Newcastle disease (ND) is one of the most prevalent and devastating viral infections in poultry with a high mortality rate. It is also responsible for a significant part of the heavy economic losses in poultry houses around the world. From the taxonomic perspective, the ND virus (NDV) is a member of the genus Avulavirus within the family Paramyxoviridae in the order Mononegavirales. Based on the taxonomy, the infectious agent causing ND is named the avian paramyxovirus-1.
The NDV contains an RNA genome with about 15 kb length coding six viral proteins in the following order: 5-NP-P-M-F-HN-L–3 ( Cao et al., 2013 ). The NDV strains are grouped in two separate classes (i.e., I and II) within a single serotype. Class I contains nine genotypes (1–9) and class II includes 18 genetic groups, 10 of which (i.e., I–IX, and XI) have been previously established while five of them (i.e., X, XII, XIII, XIV, and XV) are new genotypes ( Diel et al., 2012 ). Virulence of all NDV isolates all around the world is monitored by veterinary organizations. According to the instructions, upon isolation of a virulent NDV strain with ICPI of 0.7 and IVPI of 1.40, a report should be sent to World Organisation for Animal Health ( OIE, 2018 ).
Virulence of NDVs is related to fusion (F) amino acid sequences at the cleavage site. However, hemagglutinin-neuraminidase (HN) protein, as a critical viral protein with multiple functions, plays a key role in forming the virulence of NDV. The protein is a surface glycoprotein with several subunits, including a cytoplasmic domain, a transmembrane region, a stalk region, and a globular head. The head is the part that is responsible for receptor binding, neuraminidase activity, and all the antigenic properties of HN protein. As a virulence factor with good binding property, the HN protein can agglutinate red blood cells (RBCs).
The mechanism by which the HN protein can agglutinate the RBCs consists of binding the HN protein to the surface receptors on the RBCs. However, the HN protein is multifunctional; therefore, several other virulence-associated activities such as the attachment, fusion promotion, deletion of the sialic acid from progeny virion particle, and neuraminidase activity have been attributed to the HN protein ( Hu et al., 2010 ).
In Iran, ND has been described as an endemic disease due to its frequent incidence in various geographical areas and different industrial poultry houses. In recent years, highly virulent NDV infections have posed challenges to a considerable number of industrial farms ( Samadi et al., 2014 ; Ghalyanchilangeroudi et al., 2018 ; Sabouri et al., 2018 ). In most cases, the predominant NDV genotype that is circulating in industrial farms in Iran was VIId ( Hosseini et al., 2014 ).
Most publications analyzed NDVs based on F protein sequences. Nevertheless, there is little information about HN protein sequences and diversities ( Esmaelizad et al., 2012 ).
The present study aimed to distinguish the genetic variation of two Iranian NDV isolates from current outbreaks by phylogenetic analysis of the HN sequences and compare them to HN sequences of NDV strains from other countries.
Material and Methods
Specimen Collection and Virus Culture. Two NDV-infected broiler flocks located in Gilan and Isfahan provinces were selected for specimen collection. Sampled chickens were displaying NDV-typical nervous symptoms and high mortality rates (more than 25%). The broiler flocks had been previously vaccinated with a routine vaccination program which includes both live and killed NDV vaccines.
Heads of the affected chickens were submitted to the Laboratory. To confirm the presence of NDV in infected chickens, the brains were cultured into the embryonated eggs to isolate the viruses and confirm them using antigen-testing. To culture the viruses, 0.2-ml aliquots of suspensions from the infected tissues were inoculated into the allantoic cavity of embryonated eggs (9-day-old). After incubation at 37 °C for three days, the allantoic fluid was harvested and evaluated using the hemagglutination assay method. Subsequently, allantoic fluids specimens with hemagglutination activity were subjected to the hemagglutination inhibition test according to the recommended procedures to further confirm the presence of NDVs ( Manual, 2018 ).
