Virus Genes (2014) 49:449–455 DOI 10.1007/s11262-014-1116-2
Genetic characterization and pathogenicity assessment of Newcastle disease virus isolated from wild peacock Sagar A. Khulape • Satish S. Gaikwad • Madhan Mohan Chellappa • Bishnu Prasad Mishra Sohini Dey
•
Received: 6 June 2014 / Accepted: 16 September 2014 / Published online: 27 September 2014 Ó Springer Science+Business Media New York 2014
Abstract The continued spread and occurrence of Newcastle disease virus (NDV) has posed potential threat to domestic poultry industry around the globe. Mainly, wild avian species has always been implicated for the natural reservoir for virus and spread of the disease. In the present study, we report the isolation of Newcastle disease virus (NDV/Peacock/India/2012) in necropsy brain tissue sample of wild peacock from North India. Complete genome of the virus was found to be 15,186 nucleotides (nts) with six genes in order of 30 -N–P-M-F-HN-L-50 , which was limited by 55-nts leader region at the 30 end and a 114-nts trailer sequence at 50 end. Sequence analysis of fusion protein revealed the dibasic amino acid cleavage site 112R-R-Q-KR-F117, a characteristic motif of virulent virus. Phylogenetic analysis placed the isolate in genotype II of Newcastle disease virus showing the lowest mean percent divergence (6 %) with other genotype II counterparts. The isolate was characterized as mesogenic (intermediate pathotype) based on the mean death time (63 h) in embryonated chicken eggs and the intra-cerebral pathogenicity index (1.40) in day-old chicks. The report emphasizes the dynamic ecology of NDV strains circulating in a wild avian host during the outbreak of 2012 in North India. Further the genotypic and pathotypical characterizations of the isolate could help in development of homologous vaccine against NDV strain circulating in avian population.
S. A. Khulape M. M. Chellappa B. P. Mishra S. Dey (&) Division of Veterinary Biotechnology, Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh, India e-mail: [email protected]; [email protected] S. S. Gaikwad Postdoctoral Fellow, OIE Reference Laboratory for Newcastle Disease, Anyang, Gyeonggi-do, South Korea
Keywords Avian paramyxovirus type I Newcastle disease virus Phylogenetic analysis
Introduction Newcastle disease (ND) is an acute infectious disease of birds causing major loss and devastation to poultry industry [1]. The causative agent, Newcastle disease virus (NDV), is a member of the genus Avulavirus in the family Paramyxoviridae. Of the nine different serotypes of avian paramyxoviruses (APMV), NDV belongs to APMV-1 and remains the most important pathogen for poultry [2]. NDV genome is a single-stranded RNA of approximately 15.2 kb nucleotides. The viral RNA encodes six structural proteins namely nucleoprotein (N), phosphoprotein (P), matrix (M), fusion (F), hemagglutinin-neuraminidase (HN), and RNA-directed large RNA polymerase (L) along with two non-structural proteins V and W [3]. The NDV strains are a diverse group of viruses in terms of antigenicity and genetic composition [4] and are classified as lentogenic, mesogenic and velogenic strains, respectively, based on its pathogenicity in chickens [5]. The contribution of viral protein for NDV virulence has been well documented for F, V, HN, NP, P and L proteins [6] but of these, F contributes important molecular determinant to NDV pathogenicity and is linked with the presence of critical amino acid motif at protease cleavage site of precursor F protein [7]. Genotype-based classification of NDV divide the viral strains into two distinct classes (I and II) based on genetic analysis of nucleotide sequence at the fusion protein cleavage site (FPCS). Class I strains of APMV-1 have been isolated mainly from wild birds and are generally avirulent, whereas class II strains have been recovered from wild and domestic birds and include
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virulent and avirulent isolates [8]. All class I viruses are 15,198 nucleotides (nts) in length, while class II viruses are further subdivided into genotypes I–IV, with 15,186 nts, and genotypes V–XI, with 15,192 nts [9]. Newcastle disease is a listed disease of the world organization for animal health (WOAH) affecting more than 230 species of birds with wide geographical distribution [10]. Wild birds have been implicated in the spread of infectious NDV to domestic bird population and are considered to be the natural reservoir of the infectious agent [4]. Although some NDV isolates from wild are found to be non-pathogenic to chickens, previous incidences like spread of virulent NDV strains from wild psittacine in the United States in the 1970s [11, 12] and cormorants in the 1990s [13, 14] warrant consistent monitoring and surveillance of wild birds for effective prevention and control programme. NDV is either endemic or a cause of regular epidemic in poultry throughout most parts of Africa, Asia, Central America and parts of South America despite the widespread vaccination programmes [3]. In India, NDV has been isolated from different avian hosts including chicken, pigeons, ducks, turkeys, guinea fowl and emus [16–20]. NDV has also been reported from recreational birds, migratory birds and captive wild birds [20, 21]. Although a variety of wild birds are susceptible to ND, the information available is limited due to the non-availability of clinical/necropsy samples and poor disease surveillance in wild avian population. Previously, NDV infection has been reported from a wild peacock under captivity [22] and more recently [23] in India. The recent outbreaks of virulent NDV in neighbouring countries, Pakistan [24–26] and Bangladesh [27], should be perceived as an epidemiological alert to re-assess the control and quarantine measures against ND. In the epidemiological context of India, different strains of NDV circulate in domestic bird population [28] that demands the characterization of NDV isolates from indigenous outbreak and necropsy examinations in rural free-range birds, commercial chickens and wild avian birds. Peacock, the national bird of India, comes under the family Phasianidae which also includes pheasants, partridges and jungle fowl. The natural habitats for peacock are streamside and cultivated areas. The species also frequents around human habitats and villages for scraps. The movement of this wild species in close proximity to domestic birds most of which are reared under backyard poultry farming always threatens to be the point of disease outbreak. In this study, NDV was isolated from a wild dead peacock’s postmortem sample and characterized genetically, pathotypically and biologically to understand its relationship with the previously established genotypes of the virus.
