Molecular epidemiology of infectious bursal disease viruses and development of a microparticle based vaccine
THESIS
Submitted in partial fulfillment of the requirements for the degree
DOCTOR OF PHILOSOPHY (PhD)
at the University of Veterinary Medicine Hannover, Germany
By
Tamiru Negash Alkie (Arsi, Ethiopia) Hannover, Germany 2013
Hannover, Germany)
Advisory Committee: Prof. Dr. Silke Rautenschlein
Prof. Dr. Beatrice Grummer (Institute of Virology, University of Veterinary Medicine Hannover, Germany)
PD Dr. Reimar Johne (Federal Institute for Risk Assessment, Berlin, Germany)
1st Evaluation: Prof. Dr. Silke Rautenschlein Prof. Dr. Beatrice Grummer PD Dr. Reimar Johne
2nd Evaluation: Prof. Dr. H. M. Hafez (Institute of Poultry Diseases, Faculty of Veterinary Medicine, Free University Berlin, Germany)
Date of oral exam: 25 April, 2013
Dedicated to
My wife, Eskedar Hailegebral
&
My children, Heldana and Yafet
Table of Contents
Table of Contents ... V Publications ... VII List of abbreviations ... IX List of Figures ... XIII List of Tables ... XIV
1. Introduction ... 1
2. Literature review ... 4
2.1. IBDV genomic organization and functions ... 4
2.2. Pathobiology of IBDV ... 8
2.2.1. Pathogenetic mechanisms ... 8
2.2.1.1. Polyprotein (PP) cleavage sites ... 8
2.2.1.2. Capsid and polymerase proteins ... 8
2.2.1.3. Nonstructural protein ... 10
2.2.2. IBDV host cell receptor and virus entry ... 11
2.2.3. Pathogenesis of infectious bursal disease ... 11
2.2.4. Clinical disease and pathology ... 14
2.3. Immune responses to IBDV ... 16
2.3.1. Innate immunity ... 16
2.3.2. Humoral immunity ... 17
2.3.3. Cellular immunity ... 18
2.4. Field evolution and molecular epidemiology of IBDV ... 19
2.4.1. IBDV reassortment ... 20
2.4.2. IBDV recombination ... 22
2.4.3. IBDV quasispecies and reversion to virulence ... 24
2.5. Diagnostic methods... 25
2.5.1. Embryo inoculation ... 25
2.5.2. In vitro virus propagation ... 25
2.5.3. Immunological methods ... 25
2.5.4. Molecular characterization ... 26
2.6. Vaccines and vaccination against IBDV ... 28
2.7. DNA vaccines ... 32
2.7.1. Stimulation of immune cells by DNA vaccines ... 32
2.7.2. IBDV DNA vaccines ... 33
2.8. Adjuvants ... 34
2.8.1. Cytokine adjuvants for avian viral vaccines ... 35
2.8.1.1. Chicken IL-2 and IFN-γ as adjuvants for IBDV-DNA vaccines ... 40
2.8.2. Toll like receptor (TLR) ligands as adjuvants ... 40
2.9. Microparticulate vaccine and adjuvant carrier systems ... 42
2.9.1. Enhancing specific immunity by PLGA MPs ... 45
3. Goals and objectives ... 47
4. Molecular evidence of very virulent infectious bursal disease virus in chickens in Ethiopia ... 48
5. Mucosal application of cationic poly(D, L-lactide-co-glycolide) microparticles as carriers of DNA vaccine and adjuvants to protect chickens against infectious bursal disease ... 51
6. Discussion and conclusions ... 81
6.1. Molecular epidemiology of IBDV field isolates ... 81
6.2. Immune responses induced by candidate IBDV DNA vaccines and correlation to protection ... 85
6.3. Do molecular adjuvants enhance protectivity of an IBDV DNA vaccine? ... 86
6.4. Cationic PLGA MPs as particulate carriers for DNA vaccine and molecular adjuvants ... 88
6.5. Promise of cationic PLGA MPs in improving an IBDV DNA vaccine ... 89
6.6. Concluding remarks and future perspectives ... 92
7. Summary ... 94
8. Zusammenfassung ... 96
9. References ... 99
10. Acknowledgements ... 143
Publications
Research articles:
NEGASH, T., E. GELAYE, H. PETERSEN, B. GRUMMER u. S. RAUTENSCHLEIN (2012):
Molecular evidence of very virulent infectious bursal disease virus in chickens in Ethiopia.
Avian Dis 56 :605-610.
NEGASH, T., M. LIMAN u. S. RAUTENSCHLEIN (Vaccine revised):
Mucosal application of cationic poly(D, L-lactide-co-glycolide) microparticles as carriers of DNA vaccine and adjuvants to protect chickens against infectious bursal disease
Oral presentations at scientific meetings:
NEGASH, T., B. GRUMMER u. S. RAUTENSCHLEIN (2012):
Molecular identification and differentiation of infectious bursal disease viruses (IBDVs) in field outbreaks.
82. Fachgespräch über Geflügelkrankheiten, Hannover, Germany; November 2012.
NEGASH, T.u. S. RAUTENSCHLEIN (2013):
Does mucosal application of cationic poly(D, L-lactide-co-glycolide) (PLGA) microparticles as carriers of DNA vaccine and adjuvants enhance immunity against infectious bursal disease virus?
23rd Annual meeting of the society for virology, Kiel, Germany; March 2013.
Poster presentations at scientific meetings:
NEGASH, T., B. GRUMMER u. S. RAUTENSCHLEIN (2011):
Molecular evidence of very virulent infectious bursal disease virus in chickens in Ethiopia.
21st Annual meeting of the society for virology, Freiburg, Germany; March 2011.
NEGASH, T u. S. RAUTENSCHLEIN (2011):
Development of new generation vaccination strategies against infectious bursal disease virus of chickens.
4th Graduate school day, Bad Salzdetfurth, Germany; November 2011.
NEGASH, T. u. S. RAUTENSCHLEIN (2012):
Mucosal application of cationic PLGA microparticles as carriers of DNA vaccines and adjuvants enhances viral clearance in SPF chickens after IBDV challenge.
XIIth Avian immunology research group conference. Roslin institute, Edinburgh, UK August 2012.
