Introduction

Intensive poultry farming has provided pre- disposing situations for the prevalence of mul- tifactorial respiratory complex in the farms. Different bacterial and viral agents may cause these infections simultaneously (Glisson, 2013). Based on Iran Veterinary Organization (IVO) statistics, considerable economic dam- age in broiler farms is related to respiratory complications (Ebadzadeh, 2015).

Newcastle disease is known as an import- ant agent in multifactorial respiratory diseas- es; on the other hand, the Newcastle disease (ND) virus plays a major role in the devel- opment of respiratory diseases especially in interactions between other pathogens  such as Ornithobacterium rhinotracheale, infec- tious bronchitis virus (IBV), Mycoplasma gallisepticum, and Mycoplasma synoviae (Hopkins and Yoder Jr, 1982; Weinack et al., 1984). Eleven serotypes of avian paramyxo- virus (APMV-1 to APMV-11) have been identified (Suarez, 2013). Newcastle disease virus, a synonym for the avian Paramyxovi- rus type 1 (APMV-1), is a non-segmented, negative-sense, single-stranded, enveloped RNA virus composed of approximately 15,200 nucleotides (Gogoi et al., 2017; Sam- son, 1988; Zhao et al., 2018).

Newcastle disease virus has a 15kb RNA genome that codes six viral proteins: an RNA directed RNA Polymerase (L), Hemagglu- tinin-Neuraminidase protein (HN), Fusion protein (F), Matrix protein (M), Phosphopro- tein (P) and Nucleoprotein (NP) (Aldous and Alexander, 2001; Lee et al., 2017).

Although different laboratory methods have demonstrated minor antigenic variation between different isolates of NDV, all NDV isolates can become neutral by 1 serotype an- tibodies as they are all from avian APMV-1 serotype. APMV-1 can be grouped into two

classes (class I and class II) which are deter- mined based on genetic and antigenic of the F gene. Class I isolates are all grouped into  a single genotype and three subgenotypes. This class is mostly isolated from both wild and domestic birds found in Africa, Asia, Europe, and America and is considered of low virulence in chicken. Class II is most-  ly found with a high rate of virulence. Ac- cording to the recent literature, the class II isolates are classified into genotypes I-XVIII (Bello and Yusoff, 2018).

Newcastle disease with different levels of virulence is contributed to a high rate of mor- bidity and mortality throughout the world (Choi et al., 2014; Kiani et al., 2016). The virus strains may be classified based on mean death time (MDT) as velogenic (highly virulent), mesogenic (intermediate virulence), or lento- genic (nonvirulent) (Brown and Bevins, 2017). Based on OIE (OIE Manual, 2018) the pathogenicity of the virus is determined by the cleavage site of F protein. Aldous pro- posed that genotyping of NDV isolates should become part of diagnostic virus char- acterization for reference laboratories by producing a 375-nucleotide sequence of the F gene, which includes the F0 cleavage site. As a standard assay to characterize NDV strains, genome sequencing and phylogenet- ic analysis of F gene are widely, utilized.The F glycoprotein is responsible for fusion be- tween the cellular and viral membranes and subsequent virus genome penetration (Ald- ous and Alexander, 2001; Glickman et al., 1988; Liu et al., 2015). The sequence of the F protein cleavage site is a major determinant of NDV pathogenicity. The cleavage sites of virulent NDV strains usually contain multi- ple basic residues, whereas avirulent strains have fewer basic residues (Xiao et al., 2012).

Behshad Beheshtian et al.                                                               Iranian Journal of Veterinary Medicine

Comparison of amino acid sequences showed that viruses with a 112-RKRQKRR-116 mo- tif at the C-terminus of the F2 protein and F (phenylalanine) at residue 117, are virulent (Collins et al., 1998; Damena et al., 2016; Hosseini et al., 2014; Panda et al., 2004).

Virulent Newcastle disease virus is now endemic in Iran. Despite the implementa- tion of the various vaccination programs in commercial poultry flocks, a considerable number of ND outbreaks have been report- ed in recent years (Ebrahimi et al., 2012; Hosseini et al., 2014).

The main aim of this study was to investi- gate the role of the Newcastle disease (ND) virus in multifactorial respiratory diseases. Moreover, the involvement of the very vir- ulent Newcastle disease virus (VVND) and lentogenic strains are analyzed in infected flocks. Also, phylogenetic analysis of the fu- sion gene of 5 virulent isolates and Iran iso- lates compared to different reference NDV genotypes was generated.

Materials and Methods

Sampling

A total number of 180 tracheal swab sam- ples were taken from 20 Newcastle vacci- nated commercial broiler flocks in the age of 9-49 days of Qazvin province during 2014-2015. In the laboratory, every 3 swab samples were pooled and finally, 60 sam- ples (3 samples/farm) were tested.