RNA Extraction and Reverse-transcription PCR (RT-PCR). Total viral RNA was extracted from the allantoic fluid using RNeasy Mini Kit (Qiagen, Germany) according to the protocol provided by the manufacturer. Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific, Canada) was used to convert the viral RNA to the complementary DNA (cDNA) based on the recommendations of the manufacturer.
Afterward, the cDNAs produced during the synthesizing step were incorporated into PCR procedures along with HN-specific primers. Two pairs of primers, specific for the HN region were used for the amplification of the complete HN gene (1730 bp). Sequences of forward primers F1 and F2 were 5’-TTC ACA ACC TCC GTT CTA CC-3’ and 5’-AAG TCT TGC AGT GTG AGT GC-3’, respectively. Moreover, the sequences of reverse primers R1 and R3 were 5’-TTG CAC TCA CAC TGC AAG AC-3’ and 5’-TCA TCT TTG AGG ATC TCA AC-3’, respectively.
Each pair of primers, capable of amplifying a part of the HN gene sequence, was incorporated into a separate PCR assay; accordingly, the whole HN gene sequence was amplified ( Esmaelizad et al., 2012 ). Therefore, the primers amplified DNA fragments with different lengths. A PCR program was set in the thermal cycler for amplification of templates which included denaturation (94 °C for 30 sec), annealing (51 °C for 30s), and extension (72 °C for 40s). All these three steps were repeated for 35 cycles; subsequently, a prolonged extension period at 72 °C for 10 min was also set as a final step after completion of the 35 cycles.
Sequences and construction of a phylogenetic tree. The PCR products were visualized in two steps; first, they were run on a 1.5% gel electrophoresis, and afterward, they were stained with ethidium bromide. A PCR purification kit (AccuPrep® PCR Purification Kit, Bioneer Co., Korea) was used to purify the amplified DNA fragments from the PCR product. Afterward, the purified PCR products were sequenced using both forward and reverse primers.
An enhanced sequencing package (BigDye Terminator Cycle Sequencing Kit), including the required materials from ABI (Applied Biosystems, USA), was used to run the sequencing reactions. The reactions were configured according to the instructions described by the manufacturer. The samples were sequenced using an ABI sequencing machine (Prism 310 Genetic Analyzer, Applied Biosystems, USA).
Subsequently, the raw results produced by the ABI machine, which were computer-produced nucleotide sequences, were checked for any errors or wrong data and edited before exporting to a digital file with FASTA format with the help of Chromas Lite software (version 2). The erroneous and ambiguous parts of the sequences were also omitted using the Mega software (version 7).
Next, the final purged sequences were used as templates for homology searching in a nucleotide online database (NCBI GenBank) with the help of its online software, BLAST (The Basic Local Alignment Search Tool). The software, as its name implies, searches for smalls fragments of the template sequences in an extensive database of the nucleotides sequences of all genes from all species based on specific algorithms. Our HN sequences were incorporated in BLAST analysis to confirm their identity. After validation of the identity of sequences, they were submitted into the GenBank database and received their accession numbers after evaluation by NCBI.
We registered two HN sequences of Iranian NDV in GenBank with accession numbers KX377650 and KX377651. Moreover, to construct a phylogenic tree, we retrieved several HN sequences registered on GenBank which were submitted by other research teams from different countries.
The strains names and accession numbers of obtained sequences were as followings: HN-6-07-Ch (GQ245845), JS/2/05/Go (EU044810), JS-1-05 (DQ469830), SSX03 (DQ234581), JS/2/98/Go (AF456430), SL03 (DQ234579), SGM01 (DQ234592), SWS03 (DQ234588), HN-7-06-Ch (GQ245854), XZ-9-08-Ch (GQ245867), GD/1/98/Go (AF456433), GPV-SF02 (AY325796), KR-102/89 (GQ507802), ZHJ-3/97 (FJ766529), YZ-23-07-Pi (GQ281092), NDV05-029 (FJ766528), QH1 (FJ751918).