List of abbreviations
aa Amino acids
Abs Antibodies
AC-ELISA Antigen capture ELISA
Ag Antigen
AIV Avian influenza virus
aMPV Avian metapneumovirus
APCs Antigen presenting cells
BF bursa of Fabricius
CAM Chorioallantoic membrane
CD Cluster of differentiation
CEFs Chicken embryo fibroblasts
chIFN Chicken interferon
cHsp90 Chicken heat shock protein 90
cIBDV Classical infectious bursal disease
CMI Cell mediated immunity
CMV Cytomegalovirus
CpG-ODN CpG-deoxynucleoside
CTAB Cetyltrimethylammonium bromide
DCs Dendritic cells
DNA Deoxyribonucleic acid
dpi Days postinfection
dsRNA Double stranded RNA
ELISA Enzyme-linked immunosorbent assay FDCs Follicular dendritic cells
GC Germinal center
GILZ Glucocorticoid-induced leucine zipper GM-CSF Granulocyte-macrophage cell stimulating factor
Gzm A Granzyme A
HA Hemagglutinin
HI Hemagglutination inhibition HIV Human immunodeficiency virus
HN Hemagglutinin-neuraminidase
hVP2 Hypervariable region of VP2 HVT Herpesvirus of Turkeys
IBD Infectious bursal disease IBDV Infectious bursal disease virus
IBD-ICX IBD immune complex
IBV Infectious bronchitis virus IFN-γ Interferon-γ
Ig Immunoglobulin
iIELs Intraepithelial lymphocytes
IL-2 Interleukin-2
IL-2R IL-2 receptor
ILTV Infectious laryngotracheitis virus
IM Intramuscular
iNOS Inducible NO synthase
JAK/STAT Januskinase/signal transducers and activators of transcription
MAB Maternal Ab
mAbs Monoclonal Abs
MDV Marek’s disease virus
MHC Major Histocompatibility complex
MPs Microparticles
mRNA Messanger RNA
ND Newcastle disease
NDV Newcastle disease virus
NF-κB Nuclear factor kappa-light-chain-enhancer of activated B cells
NK cells Natural killer cells
NO Nitric oxide
nt Nucleotide
OAS 20-50-oligoadenylate synthetase
P Projection
PAMPs Pathogen-associated molecular patterns PBL Peripheral blood lymphocytes
PCR Polymerase chain reaction
PEI Polyethylenimine
PFN Perforin
pi Postinfection
PI3K)/Akt Phosphatidylinositol 3-kinase
PKR Protein kinase R
PLGA poly(D, L-lactide-co-glycolide)
PP Polyprotein
PRRs Pathogen recognition receptor
pVP2 Precapsid VP2
qRT-PCR Quantitative RT-PCR
rAdV Recombinant Adenovirus
RFLP Restriction fragment length polymorphism rFPV Recombinant fowlpox virus
RNA Ribonucleic acid
RT-PCR Reverse transcriptase PCR
SPF Specific pathogen free
SRs Scavenger receptors
SVPs Subviral particles
Th1 T helper-1
TLRs Toll-like receptors
tMRCA Time of the most recent common ancestor TNF-α Tumor necrosis factor-α
Tregs Regulatory T-cells
VDAC2 Voltage-dependent anion channel 2 VLPs Virus-like particles
VNT Virus neutralization test
VP2 Virus protein 2
vvIBDV Very virulent IBDV
vvVP2 Very virulent VP2
List of Figures
Chapter 2
Fig. 1. Method of vaccine (DNA/protein) microencapsulation in PLGA microspheres using the w/o/w emulsion solvent evaporation method ... 44 Chapter 5
Fig. 1. Quantification of postchallenge chIFN-α mRNA expression levels in the bursa at 3 (A), and 7 dpi (B); and chIL-4 mRNA expression at 3 (C) and 7 dpi (D). ... 78 Fig. 2. Quantification of postchallenge intrabursal CD4+ T-cells at 3 (A),
and 7 dpi (B); and CD8+ T-cells at 3 (C) and 7 dpi (D). ... 80
List of Tables
Chapter 2
Table 1. Functions of IBDV proteins ... 7
Table 2. Examples of natural reassortant IBDV isolates ... 21
Table 3. Examples for natural recombinant IBDV isolates ... 23
Table 4. Experimental IBDV vaccines ... 31
Table 5. Improvement of the protective efficacy of avian viral vaccines after co-administration of plasmid encoded avian cytokines ... 38
Table 6. Examples for avian cytokines delivered by live viral vectors as adjuvants for avian viral vaccines ... 39
Chapter 5 Table 1. Experiment 1: Evaluation of protection conferred by IBDV DNA vaccinations in comparison to a live IBDV vaccine ... 70
Table 2. Experiment 2: Evaluation of protection induced by IBDV DNA vaccination in combination with molecular adjuvants ... 72
Table 3. Experiment 3: Study design showing experimental groups and the timing of vaccine and adjuvant application ... 74
Table 4. Experiment 3: Determination of the protective efficacy of MP based DNA vaccine and adjuvants after mucosal application to SPF chickens ... 75
1. Introduction
Immunosuppressive pathogens are major constraints of poultry production. They can affect the efficacy of the immune system, which leads to vaccination failure and increased susceptibility to many pathogens. Infectious bursal disease virus (IBDV) is a relevant immunosuppressive virus of chickens. It is a dsRNA virus targeting primarily the immature IgM+ B-cells residing in the bursa of Fabricius (BF), which is a primary lymphoid organ in avian species. Worldwide, the poultry industry has encountered heavy economic losses associated with very virulent (vv) IBDV strains during the last several years. These strains may cause high mortality in affected chicken flocks and severe immunosuppression that involves both innate and adaptive immune responses.
During recent years, significant progress has been made to understand the molecular epidemiology of vvIBDV. After the emergence of the virus in Europe in the late 1980’s, it was reported to have spread to several poultry producing regions worldwide. By the mid-1990’s, almost 80% of the World Organization for Animal Health (OIE) member countries have reported the occurrence of vvIBDV based on pathological and molecular investigations from field outbreaks. A recent study addressing the global molecular epidemiology of IBDV from four continents including Africa showed that 60-76% of IBDV isolates were vvIBDVs. However, limited information is available regarding the molecular epidemiology of IBDV, particularly that of vvIBDV in Africa. Although an acute infectious bursal disease (IBD) infection was suspected in major field outbreaks involving commercial farms and breeding centers in Ethiopia in recent years, the nature and epidemiology of the virus remain unknown.
Effective vaccination programs in combination with biosecurity are vital to control IBD. Inactivated vaccines are applied mainly to breeder flocks and may require multiple boosts to induce strong humoral immunity. Conversely, live attenuated vaccines are applied to layers and broilers and stimulate not only humoral immunity but also cell mediated immunity (CMI). Major challenges of live IBDV vaccines
include the possible risk of reversion to more virulent IBDVs and generation of chimeric viruses by exchange of viral segments between vaccinal and wild IBDV strains.
New generation IBDV-subunit vaccines may circumvent some of these problems and provide protective levels of immunity. DNA vaccines may be good candidates due to their ability to elicit both humoral and cellular immunity. Nevertheless, they did not provide protective levels of immunity in many studies and may require well characterized adjuvants to enhance their immunogenicity and protection. A number of studies have demonstrated that co-administration of chicken interleukin-2 (chIL-2) and synthetic unmethylated oligodeoxynucleotides (ODN) containing cytosine- guanosine in succession (CpG-ODN) improved IBDV DNA vaccine efficacy. CpG- ODN stimulates the host defense system by mimicking host invasion by pathogens to keep antigen presenting cells (APCs) in a state of alert. Generally, both molecular adjuvants have multifunctional immunoregulatory properties and are known to activate dendritic cells, macrophages, B- and T-cells and they can enhance the expression of costimulatory molecules by these cells. This may enhance Ag presentation and subsequent immunological responses to the co-administered IBDV DNA vaccines.
Despite measurable progress that has been made in developing DNA vaccines, further improvements are required for possible large scale applications. Vaccines and adjuvants can be made more effective by employing appropriate particulate delivery systems to enhance the uptake and processing by APCs. Microparticles (MPs) prepared from biodegradable poly(D, L-lactide-co-glycolide) (PLGA) polymers are one of the most extensively characterized particulate carriers for delivering vaccines and adjuvants either parenterally or mucosally to enhance systemic and mucosal immunity.
The goals of this study were to determine the molecular epidemiology of recent IBDV isolates in Ethiopia and furthermore to develop and improve IBDV DNA vaccines by using adjuvants and MPs as mucosal vaccine and adjuvant carriers.
The objectives of the first part of this study were to evaluate the pathogenicity of Ethiopian IBDV field isolates experimentally and to characterize their molecular nature by investigating the hypervariable region of the virus protein (VP) 2 (hVP2) and the 5` two thirds of VP1.
The objectives of the second part of the thesis were to assess the immunogenicity and protective efficacy of candidate recombinant IBDV DNA vaccines that encode the vvIBDV-VP2 genes of a selected Ethiopian and reference IBDV strain; we included the molecular adjuvants, CpG-ODN and plasmid encoded chIL-2 to enhance protection. Furthermore, we evaluated cationic PLGA MPs as a carrier for mucosal delivery of the DNA vaccine and adjuvants to improve the efficaciousness of the IBDV DNA vaccine.
2. Literature review
IBDV is one of the economically most important immunosuppressive viruses of chickens. IBDV is an Avibirnavirus and belongs to the family of Birnaviridae (BROWN 1989). Two serotypes of the virus have been described. Serotype 1 IBDV strains are pathogenic to chickens (MÜLLER et al. 2003; VAN DEN BERG et al. 2004), whereas serotype 2 strains are non-pathogenic (MCFERRAN et al. 1980). Serotype 1 IBDV isolates comprise the variant, calssical virulent and vvIBDV strains, which greately differ in their pathogenicity to chickens. Variant IBDVs do not cause mortality, whereas the classical strains cause up to 20% mortality (MÜLLER et al. 2003).
vvIBDV causes mortality exceeding 50% in susceptible chickens (CHETTLE et al.
1989; BERG et al. 1991; MÜLLER et al. 2003).
2.1. IBDV genomic organization and functions
IBDV is a non-enveloped virus with a bipartite dsRNA genome (DOBOS et al. 1979;
MULLER et al. 1979). The main open reading frame of genome segment A encodes a polyprotein (PP) (NH2-pVP2-VP4-VP3-COOH) that is cleaved by the virus encoded protease into pre-capsid virus protein (VP)2, VP4 and VP3 within infected cells (BIRGHAN et al. 2000; LEJAL et al. 2000). The pre-capsid VP2 undergoes defined sequential C-terminal cleavage by VP4 (viral protease) (SANCHEZ u. RODRIGUEZ 1999), host protease (puromycin-sensitive aminopeptidase) (IRIGOYEN et al. 2012), and by the endopeptidase activity of VP2 (LUQUE et al. 2007; IRIGOYEN et al.