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Materials and methods Virus identification and isolation The necropsy sample comprising brain tissue from a dead wild peacock was received from the District Veterinary Polyclinic, Rewari of Haryana state India. The brain tissue was triturated in phosphate-buffered saline (pH 7.2). The virus isolation was done in 9-day-old specific-pathogenfree (SPF) embryonated chicken eggs (Venky’s India Pvt. Ltd., Pune, India) through allantoic cavity inoculation of the tissue suspension. A total of three blind passages were given, and hemagglutinating titres for each batch of allantoic fluid were determined. For specific identification of the NDV isolate, reverse transcriptase polymerase chain reaction (RT-PCR) using RNA extracted from virus-infected allantoic fluid was done by specific amplification of partial sequence of fusion protein gene using reported degenerate primers [15]. The virus isolate was designated as NDV/Peacock/2012/India. Pathogenicity testing The mean death time (MDT) and intra-cerebral pathogenicity index (ICPI) analysis were determined using 9-dayold SPF embryonated chicken eggs and day-old SPF chickens, respectively, following standard protocols [29]. Viral RNA extraction and RT-PCR Viral genomic RNA of NDV/Peacock/2012/India isolate was extracted from infected allantoic fluid using Trizol reagent (Invitrogen, USA) as per manufacturer’s guidelines. cDNA synthesis was performed using Thermoscript first strand synthesis kit (Invitrogen, USA). Genome sequencing The complete genome sequence of NDV/Peacock/2012/ India was sequenced by overlapping polymerase chain reaction strategy using reference primers available at the Recombinant DNA laboratory (primers available upon request) to cover the full-length genome of NDV/Peacock/ 2012/India. The PCR reaction was carried out in Mastercycler (Eppendorf, Germany) using DreamTaq DNA polymerase (Thermo Scientific, USA). The amplified product was purified using QIAquick gel extraction kit (Qiagen, Germany) as per manufacturer’s instructions. The overlapping PCR products were cloned in pTZ57R vector (Thermo Scientific, USA). Three positive clones for each overlapping fragments were sequenced in forward and reverse direction and compared with the sequence obtained from uncloned PCR products to achieve maximum
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consensus and to reduce nucleotide incorporation errors of Taq DNA polymerase. Sequence analysis The sequence editing and assembly were done using Serialcloner programme (version 1.3). The complete sequence of the NDV/Peacock/2012/India isolate was submitted to GenBank (Accession No. KJ769262). Complete genome sequences of 43 previously characterized AMPV-1 from the Asian subcontinent and other parts of world were compared to ascertain the phylogenetic relationship of NDV/Peacock/ 2012/India. The sequence alignment was done at Mobyle@Pasteur (http://mobyle.pasteur.fr) [30] using Muscle modules [31]. Muscle alignment was done for the F gene and concatenated full-length sequence of selected NDV strains. Bayesian approach was used to reconstruct the phylogenetic tree using aligned sequences with GTR ? C ? I evolutionary model as determined by JModeltest [32]. Bayesian analysis was carried out with BEAST software package [33]; for each dataset, three independent runs were conducted with Markov chain Monte Carlo (MCMC) chain length of forty million states sampled at every 2,000 generations using relaxed molecular clock and default parameters of prior model. Trees were summarized to construct the maximum clade credibility (MCC) tree after 10 % burn-in using programme TREEANNOTATOR. The resulting MCC tree was visualized and edited in FIGTREE software. Pairwise sequence comparison (PASC) was performed using aligned sequence dataset for coding sequence of each gene and concatenated genome of NDV. Mean nucleotide distances between the isolate and viral strains from each genotype were calculated using PASC in MEGA6 software [34].