A phylogenetic tree was constructed by importing the retrieved HN sequences along with our two sequences into the MEGA software (version 7.0) ( Kumar et al., 2016 ) and running the phylogenetic tree analysis by setting the specific algorithms. The algorithm which was used in the software for the construction of the tree was the Neighbor-Joining (NJ) ( Offeddu et al. ) method (1000 replicates for bootstrap).
Furthermore, a matrix table containing evolutionary distances was computed by the Pairwise Distance method. According to the configurations which were set in the MEGA software, simulated trees with the lowest possibility (Bootstrap values lower than 50) were omitted from the final results. The constructed tree was interpreted based on the values which were calculated according to the NJ method. The values above 70 were supposed to be well-supported by the sequence alignments, while the lower values were not.
The RT-PCR amplification of viral RNA using HN-specific primers resulted in the amplification of two viral RNA fragments with 1000 bp and 730 bp lengths. Each of the amplicons is in fact an amplified part of the original viral HN sequences. Afterward, the PCR-amplified products (HN gene) were sequenced. Molecular phylogeny of the Iranian NDV strain isolates as well as their similarities and possible relationships to other representative NDV strains from other countries were assessed based on genetic variations on the HN gene sequence.
It was found that the HN gene of the Iranian NDV isolates has high sequence similarity. The search for homology between the HN gene sequences of our isolates and those of other Iranian isolates showed a high level of similarity within the range of 98.9-100%. Moreover, the constructed phylogenetic tree revealed a close molecular relationship among our two Iranian NDV isolates (95.34%) and HN sequences which are from China, SGM01 (DQ234592).
Despite the fact that there were close phylogenetic relationships between Iranian NDV isolates and several genotypes of NDV, such as V, VI, and VIII, the closest genetic association was found with the VIId genotype. Isolates of this study were clustered in one monophyletic clade with a common internal node with several genotypes of NDV (Figure 1). The difference between the La Sota vaccine strain and these two NDVs in antigenic sites are summarized in Table 1.
In the present study, two NDVs were isolated from vaccinated broilers in two different geographical locations. Based on the sequence and phylogenetic analysis of the F gene of these two viruses, they were close to VIId genotypes that had been previously reported in Iran ( Kiani et al., 2016 ). However, limited information is available about the HN gene sequence of Iranian NDVs ( Esmaelizad et al., 2012 ).
In this study, amplification of the complete HN gene was performed to find variations in the HN gene of the NDVs circulating in Iran. The ND is one of the most prevalent and economically important viral diseases of poultry in Iran. In order to clear the molecular footprint and study the genetics of the NDV deeply, the phylogenetic research on isolated strains of NDV in Iran seems to be of great importance.
Results of genetic studies can present us with a clear structure of the genetic contents of the virus which, in turn, provide us with critical information about the most important genes (virulence genes) as well as the taxonomy and even geographical origin of the virus. Viral particles of NDV, similar to most of the other viruses, have an envelope consisting of specific glycoproteins that act as an attachment facilitator, protection, and virulence agent for the virus.
The NDV, as a highly infectious virus, benefits from its structural virulent glycoprotein called HN which is named based on its hemagglutination and neuraminidase activity. The protein helps the viral particles to attach to host cells and attack the protective proteins on target cells. However, it was generally accepted that the two glycoproteins of NDV, i.e., HN and F, are the major viral agents responsible for its virulence ( Diel et al., 2012 ; Cao et al., 2013 ).
