Molecular epidemiology of IBDV field isolates

Molecular approaches allow the identification and differentiation of IBDV strains circulating in chicken populations and associate recent and past isolates (LE NOUEN et al. 2005). The molecular epidemiology of IBDV has been studied in many geographical areas and IBDV evolution well documented. Particularly, serotype 1 IBDV strains have been circulating in many poultry operations in North and South America, Europe, Asia and in African countries (YAMAGUCHI et al. 1997a;

ZIERENBERG et al. 2000; VAN DEN BERG et al. 2004; JACKWOOD u. SOMMER 2005; JACKWOOD u. SOMMER-WAGNER 2007; JUNEJA et al. 2008; HE et al.

2012b; KASANGA et al. 2012).

In this study, several IBDV isolates were collected during field outbreaks in different types of poultry flocks in Ethiopia. Experimental inoculation of chickens with most of these isolates allowed us to confirm the pathogenicity of the viruses. Inoculated chickens showed significant bursal lesions and an extensive IBDV Ag load in the bursa. The characterization of the nucleotide (nt) sequences of the hVP2 and the 5’

two thirds of VP1 of 11 isolates demonstrated that the Ethiopian isolates showed

close homology to vvIBDV isolates from other countries. Based on a recent study, it is expected that, worldwide, about 60 to 76% of IBDV isolates are of vvIBDV genotype (JACKWOOD u. SOMMER-WAGNER 2007; HE et al. 2012b). The rest of the isolates are classical and variant strains based on their hVP2 characteristics (JACKWOOD u. SOMMER-WAGNER 2007; HE et al. 2012b). Although phenotypic characterization of IBDV in experimentally infected SPF chickens is the gold standard to determine IBDV virulence, genotypic features can support IBDV grouping (VAN DEN BERG et al. 2004). vvIBDV has shown a rapid global spread; even in the USA, where the classical and variant strains dominated field outbreaks for nearly five decades, vvIBDV emerged in 2008 (STOUTE et al. 2009).

Almost all poultry producing regions report the co-existence of two or more strains of varied pathogenicity (JACKWOOD u. SOMMER-WAGNER 2007; HE et al. 2012b).

Interestingly, only vvIBDV was identified in Ethiopia during the period of our study. A recent countrywide study reported IBDV seropositivity rates in backyard chickens to be close to 92% (CHAKA et al. 2012; JENBREIE et al. 2012). Detailed molecular studies of IBDV in this segment of the chicken population may provide further information about IBDV epidemiology.

Phylogenetic approaches have been particularly useful for studying the origin and subsequent evolution of a virus. Based on the VP2 nt sequence, phylogenetic analysis revealed the formation of a cluster by Ethiopian IBDV strains; yet this cluster reclines in one major monophyletic lineage with other vvIBDVs. It was expected that vvIBDV strains of the same geographic origins may cluster together, yet some minor non-significant clustering outside the main cluster was reported among isolates of the same origins (CORTEY et al. 2012; SILVA et al. 2012). This may indicate a strong association between geographical origin and viral diversity. In contrast to this observation, vvIBDVs isolated from the major chicken producing regions of China showed very divergent phylogenetic clusters based on their VP2 sequences (HE et al. 2012b). This may highlight the circulation of vvIBDVs with diverse genetic mutations. Several recent IBDV field strains from USA did not form clusters with most of the IBDV VP2 sequences retrieved from databases, implying the virus is under

continuous evolution (DURAIRAJ et al. 2011). Despite vvIBDV isolates maintaining the identical virulence marker aa in their VP2 regions, they show differences in their pathogenicity to SPF chickens even under standardized experimental conditions.

This suggests the importance of the role of VP1 in IBDV virulence (SILVA et al.

2012), which should be besides VP2 regularly included in the molecular analysis of IBDV strains. Normally, the VP1 gene of IBDV strains shows multiple phylogenetic lineages, additionally providing evidence of its contribution in IBDV pathogenicity (JACKWOOD et al. 2012).

