Molecular Characterization of Local Isolates of Infectious Bursal Disease Virus from Broiler Chickens in Iraq

Infectious bursal disease (IBD) is an acute, highly contagious viral disease of young chickens, the causative agent belongs to the genus Avibirnavirus within the family Birnaviridae and is a nonenveloped icosahedral, bisegmented, double stranded RNA virus (Ismail and Saif 1990). IBDV can be differentiated into two serotypes, pathogenic serotype-1 and non-pathogenic serotype-2 strains (McFerran et al., 1980). Pathogenic serotype-1 IBDV field strains can be classified into classical, variant and very virulent (vv) strains (Snyder 1990). IBDV genome consists of two segments of dsRNA. The larger segment A (3.2 kb) encodes for viral proteins VP2, VP3 and VP4, which are produced by auto proteolysis of a 106 kDa precursor polyprotein from a single large ORF (Azad et al., 1987). Segment A also encodes for VP5, a 17 kD a protein from small ORF partly overlapping with the polyprotein (Bayliss et al., 1990). Genome segment B (2.9 kb) encodes for RNA polymerase, a 90 kDa protein VP1 (Azad et al., 1985). The VP2 is a main mass protective antigen of IBDV and have the antigenic region responsible for the binding of neutralizing antibodies (Eterradossi et al., 1998). In the years 1986 and 1987, vaccination failures were published in different parts of the world. As a result, acute IBD outbreaks due to very virulent IBDV (vvIBDV) strains were recorded in Europe, Asia and Africa (Chettle et al., 1989; van den Berg et al., 1991; Nunoya et al., 1992;). These vvIBDVs were capable of neutralization the high maternal antibody level induced by classical serotype-1 IBD vaccines and could cause mortality up to 60–100% (Brown et al., 1994).

In Iraq, where the poultry industry is in process of great economic transformation, a protective plan is crucial to avoid virus dissemination and disease emergence. To date, diagnosis in Iraq has mainly been based on clinical signs, gross lesions, histopathology, isolation and ELISA. These techniques and tests are all laborious, time consuming, and in some cases expensive and require specialized laboratory tests. Detection of IBDV in clinical samples by RT-PCR is superior to the conventional techniques, in that it has greater sensitivity and specificity (Jackwood and Sommer, 1998). Although IBDV is often isolated and causes major losses to the poultry industry, no data is presently available about the type of IBDV strains in Iraq. The objectives of this study were to characterize IBDV field strains in commercial broiler flocks in Iraq using RT-PCR to detect vvIBDV using a specific primer and to evaluate comparatively the Iraqi RT-PCR with those previously published in the world. 

During the period from July 2012 to June 2013, IBD was suspected in 7 commercial broiler flocks (Tikrit province north of Baghdad) on the basis of clinical signs and postmortem findings. Bursal tissue was homogenated in sterile phosphate buffered saline (pH7.2) with penicillin and streptomycin (1 mg/ml each). The homogenate was then frozen, thawed three times and centrifuged at 3,000xg for 10 min. The supernatant was inoculated into 9-day-old embryonated chicken eggs via chorioallantoic membrane (CAM) route using 100μl sample per egg. The inoculated embryos were incubated at 37°C and candled daily for 5 days. Embryos that died within 24 h post-inoculation were considered as non-specific and discarded. Those showing embryonic death after 24 h up to Day 5 post-inoculation were considered infected and chilled the CAM homogenates plus the allantoic fluid were harvested and used as a source of stock virus for further analysis.

This study was done at the genetic engineering laboratory, Biology department, College of Science,University of Tikrit.

The extraction of RNA was made by using total RNA Mini Kit (Geneaid Company USA) according to kit protocol. Complementary DNA was generated from RNA using the reverse transcriptase RevertAid^TM (AccuPower® Rocket Script). The RNA was first incubated at 95°C for 3 min and placed on ice for at least 3 min in the presence of the gene specific primer (5-GTAACAATCACACTGTTCT CAG-3), which binds nucleotides 673 - 923 of the positive strand of IBDV segment A (Li et al., 2009). The RNA was reverse transcribed in a final volume of 20 μl containing reaction buffer, 1 mM of each dNTP (Thermo Scientific), 20U URiboLock^TM RNase Inhibitor and 200 units RevertAid^TM Reverse transcriptase. Reverse transcription reactions were performed at42°C for 60 min and the reverse transcriptase inactivated at 70°C for 10 min.

