Genetic characterization and phylogeny of pigeon paramyxovirus isolate (PPMV-1) from Pakistan
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Knowing the genome characteristics of circulating Newcastle disease viruses [avian paramyxoviruses (APMV-1) and pigeon paramyxoviruses (PPMV-1)] is important to devise appropriate diagnostics and control strategies. APMVs originating from chicken and wildlife in Pakistan are well-elucidated; nevertheless, molecular characterization for the circulating PPMV-1 is largely unknown.
Here, we have performed fusion (F) and hemagglutinin (HN) gene based characterization of PPMV-1 isolated from an outbreak in a pigeon flock. With F0 proteolytic cleavage site (112RRQKR↓F117), characteristic of velogenic/mesogenic serotype, the complete F and HN gene based sequence analysis of the isolate revealed evolutionary relationship to genotype VI. Further analysis of hyper-variable region of F-gene demonstrated clustering of the study isolate with genotype VIb. The deduced residue analysis for both F and HN protein showed a number of substitution mutations in the functional domains distinct from representative strains of each genotype including the vaccine strains; some of them were found exclusive to the study isolate.
Though limited and preliminary data, the findings enhance our knowledge towards circulating strains of PPMVs in Pakistan. Further studies are needed to ascertain its potential for transmission in the wild birds, commercial and backyard poultry and its subsequent shedding into the environment.
KeywordsPigeon paramyxovirus type 1 (PPMV-1) Newcastle disease virus Pigeon Pakistan
Pigeon Paramyxovirus type 1 (PPMV-1) is an antigenic and host variant of classical Newcastle Disease virus (NDV) or Avian Paramyxovirus type 1 (APMV-1) that causes Newcastle Disease (ND)-like infection and pathology in pigeons (Ujvari et al. 2003). Including the earliest known PPMV-1 (Iraq78) originating from the Middle-East (ME), and the strains responsible for third panzootic in 80 s involving a pigeon-adopted APMV-1 are considered likely to be derived from these ME viruses (Kaleta et al. 1985; Ujvari et al. 2003) and as yet continue to circulate around the globe. The virus has an approximate genomic length of 15192 nt and, despite characteristic F protein cleavage site for velogenic strains (112GRQKRF117 or 112RRKKRF117 or 112RRQKRF117), differences in pathogenicity index range from moderate to no virulence for chicken (Collins et al. 1994; Dortmans et al. 2010). However in recent years, some PPMV-1 have been reported to be highly pathogenic for chicken after passage either in chicken or chicken embryo indicating their potential to cause ND outbreaks (Dortmans et al. 2011). Today, ND caused by either virulent APMV-1 or PPMV-1 is considered endemic to feral and domestic pigeon (Columbiformes) worldwide (Aldous et al. 2004). Clinical symptoms differ depending upon host immune titre and virulence of isolate involved (Dortmans et al. 2011). Most of the times, the observed clinical symptoms relate to neurotropic form of ND including tremors, torticollis and disturbed equilibrium, however naturally infected pigeon also exhibit respiratory symptoms such as sneezing, coughing and tracheal rales (Marlier and Vindevogel 2006).
Since the establishment of well-organized poultry sector in Pakistan to-date, there have been a number of epidemics of ND in commercial poultry, wild birds and domestic pigeons. Although, APMV-1 from commercial poultry and wild birds have been isolated and well-characterized as genotype VI and VII (Munir et al. 2012; Shabbir et al. 2012, 2013a, b), despite a number of clinically suspected outbreaks in pigeons including vaccinated ones (Lab data, not published), there is absolute paucity of information pertaining to circulating lineage of PPMV-1 and its nucleotide and amino acid profile to reference and vaccinal strains. We analysed complete F and HN genes of an isolate recovered from ND outbreak in a pigeon flock. The obtained sequences were processed for phylogeny and amino acid residue analyses giving an insight towards genetic diversity of the indigenous strain and its comparative evolution to those reported earlier.
A Newcastle disease outbreak occurred in a flock of racing pigeons (n = 56) in district Lahore, Pakistan (31.5790°N, 74.3096°E) during December, 2014. Four days post appearance of clinical symptoms, 13 died and 29 were morbid. The clinical symptoms observed in affected pigeons were circling movement and tremors while some birds were also exhibiting mild respiratory symptoms such as sneezing, coughing and nasal discharge. Necropsy was performed and samples (trachea, lungs, and brain) were processed for isolation of NDV in 9 day old embryonated chicken eggs (ECE). Presence of agglutinating virus was confirmed in harvested allantoic fluid by spot agglutination assay using 10 % chicken red blood cells (RBCs). Later, identity of the isolate was confirmed as NDV through standard haemagglutination inhibition assay using specific antisera.
