Chickens are the most commonly affected avian species. Clinically, five NDV pathotypes were categorized. Additionally, in vivo testing in embryos or chickens/pigeons can be done to determine strain pathogenicity, including mean death time (MDT), intravenous (IV) pathogenicity index (IVPI), and intracerebral pathogenicity index (ICPI). The MDT is the time to death, measured in hours, after inoculation of ECEs. The MDT for the NDV strains, <60, 60–90, or >90 h, was considered velogenic, mesogenic, or lentogenic strains, respectively (2, 33). The IVPI involves scoring illness (0 = normal; 1 = sick; 2 = paralyzed or nervous signs; and 3 = death) after IV inoculation of 6-week-old chickens. The velogenic strains have an IVPI score of 2–3, mesogenic 0–0.5, and lentogenic of zero (34). Now, the definitive in vivo assessment of NDV virulence is based on the ICPI test. It is considered as the most sensitive and widely used test for measuring the virulence in ten 1-day-old chicks (2, 35). The ICPI involves scoring sick or dead (0 = normal; 1 = sick; and 2 = dead). Scores with an ICPI of 1.5–2 is considered velogenic, 1–1.5 mesogenic, and 0–0.5 lentogenic. ICPI score of ≥0.7 is considered “notifiable” to the OIE (2). Particular drawbacks of pathogenicity indices are in the interpretation of pathotype results. A previous study (36) reported 10 NDV isolates from pigeons to have ICPI values (1.2–1.45) and IVPI values (0–1.3), classifying the isolates as virulent. However, MDT was low (98 h) in lentogenic strains. Indeed, not all virulent strains have an MDT <60 h. The in vivo tests on NDV isolates from species other than chickens can present some problems and may not produce accurate interpretations until passaged in chickens or ECEs (37). The course of the NDV infection can vary widely depending on the virulence of the virus. Several studies were conducted in Egypt on the in vivo pathogenicity of NDV field isolates (Table 1). Earlier, in a surveillance among more than 100 chicken flocks at Sharkia Province in late 1980s, 26 NDVs were isolated, velogenic (n = 15), mesogenic (n = 9), and lentogenic (n = 2) isolates, from vaccinated birds, besides one mesogenic from egg shell and other lentogenic from drinking water. The velogenic NDVs showed IVPI (1.5–1.8) in chickens; however, higher figures of ICPI (2.3–3.7) were recorded in pigeon squabs (38). The values of pathogenicity indices of Egyptian NDV isolates (Table 1) revealed that the majority of NDV strains were velogenic. The MDT (36–60 h), IVPI values of 1.5–3, and ICPI scores (1.6–2.0) are indicative for velogenic nature NDV isolates. However, for mesogenic strains, the MDT and ICPI were >60 h and 0.5–1.5, respectively. Besides, 96–108 h and 0.38–0.44 for MDT and ICPI, respectively, indicate the lentogenic nature of strains. The aforesaid findings are consistent with the sequence analysis of F protein cleavage site. In contrast, Nagy et al. (51) characterized 13 NDV isolates from vaccinated chicken flocks during 2014–2017; two of them, Ck/ME3/Eg/16 and Ck/ME5/Eg/16, had the 112GRQGRL117 cleavage motif characteristic to lentogenic strains, although they showed MDT <36–48 h and high ICPI (1.89–2.00) congruent for velogenic pathotype. Similarly, Naguib et al. (53) recorded that the sequence of isolate R1973/11 represents a cleavage site of lentogenic viruses; however, it had an ICPI of 1.88, clearly categorizing the isolate as velogenic. The applied molecular pathotyping by RT-qPCR using the primers and probes specific for avirulent and virulent pathotypes revealed that two Egyptian NDV isolates during 2011 were positive for lentogenic and virulent pathotypes, indicating mixed infection.

