Molecular evaluation and genetic characterisation of Newcastle disease virus's haemagglutinin‐neuraminidase protein isolated from broiler chickens in Iran

Abstract Background Newcastle disease (ND) virus (NDV) is one of the major pathogens in poultry farms that causes severe economic damages to the poultry industry, especially broiler chicken and turkey farms. Despite the endemicity of ND and its many epidemics in the country, the nature of the Iranian strain of the Newcastle virus is still largely unknown. This study aimed to characterise and evaluate NDV isolates obtained from commercial poultry farms in Iran in 2019 through haemagglutinin‐neuraminidase (HN) gene sequencing. Method HN gene of each NDV isolate was amplified and sequenced using specific primers followed by phylogenetic analysis of full length of HN gene open reading frame and amino acid (aa) sequence of HN. Results Phylogenetic analysis of the HN gene showed that the virus is very closely related to genotypes VII and III. Analysis of HN gene nucleotide sequences showed that all isolates encode proteins with a length of 571 aa. Conclusion Results of the present study are useful for a better understanding of molecular epidemiology of indigenous NDV strains and determining important molecular differences between fields and commonly used vaccine strains related to main immunogenic proteins.


INTRODUCTION
of Avaluvirus and Paramyxoviridae, which can cause highly infectious, acute ND in poultry. The virus is the most common type in poultry (Dimitrov et al., 2017;Mayo, 2002;Shafaati et al., 2013). Envelope virus with a single-stranded, non-segmented RNA genome of negative sense (Bello et al., 2018a;Hashemzadeh et al., 2015). The NDV genome contains six major structural genes (3′-NP-P-M-F-HN-L-5′), and it has two minor proteins called W and V, which is achieved through the process of RNA editing on the P gene by adding guanine nucleotides (Abdisa & Tagesu, 2017;Ganar et al., 2014;Motz et al., 2013). Viral replication, transcription, translation and protein processing occur in the host cell's cytoplasm, while virus particles are assembled in the plasma membrane by budding (Bi et al., 2019). The three main pathotypes, including velogenic, mesogenic and lentogenic, of NDV are calculated based on the inclusion of the intracerebral pathogenicity index, the intervein pathogenic pathogenicity index, the mean death time and the cleavages of the F proteins (Gowthaman et al., 2016;Mayahi & Esmaelizad, 2017;Suarez et al., 2020). The two proteins, haemagglutinin-neuraminidase (NA; HN) and F, are the main virulence factor of the virus (B. Liu et al., 2015;Ren et al., 2019). Two glycoproteins F and HN play essential roles in the assembly and development of envelop viruses and determining tropism in the host and tissues (Jin et al., 2017). HN glycoprotein has activities such as hemagglutination (HA), NA and stimulates F protein activity (Soltani et al., 2019). The binding of the HN protein to the sialic acid receptor initiates protein F action, which leads to the fusion of the virus membrane and the host cell membrane (Liu et al., 2019b). NDV virus isolates have a high mutation potential that enables several genotypes of viruses to grow at the same time. Newcastle virus strains' virulence is determined by tissue or organ tropism, the host immune system and/or replication impact (Fan et al., 2017). Full genome and structural genes for 20 Avulavirus species have been recently published in an extensive comparative study (Munir & Shabbir, 2018 (Mayahi & Esmaelizad, 2017;Miller et al., 2015;Orynbayev et al., 2018).
Despite the prominent role of HN glycoprotein in pathogenicity and induction of host immune responses by NDV, there is limited information on the molecular properties of this glycoprotein and its encoding gene (Soltani et al., 2019b

HA test
The HA assay was performed using the Alexander and Chettle (1977) method. Briefly, a total of 25-µl phosphate buffer saline (PBS) was added to all wells of a 96-well microtiter plate (U-shaped well). Next, 25 µl of viral suspension was added to the first well. The virus suspension was diluted into the last well by a multichannel micropipette. In each well, a 25-µl PBS solution was added. A total of 25 µl of 1% chicken red blood cells (RBCs) was then added to each well. The plates were then incubated at room temperature and observed after 30 min.

Hemagglutination inhibition (HI) test
The HI assay was used to titrate the antibody response to a viral infection using the Alexander and Chettle (1977)   All chemicals, unless differently stated, were provided by Cinnagen.