Results Pathogenicity assessment NDV/Peacock/2012/India (Accession No. KJ769262) isolate exhibited a MDT of 63 h in embryonated chicken eggs, while the ICPI value in day-old chicken was 1.40. The isolated virus was found to be mesogenic by WOAH standard criteria. Whole genome sequence characteristics The genome length for NDV/Peacock/2012/India was found to be 15,186 nts and is similar to the NDV strains from ‘‘early’’ genotype described previously [8]. The genome organization for the isolate is in the order of 30 -NPP-M-F-HN-L-50 . The salient aspects of the viral genes such as gene length, length of ORF, along with deduced
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properties of respective proteins are summarized in Table 1. The sequence analysis of each gene start (GS) and gene end (GE) shows the presence of a consensus sequence characteristic of NDV. The pairwise comparison shows the concatenated genome of the virus bear very low mean divergence (6 %) with representative genotype II viruses than with any other genotypes ([50 % mean divergence) (Table 2). The full length F gene showed only 4.4 % mean divergence with genotype II strains while divergence ranged from 11 to 19.3 % with all other genotypes. The FPCS sequence motif has polybasic amino acid residues 112R-R-Q-K-R116 with phenylalanine (F) at residue 117 similar to that of mesogenic and velogenic strains of NDV. The unique substitution sites L69M and D82E characteristics of genotype II viruses were also observed in the deduced fusion protein sequence. The deduced length of HN protein of NDV/Peacock/ 2012/India was 577 amino acids, which is a common feature of genotype II viruses [24]. The HN protein also displayed 13 conserved amino acid residues (174,175,198, 236, 258, 299, 317, 401, 416, 498, 516, 526 and 527) critical for binding with sialic acid containing receptor that are also present and conserved in NDV/Peacock/2012/ India. The isolate was also found to possess K263N and E332G substitution in the neutralizing epitopes with all other amino acid positions important for antigenic properties remaining unchanged. Phylogenetic analysis The Bayesian approach comprises estimation of the posterior probabilities using specific evolutionary model. The model for a particular sequence dataset is determined based on AICs, BICs scores [35, 36]. Based on the lowest AICs score and BIC value, a discrete gamma distribution model with different evolutionary rates among the sites deduced by JModeltest programme was used for phylogenetic analysis of F and concatenated genome. The phylogenetic relationship of NDV/Peacock/2012/India with other members of AMPV1 was obtained by comparing the complete coding region of F gene and the concatenated genome. MCC tree for the concatenated genome based on 45 genomes scale data is depicted in Fig. 1a, b. Two viral strains from class I were used as out-group to root the tree. MCC tree showed that isolate NDV/Peacock/2012/India belongs to genotype II. The tree topology for concatenated and fusion gene phylogenetic trees remained similar (Fig. 1a, b).
Discussion An NDV isolate from a wild peacock was characterized in the present study. The virus was successfully recovered in
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Table 1 Genome and predicted characteristics of the encoding viral proteins of NDV/Peacock/2012/India isolate Genes
Hexamer phasing positiona Gene start
b
IGS
Total length
30 UTR
ORF
50 UTR
AcAAUAC
Gene end
UAbCGGG
mRNA characteristics
c
Protein characteristics Size (AA)
MW (kDa)
pI
1,746
66
1,470
210
2
489
53.2
P
AAUA CG
AAcUACG
1,451
83
1,188
180
1
395
41.9
6.68
P/V
AAUAbCG
AAcUACG
1,452
83
7,20
703
–
221
23.4
6.74
P/W
AAUAbCG
AAcUACG
10.11
NP
b
5.65
1,453
83
684
686
–
227
24.5
M
AAUA CG
AAcCACG
1,241
34
1,095
112
1
364
39.7
9.63
F
AACAbCG
AcCUACG
1,792
46
1,662
84
31
553
59.1
8.49
HN L
AUAbCGG AGGACAb
AAAAAAc AAAAAAc
2,002 6,703
91 11
1,734 6,615
177 77
47 –
577 2,204
63.4 24.8
8.06 6.97
b
a
Hexamer-phasing position is a unique property of Paramyxoviruses which have genome length in multiples of six. The ‘‘hexamer phasing’’ is where a stretch of six nucleotides on the viral genome gets associated with a single subunit of nucleoprotein (NP) for its efficient replication
b
and c denote gene start and gene end sites, respectively; 30 leader (55 nucleotides) and 50 trailer (114 nucleotides) regions are also to be included for the total length of the virus
Table 2 Pairwise sequence comparison of NDV/Peacock/2012/India with other genotypes of NDV Nucleotide sequence
Genotypes of NDV I
II
III
IV
V
VI
VII
9.4
8.3
13.8
14.9
14.1
12.1
14.5
P
11.7
9.9
12.7
16.7
19.1
18.2
16.5
M
9.8
2.8
12.7
12.5
17.8
17
16.3
F
11.1
4.4
13.1
13
17.8
19.3
17.1
HN
11.3
7.1
11.9
11.9
20.1
21
17.7
8.9
3.8
11.3
11.5
16
16
14.6
6
56.3
56.2
65.9
66.7
64.2
NP
L Concatenated genome
58.8
Values indicate mean percent divergence of the NDV/Peacock/2012/ India isolate for individual genes and concatenated genome with other NDV
allantoic fluid after passaging in SPF chicken eggs. The standard in vivo pathogenicity indices for the isolate was MDT of 63 h and ICPI of 1.40, which were found to be consistent with the range assigned for mesogenic virus strains namely MDT of 60–90 h and ICPI value of 1–1.5. Thus, the virus isolate was found to be a mesogenic strain from the results of in vivo pathogenicity assessment test. Since the virus has been isolated from a brain tissue, the virulence of the virus is in accordance with the MDT and ICPI test. Complete genome sequencing was done for the isolate, and the sequence was deposited in GenBank with accession no. KJ769262. The total length of the viral genome was found to be 15,186 nts similar to NDV strains from early genotypes (1930–1960). The genome sequencing revealed the presence of a polybasic amino acid motif 112R-R-Q-KR-F117 at FPCS, a primary molecular determinant of virulence. For lentogenic NDV strains, FPCS has the sequence 112G-R(K)-Q-G-R-L117, while the mesogenic and