In Egypt, NDV infection results in variable mortalities and clinical findings. The clinical signs observed in commercial broiler chickens were severe depression, green diarrhea, paresis, and death within 48–72 h after the onset of the disease. Other signs including severe conjunctivitis, facial swelling, and birds standing dull with drooping wings were recorded in many studies. Besides, a drop in egg production reached 50% in layer flocks. Necropsies revealed congestion of the meningeal blood vessels and signs of septicemia in the form of congested subcutaneous (SC) blood vessels; congestion of the liver, spleen, and lungs; and gallbladder enlargement. Tracheitis and airsacculitis were seen in the respiratory tract. Hemorrhages on the tips of the proventriculus gland, greenish mucous content in the gastrointestinal tract, elliptical raised ulcers in the intestine, and enlarged hemorrhagic cecal tonsils were also observed (40, 110, 111). Variable mortalities (10–100%) were recorded in backyard and commercial vaccinated and non-vaccinated broiler poultry flocks (9, 49, 111–113). The clinical signs were respiratory distress and elevated mortality with nervous manifestation and deaths occurring with 24–48 h after the onset of clinical signs (48).

National efforts to update the knowledge about NDV/AIV prevalence and an active surveillance undertaken on 195 broilers and layers farms from 18 Egyptian provinces resulted in 41/195 (21%) positive for matrix gene of NDV and 24/195 (12%) positive for virulent NDV (44). During 2014–2015, the NDV genotype VII was reported with a percentage of 37.8% (114). There was a similar incidence of 37.5% in chicken flocks of 10- to 240-day-olds located in different districts of Sharkia province (115). Lower prevalence (12.5–16.2%) was recorded (49, 52, 116). A higher incidence of NDV with a percentage of 57.5% was detected during 2012–2015 (46) and 45.46% during 2019 (117). The highest incidence of NDV with a percentage of 86.2% was recorded in commercial chicken flocks during 2012–2015 (48). Moreover, Moharam et al. (9) screened 26 chicken flocks (backyard and commercial) during 2015–2016. Small-scale holders including farms keeping layers (n = 9), broilers (n = 4), or Balady chickens (n = 3) situated in provinces Beheira and Monufia of the Nile delta region. Commercial broiler farms were located in the provinces of Giza (n = 9) and Monufia (n = 1). Although all flocks were ND vaccinated, a virus was in both holders with a percentage of 84.6%. However, virulent NDV was detected in 30.76%. A recent study was conducted on 120 poultry flocks from 10 Egyptian provinces in the Egyptian Delta region during 2015–2019. The highest prevalence of virulent NDV was reported in broiler flocks (41.1%; 37/90), followed by layer flocks (38.4% 5/13). Baladi and Sasso chickens represented a confirmed NDV of 71.4% (5/7) and 33.3% (1/3), respectively (10).

Regarding the prevalence of NDV according to localities, several studies were conducted to investigate the geographic prevalence of NDV in different provinces in Egypt. The overall incidence of NDV in Egyptian provinces during 2012–2019 (Figure 2) ranged from 8.3% in Luxor to 100% in many regions: Port Said, Damietta, Gharbia, Menofia, Qalubia, and Minya (44, 46, 47, 52). The incidence of NDV in Sharkia ranged from 14% (52) to 50% (10). No NDV was detected in some provinces: Qaluibia, Menofia, Matrouh, Qena (52), Giza (10), and Aswan (44). The variations mentioned above in the prevalence of NDV along the Egyptian provinces could be attributed to (i) individual concept of NDV surveillance either in domesticated or wild, free-living, and migratory birds; (ii) difference of testing procedures and sample size; (iii) variable distribution of poultry farm population and levels of biosafety and biosecurity; (iv) absence of efficient and reliable surveillance systems that are needed to document the disease status of a population at a given time; and (v) different factors like the disease awareness of persons reporting suspect cases. In this framework, and according the OIE, any national surveillance scheme for an animal/avian infection may be constructed on two different surveillance approaches: active as the regular periodic samples' collection by veterinary health authorities and passive surveillance, which is distinct from active surveillance, as birds are only tested if they show clinical signs, and then they are detected and reported to the national authorities (118). Besides mandatory standard parameters of ND prevalence, studies must be approved and applied.

