- Email: [email protected]
Molecular characterization of Newcastle disease virus strains isolated from different outbreaks in Northeast India during 2014–15
Accepted Manuscript Molecular characterization of Newcastle disease virus strains isolated from different outbreaks in Northeast India during 2014-15 Barnali Nath, Nagendra N. Barman, Sachin Kumar PII:
S0882-4010(15)30172-8
DOI:
10.1016/j.micpath.2015.11.026
Reference:
YMPAT 1726
To appear in:
Microbial Pathogenesis
Received Date: 28 October 2015 Revised Date:
23 November 2015
Accepted Date: 25 November 2015
Please cite this article as: Nath B, Barman NN, Kumar S, Molecular characterization of Newcastle disease virus strains isolated from different outbreaks in Northeast India during 2014-15, Microbial Pathogenesis (2015), doi: 10.1016/j.micpath.2015.11.026. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Molecular characterization of Newcastle disease virus strains isolated from different
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outbreaks in Northeast India during 2014-15.
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Barnali Nath1, Nagendra N. Barman2, Sachin Kumar1*
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Guwahati, Assam 781039, India.
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Agricultural University, Khanapara, Guwahati, Assam-781022, India.
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Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati,
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Department of Veterinary Microbiology, College of Veterinary Sciences, Assam
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* Corresponding author: Mailing address: Department of Biosciences and Bioengineering,
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Indian Institute of Technology Guwahati, Guwahati, Assam 781039, India.
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Phone: (91) 3612582229; Fax: (91) 3612582249
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E-mail: [email protected]
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ACCEPTED MANUSCRIPT Abstract:
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Newcastle disease virus (NDV) isolates recovered from different outbreaks in chicken flocks
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in Assam during 2014-15 were genotypically and pathotypically characterized. Nucleotide
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sequence analysis of fusion (F) and hemagglutinin protein genes showed a close similarity
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with genotype XIII strains of NDV. Amino acid sequence of F protein showed a virulent
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cleavage site
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embryonated chicken eggs showed a virulent pathotype of the isolated NDV strains. The
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study will help us to understand the biology of circulating strains of NDV in Northeastern
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part of India.
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R-R-Q-K-R-F117. Furthermore, pathogenicity test in one-day-old chicks and
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Keywords: Newcastle disease virus; Outbreak; Pathogenicity; Virulent. 2
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Short Communication: Newcastle disease (ND) is a highly infectious viral disease of domestic and wild avian
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species. ND was first reported from Java, in 1926 [1] and subsequently from the different
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parts of the world [2-8]. ND is endemic in many developing countries, including India. The
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Indian poultry industry suffers severe economic losses due to recurring episodes of ND
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outbreak in both vaccinated and unvaccinated flocks [9-12].
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ND is caused by Newcastle disease virus (NDV) which belongs to the genus
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Avulavirus under the family Paramyxoviridae [13]. The genome of NDV comprises of a non-
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segmented, negative-sense, single-stranded RNA. The genome size of NDV varies between
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15,186 to 15,198nucleotides [14-18]. The typical NDV genome consists of six essential genes
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encoding nucleocapsid (N), matrix protein (M), phosphoprotein (P), fusion protein (F),
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haemagglutinin-neuraminidase protein (HN), and large polymerase protein (L). The F protein
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is considered to play a vital role in the virulence of NDV strains [17, 19]. The F protein helps
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in the entry of the virus into the host cell by mediating fusion of the viral envelope with the
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plasma membrane [13]. The virulence of NDV depends upon the amino acid sequence at the
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cleavage site of the F protein [20, 21]. The consensus amino acid sequence of the F protein
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cleavage site of velogenic and mesogenic strains is 112R/K-R-Q-R/K-R↓F117; whereas, that of
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lentogenic strains is
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virion to sialic acid-containing cell surface receptors [22]. HN also has neuraminidase
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activity which cleaves sialic acid from sugar side chains and releases progeny virions from
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the surface of infected cells [23]. The HN protein determines the tropism as well as the
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virulence of NDV by interacting with the F protein for fusion promotion [24-26].
