Prevalence of Very Virulent Infectious Bursal Disease Virus (IBDV) in Egyptian Delta

Infectious Bursal Disease (IBD) serotype 1 viruses continue to cause direct and indirect significant economic losses to poultry industry. The direct economic impact of IBDV is due to the high mortality rates [1, 2]. The indirect economic impacts is due to IBDV-induced immunosuppression of infected birds [3], Infectious bursal disease viruses are non-enveloped, icosahydral member of the genus Avibirnaviridae [4,5]. The double stranded RNA genome of IBDV is composed of 2 segments; A and B codes for five viral proteins (VP). The larger segment, A, encodes VP2, VP4 and VP3 in large Open Reading Frame (ORF). In addition, segment A also contain a small ORF partially overlapping the other ORF, which encodes VP5 [4, 6]. Although the non-structural protein VP5 is not essential for the virus replication, recent evidence indicates that it could be related to the virulent phenotype and adaptive capacity of the virus. The VP5 gene is also of evolutionary interest because it has an open reading frame that terminally overlaps with the VP2-VP4-VP3 polyprotein coding region [7]. The smaller segment, B contain one ORF encoding VP1; the RNA-dependant RNA polymerase. The major structural proteins of the virion are VP2 and VP3, both of which are constituents of the IBDV capsid. VP2 carries the major neutralizing epitopes[7,8]. VP2 protein contains important neutralizing antigenic sites and elicits protective immune response and most of the amino acid (AA) changes between antigenically different IBDVs are clustered in the hypervariable region of VP2. Thus, this hypervariable region of VP2 is the obvious target for the molecular techniques applied for IBDV detection and strain variation studies. Neutralizing monoclonal antibodies against VP2 can be used to differentiate the serotypes and strains [9, 10]. The VP2 is also responsible for antigenic variation [11-12] and virulence [13,14]. Two distinct serotypes, I and II, have been identified [15-17]. All known pathogenic IBDV strains belong to serotype I. pathogenic IBDV serotype I isolates are commonly grouped based on antigenic and pathogenic properties in one of 6 categories, mild, intermediate, intermediate plus, classical, variant and very virulent as described by[15]. Reverse transcription polymerase chain reaction (RT-PCR) using various primers is applied for detection of IBDV [18]. Studies of nucleotides and deduced amino acids sequence changes occurring in segment A have been adopted to differentiate or correlate between IBD viruses either field or vaccine strains[19,20-21]. Several reports have classified the Egyptian IBDV isolates as classical IBDV [22, 23-24]. On the other hand, some reports have provided partial evidence of the presence of antigenically variant IBDV strains in Egyptian flocks [25-27]. In 2002 direct detection of IBDV antigens in bursal homogenates using monoclonal antibodies against classical and variant epitope markers provided evidence of the presence of antigenically variant IBDV strains in Egyptian flocks [27]. A year later variant IBDV was isolated [28]. In 1999, a new Egyptian IBDV strain, designed 99323, was isolated and identified [29]. The nucleotide sequencing of variable region of gene encoding VP2 further showed an atypical antigenic profile of strain 99323 related to some critical amino acids changes. The nucleotides sequence of 99323 isolate was mostly similar with to that identified 2 Egyptian isolates; Kal2001 and Giza2000. Sequence analysis of the of Kal2001 showed sequence relatedness to vvIBDV strains with sequence homology ranging between 98.8 and 99.5% while, Giza2000 showed relatedness to vvIBDV strains with sequence homology ranging between 98.1 and 98.3%. Variant and highly virulent forms have been the cause of significant economic losses. Vaccination failures were described in different parts of the world. The inception of very virulent IBD created the need for a better characterization of the circulating strains so that, the vaccination schedule could be adapted faster to a new epidemiological situation [15]. This study aims at detection and characterization of the circulating IBD viruses in commercial broiler flocks by RT-PCR/ Sequencing Analysis., In addition a new method to select differential virus's strains for specific VP2 gene sequencing analysis, computerized analysis through Totallab Quant®. Software (Version 1.0.1, NonLinear Co., 2011) were used to calculate IBD viruses strains DNA content through log curve of DNA ladder fragments.