Columbiformes birds including pigeons and doves can be infected with NDV; however, the disease in pigeons is mainly caused by pigeon-specific viruses, e.g., PPMV-1. Pigeons and doves had been implicated as amplification or reservoir hosts, as they were frequently infected with virulent strains of NDV (119, 120). Since 1981, the clinical pictures in pigeons were consistent with ND, and then the virus infection was serologically confirmed in diseased pigeons along the Nile delta provinces in 1984 (26). Compulsory vaccination of racing pigeons is a local act in some countries. However, control of NDV in wild pigeons is almost impossible (121). Thus, pigeons are considered threatening carriers for the poultry industry (122). Pigeons in Egypt are not regularly vaccinated. As described by Mansour et al. (123), Elgendy et al. (124), and Rohaim et al. (54), pigeons suffered from NDV and AIV infections. In previous records of pigeons, the NDV infection caused a variable range of mortalities and morbidities (125, 126). Abou Hashem (127) did isolate 24 antigenically similar PPMV-1 viruses from pigeons in the Egyptian provinces of Sharkia (n = 14) and Dakalia (n = 10). Experimental IV or intraocular (IO) inoculation of pigeons revealed diarrhea and nervous signs [3–11 days post-infection (dpi)] followed by 100% death. The virus was transferred by contact with birds and caused mortality of 60–80%. The prevalence of PPMV-1 in Dakhlia province showed 18/100 pigeons suffering from diarrhea and nervous signs. When pigeons were infected experimentally with field viruses, the re-isolation of PPMV-1 was up to 13 days pi; and using HI, the relationship between PPMV-1 and the LaSota strain was confirmed (128). Moreover, Amer et al. (129) explored the virulence and transmissibility of NDV experimentally in pigeons, as 5% of the infected birds showed greenish diarrhea and 95% had positive virus isolation, with a detection rate of 70% in contact pigeons. In Kafr El-Sheikh province during 2014, wild pigeons showed dullness, lethargy, and neurological signs in addition to petechial hemorrhages in the heart and brain, congested lungs and liver, and enlargement of spleen upon necropsy. Molecularly, the detected virus (Pigeon/Egypt/VRLCU/2014) belonged to genotype VI, a well-known lineage representing PPMV-1 in pigeons. Biologically, as shown in Table 1, the virus had ICPI and MDT of 1.2 and 86 h, respectively (54). In 2014, a pooled brain sample from three diseased free-living pigeons (torticollis and whitish green diarrhea) in Desouk, Kafr El-Shiekh province, Egypt, was identified by HI and partial F-gene sequencing as genotype VI (130). One study focused on the outbreaks of influenza and paramyxovirus co-infections among clinically diseased pigeons (7), where mortalities varied from 10 to 92.5% in both single and mixed virus infections, with an indicative clinical picture of PPMV-1 and/or AIV, as pigeons showed tremors (83.3%), torticollis (17.7%), wing and leg paralysis (25%), and greenish diarrhea. Also, respiratory signs were observed only in few naturally infected pigeons. NDV was detected in 67.8% of diseased pigeons, mostly in pigeons aged ≤ 1 month. Mesogenic NDVs are rarely traced in prevalence along the history of ND. Nevertheless, Hamouda et al. (131) utilized restriction fragment length polymorphism (RFLP) to identify nine strains (mesogenic/lentogenic PPMV-1); 27 (mesogenic/lentogenic NDV) in the Sharkia province, in addition to one velogenic strain for each NDV and PPMV-1, were also detected. In another study, brain samples were collected from 12 pigeon farms with severe neurological symptoms and greenish diarrhea and showed a mortality rate of 3.3–38.5%. The ICPI values were 1.41–1.51, and MDT was 64–69 h (Table 1). However, IVPI in chickens was zero, indicative of moderate virulence (mesogenic nature) in chicken. Phylogenetically, all tested viruses were in genotype VI (56). Viruses of genotype VI had also been circulating in apparently healthy pigeons (132, 133), and in the study of Sabra et al. (55), it was confirmed that at least sub-genotype VIg is probably maintained in healthy captive pigeons in Egypt, with an ICPI value of 1.31 (Pigeon/Egypt/Giza/11/2015).