How the vvIBDV strains evolved in Ethiopia remains unclear. Literature suggests that international trade of live poultry and poultry products may facilitate the global spread of IBDV (COBB 2011). IBDV may spread through contaminated equipment (FLENSBURG et al. 2002; JACKWOOD u. SOMMER-WAGNER 2010). The high tenacity of the virus and its resistance to several disinfections and virucidal procedures may contribute to the rapid distribution of the virus (VAN DEN BERG et al. 2000b; GARRIGA et al. 2006). The risk of transmission via infected free living wild birds (JEON et al. 2008) can not be excluded as IBDV or IBDV-specific Abs were detected in other avian species and in backyard chickens (KASANGA et al. 2008).

Almost all acute disease outbreaks in backyard chickens in developing countries remain undiagnosed. vvIBDV isolates from wild birds and backyard chickens were shown to be highly pathogenic for SPF chickens under experimental conditions and maintain virulence marker aa residues across their VP2 and VP1 genes (HERNANDEZ-DIVERS et al. 2008).

Ethiopian IBDV isolates appear clonal and are very virulent. In the case of RNA viruses, biological events including genetic reassortment or recombination alter the phenotypes and genotypes of circulating viruses and compromise their genetic stability. Also the first vvIBDV that had caused severe disease outbreak in Europe was linked to the emergence of segment reassortant IBDV between a newly appeared IBDV, which combined its very virulent VP1 segment with an endemic IBDV bearing a very virulent VP2 segment (HON et al. 2006). Natural homologous intragenic recombination apart from reassortment may lead to new variants of IBDV

(HON et al. 2008; HE et al. 2009a). Likewise, Ethiopian strains in our study showed aa residues within the region of VP1 that are characteristic of attenuated as well as vvIBDV, so we can not rule out the possibility of recombination within this gene segment.

While prototype vvIBDVs present glycine (G) at position 254 (loop PDE) of the minor hydrophilic peak 1 domain of the hypervariable region of VP2, we found a serine (S) residue in all Ethiopian vvIBDV isolates at this position. A serine (S) residue at this position was detected in most recent vvIBDV field isolates from Africa, Europe and Asia from chickens vaccinated with classical live IBDV vaccines (KASANGA et al.

2007; MARTIN et al. 2007). Recent study revealed that most other IBDV isolates show frequent aa exchange at this position as well (ICARD et al. 2008; DURAIRAJ et al. 2011). This indicates a significant role of this domain in determining IBDV antigenicity as demonstrated by previous studies (BAYLISS et al. 1990; HEINE et al.

1991; LANA et al. 1992; DORMITORIO et al. 1997). Also the crystal structure of the VP2 protein suggests selection pressure associated with the hydrophilic domains due to their location at the outermost exposed part of the VP2 protein (COULIBALY et al.

2005). A neutralizing Ab escape mutant virus at position 254 (S) was generated from Del-E IBDV by site-directed mutagenesis (JACKWOOD u. SOMMER-WAGNER 2011). Chickens vaccinated with a live Del-E vaccine and challenged with the mutant developed severe bursal lesions, whereas those vaccinated with Del-E and challenged with a homologous virus were fully protected (JACKWOOD u. SOMMER-WAGNER 2011) showing the contribution of this particular aa residue in vaccination failure.

Vaccination failure associated with the application of plaque purified and cloned live IBDV vaccines during and in subsequent IBD outbreaks in Ethiopia (personal communication with poultry producers and veterinarians) may be due to the lack of cross protection due to aa exchanges at the hydrophilic domains of field isolates compared to the vaccinal strains. Vaccines on the basis of IBDV quasispecies (JACKWOOD u. SOMMER 2002) may in contrast to a cloned live vaccine provide a

better cross protection in the face of heterologous IBDV field challenges counteracting at least in part the development of Ab escape mutants.

6.2. Immune responses induced by candidate IBDV DNA vaccines and correlation to