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velogenic viruses have the sequence 112R-R-Q-K(R)-RF117. The MDT and ICPI results were consistent with the deduced sequence of FPCS region and placed the virus in intermediate pathotype of virus i.e. mesogenic strain. Traditionally, mesogenic virus strains have been associated with respiratory and nervous signs in infected birds but usually with low mortality. The death of peacock in this case may be due to the absence of neutralizing antibodies to NDV. The clinical signs, including torticollis, paralysis of wings before death and isolation of the virus from brain tissue, also correlated with the mesogenic nature of the virus. This is the first Indian report of complete genome sequencing and pathotypic characterization of a mesogenic NDV isolate from a wild dead peacock which is commonly observed near domestic cultivation and wetlands. Phylogenetic analysis of the viral isolate was done to interpret the historical relationships among different NDV strains based on the information contained in their genome sequence. These interpretations are conditional and are based on use of a particular statistical model, which attempts to describe relationships in given dataset by confidence, reliability or robustness [37]. In this report, we applied bayesian approach combined with stochastic estimation procedures such as MCMC (Markov chain Monte Carlo) which involves determination of parameter space in terms of topology, branch length and substitution rate based on prior probability, likelihood function and dataset [38]. The phylogeny was reconstructed using all six concatenated genes and for the fusion gene of NDV. Concatenated genome offers better resolution and lower statistical error than a single gene for larger sample size [39]. The phylogenetic analysis placed the isolate NDV/Peacock/2012/India within genotype II in class II of APMV type I with the highest posterior probability value. The NDV isolate was also placed as a different clade in
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Fig. 1 Maximum clade credibility (MCC) trees of NDV/Peacock/ 2012/India isolate with other known NDV strains. Analysis was conducted in BEAST software package with relaxed molecular clock and default parameters for prior model to obtain tree with maximum
posterior probability value at each node. The class I, type I Avian paramyxovirus strains were used as out-group to root the tree. MCC tree of (a) the concatenated full length sequence and (b) the complete open reading frame of the F gene of selected NDV strains
genotype II viruses (Fig. 1a, b) as evidenced by [4 % sequence divergence from other NDV strains in genotype II viruses (Table 2). This is consistent with the fact that genotype II viruses are reported to show inconsistent phylogenetic relationships while, genotypes III, IV, V, VI and VII were monophyletic in nature [40], and hence, the MCC tree for F gene and concatenated whole genome was evaluated, which showed a similar topological distribution. The genotype II viruses were known to be circulating throughout the globe since 1930s, which comprise lentogenic, mesogenic and virulent strains of NDV. Some of the well-known lentogenic and mesogenic vaccine strains are also classified under genotype II such as LaSota and Beaudette C. NDV is endemic in India, and genotype II viruses are found to be in constant circulation along with other virulent genotypes VI and VII [28]. Earlier, virulent NDV (genotype VI) was detected in a peafowl from the same geographical location around the same time frame when samples were assessed for this study [23]. Presence of more than two genotypes of viruses in an outbreak has been substantiated by earlier reports [41, 42], and this study helps to look into the contemporary virus strain that is circulating in the wild peacock population in Haryana state of North India.
The backyard poultry system and commercial poultry industry share approximately equal bird population in India [43]. Backyard poultry is characterized by small flocks with limited biosecurity measures, poor management and poor or an absent disease prevention programme [44]. As such, backyard poultry raising is a supplementary activity and contributes significantly to incomes and home food consumption in rural areas of many developing countries [45]. Limited biosecurity measures in backyard poultry and live bird market create opportunities for wild and domestic birds to intermingle at local wetlands and help in transmission of disease and emphasize the role of wild birds as one of the potential transmitters. Additionally, unrestricted movement, close proximity and sporadic encroachment of wild birds with backyard poultry have been seen as a hotspot for virus spread and disease transmission of avian viral diseases [46]. Recently, healthy backyard poultry [24] and birds living around poultry farms [47] were reported to carry virulent NDV strains. In earlier instances, NDV isolated from peacock in India [22, 23] involved characterization of the virus based on the FPCS region. The classification of NDV based on FPCS many times fails to give a clear picture of the virus pathotype and sometimes may be misleading [48]. Hence, a
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need for complete genome analysis is always beneficial as it is coupled with an ever decreasing cost of sequencing [39]. Most of the genotypes of NDV have been assigned with molecular signature for F gene in a recent report by Almeida [49] apart from genotype II. The genotype II NDV comprises many lentogenic and mesogenic vaccine strains including virulent strain like chicken/U.S.(TX)GB/1948, and hence, there is a need for complete genotypic and pathotypical characterizations of genotype II NDV isolates that may be derived from live/dead domestic, backyard or wild avian host to understand the evolutionary and epidemiological aspect of the disease. Genotype II strains like LaSota, B1 have been in use for routine vaccination of commercial poultry flocks. For this virus isolate, the HN gene receptor binding and antigenic recognition sites are conserved. Also, the presence of a polybasic FPCS sequence motif in the F gene coupled with the MDT and ICPI values being in the range of mesogenic category offers an attractive target for assessment of immune response in commercial chicken and evaluation of the virus for protection against virulent virus. Further characterizations regarding adaptation of this virus in cell line and thermo-stability assays are underway. Acknowledgments The authors wish to thank Dr. A. K. Sharma, Wildlife Centre, Indian Veterinary Research Institute with his help in collection of the sample. This research work was supported by grants from the National fund for basic and strategic research in agriculture of ICAR (NFBSFARA/BS-3010) and Department of Biotechnology, Government of India (DBT-JRF/2012-13/107 and BT/PR15373/ AAQ/57/116/2011).