To follow the transmission dynamics of avian avulavirus (velogenic viscerotropic ND-genotype VII), intranasally (IN) or intramuscularly (IM) infected 8-week-old non-vaccinated native Egyptian Balady pigeons were kept in contact with non-vaccinated commercial Arbor Acres broiler chickens (4 weeks of age). The IM-infected birds had 100% mortality for chickens and 53.3% for pigeons, whereas the mortalities of IN-infected birds were 70 and 6.6% for chickens and pigeons, respectively. The viral shedding was higher in the oropharynx compared with the cloaca for both IN- and IM-infected pigeons. The IN-infected pigeons continued shedding the virus from the oropharynx at 4–16 dpi, while IM-infected pigeons had no oropharyngeal shedding at 11 dpi. Contact chickens had typical ND clinical picture, with mortalities of 40–60% and with higher virus shedding titers upon contact with IM-infected pigeons compared with IN-infected ones (134). It is worth mentioning that PPMV-1 strains could be isolated from the intestinal tract of infected pigeons suffering from greenish diarrhea. Those infected pigeons enhance the spread of the disease due to potential exposure of other birds to contaminated food with the pigeon fecal material (121, 135). Doves are common birds that come into contact with chickens in Egypt, on either open rural farms or live bird markets (LBMs), transmitting NDV to other bird species. When doves were inoculated with NDV using several routes, they were highly susceptible and showed nervous manifestations and congested organs. They can also shed the virus and transmit it to contact-susceptible chickens. The velogenic viscerotropic NDV strain had been detected in cloacal swabs (15/140) from free-flying doves in different localities in Egypt (136). During 2014–2015, NDV was detected in doves and characterized as genotype VII from samples collected from Gharbia (NDV/Dove/Bassioun/Egypt/MS2/2014KR082486) and Kafr El-Sheikh (NDV/dove/Desouk/Egypt/MS5/2015KT006286).

Taken together, pigeons and/or doves play significant roles in introduction, maintenance, transmission, epidemiology, and distribution of emergent NDV viruses in Egypt. It is of high value to track pigeons, at least those in close vicinity to poultry farms. The disease caused by NDVs of pigeon origin varies according to several factors, including host, environmental, and co-infection scenarios as well as lack of hygienic precautions.

Quails had been introduced to the commercial poultry sector in Egypt mainly for food consumption and were considered carriers and/or susceptible hosts to NDV (58, 137, 138). In Egypt, El-Zanaty and Abd El-Motelib (139) isolated the viscerotropic velogenic ND from quails of the Assiut province. In the Suez Canal University, a farm of 5,000 quails had 1.6% mortality; preliminary diagnosis suggested ND. Diseased birds, 3 weeks old, showed mild respiratory and nervous signs. Dead ones showed focal hemorrhagic lesions in the respiratory system and hemorrhagic spots on the liver, spleen, kidneys, and heart, without any obvious lesions in the digestive tract. Seroconversion confirmed NDV antibodies (1:32–1:256). The NDV was successfully isolated with a percentage of 75%. Hemagglutination assay (HA) titers ranged from 1:8 to 1:2,048, while MDT/minimal lethal dose (MLD) of three strains was 70, 80, and 75 h (mesogenic). The experimental ND infection resulted in 25% deaths after 1 week (58). Aly (140) investigated PPMV-1 isolates experimentally in quails and revealed that it could cause mild infection with 5% mortality in quails, but contact pigeons displayed greenish diarrhea and nervous signs (25%) followed by deaths (20%).