2009) to release the mature VP2 protein. Other small peptides released during the PP processing such as pep46 (46 aa) remain associated with the outer capsid (CHEVALIER et al. 2005), but can not be visualized in IBDV particles by X-ray crystallography (COULIBALY et al. 2005). The N-terminus moiety of pep46 bears a positively charged hydrophobic domain and may be responsible for virus penetration into the cytoplasm of infected cells (GALLOUX et al. 2007). The C-terminal moiety of pep46 may assist in the formation of larger pore sizes to enhance viral entry into infected cells (GALLOUX et al. 2010).
The VP2 crystal structure indicates three domains: the base (B), shell (S), and projection (P) domains (COULIBALY et al. 2005; GARRIGA et al. 2006; LEE et al.
2006). The B and S domains are formed by the conserved N-and C-terminal stretches of VP2. The P domain is the middle part containing the host cell receptor binding motifs and the hypervariable region of VP2 (hVP2). The hVP2 harbours antigenic major hydrophilic peak A (amino acid; aa 212-224) and B (aa 314-325) (SCHNITZLER et al. 1993) that form loops PBC (aa 219-224) and PHI (aa 316-324) (COULIBALY et al. 2005), respectively. The minor hydrophilic peak 1 (aa 248-254) and peak 2 (aa 279-290) in the hVP2 form loops PDE (aa 249-254) and PFG (aa 279- 284) (COULIBALY et al. 2005).
The IBDV outer capsid is composed of a single shell of 260 trimeric spikes formed by the P domain of VP2 radially projected from the capsid, and organized in a T=13 icosahedral lattice (BOTTCHER et al. 1997). This organization is determined by the electrostatic interactions of precapsid VP2 with the VP3 C-terminal residues (SAUGAR et al. 2010). The expression of the mature VP2 alone as a recombinant protein forms dodecahedral T=1 subviral particles (SVPs) containing 20 VP2 trimers (CASTON et al. 2001; GARRIGA et al. 2006), whereas precapsid VP2 expression forms a tubular structure (CASTON et al. 2001).
The last five acidic residues at the C-terminus of VP3 interact with pVP2 during particle morphogenesis for correct capsid assembly (CHEVALIER et al. 2004;
SAUGAR et al. 2005; LUQUE et al. 2009). The sixteen C-terminal residues of VP3 interact with VP1 (TACKEN et al. 2002; MARAVER et al. 2003; GARRIGA et al.
2007). VP3 binding to the genomic dsRNA and VP1 forms the ribonucleoprotein complex (LUQUE et al. 2009).
The VP5 protein is encoded by another open reading frame on segment A that partially overlaps with the 5’ end of the PP gene. It is a host membrane-associated and highly basic protein with a cytoplasmic N-terminus and an extracellular C- terminal domain (LOMBARDO et al. 2000).
Segment B encodes the polymerase (SAUGAR et al. 2010), which is present in the virion as a free protein or covalently linked to the 5′ ends of both genome segments (VPg) (MÜLLER u. NITSCHKE 1987). PAN et al. (2007) has recently characterized the VP1 crystal structure. VP1 has three domains: the N- terminus (aa 1-167), central polymerase (aa 168-658) and C-terminal (aa 659-878) regions. The N-terminus of VP1 is involved in protein priming as it possesses the putative guanylylation site (XU et al. 2004; PAN et al. 2007). The central polymerase domain folds into a right-hand shape (fingers-palm-thumb) structure. The five RNA polymerase motifs (C, A, B, D and E) are located in the palm region of the polymerase. Each of the aa motifs function during virus replication, for example, in nucleotide recognition and binding ( e.g. motifs A, B & F), phosphoryl group transfer (A & C), in a metal ion like Mn2+ and Mg2+ binding (C), nucleotide guidance to active sites (D), and primer gripping (E) (PAN et al. 2007). Motif C forms the polymerase active site (SHWED et al. 2002;
PAN et al. 2007). The finger sub-domains contain polymerase motifs F and G that are involved in virus replication (PAN et al. 2007).
The 5’ non-coding regions of both segments contain promoter elements (NAGARAJAN u. KIBENGE 1997) as well as 18S rRNA binding element and play roles in the initiation of virus replication (MUNDT u. MÜLLER 1995).
No N-linked glycosylation of any of the virion proteins has been detected. The biological functions of the different proteins of IBDV are summarized in table 1.
Table 1: Functions of IBDV proteins
Proteins Functions References
VP2 Host receptor binding (OGAWA et al. 1998)
Contains neutralizing epitopes (AZAD et al. 1987) Virulence determinant (BRANDT et al. 2001) Cell culture adaptation (MUNDT 1999)
Apoptosis (FERNANDEZARIAS et al.
1997)
Endopeptidase activity (IRIGOYEN et al. 2009)
VP3 Chaperone activity (CHEVALIER et al. 2004)
Antiapoptosis by interacting with PKR (BUSNADIEGO et al.
2012)
Suppresses hosts RNA silencing mechanism (VALLI et al. 2012) Transcriptional activator (TACKEN et al. 2002) Forms ribonucleoprotein complex (LUQUE et al. 2009) VP4 Viral protein processing (viral protease) (BIRGHAN et al. 2000)
Trans-activate VP1 synthesis (BIRGHAN et al. 2000) Suppresses type I IFN by interacting with
GILZ
(LI et al. 2013b)
VP5 Early antiapoptotic effects (LIU u. VAKHARIA 2006) Late apoptotic effects (LI et al. 2012)
VP1 Viral polymerase (SAUGAR et al. 2010)
Virulence determinant (LE NOUEN et al. 2012)
2.2. Pathobiology of IBDV
2.2.1. Pathogenetic mechanisms
Early studies revealed segment A as the sole determinant of IBDV virulence.
Nevertheless, the molecular basis of IBDV pathogenicity likely depends on the synergism between both segments of the virus (BOOT et al. 2000; BRANDT et al.
2001; BOOT et al. 2005; ESCAFFRE et al. 2012).
2.2.1.1. Polyprotein (PP) cleavage sites
To determine the molecular determinants of IBDV virulence, segment A and B of several strains were sequenced and analysed (XIA et al. 2008). vvIBDV isolates with aa substitutions adjacent to the pVP2 maturation site (aa 441–442), close to the VP2–VP4 (aa 512–513) and VP4–VP3 (aa 755–756) cleavage sites (BROWN u.
SKINNER 1996; CHEVALIER et al. 2004; XIA et al. 2008) and at the C-terminus of VP3 (CHEVALIER et al. 2004a) were reported. An aa substitution at the predicted protease active-site of the VP4 gene of one of the European highly virulent strains UK661 was suspected to contribute to its virulence. Generally, these mutations may speed up the proteolytic activity of VP4, the processing and maturation of VP2, and the capsid assembly efficiency. This may enhance the replication of vvIBDV and lead to higher yields of virus particles in the host tissues (YAMAGUCHI et al. 1997b;
CHEVALIER et al. 2004; XIA et al. 2008).
2.2.1.2. Capsid and polymerase proteins
Exposed at the virion surface, VP2 contributes to IBDV virulence (YAMAGUCHI et al.
1996b; BOOT et al. 2000). Most vvIBDV isolates have key aa marker of virulence at the hVP2 region (BROWN et al. 1994; BROWN u. SKINNER 1996; YAMAGUCHI et al. 1997a; ISLAM et al. 2001). Specific aa residues responsible for tissue culture
adaptation, virulence and cell tropism have been mapped onto the VP2 gene using reverse genetics (MUNDT u. VAKHARIA 1996). Amino acid mutations at positions 253 (Q →H), 279 (D →N) and 284 (A →T) in the VP2 were generated by reverse genetics and completely attenuated the vvIBDV isolates. The modified viruses propagated well in cell-culture and showed reduced pathogenicity in chickens (LIM et al. 1999; MUNDT 1999; VAN LOON et al. 2002). The adaptation of vvIBDV strain OKYM to cell culture introduced comparable aa mutations at positions 279 (D →N) and 284 (A →T) (YAMAGUCHI et al. 1996b). These mutations were also detected in other cell culture-adapted classical and vvIBDV strains (YAMAGUCHI et al. 1996a).