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G/E-K/R-Q-G/E-R↓L117. HN protein helps in the attachment of the
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NDV isolates can cause a wide range of clinical disease in chickens, ranging from
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asymptomatic to highly fatal. NDV isolates have been classified into three major pathotypes
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mesogenic (moderately virulent) and velogenic (highly virulent). Velogenic viruses are
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further divided into viscerotropic and neurotropic based on its predilection site in the intestine
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and central nervous system, respectively [27]. Both live and inactivated vaccines against ND
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are commercially available. Lentogenic NDV strains B1 and LaSota are efficiently used as
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live attenuated vaccines worldwide including India [27, 28]. Mesogenic NDV strains R2B,
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Mukteswar, Roakin and Komarov are used in countries where ND is endemic and where the
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risk of its outbreak is high.
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In developing countries, the conventional live vaccines against NDV are not effective
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due to inappropriate cold chain and inappropriate storage condition. The problem aggravates
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more for the poultry production in tropical countries [29]. Inactivated vaccines are not that
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effective and occasionally cause necrosis because of adjuvants present in the vaccine
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formulation [30]. Moreover, recent studies from different countries suggested that there has
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been a substantial antigenic shift in the NDV strains circulating in poultry population [31-35].
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The emergence of newer strains and failure of the vaccination are the major reasons of its
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outbreak in developing countries [29].
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Geographically, ND imposes a serious threat to the poultry industry and economy of
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the Northeast part of India because poultry farming is a major source of people’s livelihood.
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In addition, poultry products are integral parts of the diet for local inhabitants. Moreover, the
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Northeast India adjoins the countries like China, Bhutan, Myanmar and Bangladesh, where
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newer strains of NDV are regularly reported [36-40]. In the present study, we report the first
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comprehensive NDV outbreak from the Northeast part of India (Figure 1). The results of our
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study will be useful to understand the biology of circulating strains of NDV in India.
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Three different NDV outbreaks have been reported during the period of June 2014 to
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February 2015 from Northeast India. The details of the outbreak have been summarized in 4
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NDV specific antibody by hemagglutination inhibition (HI) assay and commercial enzyme-
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linked immunosorbent assay (ELISA) [IDEXX, USA]. Infected tissue samples such as brain,
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bursa, lungs, liver, kidney, spleen and intestine were collected from ailing and/or dead birds.
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Collected tissues were fixed in 10% neutral buffered formalin for approximately 48 hours.
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Formalin-fixed tissue samples were routinely processed for hematoxylin and eosin staining
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after paraffin embedding and sectioning (3µm) following standard procedure [41].
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Tissue samples containing viruses were inoculated in the allantoic cavities of 9-days-
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old specific pathogen free embryonated chicken eggs and infected allantoic fluids were
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collected 48 hours post inoculation. The presence of virus in collected allantoic fluids was
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confirmed by hemagglutination assay using 1% chicken RBC. Moreover, the isolated virus
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strains were plaque purified using chicken embryo fibroblasts (CEF) following standard
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protocol [42]. Briefly, CEF were infected with virus at a multiplicity of infection (MOI) of
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0.01 and incubated at 37°C in Dulbecco's Modified Eagle Medium (DMEM) with 5% fetal
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calf serum. The MOI of the NDV strains were established using the standard protocol [12].
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The viral genomic RNA was extracted from the homogenized tissue samples using
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TRIzol® Reagent (Invitrogen, Grand Island, NY, USA). Reverse transcription (RT) was
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performed following the manufacturer’s protocol using a SuperScriptTMIII RT enzyme
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(Invitrogen, Grand Island, NY, USA) by NDV gene specific forward primer (NDV 4163
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forward: 5´ AGC CTG CTA TCC YAT AGC AAA TGC 3'). The cDNA synthesised by the
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RT reaction was further amplified by PCR using NDV F and HN gene specific primer pairs
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(F 4544 forward: 5´ GCT GCT AGC ATG GGC TCC AGA CCT TC 3´ and F 6205 reverse:
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5´ CGT GGT ACC TCA CAT TTT TGT AGT GGC 3´; HN 6412 forward: 5´ CGG GGT
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ACC ATG GAC MGC GCM GTT AG 3´ and HN 8147 reverse: 5´ CGG GAT CCC TAR
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CCA GAC CTG GCT TCT C 3´ where M stands for A/C, R stands for A/G. The number in
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NDV). Degenerate consensus primers were designed using available GenBank sequences of
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different strains of NDV (GenBank accession numbers: NC_002617, FJ986192, AY562988,
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JF950510). The amplified 1735 and 1661 base pairs (bp) of F and HN genes, respectively
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were purified and cloned into pGEM®-T vector (Promega, Madison, WI, USA). The cloned
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gene products were sequenced by the Sanger sequencing method and analyzed by DNA Star
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software. The evolutionary relationship of isolated NDV strains was analyzed with available
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GenBank sequences using MEGA 5.2.2 software.