Japanese quails in Egypt were also susceptible to infection with NDV genotype VII, where the virus caused 33% mortality in quails and 100% mortality in chickens, with a typical ND picture, which was more severe in chickens compared with quails (141). The low death rates accompanied with nervous involvement and different shedding patterns are shared observations in partially resistant birds such as pigeons (142), cormorants (143), and ducks (144) upon infection with virulent NDV viruses. Also, vaccination protected quails against NDV infection (145, 146).

Migratory and non-migratory free-flying wild birds can play significant roles in NDV potentiation, transmission, and spread. It is believed that most wild birds got NDV as a direct result of spillover from domestic poultry species (24). Few exceptions are noticed, as NDVs are endemic in migratory birds, such as PPMV-1, which had taken wild pigeons as a reservoir/adaptation host, which lead to (i) emergence of highly virulent forms of ND in other avian species but not pigeons, (ii) global dissemination of such viruses, and (iii) increasing threats to the commercial poultry sector (23).

In 1976, 9/386 NDV isolates were identified from cloacal swabs of migratory birds in Northern Egypt (Bahig, Burg El-Arab, and Ikingi Mariut) (147). The MDT in chicken embryos was 59.2–77.6 h. But the pathogenicity indices in pigeons were 0.14–1.98 and 1.95–3.02 for IM and IC applications, respectively. Intracloacal application showed that all isolates are lethal to susceptible chickens, which suffered from provoked neurological signs and intestinal lesions (velogenic features of NDV) (57). Most recently, Rohaim et al. (148) isolated and identified vaccinal APMV-1 (5/297 oral and cloacal swabs) during a survey of apparently healthy wild birds in eight Egyptian provinces during early 2014 to late 2015. The APMV-1 isolate from teal (NDV/Teal/VRLCU-EG/2015) had an MDT of 96 h and an ICPI of 0.4375, harbored the GRQGRL motif at its F protein cleavage site, and belonged phylogenetically to genotype II (100% identity with the LaSota vaccinal strain), which collectively indicate its lentogenic nature and highlight the potential reverse spillover of NDV live vaccines from domestic poultry to wild birds (148). Furthermore, El Naggar et al. (59) also characterized NDV (4/112) of wild bird origin in Egypt. Teal (n = 2), quail (n = 1), and cattle egret (n = 1) tested positive for NDV, whereas house sparrow samples were negative. The ICPI and MDT ranged at 1.6–1.83 and 63.2–65 h, respectively (Table 1), suggestive of the velogenic potential of the four isolates. They were molecularly clustered into VII genotype and had the RRQKRF polybasic motif at the F protein cleavage site. Under experimental conditions, the previously mentioned four isolates did cause a pantropic infection in chickens, and the LaSota-vaccinated one failed to survive the disease (59).

Waterfowls, including ducks and geese, are less susceptible to NDV infection (24), as many NDV strains of different virulence had been isolated from either diseased or clinically healthy ducks, which raise the question of whether ducks/geese are only natural reservoirs or susceptible host to NDV (60, 61). Thus, the interest on natural infection of those birds with NDV has greatly increased (62–65). Prolonged viral shedding of waterfowl increased the risks of NDV of waterfowl origin (66). In Egypt, IM inoculation of Muscovy ducks with NDV genotype VII leads to only 5% mortality accompanied with higher and prolonged cloacal shedding compared with tracheal one. In contrast, IN inoculation did not cause deaths in ducks but elevated the tracheal shedding. The contact chicken had severe symptoms with very high mortality rates, emphasizing the fact that ducks are effective carriers of NDV (67). Besides, a virulent NDV of genotype VII was identified in 2/6 duck farms during 2017–2018 (68). Unexpectedly, NDV was also seen in co-infection reports, including two duck farms, but consistent with worldwide NDV reports from ducks (1, 64, 69). The role of ducks as a carrier of virulent NDV in Egypt remains to be investigated (68).