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Genetic characterization and pathogenicity assessment of Newcastle disease virus isolated from wild peacock Sagar A. Khulape • Satish S. Gaikwad • Madhan Mohan Chellappa • Bishnu Prasad Mishra Sohini Dey
•
Received: 6 June 2014 / Accepted: 16 September 2014 / Published online: 27 September 2014 Ó Springer Science+Business Media New York 2014
Abstract The continued spread and occurrence of Newcastle disease virus (NDV) has posed potential threat to domestic poultry industry around the globe. Mainly, wild avian species has always been implicated for the natural reservoir for virus and spread of the disease. In the present study, we report the isolation of Newcastle disease virus (NDV/Peacock/India/2012) in necropsy brain tissue sample of wild peacock from North India. Complete genome of the virus was found to be 15,186 nucleotides (nts) with six genes in order of 30 -N–P-M-F-HN-L-50 , which was limited by 55-nts leader region at the 30 end and a 114-nts trailer sequence at 50 end. Sequence analysis of fusion protein revealed the dibasic amino acid cleavage site 112R-R-Q-KR-F117, a characteristic motif of virulent virus. Phylogenetic analysis placed the isolate in genotype II of Newcastle disease virus showing the lowest mean percent divergence (6 %) with other genotype II counterparts. The isolate was characterized as mesogenic (intermediate pathotype) based on the mean death time (63 h) in embryonated chicken eggs and the intra-cerebral pathogenicity index (1.40) in day-old chicks. The report emphasizes the dynamic ecology of NDV strains circulating in a wild avian host during the outbreak of 2012 in North India. Further the genotypic and pathotypical characterizations of the isolate could help in development of homologous vaccine against NDV strain circulating in avian population.
S. A. Khulape M. M. Chellappa B. P. Mishra S. Dey (&) Division of Veterinary Biotechnology, Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh, India e-mail: [email protected]; [email protected] S. S. Gaikwad Postdoctoral Fellow, OIE Reference Laboratory for Newcastle Disease, Anyang, Gyeonggi-do, South Korea
Keywords Avian paramyxovirus type I Newcastle disease virus Phylogenetic analysis
Introduction Newcastle disease (ND) is an acute infectious disease of birds causing major loss and devastation to poultry industry [1]. The causative agent, Newcastle disease virus (NDV), is a member of the genus Avulavirus in the family Paramyxoviridae. Of the nine different serotypes of avian paramyxoviruses (APMV), NDV belongs to APMV-1 and remains the most important pathogen for poultry [2]. NDV genome is a single-stranded RNA of approximately 15.2 kb nucleotides. The viral RNA encodes six structural proteins namely nucleoprotein (N), phosphoprotein (P), matrix (M), fusion (F), hemagglutinin-neuraminidase (HN), and RNA-directed large RNA polymerase (L) along with two non-structural proteins V and W [3]. The NDV strains are a diverse group of viruses in terms of antigenicity and genetic composition [4] and are classified as lentogenic, mesogenic and velogenic strains, respectively, based on its pathogenicity in chickens [5]. The contribution of viral protein for NDV virulence has been well documented for F, V, HN, NP, P and L proteins [6] but of these, F contributes important molecular determinant to NDV pathogenicity and is linked with the presence of critical amino acid motif at protease cleavage site of precursor F protein [7]. Genotype-based classification of NDV divide the viral strains into two distinct classes (I and II) based on genetic analysis of nucleotide sequence at the fusion protein cleavage site (FPCS). Class I strains of APMV-1 have been isolated mainly from wild birds and are generally avirulent, whereas class II strains have been recovered from wild and domestic birds and include
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virulent and avirulent isolates [8]. All class I viruses are 15,198 nucleotides (nts) in length, while class II viruses are further subdivided into genotypes I–IV, with 15,186 nts, and genotypes V–XI, with 15,192 nts [9]. Newcastle disease is a listed disease of the world organization for animal health (WOAH) affecting more than 230 species of birds with wide geographical distribution [10]. Wild birds have been implicated in the spread of infectious NDV to domestic bird population and are considered to be the natural reservoir of the infectious agent [4]. Although some NDV isolates from wild are found to be non-pathogenic to chickens, previous incidences like spread of virulent NDV strains from wild psittacine in the United States in the 1970s [11, 12] and cormorants in the 1990s [13, 14] warrant consistent monitoring and surveillance of wild birds for effective prevention and control programme. NDV is either endemic or a cause of regular epidemic in poultry throughout most parts of Africa, Asia, Central America and parts of South America despite the widespread vaccination programmes [3]. In India, NDV has been isolated from different avian hosts including chicken, pigeons, ducks, turkeys, guinea fowl and emus [16–20]. NDV has also been reported from recreational birds, migratory birds and captive wild birds [20, 21]. Although a variety of wild birds are susceptible to ND, the information available is limited due to the non-availability of clinical/necropsy samples and poor disease surveillance in wild avian population. Previously, NDV infection has been reported from a wild peacock under captivity [22] and more recently [23] in India. The recent outbreaks of virulent NDV in neighbouring countries, Pakistan [24–26] and Bangladesh [27], should be perceived as an epidemiological alert to re-assess the control and quarantine measures against ND. In the epidemiological context of India, different strains of NDV circulate in domestic bird population [28] that demands the characterization of NDV isolates from indigenous outbreak and necropsy examinations in rural free-range birds, commercial chickens and wild avian birds. Peacock, the national bird of India, comes under the family Phasianidae which also includes pheasants, partridges and jungle fowl. The natural habitats for peacock are streamside and cultivated areas. The species also frequents around human habitats and villages for scraps. The movement of this wild species in close proximity to domestic birds most of which are reared under backyard poultry farming always threatens to be the point of disease outbreak. In this study, NDV was isolated from a wild dead peacock’s postmortem sample and characterized genetically, pathotypically and biologically to understand its relationship with the previously established genotypes of the virus.