In this study, the infected flocks have been selected based on the presence  of  both the clinical and postmortem respira- tory signs. These flocks are also investi- gated for the presence of some respiratory pathogens such as infectious bronchitis virus (IBV), avian influenza virus (AIV), avian metapneumovirus (aMPV), and my- coplasmas.

RNA extraction and RT-PCR

RNA was extracted by RNA kit (Cinna- Gen Co., IRAN) according to the manufac- turer’s manual. Then, the cDNA was made by Random Hexamer (RH) and Revert Aid first strand cDNA Synthesis Kit (Fermen- tas-Thermo Fisher Scientific, Canada). Part of the F gene which includes the cleavage site sequence was amplified by a pair of primers with the sequence of 5-TTGATGG- CAGGCCTCTTGC-3 and 5-GGAGGAT- GTTGGCAGCATT-3 (Kant et al., 1997).

PCR was carried out in a 50 µl reaction volume consisting of 5 µl of 10 × PCR buffer, 1 µl of 10 mM dNTPs, 1.25 µl of each primer (10 pmol/µl ), 0.25 Taq DNA polymerase (5U/ µl), 1.5 µl 50mM MgCL2,

33.75 µl of dH2O, and 6 µl cDNA dilution, and was programmed in the following con- dition: 94 ˚C for 3 min followed by 35 cy- cles of 95 ˚C for 30 sec, 53 ˚C for 30 sec, 72˚ C for 60 sec, and a final extension at   72 ˚C for 15 min. The PCR products were electrophoresed by 1% agarose gel and vi- sualized under UV (Hosseini et al., 2014).

Sequencing and phylogenetic analysis

Twelve isolates were considered for se- quencing and further analysis. PCR prod- ucts were cut out from the gel and purified by PCR AccuPreb® PCR Purification Kit (Bioneer Co., South Korea) according  to the manufacturer’s instruction. Purified RT- PCR products were sequenced in both for- ward and reverse directions by ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, USA) and run on an ABI Prism 310 Genetic Analyzer. Analyses, sequence assembling, and editing were done by the CLC sequence viewer (CLCbio). The nucleotide sequence of the F protein gene determined in this study was compared to the NDV sequence

Iran J Vet Med., Vol 14, No 2 (Spring 2020)                                                                                        137

data available in the National Center for Bio- technology Information database, and the phylogenetic relationship was established. Sequences were aligned by CLUSTAL W. Distance-based neighbor-joining trees were constructed and designed with the use of the Tamura-Nei model available in the program MEGA5, version 5 (Hosseini et al., 2014; Tamura et al., 2011). The phylogenetic tree was assessed by 1000 bootstrap replicates. Bootstrap support of C70% is shown near the nodes in the phylogenetic trees. The dif- ference in nucleotide sequence and percent- age similarity was estimated with the use of the CLC Main Workbench (CLCbio) (Hos- seini et al., 2014).

Results

ND viruses were detected in 26 of 60 (43%) samples and 12 of 20 (60 %) flocks. There was no significant correlation between age and the presence of NDV (P>0.05). All the positive flocks and birds had common respiratory signs and lesions of ND such as moderate to severe mucopurulent  exudate on the trachea, gasping, coughing, drooping wings, petechiae in the proventriculus, green and white watery diarrhea. In positive flocks, also, the other respiratory pathogens such as infectious bronchitis, avian influenza, and pneumoviruses were detected too (Data is not given).

The sequencing of the F gene revealed that 7 isolates (58.33 %) belonged to the lento- genic and 5 isolates (41.66 %) belonged to the velogenic group due to the motif cleav- age site of 112-RRQKRF-117 and also due to the presentation of the phenylalanine (F) at region 117. All 5 velogenic  isolates  in this study were classified into genotype II, subgenotype VIId.

Based on the phylogenetic analysis, this study revealed that the IR/H1248, 1/15, as vaccinal strain, bears a high similarity to the B1 and LaSota with  the rate of 100 % and  a considerable homogeneity to  the  strains of Chicken/Iran/SMV-3/2011(KU201410), IR-HGT2012.1   (JX131357.1),   and  Chick-

en/Iran/SMV-8/2013(KU201415) with the rate of 83.7 %. In the other hand, some velo- genic strains, such as the IR/H1248,10.15 possesses the closeness of 83.7 %  to  B1 and Lasota strains and a very high homo- geneity, with the rate of 100%, to chicken/ Iran/SMV-3/2011(KU201410),     IR-HGT

2012.1 (JX131357.1), chicken/Iran/SMV- 8/2013(KU201415) strains that were previ- ously identified in Iran (Table 1 and Figure 1).

