Download Title: Molecular characterization of infectious bursal disease viruses

Title: Molecular characterization of infectious bursal disease viruses from Pakistan
Authors: Muhammad Zubair Shabbir1, Muhammad Ali2, Muhammad Abbas3, Umer Naveed
Chaudhry4, Zia-ur-Rehman5, Muhammad Munir6
Affiliations: 1University of Veterinary and Animal Sciences, Lahore Pakistan; 2Bahauddin
Zakariya University, Multan, Pakistan; 3Livestock and Dairy Development Department,
Punjab Pakistan; 4Roslin Institute, University of Edinburgh Scotland (UK); 5Institute of
Pathophysiology and Immunology, medical university of Graz, Austria; 6The Pirbright
Institute, Woking Surrey, GU240NF, United Kingdom
CORRESPONDING AUTHOR:
Muhammad Zubair Shabbir
Assistant Professor
Quality Operations Laboratory
University of Veterinary and Animal Sciences
Lahore 54600, Pakistan
E. mail: [email protected]
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ABSTRACT:
Since the first report of infectious bursal disease in Pakistan in 1987, outbreaks are common
even in vaccinated flocks. Despite appropriate administration of vaccines, concerns arise if
the circulating strains are different from the ones used in vaccine. Here, we sequenced the
hypervariable region (HVR) of the VP2 gene of circulating strains of infectious bursal
disease viruses (IBDV) originating from outbreaks (n = 4) in broiler flocks in Pakistan.
Nucleotide sequencing followed by phylogeny and deduced amino acid sequence analysis
showed the circulating strains to be very virulent (vv), and identified characteristic residues at
position 222(A), 242(I), 256(I), 294(I) and 299(S). In addition, a substitution at position
221(Q→H) was found exclusive to Pakistani strains in our analysis, though a larger dataset is
required to confirm this finding. Compared to commonly used vaccine strains in Pakistan,
substitution mutations were found at key amino-acid positions in VP2 that may be
responsible for potential changes in neutralization epitopes and vaccine failure.
Key words: Infectious bursal disease virus, Hypervariable region, VP2, vvIBDV, Pakistan
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Infectious bursal disease (IBD) is a highly contagious viral disease of birds characterized by
lesions in the bursa of Fabricius, immunosuppression, severe morbidity and mortality. The
etiological agent, infectious bursal disease virus (IBDV), belongs to the family Birnaviridae
and has a double stranded bi-segmented genome (RNA) comprised of segment A [(3,300
nucleotides (nt) in length], and segment B (2,800nt in length). Segment A encodes for two
proteins named VP2 (capsid protein, 48kDa) and VP3 (ribonucleoprotein, 32-35kDa), the
viral protease VP4 (24kDa), and a non-structural protein VP5 (17-21kDa). Segment B, on the
other hand, encodes RNA-dependent RNA polymerase, VP1 (90kDa) (Mundt and Muller,
1995). There are two distinct serotypes of IBDV. Viruses in serotype 1 are classified as
classical virulent, antigenic variant, and very virulent (vv). The viruses in serotype 2 are
considered to be non-pathogenic for chickens (Ismail et al., 1988).
Following the first detection of infectious bursal disease in 1987 in Pakistan (Khan et al.,
1988), a number of disease outbreaks have been observed, even in vaccinated flocks.
Attempts have been made to characterize IBDV strains through restriction-endonuclease (RE)
assays in Pakistan (Lone et al., 2009; Zahoor et al., 2011). However, multiple genetic events
have been identified recently that have led to alterations in IBDV sequences, including
sequences used in the RE assay (Hoque et al., 2001; Parede et al., 2003; Jackwood and
Sommer-Wagner, 2007). RE-assays are therefore not adequate to determine the on-going
genetic diversity and molecular epidemiology of vvIBDVs.
To better expand our
understanding of the molecular epidemiology of circulating strains of IBDV in Pakistan, we
determined the nucleotide and translated amino acid sequences of the VP2 gene from IBDV
viruses originating from four outbreaks in broiler flocks, and compared the sequences to
those reported elsewhere around the globe.
