Isolation of novel variants of infectious bursal disease virus from different outbreaks in Northeast India

Accepted Manuscript Isolation of novel variants of infectious bursal disease virus from different outbreaks in Northeast India Sudhir Morla, Pankaj Deka, Sachin Kumar PII:

S0882-4010(16)30035-3

DOI:

10.1016/j.micpath.2016.02.004

Reference:

YMPAT 1776

To appear in:

Microbial Pathogenesis

Received Date: 17 January 2016 Revised Date:

3 February 2016

Accepted Date: 3 February 2016

Please cite this article as: Morla S, Deka P, Kumar S, Isolation of novel variants of infectious bursal disease virus from different outbreaks in Northeast India, Microbial Pathogenesis (2016), doi: 10.1016/ j.micpath.2016.02.004. 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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Isolation of novel variants of infectious bursal disease virus from different outbreaks in

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Northeast India.

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Sudhir Morla1, Pankaj Deka2, Sachin Kumar1*

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Guwahati, Assam 781039, India.

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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 Agricultural

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* Corresponding author: Mailing address: Department of Biosciences and Bioengineering, Indian

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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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Key word: Infectious bursal disease virus; Outbreak; Pathogenicity; Virulent.

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Abstract: Infectious bursal disease virus (IBDV) is a highly infectious disease of young chicken

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that predominantly affects the immune system. In the present study, we are reporting first

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comprehensive study of IBDV outbreaks from the Northeastern part of India. Northeast India

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shares a porous border with four different countries; and as a rule any outbreak in the

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neighboring countries substantially affects the poultry population in the adjoining states.

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Nucleotide sequence analysis of the VP2 gene of the IBDV isolates from the Northeastern part of

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India suggested the extreme virulent nature of the virus. The virulent marker amino acids (A222,

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I242, Q253, I256 and S299) in the hypervariable region of the Northeastern isolates were found

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identical with the reported very virulent strains of IBDV. A unique insertion of I/L294V was

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recorded in all the isolates of the Northeastern India. The study will be useful in understanding

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the circulating pathotypes of IBDV in India.

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Short communication: Infectious bursal disease virus (IBDV) is the causative agent of a highly contagious

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disease of young chicken. IBDV replicates in developing B lymphocytes in the bursa of

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Fabricius leading to its destruction and bursal inflammation [1]. The IBDV infection can cause

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immunosuppression and decrease in vaccine response, making chicken susceptible to secondary

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bacterial infection [2, 3]. Variant and classical forms are the two subtypes of IBDV [4]. The

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classical subtypes have been divided into three pathotypes: attenuated, virulent and very virulent

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(vv). Two distinct serotypes of IBDV are reported till date. Serotype I contains virulent viruses

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while serotype 2 are avirulent to chicken [5]. Both the serotypes are reported from domestic and

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wild birds. IBDV infection in chickens is mostly asymptomatic but the disease is more

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pronounced in birds of age group of three weeks [6, 7]. Moreover, the maternal antibody

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response has limited role in protecting the birds against very virulent IBDV (vvIBDV) infection

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[4].

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IBDV belongs to the genus Avibirnavirus under the family Birnaviridae. The genome of

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IBDV consists of dsRNA, which is divided into segments A and B [8]. The segment A consists

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of two partially overlapping open reading frames (ORF), the first encodes for VP5 (17kd) which

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plays a role in viral release while the other encodes for polyprotein (VP243) [9]. The polyprotein

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VP243 later self cleaved by viral protease VP4 into VP2 (48kDa), VP3 (33-35kDa) and VP4

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(24kDa) [3]. The segment B encodes for VP1 (90kDa), an RNA-dependent RNA polymerase

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[10]. The VP2 and VP3 form the viral capsid, out of which VP2 is the major contributor of viral

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neutralizing antibodies and elicits a protective immune response in the host [11]. IBDV has a

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hyper variable region (HVR) between 206 to 350 amino acid of VP2 protein. This region is

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known as the major region of IBDV to elucidate neutralizing antibodies response [12]. Due to its

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antigenicity, HVR region of IBDV strains are used for its characterization. The modulation of

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amino acid sequence in the HVR region of IBDV leads to emergence of its pathogenic variant

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that can evade the host immune response [13, 14].

