Considering the capability of the high evolutionary rate in NDV strains, new genotypes and subgenotypes have been described in recent decades globally, so it is possible that many more will be recognized by further investigations in the future. It is a fact that RNA replication is a complicated process in nature. During the recombination process, these viruses can generate new variants [19]. Therefore, genome sequence data will be one of the best tools for tracking the evolution of the viruses to monitor the field isolates in the regions in which such kinds of diseases are endemic.
According to these crucial roles of the HN protein in protective immunity and escape of vaccine immunity [9], this study was conducted to analyze the complete HN gene of 11 ND isolates by detecting the possible genetic variations in antigenic epitopes of HN protein among Iranian field isolates and five commonly used vaccine strains (Lasota, B1, VG/GA, V4, and PHY-LMV42).
The phylogenetic tree clustered the Sequences of a 1716-bp fragment of the HN gene coding region from each of the given NDV isolates into the representative strains of genotype VII and subgenotype VII.1.1. Although some of the poultry farms used for sampling are located more than 1000 km apart, we did not identify any subgenotype other than VII.1.1 in commercial broiler farms. According to the procedure of Malirat et al. (2007), the genotype variation of 2-5% in the phylogenetic interpretations is consistent with the fact that the viruses can originate from a similar geographical zone [20]. The evolutionary distance between nucleotide sequences of the isolates and the other sequences deposited by neighboring countries belonging to the class II genotype has been found more than 5% (Table 3). Furthermore, the opinion of the previous study by Dimitrov et al. (2019) has noted that group distances less than 5% represented the same subgenotype [2]. The evolutionary distance analysis among all isolates presented in this study as a group showed a range between 99-100% (Table 3). What is more, these results seem to suggest that the novel subgenotype VIIL is unique to the area. However, Dimitrov et al. (2019) have proposed a new classification system by representing the entire VII (b+d+e+J+L) subgenotypes as one group named VII.1.1 [2]. This idea argues strongly that VII.1.1 is the most predominant subgenotype of NDV in this part of the Middle East.
According to the different lengths of the HN protein [21], most of those NDV isolates belong to class І genotype, and class II subgenotype І possess 616 aa. In addition, class II genotype II viruses have 577 aa, whereas genotypes III, IV, V, VI, VII, VIII, and IX contain 571 aa. As expected, all 11 isolates belonging to genotype VII indicated HN proteins containing only 571 aa residues.
It is a fact that most NDV strains cannot tolerate the heat and will lose their infectious ability when they exposure to 50°C for 30 min. However, several thermostable NDV strains, which usually belong to genotype I with low pathogenicity to chickens, have been identified that are able to keep their infectivity and HA titer at 56°C for at least 30 min [22].
Yet, the molecular mechanism of HN protein thermostability is still unknown [23]. As shown in Fig. 2, the results demonstrated mutations S315P, I369V, and V369A among the VII.1.1 isolates. These results are consistent with the recent study [23], which revealed that residues 315, 329, and 369, especially mutations S315P and I369V, could significantly enhance the viral thermostability, HA, and NA activities.
It was previously suggested that the proline residues at specific sites, especially in b-turns or loop regions, play a crucial role in protein stabilization [24]. The pyrrolidine rings of these residues could affect the protein thermostability by restricting the backbone bond rotation of protein, thereby; reducing the conformational entropy throughout the protein unfolding process and consequently increasing the protein thermostability features [25]. Furthermore, the 3D structure of Pro315 and Val369 revealed that these residues placed close to Arg416 as well as Glu401 and Tyr526 (Fig. 3), which were known essential for receptor binding activity, fusion promotion, and neuraminidase activity in sialic acid binding sites of NDV strains [26]. Moreover, Omony et al. (2016) showed that a higher proportion of amino acids Val, Leu, and Ile might be identified within the HN protein of heat-resistant NDV strains [27]. The results showed 44, 47, and 34 amino acid residuals of Val, Leu, and Ile at the HN stalk and globular regions of the VII.1.1 subgenotype compared with 40, 47, and 33 amino acid residuals from the Lasota strain, respectively. Overall, these data suggested that the VII.1.1 isolates might contribute to poss higher thermostability features than the other NDV subgenotypes across this area of the Middle East. However, whether these mutations in amino acid sequences of the HN protein affect the thermostability of the VII.1.1 subgenotype or not remains to be further studied.
