Introduction

Newcastle disease (ND) is a highly contagious viral disease of most avian species caused by the virulent strains of Newcastle disease virus (NDV). Etiologically, NDV (avian paramyxovirus type-1) is a member of the Avulavirus genus of subfamily Paramyxovirinae (Afonso et al., 2016). Pathogenicity of the virus amongst avian species is variable. Poultry are most susceptible to NDV and severity of the disease may vary from mild infection with no apparent clinical signs to highly pathogenic form with 100% mortality (Alexander, 2003). NDV genome is about 15.2 kb in length which codes for six major proteins including nucleoprotein (NP), phosphoprotein (P), matrix (M), fusion (F), hemagglutinin-neuraminidase (HN), and large RNA polymerase (L) (Wang et al., 2017). HN and F are glycoproteins that allow the attachment and fusion of the virus to the host cells for initiating an NDV infection (Xiao et al., 2012). The F protein is usually chosen for comparative analysis and molecular determination of NDV, because it is more likely to show genetic variation compared to other internal nucleocapsid genes (Cattoli et al., 2010). NDV strains are classified into two distinct classes (class I and class II) within a single serotype, according to the phylogenetic analysis (Czeglédi, 2006). These two classes can then be further classified into genotypes. Class I viruses contain a single genotype, whereas class II viruses contain 21 (I–XXI) genotypes (Dimitrov et al., 2019). Class I viruses are frequently isolated from wild birds and live poultry markets and are generally avirulent (Kim et al., 2007; Zhu et al., 2014), whereas class II viruses have been responsible for the spread of ND. Among class II NDVs, genotype VII is one of the most prevalent genotypes circulating worldwide. Based on the updated classification and nomenclature of NDV, previously identified NDV VII genotype was divided into sub-genotypes VII.1.1 and VII.1.2. The sub-genotype VII.1.1 combines the former sub-genotypes VIIb, VIId, VIIe, VIIj, and VIIl. The former sub-genotype VIIf was classified as sub-genotype VII.1.2. Sub-genotypes VIIa, VIIh, VIIi, and VIIk were merged into a single sub-genotype, namely VII.2 (Dimitrov et al., 2019). Based on the clinical signs and severity of the disease, NDV strains are classified into four main pathotypes: asymptomatic enteric (avirulence), lentogenic (low-virulence), mesogenic (intermediate-virulence), and velogenic (high-virulence). Velogenic strains can be further categorized into two types: viscerotropic and neurotropic (James, 2017).

Notwithstanding virulent forms of ND controlled by biosecurity protocols and vaccination, outbreaks still occur and velogenic strains are endemic in the poultry flocks of Iran, which induce a large scale economic losses (Hosseini, et al., 2014; Mehrabanpour et al., 2014). Despite routine vaccination, a combination of killed and live vaccines as a controlling practice, there have been several reports on its occurrence in commercial and non-commercial poultry farms in recent years (Hosseini et al., 2014; Sabouri et al., 2016).

Poultry production is one the most important agro-industries in the north part of Iran. Mazandaran province, with 30% of total production, is the most important producer of day-old broiler chicks in Iran (IRNA, 2019). During the last decade, monitoring the NDVs suggested different genotypes of NDVs have been responsible for ND outbreak in Iran. In the current study, we aimed to define the most prevalent NDV genotypes and sub-genotypes circulating in commercial broiler and backyard chicken farms located in Mazandaran province, Iran.

Materials and Methods

 Study Design

NDV develops a broad range of clinical manifestations such as respiratory signs, nervous signs, and greenish watery diarrhea. In this survey, we only focused on rural and commercial meat-type chicken farms with a history of neurological symptoms. All samples were prepared from private poultry clinics in north of Iran during 2017-2018. Only fresh carcasses were considered. Collectively, twenty-five samples (sixteen samples from broiler farms and nine samples from backyard farms) compatible with our desired criteria were chosen for further analysis.

Sample Preparation

The whole brain of affected chickens was extracted under a Class II (laminar flow) biological safety cabinet and transferred to a sterile 15-mL falcon tube. Tissues were halogenated in phosphate buffered saline (PBS), pH 7.4, containing antibiotics of penicillin (2000 U/mL) and streptomycin (2 mg/mL), and filtered with a 0.22 µm syringe filter. Then, the filtered liquid was divided into two aliquots for RNA extraction and virus amplification, and kept at -70^oC (Alexander, 2012).

Virus Propagation

To prepare virus, about 200 μL of each sample was inoculated in the allantoic cavity of 8- day old embryonated eggs and incubated at 37ºC for seven days and candled twice daily. For each sample inoculation, 3 eggs were selected. Any death after 24 hours was recorded and subjected to further analysis. Briefly, the allantoic fluid was collected by a sterile syringe carefully and stored in a sterile 15 mL falcon tube and stored at -70^oC. All positive samples in HA testing were subjected to three passages in embryonated egg.