A single aa mutation at position 253 (H →Q/N) in VP2 markedly increased the virulence of an attenuated IBDV strain (JACKWOOD et al. 2008).
Substantial molecular evidence showed the role of segment B in IBDV pathogenicity as well (LIU u. VAKHARIA 2004; BOOT et al. 2005). Field reassortant IBDV isolates comprising segment A of vvIBDV and segment B of attenuated strains show reduced pathogenicity under field conditions and when evaluated experimentally in susceptible chickens compared to typical vvIBDV isolates (LE NOUEN et al. 2006).
This field observation of reduced pathogenicity was proven with a classical virulent IBDV that was genetically modified to contain the VP2 gene from a vvIBDV strain.
This virus failed to cause morbidity and mortality in SPF chickens unless the genetically modified virus contained a typical segment B from vvIBDV (LIU u.
VAKHARIA 2004). Recombinant IBDVs generated by exchanging different regions of VP1 of a vvIBDV with their counterparts of VP1 of an attenuated IBDV in a vvIBDV segment A background showed reduced virulence in SPF chickens (LE NOUEN et al. 2012). These viruses replicated to reduced virus titers in the bursa indicating the important role of VP1 in vvIBDV virulence. Putative virulence marker aa residues across the VP1 protein were predicted (YU et al. 2010). JACKWOOD et al. (2012) recently described the presence of aa motif TDN at positions 145, 146 and 147 in the VP1 gene of all vvIBDVs tested, and their absence in non-vvIBDV isolates. An IBDV strain designated as 94432 that maintained all virulence markers aa in its VP2 gene like other prototype vvIBDVs failed to cause mortality in chickens. The presence of
threonine (T) at position 276 in the exposed hydrophobic groove of the finger domain of VP1 has been then shown to contribute to the reduced pathogenicity. Exchanging this aa at position 276 from T →V restores the pathogenicity of the molecularly cloned virus (ESCAFFRE et al. 2012).
2.2.1.3. Nonstructural protein
VP5 is believed to play an important role in IBDV pathogenicity; nonetheless it is not essential for viral replication (MUNDT et al. 1997). In vitro, the VP5 protein showed extensive accumulation within the plasma membrane of infected cells at later time points during IBDV replication (LOMBARDO et al. 2000). VP5 may have anti- apoptotic effects on infected cells during early time points (LIU u. VAKHARIA 2006), and at the same time it may trigger apoptosis at later stages of IBDV replication in infected cells (YAO u. VAKHARIA 2001). Furthermore, it was suggested that VP5 plays a significant role in virus release from infected cells by triggering cell death (WU et al. 2009).
IBDV infection induces the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway during the early phase of IBDV replication in DF-1 cells. The regulatory subunit of PI3K may suppress premature apoptosis of infected cells, which may sustain IBDV replication and production of large quantities of infectious virus progeny (WEI et al. 2011). When PI3K was inhibited most of the infected cells showed early apoptotic signatures (WEI et al. 2011). Voltage-dependent anion channel 2 (VDAC2) and VP5 (aa residues from 1-50) interaction later during IBDV infection may be responsible for the release of infectious virus particles from infected cells (LI et al. 2012). VDAC2 is a host molecule, which forms pores in the outer mitochondrial membrane and is involved in apoptosis (SHOSHAN-BARMATZ et al.
2010). Blocking VDAC2 by small interfering RNA inhibited IBDV-induced apoptosis, reduced virus release and virus titer.
2.2.2. IBDV host cell receptor and virus entry
IBDV may bind to host cell proteins such as N-glycosylated polypeptide(s) expressed on the cell membrane of immature IgM+ B-cells during viral entry process (OGAWA et al. 1998; LUO et al. 2010). A pore forming peptide of the virus (pep46), which is associated with the outer capsid of the IBDV particle, may facilitate viral entry into the cytoplasm of infected cells (GALLOUX et al. 2007; GALLOUX et al. 2010). A lipid raft mediated endocytic mechanism was suggested based on the results of an in vitro study to support entry of attenuated IBDV to the cells (YIP et al. 2012). An in vitro binding assay showed the interaction of subviral particles (SVPs) derived from the mature VP2 with the chicken heat shock protein 90 (cHsp90) expressed on the surface of DF-1 cells (LIN et al. 2007). This binding of IBDV to cHsp90α was inhibited by cHsp90α interfering microRNAs that resulted in reduced virus titer (YUAN et al.
2012). The binding of VP2-SVP with α4β1 integrin suggested an integrin-binding domain in VP2 of IBDV, which was later confirmed that aa residues at positions 234- 236 are responsible (DELGUI et al. 2009). This integrin-binding motif is conserved in all IBDV strains. A single point mutation within this motif might completely abrogate the binding of SVPs to cells and virus infectivity (DELGUI et al. 2009). Immature B- cells have abundant α4β1 on their surfaces (ROSE et al. 2002).
2.2.3. Pathogenesis of infectious bursal disease
Natural IBDV infection occurs by the oral route. Other mucosal routes have been demonstratedfor experimental IBDV infection. The mononuclear phagocytic cells and lymphoid cells of the gut mucosa may serve as targets for IBDV infection and replication (MÜLLER et al. 1979). Infected macrophages transport the virus to the bursa of Fabricius (BF), the prime target organ for extensive IBDV replication in the cytoplasm of intrabursal IgM+ B-cells (KAUFER u. WEISS 1980; HIRAGA et al.
1994). Virus dissemination to other lymphoid organs such as to the thymus, bone marrow, spleen, Peyer's patches, cecal tonsils, and Harderian glands may take place
mainly during vvIBDV infection of susceptible chickens (ETERRADOSSI u. SAIF 2008). The cecal tonsils and bone marrow may serve as non-bursal lymphoid tissues supporting virus replication at later time points (ELANKUMARAN et al. 2002).
As early as 48 hr pi, IBDV infection induces prominent inflammation in the BF. By day 3 to 4 pi, all bursal IgM+ B-cells are infected and show cytolytic changes (CHEVILLE 1967). vvIBDV strains such as UK661 can infect Bu-1+ cells and IgY+ B-cells in several lymphoid tissues indicating both immature and mature B-cells can be infected (WILLIAMS u. DAVISON 2005), whereas classical virulent and variant IBDVs mostly target immature B-cells. Between 7 and 21 dpi, IBDV infection results in significant reduction in the number of the B subpopulation compared to the A subpopulation of IgM+ B-cells as determined by flow cytometric analysis (PETKOV et al. 2009). These two phenotypes differed on their cell size and granularity as well as showed differential expression levels of Lewis(x), IgM, Bu-1b, and MUI78 surface antigens. It becomes apparent that macrophages are infected with IBDV and in vitro studies indicated rapid virus replication with altered in vitro phagocytic activity of such cells (KHATRI u. SHARMA 2009b). Other cells like the bone marrow-derived mesenchymal stem cells may be infected with IBDV (KHATRI u. SHARMA 2009b).
The haemopoietically derived reticular cells, which reside in the antigen-trapping zone of the spleen (ellipsoid and periellipsoidal white pulp), were found to be more susceptible to IBDV. The reticular cells of mesenchymal origin, which reside in the bursal cortex, and periarteriolar lymphoid sheaths, germinal center (GC) and red pulp of the spleen were relatively resistant to IBDV (BIRO et al. 2011). Bursal follicular dendritic cells disappeared during IBDV infection probably due to lack of an intact B- cell microenvironment (JEURISSEN et al. 1998; KABELL et al. 2006).
Generally, the sequellae of IBDV infections such as severity of clinical signs, organ lesions and immunosuppression correlate with the status of immunity, age and genetic background of affected chickens and with the virulence of the infecting virus strain (BERG 2000). SPF chickens infected with vvIBDV develop an earlier onset of mortality and more severe bursal lesions compared to broiler chickens with MAB and vaccinated chickens (HASSAN et al. 2002; ARICIBASI et al. 2010). Infections by
virulent IBDV resulted in an earlier onset and higher production of IFN-γ in the bursa of young SPF chickens compared to their older counterpart. Massive interferon-γ (IFN-γ) production from T-cells infiltrating the bursa during an acute IBDV infection (ELDAGHAYES et al. 2006; RAUW et al. 2007) further activates macrophages to release proinflammatory cytokines such as interleukin-6 (IL-6) and also nitric oxide (NO), which may aggravate bursal lesions (KIM et al. 1998). IFN-γ is suggested to be a potent apoptosis inducer in IBDV infected or adjacent healthy B-cells (LAM 1997;
TANIMURA u. SHARMA 1998). A massive mast cell influx detected in the bursa of SPF chickens infected with vvIBDV may aggravate bursal lesions as typical indicators of acute hypersensitivity responses were observed in the bursa of such chickens (WANG et al. 2008; WANG et al. 2012a). These cytokine mediated bursal lesions may result in an early onset of severe immunosuppression in younger chickens (RAUTENSCHLEIN et al. 2007). Similar patterns of cytokine production and bursal lesions were detected in SPF layer type chickens infected with virulent IBDV compared to infection of age-matched 3 weeks old broiler type chickens (ARICIBASI et al. 2010).
