The present study was conducted to characterize a new field isolates of infectious bursal disease virus (IBDV) by reverse

transcription-polymerase chain reaction (RT-PCR) and partial sequencing of VP2 gene. The virus could be detected in five of

seven field samples from broiler chickens in Iraq. Nucleotide sequences of field isolates were compared with 33 reported

IBDV strains from different parts of the world. Nucleotide substitutions at I242, Q249, Q252, I256, D279, A284, I294,

S326 and S330 specific for very virulent (vv) strains, were maintained in all the field isolates. Deduced amino acid substitutions

A222, I242, Q249, Q252, I256, D279, A284, I294, S326 and S330 specific for vvIBDV strains were also present in all the

isolates. Phylogenetic analysis showed that all the field isolates in the present study were closely related to reported UK (XZh)

and Japan (OKYM) field isolates. Further comprehensive investigations will provide more information on the distribution,

variability, phylogenetic relationships of different vvIBDV isolate in Iraq and other parts of the worlds

Ali, G.H.

Hasan, I.I.

Mohammed, M.H.

UKM Journal Article Repository

INFECTIOUS BURSAL DISEASE VIRUS FROM BROILER CHICKENS IN IRAQ 97Malays. Appl. Biol. (2014) 43(2): 97–103* To whom correspondence should be addressed.MOLECULAR CHARACTERIZATION OF LOCAL ISOLATESOF INFECTIOUS BURSAL DISEASE VIRUS FROMBROILER CHICKENS IN IRAQMAJED H. MOHAMMED*, BALQEES H. ALI and ISMAEL I. HASANCollege of Veterinary Medicine, University of Baghdad, Baghdad, Iraq*E-mail: majed65bag@hotmail.comABSTRACTThe present study was conducted to characterize a new field isolates of infectious bursal disease virus (IBDV) by reversetranscription-polymerase chain reaction (RT-PCR) and partial sequencing of VP2 gene. The virus could be detected in five ofseven field samples from broiler chickens in Iraq. Nucleotide sequences of field isolates were compared with 33 reportedIBDV strains from different parts of the world. Nucleotide substitutions at I242, Q249, Q252, I256, D279, A284, I294,S326 and S330 specific for very virulent (vv) strains, were maintained in all the field isolates. Deduced amino acid substitutionsA222, I242, Q249, Q252, I256, D279, A284, I294, S326 and S330 specific for vvIBDV strains were also present in all theisolates. Phylogenetic analysis showed that all the field isolates in the present study were closely related to reported UK (XZ-h) and Japan (OKYM) field isolates. Further comprehensive investigations will provide more information on the distribution,variability, phylogenetic relationships of different vvIBDV isolate in Iraq and other parts of the worlds.Key words: Sequence analysis, VP2 hypervariable region, very virulent infectious bursal disease virus, IraqINTRODUCTIONInfectious bursal disease (IBD) is an acute, highlycontagious viral disease of young chickens, thecausative agent belongs to the genus Avibirnaviruswithin the family Birnaviridae and is a non-enveloped icosahedral, bisegmented, doublestranded RNA virus (Ismail and Saif 1990). IBDVcan be differentiated into two serotypes, pathogenicserotype-1 and non-pathogenic serotype-2 strains(McFerran et al., 1980). Pathogenic serotype-1 IBDVfield strains can be classified into classical, variantand very virulent (vv) strains (Snyder 1990). IBDVgenome consists of two segments of dsRNA. Thelarger segment A (3.2 kb) encodes for viral proteinsVP2, VP3 and VP4, which are produced by autoproteolysis of a 106 kDa precursor polyprotein froma single large ORF (Azad et al., 1987). Segment Aalso encodes for VP5, a 17 kD a protein from smallORF 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 massprotective antigen of IBDV and have the antigenicregion responsible for the binding of neutralizingantibodies (Eterradossi et al., 1998). In the years1986 and 1987, vaccination failures were publishedin different parts of the world. As a result, acute IBDoutbreaks 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 