We studied four outbreaks that occurred between 2009-2010 in broiler flocks with a high
morbidity (70 – 90%) and mortality (40-60%). Farms were located in one of the poultry
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dense areas in Punjab province, the area in and around Lahore and Multan district. Each farm
(n = 30,000 birds/farm) had a history of vaccination with intermediate and hot (intermediate
plus) strains of IBDV. The age of the birds ranged from 22–27 days, and birds exhibited
symptoms such as anorexia, fever, huddling, whitish diarrhoea and pasting around vent.
Necropsy showed typical inflammatory lesions in the bursa of Fabricius, and haemorrhages
on the breast and thigh muscles.
Pools of bursa were collected from each outbreak and minced using a sterile mortar and
pestle. A 20% suspension was made with sterile normal saline supplemented with penicillin
(10,000IU) and streptomycin (1,000µg) followed by centrifugation at 4,000rpm for 15
minutes. The filtered supernatants (0.22µm, Milipore, USA) were adsorbed on the FTA
QIAcard (Qiagen, Hilden, Germany) and shipped to Swedish University of Agricultural
Sciences, Uppsala, Sweden for processing. The genetic material was eluted from four 3mm
punches using 100uL of elution buffer that contained RNA processing buffer (10 mM TrisHCl and 0.1 mM EDTA with pH 8.0), 800U/ml RNaseOutTM (Invitrogen, Carlsbad, U.S.A.)
and 2mM DTT [(1,4-Dithiothreitol) (Sigma-Aldrich, St. Louis, U.S.A.)]. The extracted
genome was stored at -20 oC until use.
The VP2 gene was amplified using specific primers and protocols as described previously
(Kataria et al., 2001). The amplified fragments were visualized using a trans-illuminator,
excised from the gel, and purified using the Wizard® SV Gel and PCR Clean-Up System
(Promega, Co., Madison, WI) according to the manufacturer’s instructions. Using the same
primers as were used for amplification, the positive PCR products were sequenced with an
ABI PRISM BigDye Terminator version 3.1 (Applied Biosystems, Foster City, CA), as
recommended by the manufacturer. Each DNA fragment was sequenced in both directions to
generate a reliable consensus sequence. Sequence assembly and editing were performed using
the SEQMAN program of DNASTAR Lasergene 8 (version 8.0.2 13; Madison, WI).
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Construction of a phylogenetic tree was done with the neighbor-joining method using the
Kimura two-parameter model in MEGA version 5.2 (Tamura et al., 2011).
We determined the nucleotide sequence and the translated amino acid sequence of the
hypervariable region (HVR) of the VP2 gene for four IBDVs, each one representing a
different outbreak. The HVR is a major conformational and neutralising antigenic domain
spanning amino acid residues between positions 211-350. Since there are the greatest
differences among serotype-1 strains of IBDV in this region of genome, the nucleotide and
deduced amino acid residues spanning this part of genome have been widely used for
diagnostics and typing as variant, classic and very virulent (Jackwood and Sommer, 1999).
The nucleotide sequence spanning the HVR of strains isolated in the present study were
compared to the genome sequences available in PubMed (AJ310185).
The Pakistani strains clustered to vvIBDVs (VV-3) reported from Asian, African and
European countries between 1997 and 2010 (Figure 1). We observed a number of
synonymous substitutions within the study strains; the strains
IBDV/Pak/Chicken/MM126/2010 (KU321592) and IBDV/Pak/Chicken/MM127/2010
(KU321594) while IBDV/Pak/Chicken/MM125/2009 (KU321593) and
IBDV/Pak/Chicken/MM128/2009 (KU321595) were 100% identical to each other. When
compared to representative strains of each known serotype and vaccine strains, the study
variants showed divergence varying from 3.8 – 18.2. Maximum similarity in nucleotide
sequences were observed (96.3 and 96.1%) between the strains in the present study and a
very virulent virus isolated from white-leghorn chicken from Japan, VV3 (OKYM, GI:
1669530). Compared to the vaccine strains used commonly in Pakistan, a divergence of 8.3 –
8.9 (Bursaplex) and 10.4 – 10.6 (Bursine) was observed (Figure 2). Despite being studied
over a period of two years representing four outbreaks at different locations, we observed a
high genetic homogeneity particularly in residue patterns (Figure 3) and clustering within one
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clade close to vvIBDVs suggesting a single origin of IBDVs. This is not unexpected as
genetic stability within the HVR of VP2 gene of IBDV has previously been reported in a
number of studies (Owoade et al., 2004; Martin et al., 2007; Jenberie et al., 2014).