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The mean death time (MDT) and intracerebral pathogenicity index (ICPI) values of
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NDV/Chicken/Hajo/01/14, NDV/Chicken/Polashbari/01/14 and NDV/Chicken/Pandu/01/15
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suggest the velogenic nature of NDV strains (Table 1). All three isolates were named
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according to the place and year of isolation. The clinical signs observed among infected birds
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are summarized in table 1. Microscopically, the liver showed severe haemorrhages along with
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multiple areas of focal aggregation of mono-nuclear infiltrating cells along with focal areas of
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coagulative necrosis and heterophils in some areas (figure 2A). Spleen showed depletion of
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lymphocytes from its nodules and congestion of the blood vessels (figure 2B). In addition, the
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extensive proliferation of fibrous connective tissue was noticed in isolated areas around
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spleen nodules. Lung showed extensive haemorrhages and congestion of the blood vessels
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(figure 2C). Proventiculus showed mild haemorrhages with hypertrophy of proventicular
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gland (figure 2D).
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Circulating NDV strains have shown increased genetic and antigenic variations,
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along with prolonged existence in the avian hosts. The complete F and HN genes of NDV
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strains (GenBank accession number KT734765, KT734766 and KT734767) are 1662 and
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1716 nucleotides (nt), respectively. The amino acid sequence identities of F and HN proteins
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of isolated NDV strains were found distinct from the strains LaSota and R2B. It has been 6
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reported earlier that the virulence of NDV is determined by the cleavage site of its F protein
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[19]. The pathotype of isolated NDV strains is virulent with
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terminus of the F2 protein and F at residue 117. Interestingly, isolates Hajo and Polashbari
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showed the presence of arginine at position 111 preceding to F protein cleavage site, which
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has been shown earlier to be a novel velogenic pathotypes of NDV [43]. The importance of
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an extra arginine at this position requires further investigation by using reverse genetics
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approach in recombinant NDV. Nucleotide sequence analysis of F protein gene of strains
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Hajo, Polashbari and Pandu showed 84.1%, 84%, and 83.5% identity with strain LaSota,
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respectively. The HN protein was 571 amino acids long, a characteristic feature of virulent
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strains of NDV [12, 44]. Nucleotide sequence analysis of HN protein gene of strains Hajo,
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Polashbari and Pandu showed 81%, 81%, and 81.7% identity with strain LaSota,
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respectively. The sialic acid binding site 234NRKSCS237 present in the HN protein was found
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to be conserved in isolated strains of NDV [45]. Glycosylation sites present in both F and HN
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proteins of NDV strains showed complete identity with that of strain LaSota except at
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position 508. The glycosylation site at position 508 has not been implicated in the
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pathogenicity of NDV [46].
R-R-Q-K-R↓F117 at the C
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Our result suggests that the circulating strain of NDV in the Northeast India is distinct
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from the vaccine strains and showed identity with the existing genotype circulating in South
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Asian countries [47-49]. On phylogenetic analysis with 72 different sequences from
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GenBank, F gene of isolated NDV strains showed clustering with the strains of genotype XIII
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in class II (figure 3A). Similarly, HN gene of isolated NDV strains showed clustering with
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the strains of genotype XIII in class II (figure 3B). It has been observed that most of the
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circulating strains in India belong to genotype VII and XIII [10, 11, 50]. The conventional
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used NDV vaccine LaSota belongs to genotype II.
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results demonstrate that chickens infected with strains Hajo, Polashbari and Pandu showed
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signs of virulent NDV. The study will be useful to design the vaccine strategy and
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understanding the pathobiology of circulating strains of NDV in Northeast India. Northeast
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part of India is geographically isolated from the rest of the country. Besides, backyard poultry
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farming, mobility of poultry meat and their products through the live bird market are the main
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contributory factors in the endemic nature of NDV in Northeast India. Presumably, a program
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aimed at a permanent eradication or control of NDV at long term should include
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methodologies which allow not only an efficient approach in domestic poultry but also in free
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range birds.