Bursal lesions with B-cell depletions lead to severe humoral immunosuppression (SHARMA et al. 1994; SHARMA et al. 2000). When susceptible neonatal chickens younger than two weeks of age are infected, they may lose the entire bursal B-cells, which result in permanent immunologic damage (HUDSON et al. 1975; WITHERS et al. 2005). Cytokine dysregulation may cause suppression of innate and cellular immunity (RAUW et al. 2007). This was elucidated as recombinant chicken IFN-γ (rchIFN-γ) inhibited an in vitro proliferation of naïve peripheral blood lymphocytes (PBL) or splenocytes (RAUW et al. 2007). Lymphocytes harvested from the blood and lymphoid organs of an IBDV infected or live IBDV vaccinated chickens showed reduced lymphoproliferative responses when stimulated by conventional mitogens (MAZARIEGOS et al. 1990; SHARMA et al. 2000). This inhibition has been detected during early time points after IBDV infection coinciding with an increased IFN-γ response. Recently, in vitro study described the interaction of IBDV VP4 with the chicken glucocorticoid-induced leucine zipper (cGILZ) that inhibited the transcription
of NF-κB with a subsequent suppression of the innate immunity (LI et al. 2013b).
Others suggested T-cells with regulatory property may mediate cellular immunosuppression (KIM et al. 1998; SHANMUGASUNDARAM u. SELVARAJ 2011). IBDV infection induced mucosal immunosuppression denoted by reduced intraepithelial lymphocytes (iIELs) and their in vitro cytotoxicity activity (KUMAR et al.
1998). A reduced intestinal secretory IgA+ B-cells were also detected (WANG et al.
2009a).
The immunological functions of both B- and T-cells may be restored during recovery of infected chickens (KIM et al. 1999; SHARMA et al. 2000). The mechanisms of B- cell functional restoration were described, whereas of T-cells remained unclear.
Bursal stem cells, which survive IBDV-induced depletion, proliferate to generate new and larger bursal follicles. They became repopulated with IgM+ B-cells, and Bu-1+
cells expressing IgM or IgY (WILLIAMS u. DAVISON 2005) and dendritic-like cells (WITHERS et al. 2005). The B-cells in these follicles may undergo immunoglobulin (Ig) gene (hyper)conversion for Ig diversity and sustain specific immunological functions (WITHERS et al. 2005; WITHERS et al. 2006). Previous study indicated the strong expression of the Lex and chB1 genes in the recovering follicles as indicators of Ig gene (hyper)conversion (IVAN et al. 2001). Medullary B-cells surviving IBDV infection form small follicles, which lack Ig gene (hyper)conversion. Birds with only small follicles do not produce Abs against IBDV or other Ags, and such chickens may be in a state of permanent immunosuppression (WITHERS et al. 2006).
2.2.4. Clinical disease and pathology
Chickens infected between 3 and 6 weeks of age develop the most severe clinical signs of IBD (ETERRADOSSI u. SAIF 2008). Susceptible chickens exposed to vvIBDV and classical virulent strains show a sudden onset of clinical disease within 2-3 days of exposure, characterized by severe depression and ruffled feathers (VAN DEN BERG et al. 2004). Chickens younger than 2 weeks of age and birds older than 6 weeks rarely develop clinical signs (VERVELDE u. DAVISON 1997).
Experimentally, broilers especially with higher MAB levels may not show clinical signs or mortality when infected with vvIBDV. Mortality peaks at 4 dpi with virulent strains. A rapid recovery after 5-7 dpi is a prominent feature of acute IBD (VAN DEN BERG et al. 2000a). In general viral shedding in the faeces of naturally infected or live vaccine vaccinated chickens can last up to 2 weeks and viral RNA can be detected by RT-PCR up to 4 weeks (KABELL et al. 2005). Age and immune status of infected chickens (SKEELES et al. 1979; ABDEL-ALIM u. SAIF 2001; IVAN et al.
2005; SAPATS et al. 2005), route of infection and nature of infecting viruses (WINTERFIELD et al. 1972; SKEELES et al. 1979; ELANKUMARAN et al. 2002) influence the development of clinical IBD and virus shedding.
On the first few days after infection, an increased in bursa to body weight ratio is usually observed due to edematous bursae. Occasionally, extensive hemorrhage throughout the entire bursa has been observed in the case of vvIBDV. Compared with a moderately pathogenic strain of the virus, the vvIBDV strains caused a greater decrease in thymic weight index. Generally, hemorrhagic inflammation of the bursa is the main pathological feature of infection by virulent strains, whereas variant strains (e.g. GLS and E/Del) cause rapid bursal atrophy mostly without an inflammatory response (LAM 1997; TANIMURA u. SHARMA 1998). IBDV-induced lymphoid cell depletion is responsible for bursal atrophy as early as 7-8 dpi (CHEVILLE 1967).
Histological bursal lymphoid depletions are comparable between vvIBDV and virulent IBDV infection in the early few days of infection (TANIMURA et al. 1995;
TSUKAMOTO et al. 1995; INOUE et al. 1999; STOUTE et al. 2009), soon followed by heterophilic infiltration. A more virulent IBDV strains caused severe lymphoid depletions in the cecal tonsils, thymus, spleen, and bone marrow. The pathogenicity may correlate with lesion production in non-bursal lymphoid organs. As the inflammatory reaction subsides, cystic cavities develop in the medulla of affected bursal follicles followed by fibrosis in interfollicular areas (ETERRADOSSI u. SAIF 2008). Proliferation of the bursal epithelial layer produced a glandular structure of columnar epithelial cell containing globules of mucin. During recovery stage, scattered foci of lymphocytes appeared in the bursal follicles.
2.3. Immune responses to IBDV
Apart from its immunosuppressive effects, IBDV infection in chickens activates all branches of the immune system. However, the level of activation varies depending on the virulence of infecting strains, age, immune status and genetic background of affected chickens.
2.3.1. Innate immunity
The influx of macrophages, heterophils and mast cells in the bursa of Fabricius constitutes the early innate immune response to IBDV (KHATRI et al. 2005;
PALMQUIST et al. 2006; RAUTENSCHLEIN et al. 2007; WANG et al. 2008). The influx of these cells may be mediated by chemokines (IL-8, iNOS) (KHATRI et al.
2005; ELDAGHAYES et al. 2006; PALMQUIST et al. 2006; RAUTENSCHLEIN et al.
2007; RAUW et al. 2007; RAUF et al. 2011a). Toll-like receptors (TLRs) such as TLR3 and TLR7 expressed by these inflammatory cells detect IBDV nucleic acids.
Their mRNA expressions have been found upregulated during an acute IBDV infection (RAUF et al. 2011a; GUO et al. 2012). These interactions between IBDV and TLR3 or TLR7 have been shown to activate the interferon (IFN) system and also induced proinflammatory cytokines (IL-6, IL-1β, and IL-18) (RAUF et al. 2011a; GUO et al. 2012). The release of these cytokines was suggested to be tightly regulated by NF-κB, whereby its expression was found to be elevated in the bursa during the early phase of IBDV infection (KHATRI u. SHARMA 2006; GUO et al. 2012). The upregulation of IFN-α/β mRNA expression was reported in lymphoid organs and PBL of chickens experimentally infected with virulent IBDV (KIM et al. 1998; RAUF et al.
2011a; MAHGOUB et al. 2012). The expression levels of these cytokines in the bursa differ in relation to the virulence of infecting strains, genetic background and age of infected chickens (ELDAGHAYES et al. 2006; RAUTENSCHLEIN et al. 2007;
ARICIBASI et al. 2010; RAUF et al. 2011a). IFNs may protect chickens against IBDV infection. This was verified experimentally whereby chickens pretreated with
recombinant chicken IFN-α/β and latter challenged with IBDV had reduced challenge virus replication and pathological lesions in the bursa (CAI et al. 2012).