Polymerase chain reaction (PCR) amplification of products intended for sequencing was carried out using a high fidelity DNA polymerase. A typical 25 μl reaction contained Buffer with MgSO4, 0.2 mM dNTPs (Thermo Scientific), 200 nM of each primers P1 (5-GTAACAATCACACTGTTCTCAG-3) and P2 (5-GATGGATGTGATTGGCTGGG-3) which binds nucleotide position 673–923 of IBDV segment A, respectively (Bayliss et al., 1990), 2.5 units (U) DNA polymerase (Fermentas) and 2μl of cDNA template. The reactions were carried out for 1 cycle at 95°C for 3 min, 35 cycles at 95°C for 30 s, 60°C for 30 s, 72°C for 1 min and 1 cycle at 72°C for 7 min. The amplified 251 base pair product contained the VP2 hypervariable region coding sequence. Amplicons were separated from reaction components using the QIAGEN Gel extraction kit, with the concentration of DNA determined spectrophotometrically using a Nano Drop Spectrophometer 1000 (Thermo Scientific). Purified amplicons were sequenced using both P1 and P2 primers by a commercial sequence provider (Macrogen) using the Big Dye terminator cycling (Applied Biosystems) condition and sequencing at 1st Base Laboratory, Malaysia.

Phylogenetic tree was constructed based on the hypervariable region of the nucleotide sequence from position 673 to 923 (Bayliss et al., 1990) and deduced amino acid from 225 to 307 of the VP2 gene. Thirty three published IBDV isolates were included in this study to ease investigation of evolutionary relationships among the isolates. Sequences were aligned using ClustalX software version 1.83. Translation to amino acid sequences were done using biology work bench (Version 3.2). The tree views were edited using Tree View X version (http://evolgen.biol.metrou.ac.ip/TE/ TE_man.html). 

The hypervariable region of VP2 gene was successfully amplified. All the amplified cDNA showed identical mobility on a 1% agarose gel. The PCR amplification was done by using primer P1-P2 generate specific DNA band of 250 bp (Fig. 1).

To determine the genetic changes, the VP2 variable region for Iraqi IBDV isolated from cDNA transcripts. Sequences of IBDV were analyzed and aligned with other various published IBDV strains from the gene bank. There was no insertion or deletion of nucleotide sequence for this strain in compared with published sequences used in this study: vvIBDV ( IqT1, IRQ1, IRQ2, IRQ3, IRQ4, IRQ5, Hen-h, HJH-8, ch/slemani, 09D214, JRMP42IR, UPM0081, AK2SC10, AK2, SC10, UPM92/94, Tasek 94, OKYM, HK46, Ehime 91, SH/ 95). caIBDV (CT), variant IBDV (GLS, Variant A, E//DEL), attenuated IBDV (Soroa, KTI99. PBG98, D78, AmerVH9907) (Fig. 2). 

Fig. 1: Hypervariable region (250pb) of IBDV VP2 gene Lane 1 – positive sample 1; Lane 2 – positive sample 2; Lane 3 – positive sample 3; M – 100 bp DNA marker (Promega, USA).

 

Fig. 2: Nucleotide sequences of HPVR of VP2 from nucleotide 673-923 (Bayliss et al., 1990) of the IqT1 compared with other published IBDV strains. A dot indicated position where the sequence is identical to others.

 

Table 1. Summary of the proposed molecular markers (amino acid residues) of IqT1 isolates with other published IBDV strains

Molecular characterization analysis of the deduced nucleotide sequences of VP2 (pos. 673- 923) showed 9 nucleotide I242, Q249,Q252, I256, D279, A284, I294,S326 and S330 were common in all vvIBDV. Alignment analysis of the deduced aa sequences showed 21 isolates had typical A222, I242, Q249, Q252, I256, D279, A284, I294, S326 and S330 of vvIBDV (Table 1). The IqT1 isolate was closely related 100 % with (IRQ1 and IRQ2), and had high homology (98.6%) with the representative vvIBDV strains (HK46, OKYM, and UK661). As compared with the attenuated vaccine strains (89.2%) (Soroa, KTI99. PBG98, D78, AmerVH9907.