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Low virulent, intermediate plus strains are used as vaccines against IBDV. The low

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virulent strains may sometime cause bursal inflammation because of the reversion to the virulent

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pathotype. In addition, the low virulent strains failed to protect birds against vvIBDV infections

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[15, 16]. In India, IBDV was first reported in 1971, after which a series of outbreaks were

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reported from different parts of India [17-20]. Currently, IBDV is endemic and a serious problem

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for the poultry industry in India. Northeastern part of India shares a porous border with China,

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Bangladesh, Bhutan and Myanmar. The new strains of IBDV are regularly reported from these

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parts of the world [21-23].

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Four different IBDV outbreaks have been reported during 2014-15 in the Northeastern

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part of India (figure 1). The details of the outbreaks are summarized in Table 1. The outbreaks

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were recorded mainly from broiler chicken of age group 2-3 weeks. Bursal samples were

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collected both from ailing and dead birds. The tissue samples were fixed with 10% formalin and

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sectioned 4µm thick for its staining with hematoxylin and eosin using standard procedure [24].

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Total RNA was isolated from the atrophied bursal tissue using TRIzol® reagent (Invitrogen,

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Grand Island, NY, USA). cDNA was synthesized using PrimeScript™ RT reagent (Clontech

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Laboratories,

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AACCTGCAAGATC3´). The cDNA was further used for the amplification of VP2 gene of

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IBDV by gene specific forward and reverse primers (VP2F 5´CTAGCTAGCATGACA-

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AACCTGCAAGATC3´ and

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using Phusion® high-fidelity DNA polymerase (New England Biolabs, USA). The PCR

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CA,

USA)

using

VP2R

IBDV

specific

primer

(5´CTAGCTAGCATGACA-

5’GGGAATTCTTACCTTAGGGCCCGGATTATG3´)

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products were purified and sequenced by BigDye terminator v 3.1 kit (Applied Biosystems,

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USA). The sequence data was analyzed using SeqMan software from DNASTAR 5.0 version

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(DNASTAR Inc., Madison, WI). The nucleotide sequences of all the isolates were submitted to

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GenBank. Phylogenic analysis was done based on the comparison of the HVR region of the VP2

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gene of the isolates with the reference strains from GenBank [25-27]. The sequences were

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aligned and assembled by MEGA6 software using the neighbour-joining method [28].

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The reported outbreak was confirmed positive for IBDV by the ELISA (IDEXX, USA).

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The serum collected from the ailing birds showed a positive titer with the commercial ELISA.

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The tissue samples collected from the ailing and dead birds showed hemorrhagic lesion in the

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bursa

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polymorphonuclear cells in the bursal follicles (figure 2). The viral RNA isolated from the tissue

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samples showed expected amplification of VP2 gene having a size of 1300 bp encoding its

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complete ORF. The genome sequence analysis of amplified PCR product showed identity with

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the reported IBDV sequence and submitted to GenBank (Accession numbers KU530208,

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KU530209, KU530210, and KU530211). All the IBDV isolates showed close identity with the

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strains circulating in the Bangladesh, China and India. The Northeastern IBDV isolates from

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India showed maximum identity with the BD 3/99 strain from Bangladesh (GenBank accession

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number AF362776). The Northeastern isolates of IBDV showed 96-99% and 95-97% VP2 gene

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nucleotide and amino acid identity, respectively with the BD3/99 strain from Bangladesh. The

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amino acid sequence of the Northeastern isolates of the IBDV VP2 protein showed high identity

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with the very virulent pathotype with all marker amino acid moieties (Table 2). Interestingly, the

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Northeastern isolates of IBDV showed insertion of a unique valine residue at position 294

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(I/L294V). Surprisingly, insertion of L240F which was previously reported in less virulent IBDV

Fabricius.