IBDV infection of chicken embryo fibroblasts (CEFs) resulted in upregulation of IFN- inducible 20-50-oligoadenylate synthetase (OAS), IFN regulatory factors, IL-6 and IL- 8 mRNA expression (LI et al. 2007). The OAS and RNase L pathway interferes with viral infection through the cleavage of viral ssRNA, which is one of the recognized viral suppressor activities of OAS (MALATHI et al. 2007). Recombinant type I IFN pretreated CEFs resisted IBDV replication and resulted in reduced viral titer after infection (O'NEILL et al. 2010; CAI et al. 2012).
Nitric oxide released by macrophages may constitute an early host defence against IBDV and promotes the killing of IBDV-infected and possibly virus-free cells (KHATRI et al. 2005; KHATRI u. SHARMA 2006; PALMQUIST et al. 2006; KHATRI u.
SHARMA 2009a).
2.3.2. Humoral immunity
Significant titers of systemic IBDV specific-Abs have been detected in the convalescent sera of chickens that are naturally or experimentally infected with IBDV (ETERRADOSSI u. SAIF 2008). All classes of Igs can be produced, but the Ab response may not protect chickens from antigenetically different IBDV strains.
Neutralizing Abs are directed against the conformation dependent neutralizing epitopes of VP2 (FAHEY et al. 1991; SNYDER et al. 1992). Abs against VP3 (BECHT et al. 1988; FAHEY et al. 1991) and conformation-independent antigenic domains of VP2 (AZAD et al. 1987) are non-neutralizing. Live and inactivated IBDV vaccines may induce vigorous Ab responses in the first few weeks postvaccination (MAAS et al. 2001; ARICIBASI et al. 2010). Compared to cell culture derived strains, bursal and embryo derived strains induce higher neutralizing Ab titers (RODRIGUEZ- CHAVEZ et al. 2002).
Humoral immunity plays a significant role in protection against IBDV. Maternal antibody (MAB) provides passive protection in the first few weeks after hatch (AL- NATOUR et al. 2004). MAB positive chickens developed significantly less bursal lesions than Ab negative chickens after IBDV challenge supporting the role of passive immunity in protection (HASSAN et al. 2002; ARICIBASI et al. 2010). MAB may interfere with the development of an active immune response after IBDV vaccination (RAUTENSCHLEIN et al. 2005a).
Although Ab mediated immunity is crucial against IBDV, an important role of the cell mediated immunity (CMI) is suggested by several groups (RAUTENSCHLEIN et al.
2002a; YEH et al. 2002).
2.3.3. Cellular immunity
During acute IBD, while bursal follicles are B-cell depleted, T-cells accumulate at the site of virus replication (TANIMURA u. SHARMA 1997; KIM et al. 1998; KIM et al.
2000; SHARMA et al. 2000). A notable influx of CD4+- and CD8+ T-cells was detected as early as 1 dpi and peaked at around 7 dpi (KIM et al. 2000). Although viral Ag was cleared by week 3 pi, T-cell influx and activation continued to week 12 pi. No T-cell depletion was detected from the bursa during IBDV infection. However, IBDV particles were detected in intrabursal T-cells (MAHGOUB et al. 2012).
Infiltrating T-cells in the bursa show markers of activation such as upregulated IL-2, major histocompatibility complex (MHC) class II molecules, and IFN-γ mRNA expression (KIM u. SHARMA 2000; RAUW et al. 2007; RAUF et al. 2011b). T-cells are not only involved in bursal recovery by killing virus infected cells, but also contribute to bursal lesions. T-cell compromised SPF-chickens had the highest viral Ag load and milder inflammatory bursal lesions compared to T-cell intact birds after IBDV infection (RAUTENSCHLEIN et al. 2002a). T-cells infiltrating the bursa after IBDV infection expressed higher levels of the mRNA for cell membrane-disrupting proteins such as perforin (PFN) and granzyme A (Gzm A), and other cytolytic molecules such as the high mobility group proteins. PFN and Gzm mediated
cytotoxic activity may contribute to rapid viral clearance from the bursa (RAUF et al.
2011b). The role of T-cells in IBDV protection was supported in vaccination studies with T-cell or B-cell compromised chickens. Chickens depleted of functional T-cells either by neonatal thymectomy or Cyclosporin A treatment showed insufficient protection against IBDV challenge after immunization with an inactivated IBDV vaccine, whereas chickens with intact T-cells had significantly higher IBDV protection rates (RAUTENSCHLEIN et al. 2002b). Chickens with severely compromised Ab- producing ability following treatment with cyclophosphamide were sufficiently protected against IBDV challenge despite the absence of detectable vaccine-induced Abs. This implies that T-cells may have role in protection (YEH et al. 2002).
2.4. Field evolution and molecular epidemiology of IBDV
The first outbreak of infectious bursal disease (IBD) that had occurred in 1957 in a broiler farm near Gumboro, the Delaware area in the USA, was caused by the classical serotype 1 IBDV (COSGROVE 1962). The variant IBDV strains then emerged in the 1980’s in IBDV-vaccinated farms in the Delmarva area and were antigenetically different from the former isolates. In the late 1980’s, vvIBDV emerged in Europe (CHETTLE et al. 1989) and rapidly spread across continental Europe and Asia (LIN et al. 1993; SHCHERBAKOVA et al. 1998), Middle East (PITCOVSKI et al.
1998), South America (DI FABIO et al. 1999), and Africa (ZIERENBERG et al. 2000).
IBDV undergoes genetic variation during its evolution to adapt to new hosts and to escape the host immune responses. Different biological mechanisms may play important roles for the emergence of novel viruses, particularly in segmented RNA viruses, such as IBDV.
Early IBDV isolates frequently showed mutations at the major hydrophilic domains particularly in the loops PBC and PHI, which affected the antigenicity of the strainsand induced vaccination failure (BAYLISS et al. 1990; HEINE et al. 1991; LANA et al.
1992; DORMITORIO et al. 1997). In the past few years, several field IBDV strains
isolated from different geographic areas showed aa substitutions at the minor hydrophilic domains mainly at position 254 (loop PDE) and 284 (loop PFG) (JACKWOOD u. SOMMER-WAGNER 2005; MARTIN et al. 2007; DURAIRAJ et al.
2011; JACKWOOD u. SOMMER-WAGNER 2011). Most of these viruses have been identified from areas where the viruses have been circulating for a long period of time (MARTIN et al. 2007). In the USA, one-third of the investigated field IBDV isolates (out of 300) failed to react with any of the described monoclonal abs (mAbs) that have been used to identify IBDV strains for the last 2 decades, which may reveal the circulation of new IBDV subtypes (DURAIRAJ et al. 2011). A new variant IBDV differing from the Delaware (Del E) variant of the Delmarva Peninsula was identified, which did not react with those mAbs (GELB et al. 2012). IBDV isolates, which contain epitopes of both variant and classical IBDVs in their VP2 genes were demonstrated, which can affect mAb reactivity (JACKWOOD 2012). This may provide an explanation for the increased antigenic and virulence diversity of the recent IBDV isolates. Atypical IBDVs, which harbour aa residues characteristics of variant, classical, and vvIBDV in their VP2 were characterized and showed atypical pathogenicity and reactivity patterns to most of the mAbs (MARTIN et al. 2007).
2.4.1. IBDV reassortment
Genetic reassortment might be accountable for the emergence of vvIBDV in the late 1980’s in Europe (HON et al. 2006). The time of the appearance of the most recent common ancestor (tMRCA) of very virulent (vv) VP2 is approximated around 1960, whereas of vvVP1 around 1980 (HON et al. 2006), in which the latter coincided with the emergence of vvIBDV in the late 1980’s (CHETTLE et al. 1989). Thus a newly appeared vvVP1 from an unidentified avian reservoir was suggested to recombine with an already existing vvVP2 to evolve to the vvIBDV genotype, which then caused massive mortality in Europe (HON et al. 2006). This indicates the independent evolutionary history of the two segments of vvIBDV (ISLAM et al. 2001; LE NOUEN et al. 2006). Recently, several natural reassortant IBDV isolates were characterized
during field outbreaks (Table 2). The most common reassortant IBDVs contain segment A of vvIBDV and segment B from attenuated strains indicating the drawbacks of extensive application of live IBDV vaccines. Attempted experimental generation of reassortant viruses by co-infecting specific pathogen free (SPF) chickens with vvIBDV and attenuated serotype 1 IBDV has failed. The process of reassortment may be more complex in the field than expected and may involve the interactions of several factors: time, environment and vaccine pressure (WEI et al.