A total of 408 Egyptian NDV F protein strains were obtained from the GenBank to be included in our analysis (335 from chicken, 52 from pigeon, 17 from other avian species, and four of unknown origin). The last ones were considered to be of chicken origin. The official molecular NDV reports started in Egypt as early as 2005 (41). However, the National Center for Biotechnology Information (NCBI) database had other Egyptian NDV strains that are assumingly from 1976, mid-1990s, and early 2000s. Consistent with the second NDV outbreak in Egypt (VII), poultry researcher focused more on NDV starting from 2011, with maximum detection in 2012 and 2014–2016 (Figures 1A,B). All recorded sequences belonged to NDV class II.

However, only 136 had a complete F protein gene, as also most of the sequences came from NDV strains of chicken origin (n = 117), followed by pigeon (n = 14), and finally few sequences from teal (n = 3), quail (n = 1), and cattle egret (n = 1). A pilot phylogenetic tree was constructed based on the recommendations of Dimitrov et al. (17), from which these criteria were mainly included: (i) alignment using complete F protein nucleotide sequences, (ii) construction using maximum likelihood method using the general time-reversible (GTR) model with Gamma distribution (G), (iii) application of the model with 1,000 bootstrap replicates and values of ≥70 were indicated above the tree branches, and (iv) involvement of independent strains with no obvious epidemiologic link.

Chicken (broiler, layer, and breeder) is hugely involved in the poultry industry of Egypt, with an investment of nearly LE18 billion at that time of recording (78). Large industrial farms follow a strict vaccination program with intensive biosecurity. However, backyard/family farms do not usually vaccinate their birds and either treat their diseased ones symptomatically or submit them for slaughter. Such a discrepancy in NDV control strategies and weak governmental imposition of field regulations contribute significantly in disseminating the NDV. In this regard, following the molecular pattern of NDV in chicken is of great value.

The NDV F sequences from chicken origin are grouped under the VII.1.1 or II genotypes. Genotype VII was reported from different parts of the world and was dominantly circulating in Egypt since 2011 (79) and was found across almost the whole country (Figures 1A,B). Previously, complete F strains were classified as VIIb (9) or VIId (6, 50). Recent literature started to follow the new classification system and predominantly described VII.1.1 from chickens (10, 51, 80). Some partial F sequences were identified as VIIj (81), or generally as VII (79, 111), and most surprisingly as genotype VI (39) or I (accession number KR535623). However, studied sequences were too short to be considered for conclusive genotypic taxonomy. Since the VII.1.1 included anyhow previously characterized members of VIIb, VIId, VIIe, VIIj, and VII1, we could assume that all previously speculated NDV VII strains from chicken are actually one genetic linage of viruses or one genotype known as VII.1.1, which is responsible for the second epizootic outbreak/wave of NDV in Egypt, and still endemic in Egypt since then (Figures 4A, 5B). Meanwhile, NDV genotype II from chicken was initially described in 2005 (41) and 2006 (42), when it was responsible of the first reported NDV outbreak in Egypt. Subsequently, it was displaced by genotype VII. Nevertheless, genotype II is still existing but to a lesser extent (51) (Figures 4A, 5A). Pigeons are brought in Egypt for several reasons, including passion, gaming, investment, and meat production. They are mostly raised in spindly wooden columns that look like a medieval siege tower. Unfortunately, pigeons are a high potential source of NDV spread at least to commercial/backyard poultry sector due to their migratory behavior, free-living system of rearing, close contact with other birds in or out LBMs, and reluctance of owners to vaccination. Despite being a carrier reservoir for NDV (134), NDV strains of chickens were more pathogenic and transmissible to chickens compared with those from pigeons (82). The NDV surveillance system in the pigeon is quite weak and does not reflect the actual situation.