The N-linked glycosylation sites are found to be necessary for some biological activity of proteins, such as proper conformation, disulfide bond formation, correct folding, and oligomerization. Several studies have previously suggested that any changes in the glycosylation sites of NDV might affect its biological functions [28]. Examination of N-linked glycosylation sites of the HN glycoprotein of NDV revealed that there are a total of six predicted sites for the addition of N-linked carbohydrates at residues 119, 341, 433, 481, 508, and 538 [28]. Four of these sites (residues 119, 341, 433, and 481) have been found functional in NDV strains with the consensus sequence motif NXS (Asparagine-X-Serine) or NXT (Asparagine-X-Threonine). X, however, can be any amino acid except proline or aspartic acid [28]. Earlier studies have illustrated that losing a glycosylation site may lead to either decreasing or increasing the virulence of a virus, depending on its location [29-31]. For example, some researchers have mentioned that the elimination of N-glycans in NDV HN (especially at position 481) could have reduced the pathogenicity of the virus [31]. These data have suggested that the VII.1.1 subgenotype might possess higher virulence than those parental viruses. Whether the new N-glycosylation site (NIS) can influence the virulence of the virus or not should be studied more.
Functional antigenic sites studies on HN glycoprotein indicated that a total of seven overlapping antigenic sites (sites 1, 2, 3, 4, 12, 14, and 23) could influence the attachment of the virion to the cellular receptor and neutralize viral infectivity [32]. Five of these sites (residues 193 to 201 as site 23; 345 to 353 as sites 1 and 14; and a domain composed of residues 494, 513 to 521, and 569 as sites 2 and 12) have been identified as the principal glycoprotein antigenic sites of HN protein, which could affect the attachment of virion to a cellular receptor and consequently neutralize viral infectivity [32]. As shown in Fig. 3, the aa substitutions G494→D and I514→V (antigenic sites 12 and 2), as well as the mutation E347→Q (sites 1 and 14), have been detected (Fig. 3). The mutation R197→ G, however, has been only identified in antigenic site 23 of sample number eighteen. In addition, the results have shown several aa substitutions so close to these antigenic sites, including; Y203→H, D342→N, T509→I, T522→I, V495→E, G570→R.
Previously, functional inhibition studies on antigenic sites 12, 2, and 23 have demonstrated that the monoclonal antibodies against these sites could influence the biological activities of NDVs by either preventing viral attachment to chick cell monolayers or decreasing HA and NA of the virus [32]. The mutant I514V (sites 12 and 2) is reported herein and previously among several virulent isolates of NDV strains [33].
Based on the opinions of the previous studies, the aa substitution in HN residue 347 is contributed to the lower antigenic relationship between field isolates and vaccine strains [34]. The mutation E347K has previously been reported in genotype VII viruses by several researchers from Asia, especially in Korea [35] and China [36]. Moreover, the recent study by Zhu et al. (2016) indicated the failure of vaccination when chicken infected with a variant strain carrying mutation E347K was vaccinated later by the Lasota strain [34]. ). In addition, the effect of an aa substitution in residue 347 on NDV HI titers was reported earlier [36]. Overall, it suggests the idea that the mutation of E347Q may lead the VII.1.1 subgenotype to the new antigenic variation and novel amino acid substitutions in the HN linear epitope. These aa residue substitutions may influence the antigenicity of HN glycoprotein.
Furthermore, sites 1 and 14 were found effective in the HA activity of NDV strains with no detectable influence on NA function [32]. The data obtained from our HI assay demonstrated the HI titers of 7-9 (Table 2). These results seem to suggest the idea that this high level of HI titers might be closely related to the aa substitutions of VII.1.1 antigenic sites.
Regarding the mechanism of interaction between the HN and F proteins, there are two sialic acid binding sites in the stalk and globular head of the HN protein: first, the residues located in the sialic acid-binding site I at positions 234-239 [37]; second, the residues are composed of four loops at the dimer interface of HN protein in the sialic acid-binding site II: residues 156 to 174, 191 to 203, 515 to 527, and 547 to 556 [38]. That HN protein is responsible for both the entire fusion procedure and the initial triggering of the F protein has previously been proven. Whereas site I triggers the interaction with F protein and modulates neuraminidase activity, site II operates to maintain contact with the target cell during the fusion process [26]. According to the multiple alignments shown in Fig. 2, the results illustrated a total of three aa substitutions F156→Y, Y203→H, and T522→I in the sialic acid-binding site II among the VII.1.1 isolates. The mutation R197→G, however, has been only observed in the binding site II of sample number eighteen. Furthermore, we have found the aa substitution I514→V among the isolates from the presented study, which was occurred at the position very close to the sialic acid-binding site II of HN protein.
Due to the fact that the HN protein of NDV probably exists in virions in both nondisulfide-linked and disulfide-linked forms, and according to the crucial role of cysteine residues in the disulfide linkage of the HN homodimers, the number of these residues and their positions have been studied in NDV strains accurately [39, 40]. The result illustrated a total of thirteen cysteine residues at positions 123, 172, 186, 196, 238, 247, 251, 344, 455, 461, 465, 531, and 542 in the VII.1.1 subgenotype. Although twelve of these residuals have been identified conserved among all 11 isolates, the cysteine at residue 123 was observed variable in comparison with the Lasota strain. The residue 123 is noticeable by its deficiency in the major part of the NDV isolates analyzed thus far. The previous study by McGinnes and Morrison (1995) has revealed the higher fusogenicity of a mutant virus than that of the ancestral virus. These authors have suggested that the mutation at residue 119, located near the Cys-123 residue in the stalk region of HN protein, might implicate better covalent linkage by enhancing the efficiency of Cys-123 in an intermolecular covalent bond. Consequently, it can increase the fusogenicity of the virus [28].