capableof neutralization the high maternal antibody levelinduced by classical serotype-1 IBD vaccines andcould cause mortality up to 60–100% (Brown et al.,1994).In Iraq, where the poultry industry is in processof great economic transformation, a protective planis crucial to avoid virus dissemination and diseaseemergence. To date, diagnosis in Iraq has mainlybeen based on clinical signs, gross lesions,histopathology, isolation and ELISA. Thesetechniques and tests are all laborious, timeconsuming, and in some cases expensive and requirespecialized laboratory tests. Detection of IBDV inclinical samples by RT-PCR is superior to theconventional techniques, in that it has greatersensitivity and specificity (Jackwood and Sommer,1998). Although IBDV is often isolated and causesmajor losses to the poultry industry, no data ispresently available about the type of IBDV strainsin Iraq. The objectives of this study were tocharacterize IBDV field strains in commercial broiler98 INFECTIOUS BURSAL DISEASE VIRUS FROM BROILER CHICKENS IN IRAQflocks in Iraq using RT-PCR to detect vvIBDV usinga specific primer and to evaluate comparatively theIraqi RT-PCR with those previously published in theworld.MATERIALS AND METHODSBursa sample collection and virus isolationDuring the period from July 2012 to June 2013,IBD was suspected in 7 commercial broiler flocks(Tikrit province north of Baghdad) on the basis ofclinical signs and postmortem findings. Bursaltissue was homogenated in sterile phosphatebuffered saline (pH7.2) with penicillin andstreptomycin (1 mg/ml each). The homogenate wasthen frozen, thawed three times and centrifuged at3,000xg for 10 min. The supernatant was inoculatedinto 9-day-old embryonated chicken eggs viachorioallantoic membrane (CAM) route using 100μlsample per egg. The inoculated embryos wereincubated at 37°C and candled daily for 5 days.Embryos that died within 24 h post-inoculation wereconsidered as non-specific and discarded. Thoseshowing embryonic death after 24 h up to Day 5post-inoculation were considered infected andchilled the CAM homogenates plus the allantoicfluid were harvested and used as a source of stockvirus for further analysis.RNA extraction and reverse transcriptionThis study was done at the genetic engineeringlaboratory, Biology department, College ofScience,University of Tikrit.The extraction of RNA was made by using totalRNA Mini Kit (Geneaid Company USA) accordingto kit protocol. Complementary DNA was generatedfrom RNA using the reverse transcriptaseRevertAidTM (AccuPower® Rocket Script). The RNAwas first incubated at 95°C for 3 min and placed onice for at least 3 min in the presence of the genespecific primer (5-GTAACAATCACACTGTTCTCAG-3), which binds nucleotides 673 - 923 of thepositive strand of IBDV segment A (Li et al., 2009).The RNA was reverse transcribed in a final volumeof 20 μl containing reaction buffer, 1 mM of eachdNTP (Thermo Scientific), 20U URiboLockTMRNase Inhibitor and 200 units RevertAidTM Reversetranscriptase. Reverse transcription reactions wereperformed at42°C for 60 min and the reversetranscriptase inactivated at 70°C for 10 min.PCR amplification and sequencingPolymerase chain reaction (PCR) amplificationof products intended for sequencing was carried outusing a high fidelity DNA polymerase. A typical 25μl reaction contained  Buffer with MgSO4, 0.2 mMdNTPs (Thermo Scientific), 200 nM of each primersP1 (5-GTAACAATCACACTGTTCTCAG-3) and P2(5-GATGGATGTGATTGGCTGGG-3) which bindsnucleotide position 673–923 of IBDV segment A,respectively (Bayliss et al., 1990), 2.5 units (U)DNA polymerase (Fermentas) and 2μl of cDNAtemplate. The reactions were carried out for 1 cycleat 95°C for 3 min, 35 cycles at 95°C for 30 s, 60°Cfor 30 s, 72°C for 1 min and 1 cycle at 72°C for7 min. The amplified 251 base pair productcontained the VP2 hypervariable region codingsequence. Amplicons were separated from reactioncomponents using the QIAGEN Gel extraction kit,with the concentration