Molecular Characterization and Epitope Mapping of Fusion (F) and Hemagglutinin (HN) Genes of Avian Paramyxovirus Serotype I from Peacocks in Pakistan

Sameera Akhtar1*, Muhammad Akram Muneer1, Khushi Muhammad1, Muhammad Yasin Tipu1, Muhammad Anees2, Imran Rashid1, Raza-urRehman3 and Irshad Hussain1 1University of Veterinary and Animal Sciences, Lahore 54000, Pakistan 2Veterinary Research Institute, Lahore 54000, Pakistan 3Poultry Research Institute, Rawalpindi 46000, Pakistan Article Information Received 05 August 2016 Revised 01 October 2016 Accepted 11 October 2016 Available online 06 April 2017

Effective diagnosis and control measures for Newcastle disease virus (NDV) requires continuous disease surveillance and understanding of field NDV strains. The NDVs, a prototype of Avian Paramyxoviruses (APMV-1), have been reported previously both in chicken and feral birds (natural reservoirs) in Pakistan; however, their molecular characterization is largely inadequate and requires continuous evaluation. In this study, sequencing and molecular analysis of fusion (F) and hemagglutinin (HN) genes of NDV strain isolated from an outbreak in a peacock flock was undertaken. Proteolytic cleavage site of F 0 showed a cleavage motif ( 112 RRQKR↓F 117 ), representative of a velogenic serotype. Moreover, based on F and HN gene nucleotide sequence analysis, the isolate clustered as genotype VII closely associated to those reported from Indonesia. Further sequence analysis of hyper-variable region within F gene revealed its clustering to sub-genotype VIIe. The inferred residue analysis of the virus revealed a number of substitution mutations in the structural and functional domains when compared to the representative strains of each genotype including the vaccine strains (genotype II and III). Interestingly, some of these mutations were found exclusive to the study isolate. Not only do these prime findings improve our understanding about currently circulating strains of NDVs but they also help us to envisage potential efforts to avoid future outbreaks in wild birds, as well as in commercial and backyard poultry.
T hroughout the world, Newcastle disease is one of the most important diseases of poultry having significant economic implications. The disease occurs both in intensively-raised commercial flocks as well as backyard poultry (Kattenbelt et al., 2006). Newcastle disease virus is categorised as a member of the family Paramyxoviridae in the genus Avulavirus. It is an enveloped virus, having a negative sense and single stranded RNA genome of approximately 15.2 Kb (Mayo, 2002). The cleavage site of the velogenic and/or mesogeneic strains of NDVs has amino acid sequence motif 112 R/KRQR/KR↓F 117 in the F protein, whereas the lentogenic viruses have amino acid sequence of 112 G/EK/RQG/ER↓L 117 (Wei et al., 2008). The clinical manifestations of NDV in susceptible birds depend on the age, host immune status, environmental stress, virulence and tissue tropism of the isolate (Dortmans et al., 2011;OIE, 2012). Clinical signs vary from in-apparent to severe; the latter is associated with respiratory, enteric and neurological signs. The hosts targeted by NDVs are more than 200 bird species that include wild, domestic and cage birds (Alexander, 2003). It is interesting to note that an avirulent virus isolated from wild birds when subjected to passages in chicken embryos transforms to a virulent form after 20 passages for chickens (Zanetti et al., 2008). Moreover, just a two-nucleotide change at the F protein cleavage site is enough to change the virus from a low virulent to a virulent strain (Gould et al., 2001).
A number of epidemics of ND in commercial poultry and wildlife have been recorded since the initiation of high density, captive husbandry system in Pakistan. Compared to isolates originating from natural reservoirs such as wildbirds, NDVs from chickens have been isolated and wellcharacterized previously Shabbir et al., , 2013a. In this study, however, analysis of

Short Communication
complete F and HN genes of an isolate recovered from ND-suspected outbreak in a peacock flock was done. The obtained sequences were processed for phylogeny and residue analyses. The study of the isolate has added to our understanding of genetic diversity among indigenous strains of NDVs originating from wild-birds in Pakistan.