RNA isolation and RT-PCR
The pGET-II cloning vector obtained from CinnaGen was used to insert all fragments.

Sequence determination
Recombinant vectors containing HN gene fragments for bilateral sequencing were sent to the German company MWG by an Iranian intermediary company. The results were aligned and analyzed by CLC Genomic3.6 and DNASIS MAX 3.0 (Hitachi Solutions America) and compared with selected sequences available in the GenBank. The phylogenetic tree was constructed using the maximum likelihood method based on the Tamura-Nei model (Tamura & Nei, 1993) with MEGA 7 software (Kumar et al., 2016). The evolutionary distance and homology of the respective coding region were also estimated using pairwise sequence comparison in MEGA 7 software (Kumar et al., 2016) and analyzed in Excel. Prediction of amino acid (aa) sequences and their alignments were also performed by MEGA 7 (Kumar et al., 2016).

Genomic features
Approximately 1980

Phylogenetic analysis and distance estimation
The entire coding region of HN gene (1716 bp), IR1 and IR2 isolates analysed in the present study were closely related to genotype III and IR3 isolates. Among available sequences of HN complete coding region, a Chinese isolate (GenBank: GU573804 (IR1 and IR3) and GenBank: JX840450 (IR2)) was found to be the most similar isolate to our isolates with 99% homology. After constructing the phylogenetic tree using the complete sequence of the open reading frame (ORF) of the HN gene, the viruses representing each genotype were clearly distinguished based on the branching patterns and topology of the trees (Figure 3). In the study of the phylogenetic tree, IR1 and IR3 isolates were found to have the exact evolutionary origin. Estimates of evolutionary distances between viruses of the present study are presented in Table 3 ( Le et al., 2018).

DISCUSSION
In the global poultry industry, ND is a critical issue. Molecular epidemiology and phylogenetic analysis of NDV in Iran and Middle East countries are essential to determine the current situation and develop control measures that need to be improved (Ahmadi et al., 2016;Hassan et al., 2016). ND varies in each region according to the factors influencing the incidence and severity of the facial disease. Due to the similarity of clinical and necropsy symptoms with other viral and microbial respiratory diseases, none of the above symptoms can be considered a specific symptom (Bello, Yusoff et al., 2018a;Bello, Yusoff et al., 2018b). In the study of Rott et al., genotyping-isolated NDV strains should be considered part of diagnostic methods in determining viral characteristics for reference laboratories, which is possible by sequencing (Bello, Yusoff et al., 2018b). In this study, the HA and HI tests were performed as conventional evaluations for NDV identification. Based on this, 10 samples of each of three positive isolates were used for the next stages of the experiment. The ability of the protein to agglutinate RBCs has been discovered. In parallel, positive results were discovered by observing the formation of RBC that precipitates at the bottom of the plate using an HI test. NDV RNA polymerase has a highly defective function. During the RNA amplification process, the occurrence of many mutations resulting from the function of this enzyme causes the emergence of new variants.
Also, selection pressure is another factor influencing the formation of new variants (Soltani et al., 2019b). The HN gene has more exposure to the immune system than other NDV genes (Soltani et al., 2019b).
Gene amplification was performed on HN fragments using primers designed in CLC Genomics Workbench 3.v. software. The presence of DNA bands showed that the nine test samples were molecularly proven to be NDV. The HN gene was completely sequenced in three isolates of the NDV to observe possible changes in antigenic epitopes of indigenous NDV and the genetic relatedness between commonly used vaccine strains and Iranian field isolates. Identity of the HN protein aa sequences among these three isolates varied from 98.9% to 100%, while the corresponding range between the Iranian field isolates and the vaccine strains was from 84.7% to 90.1%. These numbers are indicative of the notion that circulating viral strains in Alborz Province