of DNA determinedspectrophotometrically using a Nano DropSpectrophometer 1000 (Thermo Scientific). Purifiedamplicons were sequenced using both P1 and P2primers by a commercial sequence provider (Macro-gen) using the Big Dye terminator cycling (AppliedBiosystems) condition and sequencing at 1st BaseLaboratory, Malaysia.Sequence and Phylogenetic analysisPhylogenetic tree was constructed based on thehypervariable region of the nucleotide sequencefrom position 673 to 923 (Bayliss et al., 1990) anddeduced amino acid from 225 to 307 of the VP2gene. Thirty three published IBDV isolates wereincluded in this study to ease investigation ofevolutionary relationships among the isolates.Sequences were aligned using ClustalX softwareversion 1.83. Translation to amino acid sequenceswere done using biology work bench (Version 3.2).The tree views were edited using Tree View Xversion (http://evolgen.biol.metrou.ac.ip/TE/TE_man.html).RESULTSAmplification of the hypervariable Region of VP2GeneThe hypervariable region of VP2 gene wassuccessfully amplified. All the amplified cDNAshowed identical mobility on a 1% agarose gel. ThePCR amplification was done by using primer P1-P2generate specific DNA band of 250 bp (Fig. 1).Sequence Analysis of VP2 regions of IBDV isolateTo determine the genetic changes, the VP2variable region for Iraqi IBDV isolated from cDNAtranscripts. Sequences of IBDV were analyzed andaligned with other various published IBDV strainsfrom the gene bank. There was no insertion ordeletion of nucleotide sequence for this strain incompared with published sequences used in thisstudy: vvIBDV ( IqT1, IRQ1, IRQ2, IRQ3, IRQ4,IRQ5, Hen-h, HJH-8, ch/slemani, 09D214,JRMP42IR, UPM0081, AK2SC10, AK2, SC10,INFECTIOUS BURSAL DISEASE VIRUS FROM BROILER CHICKENS IN IRAQ 99Fig. 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).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).Molecular characterization analysis of thededuced nucleotide sequences of VP2 (pos. 673-923) showed 9 nucleotide  I242, Q249,Q252, I256,D279, A284, I294,S326 and S330 were common inFig. 2 continue...100 INFECTIOUS BURSAL DISEASE VIRUS FROM BROILER CHICKENS IN IRAQFig. 2: Nucleotide sequences of HPVR of VP2 from nucleotide 673-923 (Bayliss et al., 1990) of the IqT1 compared withother published IBDV strains. A dot indicated position where the sequence is identical to others.Fig. 2 continued...INFECTIOUS BURSAL DISEASE VIRUS FROM BROILER CHICKENS IN IRAQ 101all vvIBDV. Alignment analysis of the deduced aasequences showed 21 isolates had typical A222,I242, Q249, Q252, I256, D279, A284, I294, S326and S330 of vvIBDV (Table 1). The IqT1 isolate wasclosely related 100 % with (IRQ1 and IRQ2), andhad high homology (98.6%) with the representativevvIBDV strains (HK46, OKYM, and UK661). Ascompared with the attenuated vaccine strains(89.2%) (Soroa, KTI99. PBG98, D78, AmerVH9907.Phylogenetic analysisA phylogenetic analysis tree resulting fromthe analysis of the deduced aa sequences wasconstructed showing the relationship between IqT1and 5 Iraqis IBDV isolates. All the strains used inthis study were clustered into four distinct branches:vvIBDV strains, classical strains, antigenic variantstrains, 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 tothe vvIBDV group; GLS, Variant A, E//DEL whichwere more closely related to variant group whileSoroa, KTI99. PBG98, D78, AmerVH9907 isolateswhich are regarded as typical attenuated and finallyca IBDV group, such as CT strain (Fig. 3).DISCUSSIONInfectious bursal disease virus is one of the seriouschallenges for the poultry industry in Iraq and isresponsible for considerable economic lossesparticularly in broiler industry (Al-Mayah andMudhar 2010). The disease has been recordedregularly in broiler chickens in spite of regularvaccination with intermediate type vaccines duringthe period from July 2010 to June 2013.The disease was recorded both in the vaccinatedand unvaccinated flocks (Mayah and Tabeekh.2010). Occurrence of the