Ten Samples were collected randomly from a commercial broiler flocks in Elbehara, KafrElshiekh and Alexandria governorates with a massive increase in reported mortalities due to clinical IBD. Gross examination of dead birds revealed hemorrhages, swelling and exudates in bursa. 10 bursae of 3,4-weeks-old chickens were collected and preserved at -20ºC until used for RNA extraction.

The vaccinal IBDV strain Bursa-Vac® (Schering-plough, USA) and known virulent IBDV in the department of microbiology (Faculty of vet. Medicine, Alexandria University) were used as control viruses in RT-PCR experiment. GenBank published classical, very virulent, vaccinal and variant sequences were selected for sequence comparisons and phylogenetic analysis (Table1).

Samples were prepared for RNA extraction by disrupting one part of each bursa in sterile saline (1:1). Bursal homogenates were pooled. Total RNA was extracted directly from filtrated supernatant using Total RNA purification Kit® (Jena Bioscience GmbH, lobstedter, Germany), according to manufacturer’s instructions.

Primer Design and Reverse Transcription / Polymerase Chain Reaction (RT-PCR).

Table 1.  IBDV strains used in sequence analysis and phylogenom

 

Novel primers recognizing conserved regions of the IBDV VP2 flanking the hypervariable region were designed after reviewing published primers and sequences[30-34]. The primer sequences were as follows; the forward primer (AUS GU: 5`-TCA CCG TCC TCA GCT TAC CCA CAT C 3`) and the reverse primer (AUS GL: 5`-GGA TTT GGG ATC AGC TCG AAG TTG C-3`). The primer pair AUS GU and AUS GL (annealing position 587-604 and 1212-1229 of IBDV segment A respectively) was reported to generate approximately 620 bp fragment within IBDV VP2. Oligos were manufactured by Metabion GmbH, (Lena-Christ-Strasse, Germany).

Briefly, the reaction mixture contained 1x of One-step RT-PCR Enzyme Mix (containing Omniscript Reverse Transcriptase, Sensiscript Reverse Transcriptase and HotStarTaq DNA polymerase), 0.2 U µL­¹ RNase inhibitor, 400 µM of each of deoxynucleotide triphosphate and 100 pmol each of primers, in a total volume of 50µL Thermo Scientific Verso™ 1-Step RT-PCR kit. (ABgene® LTD) (Cat No.AB1454v7.ABgeneHouse, BlenheimRoad, Epsom, Surrey. KT199AP, UK). Containing 2.5mM magnesium chloride (MgCl2). The PCR reaction was performed in thermal cycler (perkin Elmer 9700) as follows: 20 min at 50ºC (RT reaction), 95ºC for 15 min (initial PCR activation); 39 three-step cycles of 94ºC for 30 sec (denaturation), 59ºC for 40 sec (annealing) and 72ºC for 1 min; then 72ºC for 10 min(final extension). Products were subject to electrophoresis in 1.2% agarose gel containing 0.5 µg mL­1 ethidium bromide.

To select differential viruses strains for specific VP2 gene sequencing analysis, computerized analysis through Totallab Quant®. Software (Version 1.0.1, NonLinear Co., 2011) were used to calculate IBD viruses strains DNA content through log curve of DNA ladder fragments. The remaining of the PCR product of the selected samples by computerized analysis through Totallab Quant®. software (Version 1.0.1, Nonlinear Co., 2011) that showed positive bands in electrophoresis were purified using the QIA quick® PCR purification kit, using the PCR purification protocol and sequenced by BigDye®Terminator v1.3 Cycle Sequencing kit (Applied Biosystem, Germany). The sequences were aligned using the (Finch TV1.4- DNA sequence analysis chromatogram trace viewer, using the GeneSifter Software (Geospiza) and the most parsimonious phylogenetic trees were constructed using the CLC sequence viewer 6.4 Program. Identification of homologies between nucleotide and amino acid sequences of the Egyptian IBDV strains and other IBDV strains published on Gene Bank was done using BLAST 2.0 and psi-BLAST search programs (National Center for Biotechnology Information (NCBI) http://www.ncbi.nlm.nih.gov), respectively. The score designated in the BLAST search have a well-defined statistical interpretation, making matches easier to distinguish from random background hits [34, 35]. The obtained nucleotide sequence comparisons and their multiple alignments with reference IBDV viruses as well as the deduction of amino acid sequences were done using the BioEdit sequence alignment editor [36], clustral 1W software for multiple sequence alignment [37], clustal V [38] and CLC Bio (CLC sequence viewer® version 6.4). The phylogenetic trees were constructed using CLC Bio (CLC sequence viewer® version 6.4). For tree reconstruction of sequences by Neighbor-joining method based on clustalW [34,39]. Bootstrapping values were calculated using a random seeding value of 111 [37]. clustalv was used when end gaps were faced. Sequence divergence and identity percents were calculated by CLC Bio (CLC sequence viewer® version 6.4). Molecular analysis of a.a sequence was done by CLC Bio (CLC sequence viewer® version 6.4.