The

histopathological

examination

showed

the

infiltration

of

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23/82 (GenBank accession number AF362773) belonging to serotype 2 was found intact in the

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Northeastern isolates of IBDV. The absence of amino acid at positions H253 and T284 suggested

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that the IBDV isolates were not originated from the attenuated vaccine strains [12, 29]. The

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Northeastern isolates of IBDV were aligned and compared with the amino sequence of reference

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OKYM vvIBDV strain (figure 3). The maximum variation of amino acids was observed in the

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strains

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IBDV/Chicken/Kamrup/51/2015 (P203S, L240F, T286I, E300A). The Northeastern isolates

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IBDV/Chicken/Kamrup/54/2015 and IBDV/Chicken/Jorhat/-PD4/2014 showed difference of two

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amino acids L240F and E300A from the reference strain OKYM. The serine rich heptapeptide

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(326SWSASGS332), a hallmark of vvIBDV was found intact, and no substitutions were recorded

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in the Northeastern isolates of IBDV [30]. On phylogenetic analysis, all the four isolates

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clustered together with the reported vvIBDV strains (figure 4).

(T209I,

A236S,

S239K,

E300P)

and

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IBDV/Chicken/Jorhat/PD5/2014

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In spite of routine vaccination, regular outbreaks have been reported frequently from

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different parts of the world due to the antigenic variations in the IBDV genome. Here we are

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reporting the first IBDV outbreak from the Northeastern part of India. The porous border and

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unique topography of Northeastern part of India has a profound effect on the disease outbreak

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[31]. It has been shown that the subtle difference in the HVR region leads to immune evasion

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and massive outbreaks of IBDV in field conditions [32, 33]. The HVR region of the VP2 gene of

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Northeastern isolates of IBDV showed antigenic variations, suggesting the emergence of IBDV

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strains that can evade the host immune response. Various conserved markers of vvIBDV such as

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Q235, hydrophilic regions, and serine rich heptapeptide in the Northeastern isolates suggested

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the very virulent nature of the isolates. In addition to all other identities with the vvIBDV, the

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presence of amino acid A300 of VP2 protein was observed in the Northeastern isolates similar to

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BD 3/99 Bangladesh strain suggesting the cross boarder circulation of the IBDV strains. The

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molecular significance of valine at position 294 in the VP2 gene of Northeastern isolates of

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IBDV requires further investigation by reverse genetics approach. The clustering of Northeastern

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isolates of IBDV with its reference south Asian and Chinese strains suggested a common pool of

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the circulating viruses in this part of the world. The role of migratory birds in the circulation of

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many viral diseases is well documented [34, 35]. Similar possibility of the IBDV outbreaks in

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the Northeastern part of Indian cannot be ruled out.

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The poultry is the major protein source largely consumed in different parts of India. The

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improper vaccination, lack of cold chain maintenance and faulty management practices are the

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major cause of vaccine failure in India. The current epidemiology and serosurveillance of IBDV

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from India has not been documented, making its control strategy a daunting task. Our

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understanding of IBDV pathogenicity and its antigenic variants has increased exponentially

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because of the availability of advance molecular biology tools. However, it will be interesting to

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analyze the complete genome sequence of more and more strains of IBDV from India in order to

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understand the molecular markers of its circulating strains.

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Conflicts of Interest:

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The authors declare no conflict of interest.

Acknowledgement:

The IBDV research in our laboratory is currently supported by the Department of Biotechnology, India (NER-BPMC/2013/134/AAB21).