Materials and methods
A ND outbreak occurred in a flock of peacock (n = 128) in district Lahore, Pakistan during 2014. Four days post appearance of clinical symptoms, 13 peacocks were dead whereas 29 were morbid. The affected birds showed circling movements and tremors while some birds were also presenting mild respiratory symptoms such as sneezing, coughing and nasal discharge. Necropsy was performed and samples (trachea, lungs, and brain) were collected and processed for isolation of NDV in 9-11 dayold embryonated chicken eggs (ECE).
Agglutinating virus in harvested allantoic fluid was identified by spot agglutination assay using 10% chicken red blood cells (RBCs) and the identity of the virus was determined by NDV specific antisera through standard haemagglutination inhibition (HI) assay. Further identification was confirmed by F-gene based reverse transcriptase polymerase chain reaction (RT-PCR) (Ling, 1997) as per optimized protocols available at Quality Operation Laboratory, University of Veterinary and Animal Sciences, Lahore.
Total RNA was extracted from allantoic fluid using commercially available RNA extraction kit as per manufacturer's instruction (QIAamp Viral RNA Mini Kit, QIAGEN, Hilden, Germany). The quantity and quality of extracted RNA was determined by (Qubit Flourometer, USA). Reverse transcription polymerase chain reaction was performed for complete F and HN genes spanning the genomic region from 4498-6330 nucleotides. Primers and protocol used for this amplification were the same as described previously (Munir et al., 2010) (Supplementary  Table I). Briefly, using QIAGEN OneStep RT-PCR Kit (QIAGEN, Hilden, Germany), a total reaction volume (25µl) comprising of 3 µl of template RNA, 5µl of 5X buffer, 5µl of Q buffer, 1 µl of 2.5 mM dNTPs, 1 µl of enzyme mix, I µl of each forward and reverse primers (10 pm/µl each) and 8 µl of nuclease free water were incubated at 50 o C for 30 and 95 o C for 15 minutes for cDNA preparation followed by 40 cycles each of 95 o C for 30 sec, 52 o C for 60 sec and 72 o C for 90 sec. The reaction was terminated by final extension at 72 o C for 10 min. Amplicons were purified using GeneJET PCR Purification Kit (ThermoFisher Scientific, USA) and sequenced in both forward and reverse directions (First BASE Lab., Malaysia) employing the same set of primers used for amplification of study genes. Sequence files were viewed in BioEdit and consensus sequence was established. The obtained sequence was blasted and matching top-hit nodes were downloaded. The obtained sequences were aligned using ClustalW programs and cut to previously known equal lengths for F and HN genes. Using MEGA6.0, phylogenetic relationships of complete F and HN genes of the study isolate were elucidated to the corresponding regions of previously characterized NDVs around the globe. The evolutionary distances were inferred and expressed based on the number of nucleotide substitutions per site. The codon positions included in the analysis were the 1st, 2nd, 3rd and noncoding. All positions containing gaps and missing data were eliminated from the data set (the "complete deletion" option). Amino acid residue analysis of representative strains of each known genotype including the vaccinal ones was compared by BioEdit.