Extracts from 10 tested bursal samples produces 620 bp amplicons (Fig. 1). For calculating and comparing DNA contents of specific VP2 gene among IBDV strains, Totallab Quant®. Software was employed. 21, 33, 18, 35, 17, 34, 14, 37, 30, 31 pg of DNA concentration of specific VP2 gene among IBDV strains number 1,2, 3, 4, 5, 6, 7, 8, 9, 10 strains (Fig. 2). Based on Totallab Quant®. Computerized analysis, IBDV strains PCR products number 2, 4, 6, 8 and 9 were selected for sequencing analysis as results of superior of DNA contents of specific VP2 gene among ten IBDV strains (Fig. 2).

Figure 1: Detection of IBDV VP2

 

Specific amplified fragment (of 620 bp) for IBDV VP2 gene using specific primer

Lane M: Marker (100 bp DNA ladder)

Lane 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10: Showing positive amplification of VP2 at (620 bp).

Lane 11: control negative

 

Figure 2: Parameters comparisons of specific amplified fragment of samples for IBD VP2 gene

 Sequencing of the selected RT-PCR products was conducted. Sequences of 608, 613, 600, 613, and 614 according to sequence information statistics for the five selected IBDV-VP2 (Table 2), Nucleotides counts and frequencies were calculated (Table 2) and the sequences were used for nucleotide analysis and deduced amino acid analysis after detection of the open reading frame (ORF).

Table 2.  Sequence information, Counts and frequencies of nucleotides

The original sequence was trimmed to remove ambiguous nucleotide sequences usually present in the beginning of the sequencing reaction. 5 isolates were selected (lane No. 2, lane No. 4, lane No. 6, lane No. 8, lane No. 9) for sequence analysis because of their superior DNA contents through linear logarithmic curve (Fig. 2) made by computerized analysis of IBDV strains (based on Totallab Quant®) of specific VP2 gene within the 10 IBDV strains. The sequences were submitted to Gene Bank database. Nucleotide sequence analysis of lane No. 4 (613 nucleotides) showed a 98% identity with Infectious Bursal Disease Virus strain Giza 2008. Lane No. 6 (614 nucleotides) returned a 98% identity with IBDV strain Giza2008. Lane No. 8. (600 nucleotide) returned a 98% identity with IBDV strain Giza2008. Lane No. 9. (613 nucleotides) returned a 97% identity with IBDV strain Giza 2008. Lane No. 2 (608 nucleotides) returned a 96% identity with IBDV strain Giza 2008. Strain Giza 2008 appears to be the closest strain with isolates in lanes No. (2, 4, 6, 8, 9). Strain Giza 2008 sequence was 99% identical to IBDV (strain Behera 2003) and was 98% identical to the very virulent (vvIBDV) Giza 2000 strain.

Nucleotide phylogenetic tree revealed that IBDV strain Giza 2008 descended from European strain 99323, while Univax branched separately from Bursine Plus vaccine and Bursavac vaccine (Fig.3). The phylogenetic tree of deduced amino acid revealed that the sequence of lane No. 2 isolate was grouped together with Giza 2008 strain which branched separately from Behera 2003 and both the vaccine strains (BuraVac andUnivax) were branched out from isolate in lane No. 2 and Giza 2008 (Fig.3).