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Acknowledgements:
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We would like to thank all our laboratory members for their excellent technical
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assistance and help. We are thankful to Dr Monika Koul for her valuable suggestions. The
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NDV research in our laboratory is currently supported by the department of biotechnology
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(NER-BPMC/2013/134/AAB21), the department of science and technology (IFA-LSBM-34),
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and the board of research on nuclear sciences (2012/20/37B/06/BRNS).
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Conflict of interest:
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The authors declare no conflict of interest.
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Reference:
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Figure legends:
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Figure 1. Geographical location of Northeast India where outbreaks occurred during 2014-15.
337 Figure 2. Histopathological examination of tissue samples isolated from liver (A), spleen (B),
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lung (c), and proventriculus (D). The liver showed severe haemorrhages along with multiple
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areas of heterophils aggregation. Spleen showed congestion of the blood vessels. Lung
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showed extensive haemorrhages and congestion of the blood vessels. Proventiculus showed
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mild haemorrhages and hypertrophy of proventicular gland. Lesions were marked with white
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arrow.
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Figure 3. Phylogenetic analysis of F and HN protein genes of isolated NDV strains. The F
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(A) and HN (B) protein genes were analyzed with 72 different NDV sequences available in
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GenBank spanning range of genotypes. Sequences were analyzed by maximum likelihood
348
method using Tamura-Nei Model. The percentage of replicate trees in which the associated
349
taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches.
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Evolutionary analysis was conducted by MEGA 5.2.2 software.
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Table 1. Details of the outbreaks of Newcastle disease virus in Northeast India.
Clinical signs observed
Vaccination status
2014
Hajo, Assam
Respiratory distress
Polashbari
2014
Polashbari, Assam
Sudden Death
Pandu
2015
Pandu, Assam
Respiratory distress, tracheal haemorrhage, caecal haemorrhage, lung congestion.
Vaccinated with strain LaSota Vaccinated with strain LaSota unvaccinated
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Hajo
F protein cleavage site 112
RRQKRF117
112
RRQKRF117
112
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% amino acid identity of F protein with strain LaSota 88.8
% amino acid identity of HN protein with strain LaSota 85.5
7
63
1.7
88.6
85.8
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58
1.7
88.1
87.8
7
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Location of outbreak
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Year of isolation
RRQKRF117
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Sample
HI Titer (log2)
MDT (hrs)
ICPI
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Highlights: Newcastle disease virus (NDV) isolates from Northeast India.
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Genotypic and pathotypic characterization of NDV isolates.
S0882-4010(15)30172-8
DOI:
10.1016/j.micpath.2015.11.026
Reference:
YMPAT 1726
To appear in:
Microbial Pathogenesis
Received Date: 28 October 2015 Revised Date:
23 November 2015
Accepted Date: 25 November 2015
Please cite this article as: Nath B, Barman NN, Kumar S, Molecular characterization of Newcastle disease virus strains isolated from different outbreaks in Northeast India during 2014-15, Microbial Pathogenesis (2015), doi: 10.1016/j.micpath.2015.11.026. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Molecular characterization of Newcastle disease virus strains isolated from different
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outbreaks in Northeast India during 2014-15.
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Barnali Nath1, Nagendra N. Barman2, Sachin Kumar1*
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Guwahati, Assam 781039, India.
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Agricultural University, Khanapara, Guwahati, Assam-781022, India.
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Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati,
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Department of Veterinary Microbiology, College of Veterinary Sciences, Assam
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* Corresponding author: Mailing address: Department of Biosciences and Bioengineering,
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Indian Institute of Technology Guwahati, Guwahati, Assam 781039, India.
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Phone: (91) 3612582229; Fax: (91) 3612582249
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E-mail: [email protected]
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Newcastle disease virus (NDV) isolates recovered from different outbreaks in chicken flocks
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in Assam during 2014-15 were genotypically and pathotypically characterized. Nucleotide
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sequence analysis of fusion (F) and hemagglutinin protein genes showed a close similarity
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with genotype XIII strains of NDV. Amino acid sequence of F protein showed a virulent
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cleavage site
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embryonated chicken eggs showed a virulent pathotype of the isolated NDV strains. The
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study will help us to understand the biology of circulating strains of NDV in Northeastern
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part of India.