The low number of NDV/pigeon paramyxoviruses detected in pigeons reflected a high level of genetic diversity. At first, Rohaim et al. (54) reported the PPMV-1 from a single case (one diseased pigeon; KU522142) in 2014 and was classified as VI (sub-genotype VIb). Consequently, two investigations in clinically affected pigeons recorded partial F protein sequences of PPMV-1. Mansour et al. (7) found three different NDV genotypes during 2013–2015 (Ia, II, and VI), where the VI sequences belonged to VIb.2, a newly emerged cluster that was described mainly in Europe, while KU522142 clustered with VIb.1/re, another classical pigeon NDV lineage from Europe. The same genotype (VIb.2) was also found in 2016 and was renamed as VIg (56). Complete F protein gene sequences of VIg were also defined in apparently healthy pigeons in 2015 (55). Based on the classification system of our study, complete F protein sequences from pigeons clustered into four groups: the newly proposed genotype XXI (XXI.1.1; n = 7), VI (n = 1), VII.1.1 (n = 5), and VII.2 (n = 1). The XXI.1.1 separated basically VIg strains from the VI genotype. The sequence, KU522142, remained in VI and assigned in the sub-genotype VI.2.1.1.2.2, while VII.1.1 and VII.2 were seen also in 2015 (83) and described in Figures 4A, 5B–E. Other strains that had a partial F protein gene (identified as genotypes I and II plus unidentified ones) need further confirmation/analysis following complete F protein gene sequencing.

Other birds, including wild ones, are considered as another introducing, disseminating, and maintaining factor of NDV in Egypt due to their migration or involvement in LBMs. They are also involved directly or indirectly in the wild–domestic–human interfaces, imposing the importance of continuous surveillance of pathogens. All five complete F protein gene sequences from quail (n = 1), Cattle egret (n = 1), and teal (n = 3) belonged to genotype VII.1.1 (59) except one sequence from teal (148), which was in genotype II (Figures 4A, 5A,B). Another 12 NDV partial F protein sequences from passerines (n = 1), dove (n = 2), house sparrow (n = 3), cattle egret (n = 2), white wagtail (n = 1), white-throated kingfisher (n = 1), and hoopoe (n = 1) were found in genotype VII, while ostrich (n = 1) was placed in genotype II.

For the HN protein, a total of 46 complete gene sequences were collected and involved in the phylogenetic tree (Figure 4B) to collect some evolutionary criteria for an important NDV surface protein in Egypt. Both Egyptian HN and F protein followed the same phylogenetic topology. They were found in genotypes VII.1.1 (strains from chicken mainly, teal n = 2, quail n = 1, and cattle egret n = 1), II (one sequence from chicken), and VI or XXI.1.1 (strains of pigeon origin).

The F protein cleavage site (112-117) is considered a major determinant factor that plays a role in virus pathogenicity/virulence (19). However, other factors, for example, the HN stem region and globular head, are also involved (12). All isolates of Egyptian NDVs had an F protein of 552aa length. The deduced aa analysis showed the relative stability of F protein cleavage site in the Egyptian NDV isolates overtime, which tend to be genotype-specific, not species-specific. NDV isolates in VII.1.1 and VII.2 (from chicken, pigeon, teal, quail, or cattle egret) had the velogenic motif RRQKRF except one chicken isolate that had the RRKKRF motif.

Genotype II strains had either the velogenic motif RRQKRF (in chicken and pigeon) or the lentogenic motif GRQGRL (in chicken and teal). Pigeon NDV strains in XXI.1.1 had the velogenic KRQKRF motif, while pigeon strains in genotype VI and I had the velogenic RRQKRF and lentogenic GKQGRL ones, respectively. Two strains from pigeon (MF614961) and chicken (MK604215) also had the lentogenic motifs GRQGRL and GKQGRL, respectively (unidentified genotype; not in Supplementary Tables 1, 2). The previously mentioned 12 NDV partial F protein sequences from wild birds had all RRQKRF residues at their cleavage site, except the ostrich isolate, which had the RNQGRL motif. As previously reported, aa changes in the cleavage site affected the fusion efficiency of the F protein (84), gradual dominance of virulent strains in quasispecies (85), and virulence of the virus (ICPI decreased in Q114R mutant viruses) as reported by Samal et al. (86). However, the clinical pathogenicity of the virus under natural conditions is usually influenced by other viral, host, and environmental factors. Interestingly, Nagy et al. (51) reported high pathogenicity indices in lentogenic motif harboring NDV isolates of chicken origin, emphasizing the importance of following the OIE recommendations in determining virulence regarding NDV of field origin.