2008).
Table 2: Examples of natural reassortant IBDV isolates
Isolates Sources of segments Country Year References Segment A Segment B
Unknown vvIBDV vvIBDV Europe 1980 (HON et al. 2006) SH95 vvIBDV Variant E China Unknown (SUN et al. 2003) 02015.1 vvIBDV Attenuated France Unknown (LE NOUEN et al. 2006) ZJ2000 Attenuated vvIBDV China 2000 (WEI et al. (2006) TL2004 Attenuated vvIBDV China 2004 (WEI et al. 2008)
CA-K785 vvIBDV Serotype 2 USA 2009 (JACKWOOD et al. 2011) KZC-104 vvIBDV Attenuated Zambia 2004 (KASANGA et al. 2012)
2.4.2. IBDV recombination
Natural homologous intragenic recombination is described for many animal viruses (LEE et al. 2013). The risk of live vaccines recombining to generate virulent natural recombinants have been well described, and disease outbreaks associated with these viruses have recently been described for infectious laryngotracheitis virus (ILTV) infections of chickens (LEE et al. 2012). Recombination may lead to antigenetically and genetically diverse IBDV populations and the emergence of novel vvIBDV groups (HON et al. 2008; HE et al. 2009a). It has the potential to alter the interactions of IBDV proteins and the orientation of the capsid domains preventing neutralization by pre-existing Abs, which lead to vaccine failure. Almost all IBDV recombinant viruses identified from field outbreaks are VP2 recombinants (Table 3).
Intrasegment recombination was also detected in segment B of two vvIBDV strains possibly due to the recombination between two vvIBDV donors (HON et al. 2008). A recently isolated IBDV strain, GX-NN-L, has reassortant characteristics, whereby its segment A derived from vvIBDV, and segment B from an attenuated strain. But interestingly, segment B contains putative aa residues typical for vvIBDV isolates (CHEN et al. 2012a). An attenuated vaccinal strain, ViBursaCE, is suggested to be a potential recombinant whereby its segment A is a mosaic between variant (Variant E) and an attenuated French vaccine strain (Rhone-Merieux, strain-CT) (HON et al.
2008).
Table 3: Examples for natural recombinant IBDV isolates
Isolates VP2 recombinants Country Year References SH-h hVP2 from vvIBDV (HLJ-5 strain)
within segment A of an attenuated (D78) strain
China Unknown (HON et al.
2008; HE et al.
2009a) KSH/KK1 hVP2 from vvIBDV (SH.92 strain)
within segment A from an attenuated (D78) strain
Korea 1992/1997 (HON et al.
2008; HE et al.
2009a) 849VB Part of segment A from attenuated
(D78), and the other part from vvIBDV (D6948)
Belgium 1987 (HON et al.
2008) Several
isolates
aa sequences at loop regions PBC and PHI from classical IBDV & aa at PDE
and PFG from variant IBDV
Venezuela Colombia
2001-2005 (JACKWOOD 2012)
Several isolates
aa sequences at loop regions PBC
from variant & PDE and PFG from classical IBDV
Mexico 2004-2011 (JACKWOOD 2012)
157776 aa at positions 294 to 299 from vvIBDV & residues from 222 to 279 from an attenuated strain
Italy 2003 (MARTIN et al.
2007) VP1 recombinants
OE/G2 Segment B recombinant between OKYM & OA/G1 vvIBDV strains
Turkey Unknown (SILVA et al.
2012) Harbin-1 Segment B recombinant between
HLJ-7 or HENAN & GZ/96 vvIBDV strains
China Unknown (HON et al.
2008; SILVA et al. 2012)
2.4.3. IBDV quasispecies and reversion to virulence
The existence of RNA virus quasispecies may have a paramount contribution to virus evolution. An RNA virus population is made up of heterogeneous viruses, which share the consensus sequence but differ from each other by one or many mutations (DOMINGO et al. 1985). In IBDV vaccine and field strains, the quasispecies phenomenon has been described by real time RT-PCR and melting curve analysis (JACKWOOD u. SOMMER 2002; HERNANDEZ et al. 2006). Pre-existing selection pressure, for example, altered host immune status may favor one clone of a virulent virus to overwhelm the virus population to maintain its endemicity (MORIMOTO et al.
1998).
Attenuated live IBDV vaccines are most frequently used to vaccinate commercial chickens. Reversion of these attenuated vaccinal strains to more virulent phenotypes under field and experimental conditions has been frequently reported (YAMAGUCHI et al. 2000; JACKWOOD et al. 2008) possibly due to a lack of IBDV polymerase fidelity during vaccine viral genome replication in the host cells. A tissue culture- adapted IBDV generated by reverse genetics from a vvIBDV strain reverted phenotypically and genotypically to the vvIBDV pathotype after inoculation into SPF chickens and maintained this pathotype afterwards (RAUE et al. 2004). Genetic reversion of vaccine strains is most likely to be one of the mechanisms that may contribute to the dissemination and persistence of virulent IBDV in the chicken population worldwide.
2.5. Diagnostic methods
2.5.1. Embryo inoculation
The inoculation of bursal homogenates from IBDV infected chickens per the chorioallantoic membrane of 9-10 days old embryonated SPF chicken eggs is the most sensitive diagnostic method for virus isolation. The embryos die mostly within 3- 5 days in the case of classical and very virulent viruses (HITCHNER 1970;
ROSALES et al. 1989). Embryos may not die from infection by the variant viruses, yet show hepatic necrosis.
2.5.2. In vitro virus propagation
Adaptation of IBDV field isolates to a cell culture system requires passages in embryonated chicken eggs and subsequent passages in cell culture system further attenuates the virus to the extent that these viruses do not induce bursal lesions (YAMAGUCHI et al. 1996a). The cell culture adapted viruses replicate in primary avian cells such as CEFs (SHI et al. 2009) and continuous cell lines of avian (QT35) and mammalian origins (Vero cells) (LUKERT u. DAVIS 1974). Compared to classic and variant strains, adaptation of the vvIBDV viruses to cell culture has been very difficult.
2.5.3. Immunological methods
ELISA and virus neutralization test (VNT) can be used to determine IBDV Ab levels.
Different IBDV-ELISA procedures have been described for routine diagnostic purposes (MARQUARDT et al. 1980; KECK et al. 1993). Polyclonal based Ag- capture ELISA is more sensitive (HASSAN et al. 1996) compared to the mAb-based ones to detect IBDV Abs (LEE u. LIN 1992; VAN DEN BERG et al. 2004).
Commercially available ELISA systems use either intact viral particles or a mixture of recombinant VP2 based proteins coated plates. VNT is mostly used for research purposes (SKEELES et al. 1979). The VN titers accurately correlate with protection of chickens against IBDV (KNOBLICH et al. 2000). Only VNT allows to differentiate serotype 1 from serotype 2 IBDVs (JACKWOOD et al. 1985).
Determination of the antigenic properties of IBDV field isolates is necessary when recurrent outbreaks are observed in poultry farms that had been previously vaccinated against IBDV. Although in vitro VN tests can be used for detection of antigenic differences between virus strains, in vivo cross protection studies are essential to determine the antigenicity of a virus and complete evaluation of host immune responses (JACKWOOD u. SAIF 1987). In vitro antigenicity is determined by the reaction patterns of IBDV isolates to panels of mAbs, which target conformation dependent neutralizing epitopes at the hydrophilic regions of VP2 (SCHNITZLER et al. 1993; BERG et al. 1996; ETERRADOSSI et al. 1997) using VNT or ELISA (SNYDER et al. 1988; VAKHARIA et al. 1994). By sequence analysis and site- directed mutagenesis, aa residues, which influence the reactivity patterns of IBDVs with specific mAbs were identified in the past (LANA et al. 1992; LETZEL et al. 2007;
ICARD et al. 2008; DURAIRAJ et al. 2011). Recombinant Abs developed from a chicken single chain variable Ab fragments (scFv) also differentiate IBDV isolates in a sandwich ELISA platform (SAPATS et al. 2005; SAPATS et al. 2006).