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R-R-Q-K-R-F117. Furthermore, pathogenicity test in one-day-old chicks and
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Keywords: Newcastle disease virus; Outbreak; Pathogenicity; Virulent. 2
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Short Communication: Newcastle disease (ND) is a highly infectious viral disease of domestic and wild avian
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species. ND was first reported from Java, in 1926 [1] and subsequently from the different
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parts of the world [2-8]. ND is endemic in many developing countries, including India. The
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Indian poultry industry suffers severe economic losses due to recurring episodes of ND
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outbreak in both vaccinated and unvaccinated flocks [9-12].
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ND is caused by Newcastle disease virus (NDV) which belongs to the genus
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Avulavirus under the family Paramyxoviridae [13]. The genome of NDV comprises of a non-
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segmented, negative-sense, single-stranded RNA. The genome size of NDV varies between
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15,186 to 15,198nucleotides [14-18]. The typical NDV genome consists of six essential genes
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encoding nucleocapsid (N), matrix protein (M), phosphoprotein (P), fusion protein (F),
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haemagglutinin-neuraminidase protein (HN), and large polymerase protein (L). The F protein
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is considered to play a vital role in the virulence of NDV strains [17, 19]. The F protein helps
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in the entry of the virus into the host cell by mediating fusion of the viral envelope with the
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plasma membrane [13]. The virulence of NDV depends upon the amino acid sequence at the
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cleavage site of the F protein [20, 21]. The consensus amino acid sequence of the F protein
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cleavage site of velogenic and mesogenic strains is 112R/K-R-Q-R/K-R↓F117; whereas, that of
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lentogenic strains is
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virion to sialic acid-containing cell surface receptors [22]. HN also has neuraminidase
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activity which cleaves sialic acid from sugar side chains and releases progeny virions from
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the surface of infected cells [23]. The HN protein determines the tropism as well as the
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virulence of NDV by interacting with the F protein for fusion promotion [24-26].
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G/E-K/R-Q-G/E-R↓L117. HN protein helps in the attachment of the
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NDV isolates can cause a wide range of clinical disease in chickens, ranging from
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asymptomatic to highly fatal. NDV isolates have been classified into three major pathotypes
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mesogenic (moderately virulent) and velogenic (highly virulent). Velogenic viruses are
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further divided into viscerotropic and neurotropic based on its predilection site in the intestine
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and central nervous system, respectively [27]. Both live and inactivated vaccines against ND
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are commercially available. Lentogenic NDV strains B1 and LaSota are efficiently used as
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live attenuated vaccines worldwide including India [27, 28]. Mesogenic NDV strains R2B,
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Mukteswar, Roakin and Komarov are used in countries where ND is endemic and where the
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risk of its outbreak is high.
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In developing countries, the conventional live vaccines against NDV are not effective
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due to inappropriate cold chain and inappropriate storage condition. The problem aggravates
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more for the poultry production in tropical countries [29]. Inactivated vaccines are not that
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effective and occasionally cause necrosis because of adjuvants present in the vaccine
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formulation [30]. Moreover, recent studies from different countries suggested that there has
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been a substantial antigenic shift in the NDV strains circulating in poultry population [31-35].
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The emergence of newer strains and failure of the vaccination are the major reasons of its
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outbreak in developing countries [29].
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Geographically, ND imposes a serious threat to the poultry industry and economy of
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the Northeast part of India because poultry farming is a major source of people’s livelihood.
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In addition, poultry products are integral parts of the diet for local inhabitants. Moreover, the
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Northeast India adjoins the countries like China, Bhutan, Myanmar and Bangladesh, where
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newer strains of NDV are regularly reported [36-40]. In the present study, we report the first
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comprehensive NDV outbreak from the Northeast part of India (Figure 1). The results of our
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study will be useful to understand the biology of circulating strains of NDV in India.
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Three different NDV outbreaks have been reported during the period of June 2014 to
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February 2015 from Northeast India. The details of the outbreak have been summarized in 4
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NDV specific antibody by hemagglutination inhibition (HI) assay and commercial enzyme-
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linked immunosorbent assay (ELISA) [IDEXX, USA]. Infected tissue samples such as brain,
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bursa, lungs, liver, kidney, spleen and intestine were collected from ailing and/or dead birds.
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Collected tissues were fixed in 10% neutral buffered formalin for approximately 48 hours.
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Formalin-fixed tissue samples were routinely processed for hematoxylin and eosin staining
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after paraffin embedding and sectioning (3µm) following standard procedure [41].