RNA Extraction and Reverse Transcriptase Polymerase Chain Reaction

Viral RNA was extracted and purified from infectious allantoic fluid and brain halogenated homogenized tissues using Viral Gene-spin™ Viral DNA/RNA Extraction Kit according to the manufacturer’s instructions (iNtRON, South Korea). Then, cDNA was synthesized using Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) and Random 6-mers (Yektatajhiz, cDNA Synthesis Kit, Iran). To define the NDV positive genome, PCR reaction was performed with primers A and B, which contains important structures such as the cleavage site (Kant et al., 1997). Afterwards, coding sequence of F protein, which includes heads and stacking elements of protein was amplified by one set primer described by Qin et al. (2008). The information and annealing degree of all primer sets are illustrated in Table 1

Table 1. Sequence, annealing temperature, and position of primers used in this study

Sequencing and Phylogenetic Analysis

To have more accurate and comprehensive information about F protein, the complete extracellular domain of protein was sequenced. The nucleotide sequences of virus isolates were investigated with Sequencher program (version 5.4.6; DNA sequence analysis software, Gene Codes Corporation, Ann Arbor, MI, USA). Sequence alignment and blast analysis were done by an online webpage. The phylogenetic tree was illustrated with MEGA7 (MEGA, version 7) and the evolutionary history was inferred using the Neighbor Joining (data not shown). Maximum likelihood methods with standard errors were also calculated based on 500 bootstrap replicates (Kumar et al., 2016; Tamura et al., 2004).

Accession Numbers

The sequence of F protein of NDV isolates (chicken/Iran/CR5/2017, chicken/Iran/CI1 /2017, chicken/Iran/CI6/2017, chicken/Iran/ CI3/2017, and chicken/Iran /CI9/2018) were submitted to GenBank under the accession numbers MK659694, MK659696, MK659698, MK659699, and MK659695, respectively.

Results

Nucleotide Sequencing and BLAST Analysis of Partial Fusion Gene

Altogether, out of twelve viral isolates from brain samples of RT-PCR-positive, five isolates (four from commercial and one from backyard farms) were randomly subjected to nucleotide sequencing as representative sequences. Nucleotide (nt) and amino acid (aa) comparisons of all studied isolates ranged from 98.85-99.75% and 99.23-99.53%, respectively. BLAST results of F gene revealed high percent of homology between NDV strains CK/KR/KR 005/00 (KY404087), GO/CH/LN15/15 (MF581297), and CK/UA/Lugansk/03 (KU710279) isolated from the Far East and Europe.

Phylogenetic Analysis

Coding region of the extracellular domain of F gene of the isolates were aligned with the corresponding region of the F gene from 291 NDV strains belonging to nineteen genotypes, downloaded from the GenBank and subjected to genetic analysis (data not shown). In the phylogenetic tree, our isolates were clustered with the strain under genotype VII (Figure 1). To confirm our preliminary results, the phylogenetic relationship was assessed using at least 4 strains for each sub-genotype VII (according to the criteria proposed by Diel et al., 2012). Our results showed that all the isolates belonged to sub-genotype VII.1.1 (VIIl) in a well-supported cluster (60% bootstrap value) and displayed 98.03-100% homology in the nucleotide level (Figure 2).

Notes: The number of base substitutions per site from averaging the overall sequence pairs between groups is shown. Standard error estimates are shown above the diagonal and were obtained by a bootstrap procedure (500 replicates). Analyses were conducted using the maximum composite likelihood model. The rate variation among sites was modelled with a gamma distribution (shape parameter = 1). The analysis involved 175 nucleotide sequences. Codon positions included were 1st + 2nd + 3rd + Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 639 positions in the final data set. Evolutionary analyses were conducted in the MEGA7.The average evolutionary distance of NDV isolates obtained in the present study with other sub-genotypes from genotype VII are shown in bold (n: number of isolates from each sub-genotype).

Analysis of the Fusion Protein

Deduced amino acid sequence of the cleavage site motif, putative neutralizing epitopes, profile of glycosylation sites and cysteine amino acid residues of studied isolates, and other Iranian NDV isolates of F protein were investigated to find any possible mutations within them (Table 3).

Figure 1.Phylogenetic tree of NDV strains based on the nucleotide sequence (42–1604) of the F gene. The tree was constructed using maximum likelihood method based on the Kimura 2-parameter model with MEGA7 program (Kimura, 1980). The analysis involved 121 nucleotide sequences and according to the criteria proposed by Dimitrov et al. (2019). Bootstrap >60% was applied for defining node. Black circle indicates the studied isolate.

Figure 2. Phylogenetic relationships of the nucleotide sequences of VII genotype of NDV isolates based on sequences of F gene. The evolutionary history was inferred by using the Maximum Likelihood method based on the Kimura 2-parameter model (Kimura, 1980). Tree construction was done using the Neighbor Joining method with the maximum composite likelihood substitution model (500 replicates) in MEGA7. The viruses obtained in this study are marked with squares.

Table 2. Estimates of evolutionary diversity among sub-genotypes of genotype VII based on partial coding sequences of the F gene

Table 3. Comparison of studied isolates and other Iranian NDV isolates