The deduced amino acid sequence of HVR of VP2 was determined and compared to
representative strains of vvIBDVs [(98/084, VV1), (97/102, VV2) and (OKYM, VV3),
classical virulent IBDV [(002/73, Aus-CV) and (GBF-1, CV)] and a number of vaccine
strains such as V887 (Malaysian Vaccines and Pharmaceuticals), Bursaplex (Embrex Inc.,
U.S.A.), IBA (Bestar Laboratories Pte Ltd., Singapore), NVRI-VOM (National Veterinary
Research Institute, NVRI, Vom, Nigeria) MB (Abic Ltd, Israel), Bursine Plus (Wyeth–Fort
DodgeAnimal Health, U.S.A.) and Ventri (Ventri Biologicals Venkateshwara Hatcheries Ltd,
Pune, India). The analyzed HVR included 145 amino acids residues from position 211 to 354
of the VP2 protein. We observed 100% homogeneity within the study strains with a pattern
characteristic of vvIBDV that includes the amino acid residues A222, I242, I256, I294 and
S299 (Brown et al., 1994; Jackwood and Sommer-Wagner, 2007). Since the presence of
H/N/Q at position 253 and the serine-rich heptapeptide
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SWSASGS332 next to major
hydrophilic region B has been found associated with cell tropism and virulence of isolates
(Jackwood et al., 2008; Qi et al., 2009), identification of 253Q and serine-rich heptapeptide
indicates the virulence of studied pathotype. Further, identification of histidine (H253),
asparagine (N279), threonine (T284) and Arginine (R330) has been previously linked to
propagation in cell culture (Mundt, 1999). Consistent with this, we found substitutions in the
study vvIBDVs as glutamine at 253 (H→Q), aspartic acid at 279 (N→D), alanine at 284
(T→A) and serine at 330 (R→S) indicating they are not cell-culture adapted but represent
four different outbreaks. Interestingly, though it needs large sample dataset to confirm, we
found histidine substitution at position 221 (Q→H) unique to strains from Pakistan when
compared to representative strains (Figure of 3D, label accordingly). We compared the
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sequences from strains isolated in the present study to three vvIBDVs: VV1, VV2 and VV3
(Figure 3). The strains showed a pattern similar to VV3 (OKYM strain), except for one
substitution at position 221 (Q→H); Q at this position was found to be common in all
representative strains. In addition, compared to VV1 and VV2, there was a mutation at
position 300 (A→E), while serine was replaced by glycine in contrast to VV2 at position 254
(S→G) (Figure 3).
Differences in the relatively well-known or conserved regions/position considered exclusive
to vvIBDVs have been observed. For example, the vvIBDVs reported from outbreaks in
Indonesia and Singapore reported a substitution mutation 222(A→S) indicating that alanine
at this position is not a unique characteristic of vvIBDVs (Parede et al., 2003; Jackwood and
Sommer-Wagner, 2007). The residue S299 is thought to be conserved among vvIBDVs;
however studies have reported asparagine (N) for vvIBDVs (Etarradossi et al., 1999) and
serine (S) for non- vvIBDVs strains (Jackwood and Sommer-Wagner, 2007). Further, even
with characteristic pattern of vvIBDVs (A222, I242, I256, I294 and S299), an IBDV outbreak
from Malaysia produced only 10% mortality in susceptible chickens (Hoque et al., 2001).
This is contrary to our findings where we have observed high morbidity and mortality with
serine at position 299. Taken together, since the mechanism of IBDV virulence remains
poorly understood, the genetic changes in HVR should be considered as an outcome of
evolution pertaining to host-virus-environment interaction rather than virulence markers per
se. (Hoque et al., 2001; Parede et al., 2003; Jackwood and Sommer-Wagner, 2007).