By analyzing the sequences of VII.1.1 subgenotype isolates, we found a substitution at Trp123→Cys in the stalk region of the HN protein (Fig. 2). This data suggested that mutation of residue 123 might cause higher fusion promotion among the VII.1.1 subgenotype isolates.
The diminished promotion activity of some aa substitutions in a series of heptad repeat (HR) regions, including HR1 (especially residuals 74, 81, and 88) and HR2 (especially residuals 96, 103, and 110), belonging to the NDV HN protein, has been reported previously [41]. These regions have been found essential for surface expression and proper folding of the protein. The HR1 and HR2 are separated by a 7-amino-acid intervening region adhering to the aH-bP-cP-dH-eP-fP-gP (H, hydrophobic; P, polar) rule. Several studies have previously proposed that these regions are essential for surface expression and proper protein folding, especially residues 69 and 77 [42]. The results demonstrated the aa substitutions at G75→S, N77→S, V81→I, S92→Y, T101→S, and T102→I. The HR1 and HR2 regions, however, were highly conserved among the isolates (Fig. 2). Furthermore, in 2003 clear evidence indicated that mutations of amino acids 124–152 (especially I133L or L140A) in the globular head of HN protein could interact with the HR2 region of F protein, consequently; it might reduce the promotion activity of the HN protein [43]. We have found the mutations of I127→V, D144→N, and A145→I within this region of the VII.1.1 isolates. Interestingly, the additional N-glycosylation site of the VII1.1 subgenotype is located within this region of the HN protein; thereby, it may influence the interaction between the HN protein and the HR2 region of F protein (Fig. 2).
On the other hand, according to the Prior epitope mapping studies, the mutation at residue 494 on the HN protein of NDV might be involved in the neutralization activities of the virus [44]. The results have been detected the aa substitution G494→D by analyzing the aa epitopes of the VII.1.1 subgenotype (Fig. 2). Taken together, these data seem to suggest the idea that several mutations, which have occurred in the various parts of the VII.1.1 genome, might contribute to influencing the fusion promotion and NA activities of the field isolates.
Despite acceptable biosecurity and vaccination programs within the Iranian poultry flocks by using various kinds of commercial vaccines such as the Lasota strain, outbreaks of ND is still a massive problem in this region of the Middle East [7]. As mentioned earlier, the sequence distances analysis among nt and aa sequences of the isolates under study has illustrated the lowest percentage of the sequence homology with the Lasota and V4 strains. Although most parts of the HN and F genes are completely conserved within the NDV isolates, annual change rates of approximately 2% have been detected in both genes among Iranian subgenotype VII.1.1 isolates. Herein, we have shown that the HN annual rate of change was indicative of high similarity to the F gene change rate estimated previously [45]. The presented study also suggests that the vaccination programs of broiler farms in which infected by mutant strains may be affected by these aa substitutions due to the fact that the current commercial vaccines, which comprise F or HN proteins of other types of viruses from another country, could not provide promising results against newly emerging genotypes [46]. Thus, it is recommended that further studies on molecular epidemiology of the circulating NDVs should be carried out within the region. It can help us to design a new neutralization map of the subgenotype VII.1.1 by constructing a functional profile of each antigenic site. The results of this comprehensive study can be used for future investigations to develop more effective and protective recombinant NDV vaccines that carry VII.1.1 surface HN protein.
In conclusion, the phylogenetic analysis of the isolates retrieved from the new ND outbreak during 2017-2020 has confirmed the novel VII.1.1 subgenotype, which was previously known as subgenotype VIIL, as the predominant subgenotype circulating in poultry farms of Iran. The results illustrated that the HN gene of Iranian NDV subgenotype VII.1.1 followed a similar pattern as the F gene [45], in which 1.9E-3 was calculated with a similar software setting. Sequence analysis between subgenotype VII.1.1 isolates and commonly used vaccinal strains revealed multiple aa residue substitutions on the HN glycoprotein of the VII.1.1 subgenotype. Two aa substitutions (S315P, I369V), previously found responsible for enhancing the viral thermostability of NDV strains, have been identified in the presented study. Furthermore, an additional N-glycosylation site at position 144 (NIS), as well as the cysteine residue at position 123, was identified in the investigation. Each of these has been previously found essential for NDV fusogenicity and pathogenicity. Identification of aa substitutions in the HN antigenic sites, especially the mutations I514V and E347Q, as well as the other mutant within HN binding sites of the VII.1.1 subgenotype, seems to suggest the idea that these mutations may significantly influence the HA and NA activities of the isolates. These multiple aa residue substitutions may increase the virulence activity of the field isolates and can be responsible for vaccination failure in commercial poultry farms.