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Materials and methods Virus identification and isolation The necropsy sample comprising brain tissue from a dead wild peacock was received from the District Veterinary Polyclinic, Rewari of Haryana state India. The brain tissue was triturated in phosphate-buffered saline (pH 7.2). The virus isolation was done in 9-day-old specific-pathogenfree (SPF) embryonated chicken eggs (Venky’s India Pvt. Ltd., Pune, India) through allantoic cavity inoculation of the tissue suspension. A total of three blind passages were given, and hemagglutinating titres for each batch of allantoic fluid were determined. For specific identification of the NDV isolate, reverse transcriptase polymerase chain reaction (RT-PCR) using RNA extracted from virus-infected allantoic fluid was done by specific amplification of partial sequence of fusion protein gene using reported degenerate primers [15]. The virus isolate was designated as NDV/Peacock/2012/India. Pathogenicity testing The mean death time (MDT) and intra-cerebral pathogenicity index (ICPI) analysis were determined using 9-dayold SPF embryonated chicken eggs and day-old SPF chickens, respectively, following standard protocols [29]. Viral RNA extraction and RT-PCR Viral genomic RNA of NDV/Peacock/2012/India isolate was extracted from infected allantoic fluid using Trizol reagent (Invitrogen, USA) as per manufacturer’s guidelines. cDNA synthesis was performed using Thermoscript first strand synthesis kit (Invitrogen, USA). Genome sequencing The complete genome sequence of NDV/Peacock/2012/ India was sequenced by overlapping polymerase chain reaction strategy using reference primers available at the Recombinant DNA laboratory (primers available upon request) to cover the full-length genome of NDV/Peacock/ 2012/India. The PCR reaction was carried out in Mastercycler (Eppendorf, Germany) using DreamTaq DNA polymerase (Thermo Scientific, USA). The amplified product was purified using QIAquick gel extraction kit (Qiagen, Germany) as per manufacturer’s instructions. The overlapping PCR products were cloned in pTZ57R vector (Thermo Scientific, USA). Three positive clones for each overlapping fragments were sequenced in forward and reverse direction and compared with the sequence obtained from uncloned PCR products to achieve maximum
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consensus and to reduce nucleotide incorporation errors of Taq DNA polymerase. Sequence analysis The sequence editing and assembly were done using Serialcloner programme (version 1.3). The complete sequence of the NDV/Peacock/2012/India isolate was submitted to GenBank (Accession No. KJ769262). Complete genome sequences of 43 previously characterized AMPV-1 from the Asian subcontinent and other parts of world were compared to ascertain the phylogenetic relationship of NDV/Peacock/ 2012/India. The sequence alignment was done at Mobyle@Pasteur (http://mobyle.pasteur.fr) [30] using Muscle modules [31]. Muscle alignment was done for the F gene and concatenated full-length sequence of selected NDV strains. Bayesian approach was used to reconstruct the phylogenetic tree using aligned sequences with GTR ? C ? I evolutionary model as determined by JModeltest [32]. Bayesian analysis was carried out with BEAST software package [33]; for each dataset, three independent runs were conducted with Markov chain Monte Carlo (MCMC) chain length of forty million states sampled at every 2,000 generations using relaxed molecular clock and default parameters of prior model. Trees were summarized to construct the maximum clade credibility (MCC) tree after 10 % burn-in using programme TREEANNOTATOR. The resulting MCC tree was visualized and edited in FIGTREE software. Pairwise sequence comparison (PASC) was performed using aligned sequence dataset for coding sequence of each gene and concatenated genome of NDV. Mean nucleotide distances between the isolate and viral strains from each genotype were calculated using PASC in MEGA6 software [34].