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[1] Rautenschlein S, Yeh HY, Sharma JM. Comparative immunopathogenesis of mild, intermediate, and virulent strains of classic infectious bursal disease virus. Avian Dis. 2003;47:66-78. [2] Lukert PD, Leonard J, Davis RB. Infectious bursal disease virus: antigen production and immunity. Am J Vet Res. 1975;36:539-40. [3] Kibenge FS, Dhillon AS, Russell RG. Biochemistry and immunology of infectious bursal disease virus. J Gen Virol. 1988;69 ( Pt 8):1757-75. [4] van den Berg TP, Morales D, Eterradossi N, Rivallan G, Toquin D, Raue R, et al. Assessment of genetic, antigenic and pathotypic criteria for the characterization of IBDV strains. Avian Pathol. 2004;33:470-6. [5] McFerran JB, McNulty MS, McKillop ER, Connor TJ, McCracken RM, Collins DS, et al. Isolation and serological studies with infectious bursal disease viruses from fowl, turkeys and ducks: demonstration of a second serotype. Avian Pathol. 1980;9:395-404. [6] Chettle N, Stuart JC, Wyeth PJ. Outbreak of virulent infectious bursal disease in East Anglia. Vet Rec. 1989;125:271-2. [7] Hirai K, Shimakura S, Kawamoto E, Taguchi F, Kim ST, Chang CN, et al. The immunodepressive effect of infectious bursal disease virus in chickens. Avian Dis. 1974;18:50-7. [8] Nagarajan MM, Kibenge FS. Infectious bursal disease virus: a review of molecular basis for variations in antigenicity and virulence. Can J Vet Res. 1997;61:81-8. [9] Wu Y, Hong L, Ye J, Huang Z, Zhou J. The VP5 protein of infectious bursal disease virus promotes virion release from infected cells and is not involved in cell death. Arch Virol. 2009;154:1873-82. [10] von Einem UI, Gorbalenya AE, Schirrmeier H, Behrens SE, Letzel T, Mundt E. VP1 of infectious bursal disease virus is an RNA-dependent RNA polymerase. J Gen Virol. 2004;85:2221-9. [11] Muller H, Schnitzler D, Bernstein F, Becht H, Cornelissen D, Lutticken DH. Infectious bursal disease of poultry: antigenic structure of the virus and control. Vet Microbiol. 1992;33:175-83. [12] Qi X, Gao H, Gao Y, Qin L, Wang Y, Gao L, et al. Naturally occurring mutations at residues 253 and 284 in VP2 contribute to the cell tropism and virulence of very virulent infectious bursal disease virus. Antiviral Res. 2009;84:225-33. [13] Durairaj V, Sellers HS, Linnemann EG, Icard AH, Mundt E. Investigation of the antigenic evolution of field isolates using the reverse genetics system of infectious bursal disease virus (IBDV). Arch Virol. 2011;156:1717-28. [14] Coulibaly F, Chevalier C, Gutsche I, Pous J, Navaza J, Bressanelli S, et al. The birnavirus crystal structure reveals structural relationships among icosahedral viruses. Cell. 2005;120:761-72. [15] Rautenschlein S, Kraemer C, Vanmarcke J, Montiel E. Protective efficacy of intermediate and intermediate plus infectious bursal disease virus (IBDV) vaccines against very virulent IBDV in commercial broilers. Avian Dis. 2005;49:231-7. [16] Muller H, Mundt E, Eterradossi N, Islam MR. Current status of vaccines against infectious bursal disease. Avian Pathol. 2012;41:133-9. [17] Sreedevi B, Jackwood DJ. Real-time reverse transcriptase-polymerase chain reaction detection and sequence analysis of the VP2 hypervariable region of Indian very virulent infectious bursal disease isolates. Avian Dis. 2007;51:750-7. [18] Juneja SS, Ramneek, Deka D, Oberoi MS, Singh A. Molecular characterization of field isolates and vaccine strains of infectious bursal disease virus. Comp Immunol Microbiol Infect Dis. 2008;31:11-23. [19] Mittal D, Jindal N, Gupta SL, Kataria RS, Singh K, Tiwari AK. Molecular characterization of Indian isolates of infectious bursal disease virus from broiler chickens. DNA Seq. 2006;17:431-9.