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Tissue samples containing viruses were inoculated in the allantoic cavities of 9-days-
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old specific pathogen free embryonated chicken eggs and infected allantoic fluids were
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collected 48 hours post inoculation. The presence of virus in collected allantoic fluids was
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confirmed by hemagglutination assay using 1% chicken RBC. Moreover, the isolated virus
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strains were plaque purified using chicken embryo fibroblasts (CEF) following standard
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protocol [42]. Briefly, CEF were infected with virus at a multiplicity of infection (MOI) of
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0.01 and incubated at 37°C in Dulbecco's Modified Eagle Medium (DMEM) with 5% fetal
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calf serum. The MOI of the NDV strains were established using the standard protocol [12].
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The viral genomic RNA was extracted from the homogenized tissue samples using
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TRIzol® Reagent (Invitrogen, Grand Island, NY, USA). Reverse transcription (RT) was
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performed following the manufacturer’s protocol using a SuperScriptTMIII RT enzyme
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(Invitrogen, Grand Island, NY, USA) by NDV gene specific forward primer (NDV 4163
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forward: 5´ AGC CTG CTA TCC YAT AGC AAA TGC 3'). The cDNA synthesised by the
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RT reaction was further amplified by PCR using NDV F and HN gene specific primer pairs
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(F 4544 forward: 5´ GCT GCT AGC ATG GGC TCC AGA CCT TC 3´ and F 6205 reverse:
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5´ CGT GGT ACC TCA CAT TTT TGT AGT GGC 3´; HN 6412 forward: 5´ CGG GGT
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ACC ATG GAC MGC GCM GTT AG 3´ and HN 8147 reverse: 5´ CGG GAT CCC TAR
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CCA GAC CTG GCT TCT C 3´ where M stands for A/C, R stands for A/G. The number in
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NDV). Degenerate consensus primers were designed using available GenBank sequences of
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different strains of NDV (GenBank accession numbers: NC_002617, FJ986192, AY562988,
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JF950510). The amplified 1735 and 1661 base pairs (bp) of F and HN genes, respectively
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were purified and cloned into pGEM®-T vector (Promega, Madison, WI, USA). The cloned
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gene products were sequenced by the Sanger sequencing method and analyzed by DNA Star
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software. The evolutionary relationship of isolated NDV strains was analyzed with available
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GenBank sequences using MEGA 5.2.2 software.
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The mean death time (MDT) and intracerebral pathogenicity index (ICPI) values of
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NDV/Chicken/Hajo/01/14, NDV/Chicken/Polashbari/01/14 and NDV/Chicken/Pandu/01/15
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suggest the velogenic nature of NDV strains (Table 1). All three isolates were named
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according to the place and year of isolation. The clinical signs observed among infected birds
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are summarized in table 1. Microscopically, the liver showed severe haemorrhages along with
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multiple areas of focal aggregation of mono-nuclear infiltrating cells along with focal areas of
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coagulative necrosis and heterophils in some areas (figure 2A). Spleen showed depletion of
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lymphocytes from its nodules and congestion of the blood vessels (figure 2B). In addition, the
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extensive proliferation of fibrous connective tissue was noticed in isolated areas around
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spleen nodules. Lung showed extensive haemorrhages and congestion of the blood vessels
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(figure 2C). Proventiculus showed mild haemorrhages with hypertrophy of proventicular
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gland (figure 2D).
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Circulating NDV strains have shown increased genetic and antigenic variations,
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along with prolonged existence in the avian hosts. The complete F and HN genes of NDV
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strains (GenBank accession number KT734765, KT734766 and KT734767) are 1662 and
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1716 nucleotides (nt), respectively. The amino acid sequence identities of F and HN proteins
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of isolated NDV strains were found distinct from the strains LaSota and R2B. It has been 6
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reported earlier that the virulence of NDV is determined by the cleavage site of its F protein
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[19]. The pathotype of isolated NDV strains is virulent with
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terminus of the F2 protein and F at residue 117. Interestingly, isolates Hajo and Polashbari
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showed the presence of arginine at position 111 preceding to F protein cleavage site, which
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has been shown earlier to be a novel velogenic pathotypes of NDV [43]. The importance of
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an extra arginine at this position requires further investigation by using reverse genetics
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approach in recombinant NDV. Nucleotide sequence analysis of F protein gene of strains
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Hajo, Polashbari and Pandu showed 84.1%, 84%, and 83.5% identity with strain LaSota,
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respectively. The HN protein was 571 amino acids long, a characteristic feature of virulent
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strains of NDV [12, 44]. Nucleotide sequence analysis of HN protein gene of strains Hajo,
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Polashbari and Pandu showed 81%, 81%, and 81.7% identity with strain LaSota,
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respectively. The sialic acid binding site 234NRKSCS237 present in the HN protein was found
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to be conserved in isolated strains of NDV [45]. Glycosylation sites present in both F and HN
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proteins of NDV strains showed complete identity with that of strain LaSota except at
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position 508. The glycosylation site at position 508 has not been implicated in the
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pathogenicity of NDV [46].