A phylogenetic analysis tree resulting from the analysis of the deduced aa sequences was constructed showing the relationship between IqT1 and 5 Iraqis IBDV isolates. All the strains used in this study were clustered into four distinct branches: vvIBDV strains, classical strains, antigenic variant strains, and attenuated strains. The branch one IqT1, IRQ1, IRQ2, IRQ3, IRQ4, IRQ5,Hen-h, HJH-8, ch/slemani, 09D214, JRMP42IR, UPM0081, AK2, SC10, AK2, SC10, UPM92/94, UK661, Tasek.94, OKYM, HK46, Ehime 91, SH/95, which belong to the vvIBDV group; GLS, Variant A, E//DEL which were more closely related to variant group while Soroa, KTI99. PBG98, D78, AmerVH9907 isolates which are regarded as typical attenuated and finally ca IBDV group, such as CT strain (Fig. 3). 

Infectious bursal disease virus is one of the serious challenges for the poultry industry in Iraq and is responsible for considerable economic losses particularly in broiler industry (Al-Mayah and Mudhar 2010). The disease has been recorded regularly in broiler chickens in spite of regular vaccination with intermediate type vaccines during the period from July 2010 to June 2013.

The disease was recorded both in the vaccinated and unvaccinated flocks (Mayah and Tabeekh. 2010). Occurrence of the disease in spite of vaccination could either be due to vaccine failure or due to emergence of new strains. Thus, the present work was undertaken to characterize the field isolates of IBDVs by RT-PCR followed by sequencing. 

Fig. 3: Phylogenetic tree based on amino acids sequence of HPVR of VP2 gene of IBDV isolates, displaying relationship of IqT1 and other published at IBDV strains.

The IBDV was detected in the bursa of all samples by RT-PCR. One field isolates was further characterized to show the sequences at nucleotide and amino acid levels. Molecular characterization of IBDV strain has been carried out by RT-PCR using hypervariable region of VP2 gene (Ismail and Saif, 1990). These differences in the nucleotide sequences were correlated with virulence and pathogenicity of the IBDV strains.

The hypervariable region of VP2 gene of IBDV is the most interesting part of this gene for many researches (Fahey et al., 1989; Eterradossi et al., 1998) due to the specific neutralization epitopes and pathotype specific monoclonal antibodies (Eterradossi et al., 1998). Sequence analysis of the VP2 gene hypervariable region of many IBDV has been applied for phylogenetic analysis because of the majority of nucleotides and amino acid difference among IBDV strains appear in this region (Escaffre et al., 2013).

Results showed that the nucleotide sequence of Iraqi isolate have 9 nucleotide at I242, Q249, Q252, I256, D279, A284, I294, S326 and S330 were common in all vvIBDV and published vvIBDV sequence of some European and Asia strains available in Gene bank (Zahoor et al., 2011).

Predicted amino acid sequences were compared with published sequence of IBDV. The Iraqi isolate showed typical similarity to vvIBDV strains 222PA, 256V-I, 294L-I, 279N-D and 299N-S was also maintained in this field isolates (Ghazali et al., 2013).

The phylogenetic analysis of the aa sequences confirmed these relationships, as the recent Iraqi IBDVs appeared significant genetically related to other vvIBDVs, with which they were associated in 95% of the bootstrap generated trees while with attenuated IBDV isolates showed 82.2% of the bootstrap.

In conclusion, this is the most comprehensive study of molecular typing of IBDV strains detected in Iraq, and also the first report showing molecular sequencing and phylogenic tree of vvIBDV, and we have seen nucleotide difference between local strain and vaccine strains thus, this study suggests preparing vaccine from autogenous strain to control IBD in local regions where outbreaks might not be controlled by commercially available vaccines. 

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