AC C

EP

TE D

M AN U

SC

RI PT

153

8

ACCEPTED MANUSCRIPT

EP

TE D

M AN U

SC

RI PT

[20] Indervesh, Tiwari AK, Kataria RS, Viswas KN, Bais MV, Suryanarayana VV. Isolates of infectious bursal disease virus from India are of very virulent phenotype. Acta Virol. 2003;47:173-7. [21] Li Z, Qi X, Ren X, Cui L, Wang X, Zhu P. Molecular characteristics and evolutionary analysis of a very virulent infectious bursal disease virus. Sci China Life Sci. 2015;58:731-8. [22] Rashid MH, Xue C, Islam MT, Islam MR, Cao Y. Risk factors associated with infectious bursal disease in commercial chickens in Bangladesh. Prev Vet Med. 2013;111:181-5. [23] Islam MT, Le TH, Rahman MM, Islam MA. Molecular characterization of two Bangladeshi infectious bursal disease virus isolates using the hypervariable sequence of VP2 as a genetic marker. J Vet Sci. 2012;13:405-12. [24] Stoute ST, Jackwood DJ, Sommer-Wagner SE, Crossley BM, Woolcock PR, Charlton BR. Pathogenicity associated with coinfection with very virulent infectious bursal disease and Infectious bursal disease virus strains endemic in the United States. J Vet Diagn Invest. 2013;25:352-8. [25] Bayliss CD, Spies U, Shaw K, Peters RW, Papageorgiou A, Muller H, et al. A comparison of the sequences of segment A of four infectious bursal disease virus strains and identification of a variable region in VP2. J Gen Virol. 1990;71 ( Pt 6):1303-12. [26] Mohamed MA, Elzanaty KE, Bakhit BM, Safwat MM. Genetic characterization of infectious bursal disease viruses associated with gumboro outbreaks in commercial broilers from asyut province, egypt. ISRN Vet Sci. 2014;2014:916412. [27] Ndashe K, Simulundu E, Hang'ombe BM, Moonga L, Ogawa H, Takada A, et al. Molecular characterization of infectious bursal disease viruses detected in vaccinated commercial broiler flocks in Lusaka, Zambia. Arch Virol. 2015. [28] Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol. 2013;30:2725-9. [29] Jackwood DJ, Sreedevi B, LeFever LJ, Sommer-Wagner SE. Studies on naturally occurring infectious bursal disease viruses suggest that a single amino acid substitution at position 253 in VP2 increases pathogenicity. Virology. 2008;377:110-6. [30] Heine HG, Haritou M, Failla P, Fahey K, Azad A. Sequence analysis and expression of the hostprotective immunogen VP2 of a variant strain of infectious bursal disease virus which can circumvent vaccination with standard type I strains. J Gen Virol. 1991;72 ( Pt 8):1835-43. [31] Nath B, Barman NN, Kumar S. Molecular characterization of Newcastle disease virus strains isolated from different outbreaks in Northeast India during 2014-15. Microb Pathog. 2015;91:85-91. [32] Vakharia VN, He J, Ahamed B, Snyder DB. Molecular basis of antigenic variation in infectious bursal disease virus. Virus Res. 1994;31:265-73. [33] Xu XG, Tong DW, Wang ZS, Zhang Q, Li ZC, Zhang K, et al. Baculovirus virions displaying infectious bursal disease virus VP2 protein protect chickens against infectious bursal disease virus infection. Avian Dis. 2011;55:223-9. [34] Lee DH, Torchetti MK, Winker K, Ip HS, Song CS, Swayne DE. Intercontinental Spread of AsianOrigin H5N8 to North America through Beringia by Migratory Birds. J Virol. 2015;89:6521-4. [35] Chan JF, To KK, Chen H, Yuen KY. Cross-species transmission and emergence of novel viruses from birds. Curr Opin Virol. 2015;10:63-9.