disease in spite ofvaccination could either be due to vaccine failureor due to emergence of new strains. Thus, the presentwork was undertaken to characterize the fieldisolates of IBDVs by RT-PCR followed bysequencing.Table 1. Summary of the proposed molecular markers (amino acid residues) of IqT1 isolates with other published IBDVstrainsIBD Isolate Strain 222 242 249 253 256 279 284 294 326 330IqT1 VV A I Q Q I D A I S SIRQ1 VV A I Q Q I D A I S SIRQ2 VV A I Q Q I D A I S SIRQ3 VV A I Q Q I D A I S SIRQ4 VV A I Q Q I D A I S SIRQ5 VV A I Q Q I D A I S SHen-h VV A I Q Q I D A I S SHJH-8 VV A I Q Q I D A I S SCh/slemani VV A I Q Q I D A I S S09D214 VV A I Q Q I D A I S SJRMP42IR VV A I Q Q I D A I S SUPM0081 VV A I Q Q I D A I S SAK2SC10 VV A I Q Q I N A I S SAK2 VV A I Q Q I N A I S SSC10 VV A I Q Q I N A I S SUPM92/94 VV A I Q Q I N A I S STasek 94 VV A I Q Q I N A I S SOKYM VV A I Q Q I N A I S SHK-46 VV A I Q Q I N A I S SEhime 91 VV A I Q Q I N A I S SSH/95 VV A I Q Q I N A I S SCT CA P V Q H V N T L S RKal2001 CA P V R H V N T L S RGLS VA T V K H V N T L S SVariant A VA Q V K Q V N A L S SE/DEL VA T V K Q V N A L S SSoroa AT P V R H V N T L L RKTI99 AT P V R H V N T L L RPBG98 AT P V R H V N T L L RD78 AT P V R H V N T L S RAmerVH9907 AT P V Q H V N T L S K102 INFECTIOUS BURSAL DISEASE VIRUS FROM BROILER CHICKENS IN IRAQFig. 3: Phylogenetic tree based on amino acids sequence of HPVR of VP2 gene ofIBDV isolates, displaying relationship of IqT1 and other published at IBDV strains.The IBDV was detected in the bursa of allsamples by RT-PCR. One field isolates was furthercharacterized to show the sequences at nucleotideand amino acid levels. Molecular characterizationof IBDV strain has been carried out by RT-PCRusing hypervariable region of VP2 gene (Ismail andSaif, 1990). These differences in the nucleotidesequences were correlated with virulence andpathogenicity of the IBDV strains.The hypervariable region of VP2 gene of IBDVis the most interesting part of this gene for manyresearches (Fahey et al., 1989; Eterradossi et al.,1998) due to  the specific neutralization epitopesand pathotype specific monoclonal antibodies(Eterradossi et al., 1998). Sequence analysis of theVP2 gene hypervariable region of many IBDV hasbeen applied for phylogenetic analysis because ofthe majority of nucleotides and amino aciddifference among IBDV strains appear in this region(Escaffre et al., 2013).Results showed that the nucleotide sequence ofIraqi isolate have 9 nucleotide at I242, Q249, Q252,I256, D279, A284, I294, S326 and S330 werecommon in all vvIBDV and published vvIBDVsequence of some European and Asia strainsavailable in Gene bank (Zahoor et al., 2011).Predicted amino acid sequences were comparedwith published sequence of IBDV. The Iraqi isolateshowed typical similarity to vvIBDV strains 222PA,256V-I, 294L-I, 279N-D and 299N-S was alsomaintained in this field isolates (Ghazali et al.,2013).The phylogenetic analysis of the aa sequencesconfirmed these relationships, as the recent IraqiIBDVs appeared significant genetically related toother vvIBDVs, with which they were associated inINFECTIOUS BURSAL DISEASE VIRUS FROM BROILER CHICKENS IN IRAQ 10395% of the bootstrap generated trees while withattenuated IBDV isolates showed 82.2% of thebootstrap.In conclusion, this is the most comprehensivestudy of molecular typing of IBDV strains detectedin Iraq, and also the first report showing molecularsequencing and phylogenic tree of vvIBDV, and wehave seen nucleotide difference between local strainand vaccine strains thus, this study suggestspreparing vaccine from autogenous strain to controlIBD in local regions where outbreaks might not becontrolled by commercially available vaccinesREFERENCESAl-Mayah1, A.A.S. & Mudhar A.S. 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Molecular characterization of local isolates of infectious bursal disease virus from broiler chickens in Iraq

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