Table 1. Nucleotide similarity in the part of the F gene among investigated virusesin this study, some vaccinal, standard and the strains that are separated in Iran

Figure 1. Phylogenetic analysis of the fusion gene for Iran isolates in comparison to sequences from viruses representative of different Newcastle disease virus (NDV) genotypes. The phylogenetic tree was generated by the neighboring-joining model with MEGA (version 5.1 beta). Numbers below branches indicate bootstrap values from 1000 replicates. Horizontal distances are proportional to the minimum number of nucleic-acid differences required to join nodes. The vertical lines are for spacing branches and labels. The analysis was based on the complete open reading frames of all gene segments. The viruses characterized in this report are indicated by black circles. The sequences were obtained from GenBank.

Discussion

Newcastle disease virus is the causative agent of a serious avian disease that can re- sult in significant economic losses to both the poultry industry and backyard chickens (Saadat et al., 2014).

In the past few decades, implementation

of extensive vaccination programs in com- mercial poultry farms, and to  some  extent in small rural poultry farms has reduced the number of epizootics outbreaks of Newcas- tle disease, however, failure of vaccination still occurs frequently in the poultry industry (Saadat et al., 2014; Samadi et al., 2014).

Newcastle disease  virus  vaccines  like  the other vaccines do not prevent vaccinat- ed animals from becoming infected with a virulent ND virus and subsequently, viral shedding can occur (Kapczynski and King, 2005; Miller et al., 2013). Despite the appli- cation of billions of doses of live, inactivat- ed, and recombinant NDV vaccines world- wide, VNDV continues to be endemic. Due to Miller’s study, superior protection can be provided when vaccines are matched to field isolates (Miller et al., 2013).

The virulence of NDV is known to be as- sociated with differences in the amino acid sequence surrounding the post-translation cleavage site of the F0 protein, with differ- ences in the cleavage sites being directly re- lated to the virulence of the strain. Most vir- ulent viruses have the amino acid sequence 112 R/KR-Q-K/R-R 116 at the C-terminus of the F2 protein and F (Phenylalanine) at resi- due 117, the N-terminus of the F1 protein. In contrast, low virulent viruses have sequences in the same region of 112 G/E-K/R-Q-G/E-R 116 and L (Lysine) at residue 117 (Collins et al., 1998; Dey et al., 2014; Hosseini et al., 2014; Panda et al., 2004).

Full sequencing is the analysis of the en- tire genomic DNA that provides the most comprehensive characterization of the ge- nome. By full fusion gene sequences,  Se- lim reported that all isolates of their study were related to genotype VIId subtype (Se- lim et al., 2018). Saboury implemented the complete coding sequence of fusion (F) and hemagglutinin-neuraminidase (HN) genes to identify VIIl sub-genotype of Newcastle dis- ease virus in Iran (Sabouri et al., 2018).

Partial sequencing has been used in most studies for its advantages which included the low cost of experimental price in comparison to the full sequencing and also it can simply

present interspecies nucleotide similarity.

In this study, the partial sequencing of the cleavage site of the F gene has been conduct- ed. This is because the amino acids of the F gene cleavage site are the “major determi- nant” to confirm the pathogenicity and con- sequently the virulence of the NDV(Miller PJ, 2013). Mehrabanpour showed that iso- lates from Iran belong to class II, genotype III viruses (Mehrabanpour et al., 2014). Hos- seini has detected 112RRQKRF117 in nine field isolates and classified them into the genotype VII, subgenotype VIId  (Hosseini et al., 2014). Kianizadeh’s study showed the virulent isolates with two pairs of arginine and phenylalanine at the N-terminus of the fusion (F) protein cleavage site, similar to other velogenic isolates of NDV character- ized earlier in Russia in 1995 (Kianizadeh et al., 2002). Boroomand et al. (2016) detected 112RRQKRF117 at the C-terminus of the F2 protein and phenylalanine at the N-terminus of the F1 protein residue 117 in three isolates and demonstrated that all isolates belong to the genotype VIId of class II NDV strains (Boroomand et al., 2016). Abdoshah report- ed that Iranian NDV isolates have RRQRRF at the cleavage site of the F Protein and be- longed to the VIIb subgenotype (Abdoshah, 2012). Ebrahimi demonstrated that genotype VII of NDV was still predominant in the do- mestic poultry of Asia. The existence of the VIId subgenotype in far-east countries, the subgenotype VIIb is circulated in Iran and Indian subcontinent countries (Ebrahimi et al., 2012). In the study of Nath, nucleotide sequence analysis of fusion (F) and hemag- glutinin protein genes revealed a close sim- ilarity with genotype XIII strains of NDV. The amino acid sequence of F protein con- firmed the virulent cleavage site (112) R-R- Q-K-R-F (117) (Nath et al., 2016). Accord-