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Figure Legends:

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Figure 1.Geographical location of the infectious bursal disease outbreaks in Northeast India.

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Figure 2. Histological sections of inflammed bursal follicles from the samples collected from the

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Northeastern part of India. The bursa of Fabricius showing generalized hemorrhages (A) and

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infiltration of mononuclear cells (B).

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Figure 3. Comparison of the hypervariable region of the Northeastern isolates of IBDV with the

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reference GenBank sequences. Key amino acids are marked with their respective position on the

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top. The major neutralizing epitope the hydrophilic regions A and B are marked with black and

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blue boxed regions. The heptapeptide SWSASGS which is unique for vvIBDV strains are

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marked with red box.

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Figure 4. Phylogenetic analysis of the Northeastern isolates of IBDV with the reference

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GenBank sequence of VP2 amino acid sequence. A total of 37 strains of IBDV covering the

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classical, antigenically variant, and very virulent (vvIBDV) were included for the analysis.

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Phylogenetic tree was constructed by the Neighbor-Joining method and bootstrapped for 1000

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replications. All bootstrapped values are labeled at major nodes.

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Table 1. Details of infectious bursal disease outbreaks form the Northeast India.

Particulars

Sample Number 54 PD4 Mangaldoi, Darrang Teok, Jorhat

PD5 Location of Majuli, Jorhat Outbreak Year of Collection 2015 2015 2014 2014 Host Broiler chicken Clinical Sign Off fed, Whitish diarrhoea, swollen feces-stained vent Post Mortem Nephrosis, haemorrhages on the leg and breast muscle and haemorrhagic atrophied bursa Lesion Vaccination Status Vaccinated in between 12-14 days of age except PD5* Age of bird at the 22-28 days of age in all except PD5** time disease condition Flock Size 1500 750 1000 500 Mortality 9.33 % 8.66% 12.5% 19.20 % System of rearing Deep litter All the samples were collected from Commercial Broiler Farm *PD5: The sample was collected from Majuli, the river island. The flock was unvaccinated. **Disease recorded at early age i.e. on 18th day onwards

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51 Sonapur, Kamrup

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Table 2. Amino acid sequence analysis of hypervariable region of VP2 protein of infectious bursal disease virus (IBDV) isolates. The reference strains of IBDV from GenBank have been taken for comparison of the Northeastern isolates of IBDV. Underlined amino acid represents the unique insertion.

Key amino acid in hypervariable region of VP2 protein 222

242

253

256

279 N

D78

P

V

H

V

2

Cef94

P

V

H

V

3

BD/3/99

A

I

Q

I

4

Harbin

A

I

5

Cu1-wt

P

V

6

OKYM

A

I

7

HuB-1

A

I

8

Variant E

T

9

UPM94

10

294

299

329

T

L

N

R

N

T

L

N

R

D

A

I

S

S

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1

284

SC

Strain

I

D

A

I

S

S

H

V

N

T

L

N

K

Q

I

D

A

I

S

S

Q

I

D

A

I

S

S

TE D

Q

Q

V

N

A

L

N

S

A

I

Q

I

D

A

I

S

S

IBDV/Chicken/Kamr up/51/2015

A

I

Q

I

D

A

V

S

S

11

IBDV/Chicken/Darra ng/54/2015

A

I

Q

I

D

A

V

S

S

12

IBDV/Chicken/Jorhat A /PD4/2014

I

Q

I

D

A

V

S

S

13

IBDV/Chicken/Jorhat A /PD5/2014

I

Q

I

D

A

V

S

S

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Figure 1.

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Figure 2.

B

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Figure 3.

Hydrophilic region A 222

242

253

256

281

Hydrophilic region B 294

299

Heptapeptide 330

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282 284

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202

360

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Figure 4.

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Highlights: Infectious bursal disease virus (IBDV) isolates from Northeast India.

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Phylogenetic characterization of IBDV isolates.

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Genotypic characterization of IBDV isolates.