The F protein signal peptide (1–31) was highly variable among the Egyptian strains, consistent with the findings of Orabi et al. (6). Other regions, including the fusion peptide (117–142); heptad repeats a, b, and c (HRa 143–185, HRb 268–299, and HRc 471–500); transmembrane (TM) domain (501–522); and cytoplasmic (CT) tail (523–553) were subjected to aa analysis, where several mutations were reported, which might affect the folding and fusion activities of the protein as described in the fusion peptide (87, 88) or the HRa, b, and c (89). The HRa is also supposed to include a potential antigenic epitope (90, 149–163); however, the Egyptian NDV isolates had only few reports of aa substitutions in this epitope (V168I and D170N). The TM domain affects the structural confirmation of the F protein, F–HN protein interaction, and fusion activity (91). Also, mutant CT domain modulates the F protein biological characters, virulence, and pathogenicity (92). D170N was reported in VII.2 and VII.1.1 (one pigeon and four chicken strains, respectively), which is a neutralizing epitope (93). Also, residues D479 and S486 are critical for the fusogenic activity (94) (Supplementary Tables 1, 2).

The HN protein of NDV is a surface glycoprotein that mediates several functions, including (i) attaching of NDV to cellular sialic acid receptors (95), (ii) promoting F protein fusion activity (96), (iii) facilitating the NDV budding by its neuraminidase (NA)-mediated receptor cleavage (97), and (iv) determining NDV tropism and virulence (98). Structurally, the NDV HN protein consists of CT tail, N-terminal TM domain, a stalk region, and a C-terminal globular head. The HN stalk forms an interaction with the F protein through a stretch of amino acids (aa positions 74–110) that forms two conserved heptad repeats (HRA and HRB) (99).

The HN protein length of Egyptian NDV isolates was 572aa except the parent NDV isolate (II) from 2005 (FJ939313), which was slightly longer (578aa). The NDV HN length affects the replication and biological properties of the virus as extended HN showed increased HA titer and receptor binding but impaired NA, fusion, and replication abilities. However, the virulence of virus was not changed (100). TM and stalk domains of the Egyptian NDV strains were highly conserved among genotypes (for example, VII.1.1, XXI.1, VI, and II), with few substitution mutations, which may affect the structure and activity of the HN protein (99, 101).

Major epitopes in the C-terminal head of HN were also investigated. As it is basically involved in antibody recognition (102), a single aa change in that major linear epitope ³⁴⁵PDKQDYQIR³⁵³ (1/1–4) allows the escape of its corresponding antigenic variant from neutralizing monoclonal antibodies, which was reported at least at position 347 (103, 104). It might explain the high virulence of chicken VII.1.1 isolates in chickens compared with others. Other antigenic epitopes within the HN protein were compared as shown in Supplementary Tables 3, 4.

Amino acid residues involved in receptor recognition (95), HAD ability, NA activity, fusion activity (105), interaction with F protein (106), head–stalk linker region (107), and predicted B-cell epitope (108) were highly conversed with few exceptions. Due to the intensive unplanned vaccination in Egypt, selection/vaccination pressure could explain the occurrence of such aa substitutions in the epitopes of Egyptian NDV HN overtime, which in turn may affect, to different levels, the vaccine efficacy, induced immunity, and virus shedding in birds as a result of antigenic variability (108). HN protein is a determinant viral factor for thermostability, as mutant NDV viruses (S315P and I369V) were more thermostable and possessed more HA titers and NA fusion activity (109).