Re-Emergence of Very Virulent IBDV in Egypt
Ashraf M. Metwally,
Ausama A. Yousif,
Iman B. Shaheed,
Walaa A. Mohammed,
Attia M. Samy
Ismail M. Reda
Infectious Bursal Disease (IBD) serotype I viruses continue to cause major economic losses in the Egyptian poultry industry despite the implementation of intensive vaccination programs. A recent increase in IBD related mortality in vaccinated farms prompted this investigation into the genetic character of the circulating IBD Virus (IBDV). Bursa and proventriculus samples were RT-PCR tested using novel primers flanking VP2 region coding the two major and two minor hydrophilic peaks. Infectious Bursal Disease virus was detected in tested samples. Phylogenetic analysis of the sequenced PCR product and deduced amino acid sequences of IBDV Giza 2008 VP2 demonstrated the continued circulation of very virulent IBDV (vvIBDV). The mutations reported in Giza 2008 demonstrate that Egyptian field viruses are isolating from their European ancestors. Some of the aa mutations have lead to a change in some of the exposed regions of the viral protein. Present findings explain the continued presence of vvIBDV in intensively vaccinated flocks.
Infectious Bursal Disease (IBD) serotype I viruses continue to cause direct
and indirect significant economic losses to the poultry industry. The direct
economic impact of IBD is due to the high mortality rates (Chettle
et al., 1989; Van den Berg et al., 1991).
The indirect economic impact is due to IBDV-induced immunosuppression of infected
birds (Allan et al., 1972), which is a leading
cause of vaccination failure and bad performance in chicken (Giambrone
et al., 1976; Giambrone, 1979).
Immunosuppression following IBDV infection is due to destruction of B-lymphocyte
precursors in the bursa of Fabricius (Hirai et al.,
1981). Histopathologic lesions occur in the bursa, spleen, thymus, harderian
gland and cecal tonsils. The first signs of infection occur in the bursa and
it is the most severely affected organ. Degeneration and necrosis of individual
lymphocytes in the medullary region of the bursa occur as early as one day post
infection (Cheville, 1967).
Infectious Bursal Disease viruses are non-enveloped, icosahedral members of
the genus Avibirnavirus of Birnaviridae (Dobos et al.,
1979; Hirai and Shimakura, 1974). 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 overlapped
the other ORF, which encodes VP5. The smaller segment, B contain one ORF encoding
VP1; the RNA-dependent 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 (Azad et al.,
1987; Becht et al., 1988). Neutralizing monoclonal
antibodies against VP2 can be used to differentiate the serotypes and strains
(Becht et al., 1988; Fahey
et al., 1989). The VP2 is also responsible for antigenic variation
(Brown et al., 1994; McAllister
et al., 1995; Snyder et al., 1988;
Vakharia et al., 1994a) and virulence (Brown
et al., 1994; Yamaguchi et al., 1996).
Two distinct serotypes, I and II, have been identified (Jackwood
and Saif, 1987; Jackwood et al., 1985; McFerran
et al., 1980). 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 Van
den Berg (2000).
Reverse transcription polymerase chain reaction (RT-PCR) using various primers
is applied for detection of IBDV (Lee et al., 1992;
Wu et al., 1992; Stram et
al., 1994). 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 (Vakharia
et al., 1992; Brown et al., 1994;
Qian and kibenge, 1994; Vakharia
et al., 1994b; Van den Berg et al., 1996;
Yamagushi et al., 1997; Sellers
et al., 1999; Yu et al., 2001; Zierenberg
et al., 2000).
Several reports have classified the Egyptian IBDV isolates as classical IBDV
(Khafagy et al., 1991; El-Sanousi
et al., 1994; Bekhit, 1996a, b).
On the other hand, some reports have provided partial evidence of the presence
of antigenically variant IBDV strains in Egyptian flocks (El-Sanousi
et al., 1994; Sultan, 1995). 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 (Metwally
et al., 2003). A year later variant IBDV was isolated (Hussein
et al., 2003).
Infectious Bursal Disease (IBD) serotype 1 viruses continue to cause major
problems in the Egyptian poultry industry. The emergence of variant and vvIBDV
has caused considerable concern regarding the vaccine control of IBD in spite
of extensive and multiple administrations of various live vaccines (Hassan
et al., 2002). In 1999, a new Egyptian IBDV strain, designated 99323,
was isolated and identified (Eterradossi et al.,
2004). The nucleotide sequencing of the 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 the 99323 isolate
was mostly similar with to that of reference European vvIBDV strain 89163 (98.0%
nucleotides identity). Abd El-Moaty (2004) identified
2 Egyptian isolates; Kal2001 and Giza2000. Sequence analysis of Kal2001 showed
sequence homology with classical IBDV strains ranging between 98.8 and 99.6%.