F I G U R E 3
Phylogenetic analysis based on the full length of HN gene open reading frame. The evolutionary history was inferred by using the maximum likelihood method based on the Tamura-Nei model (Tamura & Nei, 1993). The tree with the highest log likelihood (-18047.1983) is shown. Previously identified HN gene sequences of ND virus (NDV) strains representing different genotypes have been provided from the GenBank with their accession numbers. Numbers indicate the bootstrap values (1000 replicates). Horizontal distances are proportional to sequence distances. Evolutionary analyses were conducted in MEGA7 (Kumar et al., 2016) (as representative of circulating viruses in Iran) were considerably different from the vaccine strains in use and, therefore, the role of antigenic differences in weak vaccine-induced protection can be presumed (Soltani et al., 2019;Soltani, Peighambari et al., 2019b). Note: The number of base substitutions per site between sequences is shown. Standard error estimate(s) are shown above the diagonal. Analyses were conducted using the Tamura-Nei model (Tamura & Nei, 1993). The analysis involved 16 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 1678 positions in the final dataset. Evolutionary analyses were conducted in MEGA5 (Tamura et al., 2011). 571 and 616aa. The relationship between the length of the HN protein and the virulence of the virus recognised was demonstrated in the past (Gaikwad et al., 2016;Zhao et al., 2013). In standard avirulent or slightly virulent strains, the HN gene protein has an extended ORF of encoding longer proteins up to 616 aas residues (Soltani, Peighambari et al., 2019b). In the present study, the carboxyl-terminal extension length and analysis of the HN glycoprotein gene did not indicate the lack of aa extension length. The length of HN glycoprotein in all three isolates was predicted to be 571 aas indicating high virulence in all viruses . It has previously been shown that genotypes of strains III-VIII, which contain the shortest length aa (571 aa) of the glycoprotein HN, are composed exclusively of visotropic vologic strains Liu et al., 2019a). As a result of the high evolutionary rate of NDV strains, new genotypes have been reported in the last few decades, and many more may be identified in the future.
It is believed the NDV's disease-prone areas in endemic geographical areas are constantly under the influence of evolution and deformation factors (Samuel et al., 2013). Immuno selection pressure is another factor influencing the formation of new variants. The HN gene is more affected by the immune selection pressure than other NDV genes and results in the production of antiviral antibodies. Compared to other NDV genes under the same immune pressure, nucleotide mutations in Bashar HN are more likely to occur (Gong & Cui, 2011). Increasing the phylogenetic and antigenic distance between common vaccine strains and existing strains may lead to the formation of new pathogenic strains of NDV. Therefore, the persistence of ND in Iran may be the reason (Zhang et al., 2012). Phylogenetic analyses, of the three Iranian isolates in this study revealed that HN gene sequencing and phylogeny belonged to genotypes III (class II) and VII (class I; Miller et al., 2015;Munir & Shabbir, 2018). Genotype VII was isolated between 1997 to 2014 in Pakistan, India, Russia and Sweden. Genotypes XII, XIII and XIV have ancestors similar to genotype VII that produce different distinct lineages in the course of its evolution (Orynbayev et al., 2018;Ramey et al., 2013;Usachev et al., 2006). To date, the circulation of genotypes of NDV strains XIIIa, XIIId, VIIj and VIId in commercial poultry (Abah et al., 2020;da Silva et al., 2020), VI in domestic pigeons (da Silva et al., 2020;Rezaei Far et al., 2017) and VII in domestic poultry (Sabouri et al., 2016)  Our results are in accordance with the report from Ahmadi et al. (2010) indicating that in northwestern Iran, the presence of velogenic NDVs belonging to genotype VII has been confirmed. Genotype VII of NDV is now regarded as the major pathogen responsible for panzootic of ND. Therefore, the development and administration of new NDV vaccines that are closely related to predominant VII viruses may confer better protection than conventional vaccines.

CONCLUSION
In this study, HN gene and glycoprotein sequencing indicate significant genetic and aa differences between the studied and native NDV isolates in Iran with common vaccine strains. The present study's findings showed variations in important antigenic sites of studied NDVs isolated from different provinces of Iran that may be responsible for vaccine failure during previous years and employed. Phylogenetic affinity of the isolates studied in the present study with NDV isolated from wild birds in Russia and isolates obtained from China probably indicate the prominent role of migratory wild birds and trade in the widespread NDV strains and the importance of measures. Moreover, the present results may also indicate the need for designing and producing new efficient NDV vaccines in Iran.

ACKNOWLEDGEMENTS
We would like to thank all colleagues who helped us in this project, for the moral support and services provided during this project.

CONFLICT OF INTEREST
All the authors of the manuscript declare no scientific and financial conflict of interests.

ETHICS STATEMENT
The authors declare human ethics approval was not needed for this study.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.

PEER REVIEW
The peer review history for this article is available at https://publons.