Total RNA was extracted from allantoic fluid using commercially available RNA extraction kit as per manufacturer’s instructions (QIAamp Viral RNA Mini Kit, Qiagen, USA). Quantity (NanoDrop, USA) and quality (QubitFlourometer, USA) of extracted RNA was measured. The extracted RNA was subjected to amplification of complete F and HN genes spanning the genomic region from 4498 to 6330 nucleotide through reverse transcription polymerase chain reaction (RT-PCR) using the primers and protocols as described previously (Munir et al. 2010). The amplified products were purified by 1.0 % gel electrophoresis, using the Wizard® SV Gel and PCR Clean-Up System (Promega, Co., Madison, WI, USA) as per manufacturer’s instructions. Using the same primers (as used previously for F and HN gene amplification) and ABI PRISM BigDye Terminator version 3.1 (Applied Biosystems, Foster City, CA, USA), the purified genomic DNAs were processed for sequencing reaction on a 3100 DNA Analyzer (Applied Biosystems, Foster city, CA, USA). Each genomic fragment was sequenced twice in both forward and reverse directions to generate a reliable consensus sequence. The consensus sequences of F and HN genes of the study isolate (Pi/MZS1-UVAS/2014) has been submitted to GenBank under the accession number KU644586 and KU644587, respectively.
The obtained sequences and sequences reported earlier (GenBank) were aligned in BioEdit version 5.0.6 (Hall, 1999) using ClustalW and cut to equal lengths. Phylogenetic relationships of complete F, HN gene and hyper-variable region of F gene of study isolate were elucidated to the corresponding region of previously characterized viruses around the globe (http://www.ncbi.nlm.nih.gov/nuccore/?term=partial+F+gene+of+Newcastle+disease+virus) at the level of genotype and sub-genotype using the MEGA version 6.0 software (Tamura et al. 2013). 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). Furthermore, comparative residue analysis of the representative strains of each known genotype was analysed through BioEdit.
Results and discussion
As the pigeon flock had a history of vaccination with lentogenic strain (LaSota), identification of cleavage motif similar to velogenic raises concerns for the type of vaccine used to vaccinate the flock and the need for post-vaccine evaluation. Substitution and subsequent mutations at fusion peptide, HR regions, trans-membrane domain and antigen neutralization sites could affect the fusion activity of NDVand, 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; Umali et al. 2014; Wang et al. 2015). Beside potential compromise in procedures used in vaccine storage and administration, the expected genetic distance between vaccine strains and the study isolate seems to be well-explained by Wang et al. (2015) through cross-HI assay. While evaluating the antigenic diversity of different strains through cross-HI assay, they reported a lower R-value (0.13–0.18) for interaction of PPMVs to LaSota than between PPMVs (VIa and VIb, 0.7) indicating an obvious antigenic difference with vaccine strain.
We characterized the circulating genotype of pigeon (PPMV-1) closely related to previously known clades of VIb. Since outbreaks of NDV in commercial poultry has been reported in one of the province of Pakistan from a closely related genotype (VIc) to the study isolate (VIb), future studies relating to disease surveillance coupled with experiments involving potential to transmit and shedding from either vaccinates or non-vaccinates are essential. Moreover, immunity barrier provided by LaSota and other vaccinal strains needs to be evaluated in the face of newly emerging NDV strains.
Conceived and designed the experiment: SA, MAM, MR and MZS. Sampling and laboratory work: SA, MYT, AR and MZS. Analyzed the data: SA, KM, AR and MZS. Wrote the manuscript: SA, AR and MZS. All authors read and approved the final manuscript.
We thank Defense Threat Reduction Agency of USA for capacity building of the faculty as well as strengthening of existing laboratory facilities utilized in this work at the University of Veterinary and Animal Sciences, Lahore Pakistan through collaborative research projects with The Pennsylvania State University, USA. Further, thanks are due to Dr. Irshad Hussain for reading and editing the revised version of the manuscript.
The authors declare that they have no competing interests.
This article does not contain any experiment or research with animals performed by any of the authors.
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