While, Giza2000 showed relatedness to vvIBDV strains with sequence homology
ranging between 98.1 and 98.3%.
Re-emergence of variant or highly virulent forms has 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 (Van den Berg,
2000). This study aims at characterization of one of the circulating IBD
viruses in broiler flocks receiving classical IBDV vaccines. In addition, this
report also describes a novel IBDV primer and its use in the molecular characterization
of a central immunogenic region of the viral VP2.
MATERIALS AND METHODS
Sampling and Sample Preparation
Samples were collected from a commercial broiler flock with a slight increase
in reported mortalities due to clinical IBD. Gross examination of the dead birds
revealed hemorrhages, swelling and exudates in bursa, with bursa/body weight
ratios averaging 1.9. Hemorrhages were also noticed on the mucosa of the proventriculus.
||IBDV strains used in sequence analysis and phylogeny
A routine IBDV vaccination program was meticulously implemented before the
increase in mortalities. One-day-old broiler chicks were vaccinated using Univax®
BD (Shering-Plough, USA) according the manufacturers recommendations.
At 16 days, the chicks were vaccinated using Bursine® Plus (Fort Dodge,
USA). Samples from bursae and proventriculi of 3 4-weeks-old chickens that succumbed
to the disease were collected and preserved in formalin for histopathology or
at -80°C until used for RNA extraction. Bursa and proventriculus samples
were collected from SPF chicks (obtained from the SPF production facility in
Fayoum, Egypt), processed and preserved as before. SPF samples served as negative
controls in the experiment.
Viruses and Reference Sequences
The vaccinal IBDV strain Bursa-Vac® 3 (Schering-Plough, USA) and virulent
SPF-chicken propagated IBDV (Yousif et al., 2006)
were used as control viruses in every RT-PCR experiment. GenBank published classical,
very virulent, vaccinal and variant sequences were selected for sequence comparisons
and phylogenetic analysis (Table 1).
Total RNA Extraction
Samples were prepared for RNA extraction by disrupting one part of each
bursa or proventriculus sample in sterile saline (1:1). Bursal homogenates were
pooled. Proventriculus samples were also homogenized and pooled as before. A
previously tested IBDV-positive bursa from a challenge virus (see above) and
SPF tissues were prepared as tested samples. The IBD vaccine included in the
experiment was reconstituted in RNAse-free water. RNA was also extracted from
bovine sera, ovine sera, plant and bacterial cells for specificity testing of
the primers. Total RNA extraction was carried out using RNeasy® Mini kit
(QIAGEN, GmbH, Hilden, Germany) according to the manufacturers instructions.
Primer Design and Reverse Transcription/Polymerase Chain Reaction (RT/PCR)
Novel primers recognizing conserved regions of the IBDV VP2 flanking the
hypervariable region were designed after reviewing published primers and sequences
(Bayliss et al., 1990; Heine
et al., 1991; To et al., 1999; Spatas
and Ignjtovic, 2000; Banda et al., 2001).
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]. Primers were used for amplification
of a 620 bp fragment within IBDV VP2. Oligos were manufactured by Metabion GmbH,
Briefly, the reaction mixture contained 1x of OneStep RT-PCR Enzyme Mix (containing Omniscript Reverse Transcriptase, Sensiscript Reverse Transcriptase and HotStarTaq DNA Polymerase), 0.2 U μL-1 RNase inhibitor, 400 μM of each of the deoxynucleotide triphosphates and 100 pmol each of primers, in a total volume of 50 μL QIAGEN OneStep RT-PCR Buffer containing 2.5 mM magnesium chloride (MgCl2). The PCR reaction was performed in the thermal cycler (Perkin Elmer 9700) as follows: 20 min at 50°C (RT reaction); 95°C for 15 min (initial PCR activation); 39 three-step cycles of 94°C for 30 sec (denaturation), 59°C for 40 sec (annealing) and 72°C for 1 min; then 72°C for 10 min (final extension). Products were subject to electrophoresis in 1.2% agarose gel containing 0.5 μg mL-1 ethidium bromide.