Results Pathogenicity assessment NDV/Peacock/2012/India (Accession No. KJ769262) isolate exhibited a MDT of 63 h in embryonated chicken eggs, while the ICPI value in day-old chicken was 1.40. The isolated virus was found to be mesogenic by WOAH standard criteria. Whole genome sequence characteristics The genome length for NDV/Peacock/2012/India was found to be 15,186 nts and is similar to the NDV strains from ‘‘early’’ genotype described previously [8]. The genome organization for the isolate is in the order of 30 -NPP-M-F-HN-L-50 . The salient aspects of the viral genes such as gene length, length of ORF, along with deduced
451
properties of respective proteins are summarized in Table 1. The sequence analysis of each gene start (GS) and gene end (GE) shows the presence of a consensus sequence characteristic of NDV. The pairwise comparison shows the concatenated genome of the virus bear very low mean divergence (6 %) with representative genotype II viruses than with any other genotypes ([50 % mean divergence) (Table 2). The full length F gene showed only 4.4 % mean divergence with genotype II strains while divergence ranged from 11 to 19.3 % with all other genotypes. The FPCS sequence motif has polybasic amino acid residues 112R-R-Q-K-R116 with phenylalanine (F) at residue 117 similar to that of mesogenic and velogenic strains of NDV. The unique substitution sites L69M and D82E characteristics of genotype II viruses were also observed in the deduced fusion protein sequence. The deduced length of HN protein of NDV/Peacock/ 2012/India was 577 amino acids, which is a common feature of genotype II viruses [24]. The HN protein also displayed 13 conserved amino acid residues (174,175,198, 236, 258, 299, 317, 401, 416, 498, 516, 526 and 527) critical for binding with sialic acid containing receptor that are also present and conserved in NDV/Peacock/2012/ India. The isolate was also found to possess K263N and E332G substitution in the neutralizing epitopes with all other amino acid positions important for antigenic properties remaining unchanged. Phylogenetic analysis The Bayesian approach comprises estimation of the posterior probabilities using specific evolutionary model. The model for a particular sequence dataset is determined based on AICs, BICs scores [35, 36]. Based on the lowest AICs score and BIC value, a discrete gamma distribution model with different evolutionary rates among the sites deduced by JModeltest programme was used for phylogenetic analysis of F and concatenated genome. The phylogenetic relationship of NDV/Peacock/2012/India with other members of AMPV1 was obtained by comparing the complete coding region of F gene and the concatenated genome. MCC tree for the concatenated genome based on 45 genomes scale data is depicted in Fig. 1a, b. Two viral strains from class I were used as out-group to root the tree. MCC tree showed that isolate NDV/Peacock/2012/India belongs to genotype II. The tree topology for concatenated and fusion gene phylogenetic trees remained similar (Fig. 1a, b).
Discussion An NDV isolate from a wild peacock was characterized in the present study. The virus was successfully recovered in
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Table 1 Genome and predicted characteristics of the encoding viral proteins of NDV/Peacock/2012/India isolate Genes
Hexamer phasing positiona Gene start
b
IGS
Total length
30 UTR
ORF
50 UTR
AcAAUAC
Gene end
UAbCGGG
mRNA characteristics
c
Protein characteristics Size (AA)
MW (kDa)
pI
1,746
66
1,470
210
2
489
53.2
P
AAUA CG
AAcUACG
1,451
83
1,188
180
1
395
41.9
6.68
P/V
AAUAbCG
AAcUACG
1,452
83
7,20
703
–
221
23.4
6.74
P/W
AAUAbCG
AAcUACG
10.11
NP
b
5.65
1,453
83
684
686
–
227
24.5
M
AAUA CG
AAcCACG
1,241
34
1,095
112
1
364
39.7
9.63
F
AACAbCG
AcCUACG
1,792
46
1,662
84
31
553
59.1
8.49
HN L
AUAbCGG AGGACAb
AAAAAAc AAAAAAc
2,002 6,703
91 11
1,734 6,615
177 77
47 –
577 2,204
63.4 24.8
8.06 6.97
b
a
Hexamer-phasing position is a unique property of Paramyxoviruses which have genome length in multiples of six. The ‘‘hexamer phasing’’ is where a stretch of six nucleotides on the viral genome gets associated with a single subunit of nucleoprotein (NP) for its efficient replication
b
and c denote gene start and gene end sites, respectively; 30 leader (55 nucleotides) and 50 trailer (114 nucleotides) regions are also to be included for the total length of the virus
Table 2 Pairwise sequence comparison of NDV/Peacock/2012/India with other genotypes of NDV Nucleotide sequence
Genotypes of NDV I
II
III
IV
V
VI
VII
9.4
8.3
13.8
14.9
14.1
12.1
14.5
P
11.7
9.9
12.7
16.7
19.1
18.2
16.5
M
9.8
2.8
12.7
12.5
17.8
17
16.3
F
11.1
4.4
13.1
13
17.8
19.3
17.1
HN
11.3
7.1
11.9
11.9
20.1
21
17.7
8.9
3.8
11.3
11.5
16
16
14.6
6
56.3
56.2
65.9
66.7
64.2
NP
L Concatenated genome
58.8
Values indicate mean percent divergence of the NDV/Peacock/2012/ India isolate for individual genes and concatenated genome with other NDV
allantoic fluid after passaging in SPF chicken eggs. The standard in vivo pathogenicity indices for the isolate was MDT of 63 h and ICPI of 1.40, which were found to be consistent with the range assigned for mesogenic virus strains namely MDT of 60–90 h and ICPI value of 1–1.5. Thus, the virus isolate was found to be a mesogenic strain from the results of in vivo pathogenicity assessment test. Since the virus has been isolated from a brain tissue, the virulence of the virus is in accordance with the MDT and ICPI test. Complete genome sequencing was done for the isolate, and the sequence was deposited in GenBank with accession no. KJ769262. The total length of the viral genome was found to be 15,186 nts similar to NDV strains from early genotypes (1930–1960). The genome sequencing revealed the presence of a polybasic amino acid motif 112R-R-Q-KR-F117 at FPCS, a primary molecular determinant of virulence. For lentogenic NDV strains, FPCS has the sequence 112G-R(K)-Q-G-R-L117, while the mesogenic and