The study flocks had a history of vaccination with commonly used vaccines in Pakistan;
however information on the extent of serological monitoring/evaluation of the flocks was not
available. Despite the use of attenuated vaccines to protect chickens, outbreaks in vaccinated
flocks are not uncommon. The vvIBDVs have the ability to break through high levels of
maternal antibodies and, in comparison to classical variant of IBDVs, can cause disease even
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in the presence of high neutralising antibodies produced by classical attenuated vaccine
strains (Eterradossi et al., 1998; Jenberie et al., 2014). Therefore, highly attenuated vaccines
that induce low level of neutralising antibodies may not provide adequate protection to
vvIBDVs (Jenberie et al., 2014). Furthermore, beside variations at various places in deduced
residue analysis of study strains to commonly used vaccine strains in Pakistan, substitution
mutations in key epitope in the VP2 capsid at positions 221 (Q→H), 222 (P/L/Q/S→A), 249
(H→Q), 284 (T→A), 299 (N→S) and 317 (N→S) were in hydrophilic regions. Four
hydrophilic loops are identified in VP2 and the occurrence of these residue exchanges
indicates potential selective pressure for evolution of IBDV upon exposure to the immune
system (Durairaj et al., 2011). It might be possible that the change in amino acids may change
the polarity of the protein, or lead to modifications in protein folding or the interaction with
other/adjacent molecules resulting in a change in the topography of the neutralizing epitopes
and subsequent vaccine failure (Jackwood and Sommer-Wagner, 2011). Vaccination with
cell-culture attenuated vvIBDV have been found efficacious, however, risk of reversion of
vaccine strains always exists (Raue et al., 2004; Rasool and Hussain, 2006). Since virulent
classical viruses that are genetically related to classical vaccine strains have been identified
from outbreaks when users fail to follow manufacturer’s recommendations (Jackwood et al.,
2008; Jenberie et al., 2014), ensuring appropriate and correct administration of dosage while
maintaining cold-chain coupled with routine serological monitoring should be practised
adequately when using cell-culture attenuated vvIBDVs as vaccines.
In conclusion, we sequenced the HVR of the VP2 gene of circulating strains of IBDVs in
Pakistan, and found that they had a residue pattern typical of very virulent viruses closely
related to Asia/Europe lineage. Interestingly, we found a histidine (H) at position 221 that is
unique to strains from Pakistan when compared to representative strains in our analysis, and a
larger dataset would be beneficial to confirm this observation.
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Acknowledgements
We acknowledge Dr Andrew Broadbent at The Pirbright Institute for helpful comments and
suggestions, and for reading and editing the manuscript prior to submission.
Author’s contributions
Conceived and designed the experiment: MZS, MA and MM. Field work and sample
collection from Pakistan: MA, MA, ZR. Genome processing in the collaborating institute at
Sweden: MM and UNC. Analysed the data: MZS, MA and ZR: Wrote the manuscript: MZS,
MA and MM. Author’s current affiliation are given in the manuscript.
Conflict of Interest
All authors declared no conflict of interest with respect to manuscript contents.
Ethical approval
All applicable international, national, and/or institutional guidelines for the care and use of
animals were followed.
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Legends:
Figure 1: Phylogenetic consensus tree for the study IBDVs representing four different
outbreaks in Pakistan. The hypervariable nucleotide sequences (VP2 gene) of the study
strains were compared with previously classified strains as very virulent (VV) subdivided in
VV-1, VV-2 and VV-3, classic variant (CV), very virulent atypical (VVA), Australian classic
(AC) and Australian variant (AV).
Figure 2: Percent similarity and divergence of nucleotide sequences of hypervariable regions
of study isolates with reference to representative strains of different pathotypes of IBDVs
including the vaccine strains
Figure 3: Alignment of deduced amino acid sequence of the hypervariable domain of VP2
spanning between amino acid positions 211 – 354. The residue profile of study viruses is
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compared with strains of IBDV representing different pathotypes. The major and minor
hydrophilic regions are boxed for understanding.
Figure: X-ray crystal structures of the VP2 protein downloaded from the Protein Data Bank
(PDB) ID 1WCD. Annotation and visualization were made in the MacPyMol v1.7.4.4.
Residues 221, 222 and 317 are labelled green, blue and yellow, respectively.
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