Sequencing and Sequence Analysis
Reverse transcription polymerase chain reaction (RT-PCR) products were purified
from gels and sequenced by the gene-sequencing unit (VACSERA, Egypt). Identification
of homologies between nucleotide and amino acid sequences of the Egyptian IBDV
strains and other IBDV strains published on GenBank was done using BLAST 2.0
and PSI- BLAST search programs (National Center for Biotechnology Information
respectively. The scores designated in the BLAST search have a well-defined
statistical interpretation, making matches easier to distinguish from random
background hits (Altscul et al., 1997). The obtained
nucleotide sequences 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 (Hall, 1999), ClustalW
software for multiple sequence alignment (Thompson et
al., 1994)), ClustalV (Higgins and Sharp, 1989)
and MegAlign (DNASTAR, Lasergene, Version 7.1.0, USA). The phylogenetic trees
were constructed using MegAlign (DNASTAR, Lasergene, Version 7.1.0, USA) for
tree reconstruction of sequences by Neighbor-joining method based on ClustalW.
Bootstrapping values were calculated using a random seeding value of 111 (Thompson
et al., 1994). ClustalV was used when end gaps were faced. Sequence
divergence and identity percents were calculated by MegAlign (DNASTAR, Lasergene,
Version 7.1.0, USA).
Three Bursa samples were fixed in 10 % formol saline, processed by the conventional
method and, stained by Haematoxylin and Eosin (Bancroft et
al., 1996). The obtained slides were examined by the light microscope
and scored on a scale from 1-5 based on lesion characteristics (Poonia
and Charan, 2000).
RESULTS AND DISCUSSION
RT-PCR and Sequence Analysis
Extracts from tested bursal and proventriculus pools produced 620 bp amplicons.
The fragment size was exactly as calculated by in silico analysis. Positive
control and negative control extracts indicated primer specificity (Fig. 1). Sequencing of the PCR product was conducted in both directions and a
sequence of 563 nucleotides was used for nucleotide analysis and deduced amino
acid analysis. The original sequence was trimmed to remove ambiguous nucleotide
sequences usually present in the beginning of the sequencing reaction. The sequence
was submitted to GenBank database (Accession number: EU584433).
Nucleotide sequence analysis of Giza 2008 IBDV VP2 returned a 97.1% identify
with 99323 and 98.9% identity with Giza2000. We were able to calculate identity
between 91.8 and 93.7% comparing Giza 2008 with the available vaccinal strain
sequences. Giza 2008 sequence was around 97% identical to the vvIBDV strains
UK661 and OKYM. Multiple nucleotide substitutions were observed along the nucleotide
sequence of Giza 2008 compared to a consensus sequence (Fig. 2).
||RT-PCR testing of control reference and selected samples for
A unique substitution (C509 T) was observed. However, compared to the consensus,
several other characteristic substitutions specific for Egyptian vvIBDV strains
isolated after 1989 and shared with the variant strains Del/E, Variant A and
GLS, were also observed [G225A, G293A, G497A]. Most of the nucleotide substitutions
that characterize the vvIBDV strains were also observed in Giza 2008 (Fig.
A consensus of 174 amino acids was used for sequence analysis of the deduced
aa sequences of Giza 2008 [correspond to the region from aa 183 to aa 356 according
to numbering of strain F52/70 (Bayliss et al., 1990)]
(Fig. 3). Analysis of the deduced amino acid sequences of
Giza 2008 in comparison with Giza2000 and 99323 showed that a single aa mutation
(A321T) in the major hydrophilic peak B was not present in Giza 2008. However,
a single aa change in the major hydrophilic peak A (Y220F) was present in 3
of 4 sequenced Egyptian strains. The vvIBDV-specific mutation (P222A in the
major hydrophilic domain A) was present in all characterized vvIBDV sequences
in this analysis including Giza 2008 (Fig. 3). Another mutation
shared by all vvIBDV strains was observed (V256I). There were no mutations similar
to any known unique variant IBDV sequences used in this comparison (Fig.
3). The aa changes lead to change in surface probability indices indicating
increased probability of surface exposure in one location (around Thr250, Ser251,
Val252) and sequestration from the surface in two other locations (Ser17, Ser18
and Gln19 as well as Ala321), data not shown.
The nucleotide phylogenetic tree of Giza 2008 VP2 and other reference classical,
very virulent, variant and vaccinal strains of IBDV revealed that all tested
reference sequences grouped together as reported previously (Eterradossi
et al., 2004) (Fig. 4). The Egyptian sequences
of vvIBDV Giza 2008 and Giza 2000 grouped together, however, Giza 2008 was located
on a separate branch with a high bootstrap value separating both branches (Fig.
