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

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.