of the avian paramyxovirus serotype 1 (APMV-1) that meets one of the virulence criteria listed below: The virus has an intracerebral pathogenicity index (ICPI) of more than 0.7 in day-old chicks or at least three basic amino acids between residues 112 and 117, particularly arginine (R) or lysine (K). The OIE defined virulent NDV strains as mesogenic and velogenic strains that met the OIE criteria (Miller and Koch, 2013).

To characterize NDV strains, most laboratories and researchers now use genome sequence phylogenetic analysis. Because of the importance of the F gene in determining virulence, the first studies focused on partial F gene sequences (Aldous et al., 2003; Collins et al., 2007; Diel et al., 2011). The molecular virulence determination is sufficient with a partial fusion protein of 274 base pairs (bp) in length surrounding the site where the F0 precursor is cleaved into F1 and F2 fragments (OIE, 2009). According to the genome length and F gene sequence within APMV-1, NDV isolates are divided into two classes: class I (nine genotypes) and class II (twenty-one genotypes) (Dimitrov et al., 2019). The identified strain in the present study (quail/Egypt/Zagazig/2021) is clustered in class II, genotype VII and sub genotype VII 1.1 with other Egyptian strains. The Egyptian NDV sub genotype VII.1.1 strains isolated from chickens and turkeys in various Egyptian provinces are strikingly similar to our sequenced strain. This was done in line with the findings of Radwan et al. (2013), who found that sub genotype VII 1.1 is the most common NDV sub genotype in Egypt. The antigenic and genetic comparison of the isolated NDV strain from quails to strains from chickens and turkeys could indicate a possible role of quail and wild birds in disease transmission (Brown and Bevins, 2017). Analysis of the quails’ strain isolated from Egypt revealed that they are somewhat divergent, with 3.73% pairwise distance between the quail/Egypt/SDU-2/2016 (VII.1.1) and our isolate quail/Egypt/Zagazig/2021. These findings showed that Egyptian quail-bird-origin NDV strains can infect commercial poultry, highlighting the need for biosecurity assessments, vaccination, and ongoing surveillance, as well as revised vaccine approaches. The findings revealed a significant rate of mutation among circulating NDVs in recent years.

CONCLUSIONS AND RECOMMENDATIONS

In conclusion, sub-genotype VII.1.1 strains of NDV are the currently circulating strains among quails’ flocks in Egypt. Phylogenetically, NDVs in quails are closely related to NDV in chickens and turkeys which carry the virulent motif of F protein cleavage site 112 RRQKRF 117 reflecting the high possibility of virus exchange between different avian species. Unfortunately, there is no vaccination against NDV in quail flocks in Egypt. Therefore, the biosecurity and proper vaccination with newly isolated mutated viruses against NDV is recommended. Further large-scale surveillance is required to identify the development of the NDV in quails and use the appropriate vaccination.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge Prof. Dr. Adel Zaghloul, the Department of Surgery, Anesthesiology and Radiology, Faulty of Veterinary Medicine, Mansoura University, Egypt, and Dr. Tamer Abdullatif, Department of Avian and Rabbit Medicine, Faculty of Veterinary Medicine, Zagazig University, for technical and scientific support.

Author’s Contribution

All authors contribured equally.

Conflict of interest

The authors have declared no conflict of interest.

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