4). The European, Asian and Egyptian vvIBDV strains, isolated before 2000,
grouped in a separate cluster within the vvIBDV group (Fig. 4).
Phylogenetic analysis of the deduced aa sequences revealed that Giza 2008 branched
separately from Giza2000 and 99323 (Fig. 5).
The pathological alterations in the bursae collected from tested flock were
more or less the same but with little differ in its degree of severity. The
main lesions in the bursa were congestion of blood vessels, edema and inflammatory
cells infiltrations in the interstitial tissues, mainly lymphocytes, accompanied
with proliferation of the connective tissues (Fig. 6a). Necrosis
of glandular epithelium was also observed. Moreover, the lymphoid follicles
appeared scattered in the interstitial tissue, depleted and atrophied with presence
of vacuoles in the cortical and medullar portion.
||Nucleotide sequences of the VP2 variable domain in the IBDV
strain Giza 2008 and other reference classical, virulent, very virulent,
variant and vaccinal IBDV strains shown in Table 1. Dots
indicate position where the sequence is identical to the consensus
There were large numbers of cyst containing serous fluids displaced and replaced
the lymphoid follicles (Fig. 6b). Some follicles were converted
to cysts contain eosinophilic necrotic cells and nuclear debris and infiltrated
by heterophils (Fig. 6c). There was necrosis of lymphocytes
and lympho-epithelial cells with presence of its nuclear debris in lymphoid
follicles (Fig. 6d). The bursal lesion score were calculated
for the bursae that were provided. The scores are presented as averages. The
pathological finding in the flock were scored from 4-5 with an average of 4.6.
The reemergence of IBDV outbreaks in vaccinated broiler flocks despite the
intensive and meticulous application of available commercial live and inactivated
IBDV vaccines is a matter of great concern to poultry producers worldwide (Van
den Berg, 2000; Kabell et al., 2005). In
Egypt the situation is exacerbated in the absence of a 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 (Zierenberg
et al., 2000) and variant IBDV has been confirmed since 2003 (Hussein
et al., 2003; Metwally et al., 2003).
||ClustalW multiple sequence alignment of the deduced amino
acid sequences of the Giza 2008 VP2 in comparison to previously characterized
Egyptian and reference strains
Nucleic acid-based methods are useful tools for direct detection and subtyping
without isolation and propagation (Stram et al.,
1994). Reverse transcription polymerase chain reaction (RT-PCR) techniques
on selected fragments of the genome, essentially the variable domain of VP2,
followed by sequencing and phylogenetic comparison represents a valuable molecular
alternative for the classification of IBDV strains (Van
den Berg, 2000).
In this study we show that vvIBDV belonging to the Egyptian strains, which
is in fact distantly related to the European strain, have succeeded in surviving
in the Egyptian environment despite the intensive vaccination programs adapted.
Others have also reported this observation (Eterradossi
et al., 2004). Phylogenetic analysis shown that Giza 2008 is isolating,
together with Giza2000, away from the vvIBDV that was initially identified in
||Nucleotide phylogenetic tree of Giza 2008 VP2 and other reference
classical, very virulent, variant and vaccinal strains of IBDV
||Phylogenetic tree of deduced amino acid sequences of Giza
2008 VP2 and other reference classical, very virulent, variant and vaccinal
strains of IBDV
||Histopathological findings of bursae recovered from dead birds
from IBDV vaccinated commercial broiler flocks in Giza
This could indicate that vaccine-directed immunological pressures are only
aiding in the evolution of the virus. Giza 2008 is genetically distinct from
vaccine and classical IBD strains.
The nucleotide and subsequent aa changes acquired by Giza 2008 VP2 have lead
to significant changes in the folding pattern of this region of the VP2 as predicted
by protein analysis (data not shown). These accumulated changes will increase
chances that more neutralization escape mutants will evolve in the near future
(Letzel et al., 2007). There is a threat of emergence
of new vvIBDV outbreaks in the foreseeable future if current vaccination programs
do not take into account the newly circulating antigenic features.
The bursal pathology recorded indicated that the lesions were not induced by
any of the intermediate or intermediate plus vaccine strains in use (Bolis
et al., 2003; Rautenschelin et al., 2003;
Abdel-Alim and Kwakab, 2006). This was supported by
our sequencing data. The retrieved viral sequences were those of vvIBDV and
not related to any of the vaccines.
In conclusion, present data demonstrate the success and continuous evolution of the vvIBDV in the Egyptian environment. It also demonstrates that there is a threat of emergence 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 vaccines and/or methodologies to combat the inevitable reemerging IBDV mutants.
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