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velogenic viruses have the sequence 112R-R-Q-K(R)-RF117. The MDT and ICPI results were consistent with the deduced sequence of FPCS region and placed the virus in intermediate pathotype of virus i.e. mesogenic strain. Traditionally, mesogenic virus strains have been associated with respiratory and nervous signs in infected birds but usually with low mortality. The death of peacock in this case may be due to the absence of neutralizing antibodies to NDV. The clinical signs, including torticollis, paralysis of wings before death and isolation of the virus from brain tissue, also correlated with the mesogenic nature of the virus. This is the first Indian report of complete genome sequencing and pathotypic characterization of a mesogenic NDV isolate from a wild dead peacock which is commonly observed near domestic cultivation and wetlands. Phylogenetic analysis of the viral isolate was done to interpret the historical relationships among different NDV strains based on the information contained in their genome sequence. These interpretations are conditional and are based on use of a particular statistical model, which attempts to describe relationships in given dataset by confidence, reliability or robustness [37]. In this report, we applied bayesian approach combined with stochastic estimation procedures such as MCMC (Markov chain Monte Carlo) which involves determination of parameter space in terms of topology, branch length and substitution rate based on prior probability, likelihood function and dataset [38]. The phylogeny was reconstructed using all six concatenated genes and for the fusion gene of NDV. Concatenated genome offers better resolution and lower statistical error than a single gene for larger sample size [39]. The phylogenetic analysis placed the isolate NDV/Peacock/2012/India within genotype II in class II of APMV type I with the highest posterior probability value. The NDV isolate was also placed as a different clade in
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Fig. 1 Maximum clade credibility (MCC) trees of NDV/Peacock/ 2012/India isolate with other known NDV strains. Analysis was conducted in BEAST software package with relaxed molecular clock and default parameters for prior model to obtain tree with maximum
posterior probability value at each node. The class I, type I Avian paramyxovirus strains were used as out-group to root the tree. MCC tree of (a) the concatenated full length sequence and (b) the complete open reading frame of the F gene of selected NDV strains
genotype II viruses (Fig. 1a, b) as evidenced by [4 % sequence divergence from other NDV strains in genotype II viruses (Table 2). This is consistent with the fact that genotype II viruses are reported to show inconsistent phylogenetic relationships while, genotypes III, IV, V, VI and VII were monophyletic in nature [40], and hence, the MCC tree for F gene and concatenated whole genome was evaluated, which showed a similar topological distribution. The genotype II viruses were known to be circulating throughout the globe since 1930s, which comprise lentogenic, mesogenic and virulent strains of NDV. Some of the well-known lentogenic and mesogenic vaccine strains are also classified under genotype II such as LaSota and Beaudette C. NDV is endemic in India, and genotype II viruses are found to be in constant circulation along with other virulent genotypes VI and VII [28]. Earlier, virulent NDV (genotype VI) was detected in a peafowl from the same geographical location around the same time frame when samples were assessed for this study [23]. Presence of more than two genotypes of viruses in an outbreak has been substantiated by earlier reports [41, 42], and this study helps to look into the contemporary virus strain that is circulating in the wild peacock population in Haryana state of North India.
The backyard poultry system and commercial poultry industry share approximately equal bird population in India [43]. Backyard poultry is characterized by small flocks with limited biosecurity measures, poor management and poor or an absent disease prevention programme [44]. As such, backyard poultry raising is a supplementary activity and contributes significantly to incomes and home food consumption in rural areas of many developing countries [45]. Limited biosecurity measures in backyard poultry and live bird market create opportunities for wild and domestic birds to intermingle at local wetlands and help in transmission of disease and emphasize the role of wild birds as one of the potential transmitters. Additionally, unrestricted movement, close proximity and sporadic encroachment of wild birds with backyard poultry have been seen as a hotspot for virus spread and disease transmission of avian viral diseases [46]. Recently, healthy backyard poultry [24] and birds living around poultry farms [47] were reported to carry virulent NDV strains. In earlier instances, NDV isolated from peacock in India [22, 23] involved characterization of the virus based on the FPCS region. The classification of NDV based on FPCS many times fails to give a clear picture of the virus pathotype and sometimes may be misleading [48]. Hence, a
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need for complete genome analysis is always beneficial as it is coupled with an ever decreasing cost of sequencing [39]. Most of the genotypes of NDV have been assigned with molecular signature for F gene in a recent report by Almeida [49] apart from genotype II. The genotype II NDV comprises many lentogenic and mesogenic vaccine strains including virulent strain like chicken/U.S.(TX)GB/1948, and hence, there is a need for complete genotypic and pathotypical characterizations of genotype II NDV isolates that may be derived from live/dead domestic, backyard or wild avian host to understand the evolutionary and epidemiological aspect of the disease. Genotype II strains like LaSota, B1 have been in use for routine vaccination of commercial poultry flocks. For this virus isolate, the HN gene receptor binding and antigenic recognition sites are conserved. Also, the presence of a polybasic FPCS sequence motif in the F gene coupled with the MDT and ICPI values being in the range of mesogenic category offers an attractive target for assessment of immune response in commercial chicken and evaluation of the virus for protection against virulent virus. Further characterizations regarding adaptation of this virus in cell line and thermo-stability assays are underway. Acknowledgments The authors wish to thank Dr. A. K. Sharma, Wildlife Centre, Indian Veterinary Research Institute with his help in collection of the sample. This research work was supported by grants from the National fund for basic and strategic research in agriculture of ICAR (NFBSFARA/BS-3010) and Department of Biotechnology, Government of India (DBT-JRF/2012-13/107 and BT/PR15373/ AAQ/57/116/2011).
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