According to evolutionary studies (e.g. phylogeny) on various strains of NDV in different areas (i.e., Asia and Europe), velogenic genotypes have spread extensively in these areas and are responsible for the outbreaks. Recently, the most prevalent genotype which was involved in many of the NDV infection outbreaks all around Europe, South Africa, and the Far East during the 1990s was named genotype VII of NDV. The VII genotype of NDV is in fact a family of subgenotypes named from VII-a to VII-h. The first members of the family, namely VII-a to VII-e, are largely spread over China, Malaysia, Kazakhstan, and Kyrgyzstan. The remaining genotypes, from VII-f to VII-h, were repeatedly isolated within African countries ( Ebrahimi et al., 2012 ).
Esmaelizad et al. (2012) studied phylogenetic analysis for the first time in Iran to find the origin of the HN gene of NDV isolates in 2012. They found an obvious sequence relationship in phylogenetic analysis among six Iranian isolates and NDV isolates from a country near Iran, Russia. Two Russian isolates, including Sterna-Astr/2755/2001 (VIIb) and VOL95 showed significant similarity with Iranian isolates. Surprisingly, both Russian NDV isolates had high homology and sequence similarity with Iranian isolates at all nucleotide positions, except only one position on Sterna-Astr/2755/2001 (Esmaelizad et al., 2012).
The HN plays an important role in pathogenesis and immunity; therefore, Shahriari et al. (2015) used the HN gene to produce a recombinant vaccine. Firouzamandi et al. (2016), have used the HN gene to improve immunity against NDV. Sometimes, the studies performed on genetic footprints lead the researchers to characterize new sub-genotypes or sublineages of viruses.
For instance, a group of researchers in one of the neighboring countries of Iran, Pakistan, found a particular difference in the phylogeny of NDV isolates when they were evaluating the sequences of HN, F, and matrix (M) genes of the NDV isolates. The isolates were obtained from outbreaks among commercial poultry and also backyard poultry in various areas in Pakistan. The phylogenetic evaluation showed the emergence of a novel divergent group of NDV under lineage 5 which are slightly different from NDV isolates previously obtained in the area (Munir et al., 2012).
In another study performed in 2010 on virulent and velogenic strains of NDV which were isolated from a backyard and healthy poultry, the researchers found a Y526Q substitution in the HN protein. The aminoacids on that position play a crucial role in determining the binding activity of the neuraminidase receptor and the fusion activity of NDV (Munir et al., 2012).
Moreover, Otim et al. (2004) reported variations in the HN sequence of freshly isolated ND samples from poultry farms in Uganda as well as the previously obtained sequences from ND isolates. The phylogenetic evaluations demonstrated that some of the new isolates differ in their genotype from the previously obtained isolates in Uganda (Otim et al., 2004). According to the results of another study, three local ND isolates could not be identified by the specific monoclonal antibodies. These local isolates also had different variations in their HN sequence in comparison to the reference ND isolates (Hu et al., 2010).
Results of the present study are in line with those of the above-mentioned studies. It was also observed that the Iranian ND isolates were different from those of other countries in terms of their HN sequence. In this study, two NDV strains that were isolated from different farms in Iran were placed within a clade with a common internal node and as a monophyletic group next to other genotypes of NDV from different countries.
However, there was a high similarity rate (94.5-94.9%) between the HN sequences of isolates in this study and an isolate from China (SGM01; DQ234592, VII). Therefore, it can be said that the Iranian isolates could also be grouped within the VIId genotype. Based on the findings of most studies conducted in Iran, NDV isolates belong to the VIId genotype (Ahmadi et al., 2014; Mehrabanpour et al., 2014).
In the present study, it was found that the NDV isolates belong to the VIId genotype regarding the results from HN phylogenetic analysis. These findings are consistent with genotyping based on fusion gene. In addition, the collected data helps us to design a recombinant vaccine based on HN sequences of Iranian NDV isolates.
Sampling and technical procedures: M. H. K.
Data analysis: M. H. B.
Bioinformatics and technical procedures: H. H.
Paper discussions: S. Ch.
Bioinformatics and data analysis: A. Gh.
We hereby declare all ethical standards have been respected in preparation of the submitted article.
No Grant Support
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