R-R-Q-K-R↓F117 at the C
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Our result suggests that the circulating strain of NDV in the Northeast India is distinct
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from the vaccine strains and showed identity with the existing genotype circulating in South
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Asian countries [47-49]. On phylogenetic analysis with 72 different sequences from
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GenBank, F gene of isolated NDV strains showed clustering with the strains of genotype XIII
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in class II (figure 3A). Similarly, HN gene of isolated NDV strains showed clustering with
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the strains of genotype XIII in class II (figure 3B). It has been observed that most of the
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circulating strains in India belong to genotype VII and XIII [10, 11, 50]. The conventional
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used NDV vaccine LaSota belongs to genotype II.
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results demonstrate that chickens infected with strains Hajo, Polashbari and Pandu showed
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signs of virulent NDV. The study will be useful to design the vaccine strategy and
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understanding the pathobiology of circulating strains of NDV in Northeast India. Northeast
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part of India is geographically isolated from the rest of the country. Besides, backyard poultry
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farming, mobility of poultry meat and their products through the live bird market are the main
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contributory factors in the endemic nature of NDV in Northeast India. Presumably, a program
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aimed at a permanent eradication or control of NDV at long term should include
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methodologies which allow not only an efficient approach in domestic poultry but also in free
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range birds.
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Acknowledgements:
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We would like to thank all our laboratory members for their excellent technical
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assistance and help. We are thankful to Dr Monika Koul for her valuable suggestions. The
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NDV research in our laboratory is currently supported by the department of biotechnology
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(NER-BPMC/2013/134/AAB21), the department of science and technology (IFA-LSBM-34),
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and the board of research on nuclear sciences (2012/20/37B/06/BRNS).
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Conflict of interest:
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The authors declare no conflict of interest.
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Reference:
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Figure legends:
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Figure 1. Geographical location of Northeast India where outbreaks occurred during 2014-15.
337 Figure 2. Histopathological examination of tissue samples isolated from liver (A), spleen (B),
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lung (c), and proventriculus (D). The liver showed severe haemorrhages along with multiple
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areas of heterophils aggregation. Spleen showed congestion of the blood vessels. Lung
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showed extensive haemorrhages and congestion of the blood vessels. Proventiculus showed
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mild haemorrhages and hypertrophy of proventicular gland. Lesions were marked with white
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arrow.
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Figure 3. Phylogenetic analysis of F and HN protein genes of isolated NDV strains. The F
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(A) and HN (B) protein genes were analyzed with 72 different NDV sequences available in
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GenBank spanning range of genotypes. Sequences were analyzed by maximum likelihood
348
method using Tamura-Nei Model. The percentage of replicate trees in which the associated
349
taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches.
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Evolutionary analysis was conducted by MEGA 5.2.2 software.
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Table 1. Details of the outbreaks of Newcastle disease virus in Northeast India.
Clinical signs observed
Vaccination status
2014
Hajo, Assam
Respiratory distress
Polashbari
2014
Polashbari, Assam
Sudden Death
Pandu
2015
Pandu, Assam
Respiratory distress, tracheal haemorrhage, caecal haemorrhage, lung congestion.
Vaccinated with strain LaSota Vaccinated with strain LaSota unvaccinated
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Hajo
F protein cleavage site 112
RRQKRF117
112
RRQKRF117
112
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% amino acid identity of F protein with strain LaSota 88.8
% amino acid identity of HN protein with strain LaSota 85.5
7
63
1.7
88.6
85.8
6
58
1.7
88.1
87.8
7
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Location of outbreak
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Year of isolation
RRQKRF117
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Sample
HI Titer (log2)
MDT (hrs)
ICPI
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Highlights: Newcastle disease virus (NDV) isolates from Northeast India.
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Genotypic and pathotypic characterization of NDV isolates.