Sequences of lane No.6, lane No.4, lane No.9. were grouped together with Giza2000 in which they branched separately from strain Giza2000. Sequence of Lane No.8 was grouped with Bursine Plus and both of them were branched out from both 99323 European strain and CEVAC IBD L vaccinal strain (Fig.3). Translation of open reading frame(ORF) for each of the five isolated sequences (lane No.2, lane No.4, lane No.6, lane No.8, lane No.9) shown a distinct mutations were observed of lane No.9 which did not presenet in the other four isolates. a unique substitutional mutaion was observed (Ser146) in lane No.8. also two substitutional mutations observed (Pro146) and (lle149) in lane No.6. the phylogenetic tree of the translated open reading fram for the five isolated vvIBDV revealed the obvious distinction between them (Fig.5). The nucleotide phylogenetic tree of group lanes No. (2,4,6,8,9) VP2 and previously isolated Egyptian very virulent and vaccinal strains of IBDV revealed that all the tested reference sequences grouped together as areported previously[29] (Fig.3). the Egyptian sequences of five isolates of vvIBD lane No.2,4,6,8 and 9 are grouped together within strain Giza 2008 and Univax vaccinal strain. however, Giza 2008 was located on a separate branch with a high bootstrapper value separating the five isolated groups from previously recorded Egyptian very virulent strains.

Phylogenetic analysis of the deduced aa sequences revealed that the isolated VP2 IBDV lane No.8. was branched separately from 99323 European strain and CEVAC IBD L vaccinal strain. The isolated VP2 IBDV lane No.6, lane No.4. and lane No.9. are grouped together with Giza2000. The isolated VP2 IBDV lane No.2. was branched separetely from Giza 2008 and strain Beh 2003. Giza 2008, strain Beh 2003 and the isolated VP2 lane No.2. are grouped together with UNIVAX and BursaVac vaccinal strains (Fig.4). The prevalence of IBDV outbreaks in vaccinated broiler flocks despite the intensive and meticulus application of available commercial live and inactivated IBDV vaccines is a matter of great concern to poultry producers worldwide[42,15]. In Egypt the situation is exacerbated in the absence of dynamic vaccine production mechanism to follow up the evolving genetic and antigenic makeup of circulating IBDV. No major change in the vaccination routines has been adopted by commercial poultry producer although vvIBDV has been identified since 1989[43] and variant IBDV has been confirmed since 2003[27-28].

Nucleic acid-based methods are useful tools for direct detection and subtyping without isolation and propagation[44]. Reverse transcriptase polymerase chain reaction (RT-PCR) techniques on selected fragments of genome, essentially the variable domain of VP2, followed by sequencing and phylogenetic comparison represents a valuable molecular alternative for the classification of IBDV strains[21].

Figure 3.  Nucleotide phylogenetic tree of the five isolated group lanes No.(2,4,6,8,9) VP2 and the reference Egyptian vvIBD strains, commercial vaccinal strains of IBDV

Figure 4.  Phylogenetic tree of deduced amino acid sequences of the vvIBDV five isolated groupe lanes No.(2,4,6,8,9) and the reference Egyptian vvIBD strains, commercial vaccinal strains of IBDV

This study show that the vvIBDV belonging to Egyptian strains, which is in fact distantly related to European strain,  and vaccinal strains, have succeded in surviving in the Egyptian enviroment despite the intensive vaccination programs adopted. Others have also reported this observations[29] phylogenetic analysis shown that VP2 IBDV lane No.2. is isolated together with strain Giza 2008 and strain Beh 2003 in which the three of them are grouped together with UNIVAX and BursaVac vaccinal strains. VP2 IBDV lane No.4., VP2 IBDV lane No.6. and VP2 IBDV lane No.9. are isolated together with strain Giza 2000. VP2 IBDV lane No.8. was separately branched from 99323 European strain and CEVAC IBD L vaccinal strain.

The five isolated IBD viruses were away from the vvIBDV that was initially identified in Egypt. This could indicate that vaccine- directed immunological pressures are only aiding in the evolution of the IBD virus. All the five isolated viruses found with close similarity with strain Giza 2008 which is genetically distinct from vaccine and classical IBD strains.The nucleotide and subsequent aa changes aquired by lane No.2. VP2, lane No.4. VP2, lane No.6. VP2, lane No.8. VP2 and lane No.9.VP2 have lead to significant changes in the folding pattern of this region of the VP2 as predicted by protein analysis[40]. These accumulated changes will increase chances that more neutralization escape mutants will evolve in the near future[41]. There is a threat of new vvIBDV outbreaks in the foreseeable future if current vaccination programs do not take into account the newly circulating antigenic features.  There is an urgent need to develop dynamic mechanisms to produce local vaccine and methodologies to combat the inevitable prevalent IBDV mutants. 

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