2.5.4. Molecular characterization
The classical methods for molecular characterization and differentiation of IBDV field isolates include RT-PCR and restriction fragment length polymorphism (RFLP), nucleotide sequence analysis, and quantitative real time RT-PCR (qRT-PCR) (JACKWOOD 2004; WU et al. 2007a). The hypervariable region of VP2 (hVP2) of IBDV between aa residues 206 and 350 shows significant aa sequence variations.
Sequencing of this region is used to categorize isolates into different pathogenic
strains (YAMAGUCHI et al. 1997b; JACKWOOD u. SOMMER-WAGNER 2005;
JACKWOOD u. SOMMER-WAGNER 2007; SREEDEVI et al. 2007).
In early studies, RT-PCR-RFLP analysis was performed mainly on hVP2 and seldom on VP1 to distinguish IBDV isolates (MEIR et al. 2001; JUNEJA et al. 2008).
According to their restriction profiles, the viruses form molecular groups or genotypes (JACKWOOD et al. 2001). However, the limitation of this method is that viruses in a molecular group or with matching RFLPs may differ in their antigenic or virulence property and require sequence analysis.
Direct sequencing of RT-PCR products of the hVP2 and 5’two thirds of VP1 may provide deeper insights into the nucleotide and predicted aa identity of strains and may determine mutations, recombination and reassortment in the viral genomes (LE NOUEN et al. 2005). The nucleotide sequences of VP2 and VP1 can be further used to construct phylogenetic trees to demonstrate genetic relationships between IBDV isolates (HON et al. 2006).
A TaqMan qRT-PCR and melting curve analysis can be used to trace mutations in the hVP2 region (JACKWOOD et al. 2003). This method allows comparing sequences between field and vaccinal strains (JACKWOOD u. SOMMER 2002; GAO et al. 2007). It determines a single nucleotide polymorphism in VP2 (WU et al.
2007a). qRT-PCR quantifies viral load (MOODY et al. 2000).
RT-loop-mediated isothermal amplification (RT-LAMP) is a rapid field test used as a screening method, particularly when direct nt sequencing facilities are unavailable (XU et al. 2009). The LAMP assay is based on the principle of autocycling strand displacement DNA synthesis performed by the Bst DNA polymerase and a set of two inner and two outer primers that recognize 6–8 regions of target DNA. LAMP has high specificity as the primers recognize six specific regions of the target amplicon (TSAI et al. 2012).
2.6. Vaccines and vaccination against IBDV
Vaccination is widely used to prevent IBD outbreaks in the field. Most of the commercially available vaccines against IBDV are live attenuated and inactivated ones; recombinant and subunit vaccines have been licensed in some countries.
Live vaccines are produced from classical and variant IBDV strains by passaging these viruses in tissue cultures or embryonated chicken eggs (YAMAGUCHI et al.
1996a; LASHER et al. 1997; JACKWOOD u. SOMMER-WAGNER 2011). They can be classified as mild, intermediate or intermediate plus vaccines based on the level of attenuation and residual virulence for SPF chickens (VAN DEN BERG et al. 2000a).
The intermediate plus vaccines are regularly applied to protect chickens against vvIBDV challenges. The Deventer formula may help to determine the optimal time for IBDV vaccination to circumvent the neutralizing activity of MAB (DE WIT 1998). Live vaccines are favourable for mass application through drinking water and can induce strong humoral and cellular immunity (MÜLLER et al. 2003; MÜLLER et al. 2012).
The proven reversion to virulence (YAMAGUCHI et al. 2000) and their residual immunosuppressive effects (RAUTENSCHLEIN et al. 2005b; RAUTENSCHLEIN et al. 2007) are major safety concern of their extensive field applications. Breeder vaccination by priming with live vaccines and boosting with inactivated oil-emulsion vaccines prior to laying ensures higher levels of MAB transfer to the progeny (MAAS et al. 2001; MÜLLER et al. 2012) and is applied in some countries.
Commercially available IBD immune complex (IBD-ICX) vaccines are found to be safe and efficacious for in ovo and posthatch vaccination of broilers (HADDAD et al.
1997; GIAMBRONE et al. 2001; IVAN et al. 2005). They are prepared by combining an IBDV-hyperimmune serum with live intermediate plus IBDV (WHITFILL et al.
1995; JOHNSTON et al. 1997). The entrapment and retention of ICX on bursal follicular dendritic cells (FDCs) and on splenic FDCs in the germinal center were suggested as the immune enhancing mechanism of such vaccines (JEURISSEN et al. 1998). The viruses are released from the ICX when the levels of MAB declined to induce specific humoral immune responses that protect chickens against challenge
virus. A recombinant neutralizing Ab has been evaluated for formulation of an IBD- ICX vaccine (IGNJATOVIC et al. 2006).
The protective effects of many recombinant IBDV vaccines were evaluated under experimental and field conditions. The polyprotein (PP), mature VP2 or immunogenic domains of VP2 of pathogenic IBDV strains were targeted to produce candidate vaccines: subunit, vectored, virus-like particles (VLPs) and chimeric virus particles.
Some of these experimental vaccines are presented in table 4.
An IBDV-VP2 subunit vaccine expressed in Pichia pastoris is licensed for commercial uses (PITCOVSKI et al. 2003). An E. coli expressed subunit vaccine has been evaluated under field conditions (RONG et al. 2007). The use of peptide epitope mimics, i.e. mimotopes as candidate IBDV vaccines have become promising strategy. Mimotopes are chemically synthesized and resembled the neutralizing epitopes of VP2. Their expression in prokaryotic expression vector resulted in a bioactive peptide that can induce significant neutralizing Abs and protection against IBDV challenge (WANG et al. 2007). These types of vaccines induce strong humoral immunity and always require adjuvants and multiple injections for inducing protective levels of neutralizing Abs. Many live vectored IBDV vaccines, which mimic natural infection have been developed and tested for efficacy. A live Newcastle disease virus (NDV) vectored VP2 vaccine has been experimentally evaluated (HUANG et al.
2004) and recently HVT-IBD vaccine was licensed for in ovo and posthatch vaccination of broilers and layers in various countries (BUBLOT et al. 2007; LE GROS et al. 2009). These vectored vaccines induce strong systemic neutralizing Ab levels and mucosal Abs, but pre-existing immunity for example against NDV-vector may affect their efficacy.
Other IBDV-candidate vaccines include virus-like particles (VLPs). These vaccines lack viral genomes and are non-infectious. They preserve the native conformation of the capsid protein and present multiple copies of these immunogenic epitopes (BRUN et al. 2011). However, the expression systems determine the nature of the VLPs. The expression of IBDV PP by a recombinant vaccinia virus in mammalian
cells resulted in true VLPs (FERNANDEZ-ARIAS et al. 1998), whereas defective VLPs were detected when the PP was expressed in insect cells by a baculovirus (HU et al. 1999; KIBENGE et al. 1999; CHEVALIER et al. 2002). The main reason for the lack of true VLP formation in the yeast and insect cells may be the absence of the host protease, puromycin-sensitive aminopeptidase that is required for the processing of the PP (IRIGOYEN et al. 2012). The VP2 icosahedral capsid had been shown to induce the higher neutralizing Ab levels and better protection against IBDV challenge than the PP-derived structures and the VPX tubules (MARTINEZ- TORRECUADRADA et al. 2003).
Candidate attenuated live IBDV vaccines generated by reverse genetics have been shown to induce strong protective immunity (BOOT et al. 2002; MUNDT et al. 2003;
ZIERENBERG et al. 2004; QIN et al. 2010; GAO et al. 2011), but vaccinated chickens developed milder bursal lesions after a challenge study. These tailored chimeric IBDV vaccines were generated to contain VP2 regions of two different strains of serotype 1 IBDV (MUNDT et al. 2003; GAO et al. 2011) or were chimeric between segment A of serotype 1 and segment B of serotype 2 IBDVs (ZIERENBERG et al. 2004). A VP5 mutant IBDV vaccine induced better protection than its molecular cloned PP counterpart (QIN et al. 2010). BOOT et al. (2002) produced a chimeric virus containing the C-terminal serotype 2 VP3 inserted into genome segment A of serotype 1 IBDV. Nevertheless, the risk of reversion to virulence of these genetically modified viruses hinders their field applications (RAUE et al. 2004).
Recombinant IBDV-VP2 vaccines may possibly be used as ‘’marker vaccines’’
(MÜLLER et al. 2012) by allowing the differentiation of infected from vaccinated animals (DIVA) by the detection of anti-VP3 Abs in naturally infected birds.