Results and discussion
Harvested virus from allantoic fluid agglutinated chicken RBCs with a titer of 1:256 and also reacted with NDV specific antiserum confirming that the virus belonged to AMPV-1 group of NDVs. The identity of the virus was further confirmed by nucleic acid based PCR technique using NDV specific primers that targeted F gene. The fact that a number of functionally important structures such as signal peptide, cleavage activation site, hydrophobic and hydrophilic regions, and transmembrane domains are located in F and HN genes of NDVs, and that the same very genes are also used for phylogenetic analysis/ comparison to determine the geographic relationship of circulating NDV strains/genotypes (Toyoda et al., 1988;Yussof et al., 2001;Aldous et al., 2003), we characterized the virus with respect to these features. Thus, epitope mapping of biologically important regions of F and HN genes of the study isolate was carried out to see as to how our isolate differed from previously reported NDV isolates available at GenBank. Since most of the sequences available in GeneBank from Pakistan comprise of 247bp of hyper-variable region spanning 4674 to 4921nt of viral genome and most of nucleotide and subsequent amino acid substitutions are located in that particular region, we used the aligned sequence of the same length while comparing study isolate to those reported previously.
Sequence comparison of the complete F and HN genes revealed that the study isolate belonged to genotype VII. Further phylogenetic analysis of hypervariable region of F gene demonstrated that the isolate belonged to sub-genotype VIIe (Supplementary Fig. 1). Nucleotide comparison of F gene of the study isolate with that of the representative strains of NDV exhibited maximum per cent similarity with genotype VII (92.8%)  (Table I). Based upon complete F and HN gene phylogeny, peacock isolate was found to be closely related to isolates of Cockatoo/Indonesia/14698/90, Ch/Sukorejo/019/10, and Ch/Banjarmasin/010/10 that were previously reported from wild cockatoo and chickens in Indonesia. However, comparative nucleotide sequence analysis of hyper-variable region of F gene revealed close association of the study isolate with previously reported Pakistan's NDV isolates as sub-genotype VIIe. Lineage 5/genotype VII is thought to be originated from the Far East with the first isolation from Taiwan in 80s (Yang et al., 1999). Since then, the presence of this genotype has been indicated from various parts of the globe (Aldous et al., 2003). Varying at sub-lineage level, a number of virulent NDVs of class II have been reported from many Asian countries including those that share border with Pakistan (Aldous et al., 2003;Tirumurugaan et al., 2011;Zhang et al., 2011). Furthermore, genetically related NDVs to sub-genotype VIIa have been previously reported from wild birds while sub-genotype VIc, VIIb, VIIe and VIIf were reported from backyard and commercial poultry Siddique et al., 2013;Rehmani et al., 2015). However, it is the first time that this genotype (VIIe) has also been identified from wild-bird (peacock) indicating that the genotype has potential to be transmitted between the two species. Amino acid residue analysis and a comparison with previously published public database of NDVs were also undertaken for F and HN genes of the study isolate. Complete F gene spanned nucleotide sequences from 4550 -6211 totalling 1662 bp which coded for 553 amino acid residues ( Supplementary Fig. 2). Likewise, HN region comprised of DNA sequences from 6418 -8133 with 1716 bp giving rise to 571 aa residues in total ( Supplementary   Fig. 3). Within the hyper-variable region of F-gene (47-421 nt), we observed multiple basic amino acid residues at proteolytic cleavage site (F 0 ) considered typical of velogenic strains. The corresponding residues at the F 2 protein and the N-terminus of F 1 protein were found to be 112 RRQKR 116 and phenylalanine ( 116 ↓F 117 ), respectively. Analysis of the deduced residues at the cleavage site for the isolate in the current study and previously-reported isolates from Pakistan indicated co-circulation of different genotypes of NDVs in the country with similar pathogenicity (based on the motif in the cleavage site). This is of much importance since invariability in residue pattern has been observed for vNDVs in a given geographical location (Samuel et al., 2013).
The sites for glycosylation and cysteine residues are believed to be conserved for F and HN proteins. However, in comparison to each other and to the representative genotype particularly of the vaccine strain, we found differences in the composition of residues for a given glycosylation site and variations in both the number and position of cysteine residues. Further, comparison of functional domains of F and HN protein to other genotypes and vaccine strain revealed several substitutions that were more often in F protein than in the HN protein. Six potential glycosylation sites [Asp(N)-X-Ser(S)/Thr(T), where, X could be any residue except proline (P) and aspartic acid (D) including 85 NRT 87 191 NNT 193 ,366 NTS 368 ,447 NIS 449 , 471 NNS 473 , 541 NNT 543 were identified in the F protein. Furthermore, 12 cysteine (C) residues located at position 27,76,199,338,347,362,370,394,399,401,424 Fig. 2). On the other hand, HN gene analysis of predicted amino acid sequences revealed a conserved pattern of amino acid residues when compared with previous NDV strains. For example,cysteine residues at positions 123,172,186,196,238,247,251,455,461,465,531 and 542 were found to be conserved. Other biologically active key residues included residues for receptor (sialic acid) binding at positions R174, E401, R416, Y526; residues for the hydrophobic core of the stalk 4HB at positions, Y85, V88, S92, L96, T99, I103, I107, L110 and I114; and stalk residues at positions R83, A89, L90, L94 and L97 involved in direct interaction with F protein (Ke et al., 2010;Yuan et al., 2011) were found to be conserved. Furthermore, five N-glycosylation sites at positions 119 NNS 121 , 341 NDT 343 , 433 NKT 435 , 481 NHT 483 , and 538 NKT 540 were identified as has been previously reported (Umali et al., 2014). When compared to most common vaccine strain used in Pakistan (genotype II and III), beside substitutions at several point in total length of HN gene, we observed substitution mutations at  Supplementary Fig. 3). The substitutions particularly in the fusion peptide, hydrophobic regions and transmembrane region of F protein and neutralizing epitopes of HN protein could result in altered fusion activity and neutralizing escape variants (Hu et al., 2010;Wang et al., 2015). Taken together, variation in nucleotide and subsequent substitutions/alternations in amino acid profile as observed in this study is consistent with previously described theories of evolution of RNA viruses particularly the APMVs (Yu et al., 2001;Umali et al., 2014).
We found amino acid substitutions with dissimilar properties and a scattered pattern of distribution. For example, 36 th amino acid residue (isoleucine) of HN gene in our study isolate is hydrophobic but it is polar (threonine) in the representative NDVs. On the other hand, 50 th aa in our isolate is polar but it is hydrophobic in previously reported isolates. Similar substitutions could be appreciated at various regions of the genes. It is interesting to note that such substitutions with amino acids having dissimilar properties have also been found, albeit at lower rate, in representative strains of NDVs. The implications of these changes in the critical regions of F and HN genes could be serious as these changes may mean escape of the virus from the established immunity. Since the peacock flock had the history of vaccination with lentogenic strain (LaSota), identification of cleavage motif similar to velogenic strain raises concerns for type of vaccine used to protect the flock and need for post-vaccine evaluation. Substitution and subsequent mutations at fusion peptide, HR regions and trans-membrane domain could affect the fusion activity of NDV (Umali et al., 2014) and alteration in antigenic epitopes particularly those that are involved in virus attachment could result in escape variants and subsequent vaccine failure (Cho et al., 2007;Wang et al., 2015).

Conclusion
We have characterized a circulating genotype of peacock which was closely related to previously known clades of VII and VIIe sub-genotype. Based on the deduced amino acid analysis, various mutations were found throughout F and HN genes of the isolate, some were exclusive to the isolate. Whether these changes have any real impact on the immunity and disease dynamics of the virus, studies ascertaining transmission of these viruses in vaccinated as well as immunologically naïve flock and their subsequent shedding are needed for understanding their potential to cause disease in the wild birds as well as in the commercial and backyard poultry.