Identification of a European IBDV variant strain.
Bel-IBDV was first analyzed by AC-ELISA in comparison with the classical strain D78 and the variant strains E/Del and GLS (Table ) by using bursa materials of infected chickens and the MAbs described above. The reactivity patterns for the classical strain D78 and the two variant strains (GLS, E/Del) were as expected and previously described (36
). Interestingly, Bel-IBDV displayed a unique pattern, suggesting that the isolated virus was a new, to date unknown antigenic IBDV variant strain. To confirm these data and to establish the tools for a detailed analysis of the antigenic determinants, pD78A derivative plasmids encoding VP2 proteins of different strains were constructed (Fig. ) and transfected into cells. Immunofluorescence analysis confirmed the AC-ELISA results: each full-length pD78A derivative encoding a certain VP2 (D78, GLS, E/Del, Bel-IBDV) displayed a unique pattern identical to that obtained with the parental viruses in the AC-ELISA (Fig. ). Thus, this MAb collection allows for differentiation of antigenic patterns, each of which is representative of one of the strains used in the study.
Reactivities of different IBDV strains by AC-ELISA in comparison to Bel-IBDV
FIG. 2. Immunofluorescence after transfection of cRNA of segment A. BHK-21 cells were cotransfected with in vitro-transcribed capped cRNAs from cDNA plasmids containing either the full-length sequences (pD78A) or a chimeric segment A (pD78-Belg, pD78A-GLS, pD78A-E/Del) (more ...) Amino acid sequence analysis of the variable region of VP2.
To understand the relative importance of residues of the P domain (the variable region of VP2) in the modulation of VP2 antigenicity, the amino acid sequences of the P domains of strains D78, E/Del, GLS, and Bel-IBDV and isolate GA198 (15
) were first aligned (Fig. ). GA198 was chosen after a GenBank search due to its unusual amino acid composition in the PHI
loop, where an asparagine was present at amino acid position 318. Since amino acids located in two of the loops in the P domain (PBC
) have been proposed to influence the formation of neutralizing epitopes (1
), these two immunodominant loops were analyzed in detail. Amino acid differences were observed in loop PBC
at a single position (P222T and P222S) and in loop PHI
at three positions (G318D, G318N, A321E, and D323E). The amino acid sequence of Bel-IBDV revealed one specific amino acid change (P222S) in the PBC
loop. These observations suggest that a few residues control VP2 antigenicity. The locations of the single loops described above are depicted on the surface of a single VP2 trimer (Fig. ) and on the whole virus capsid (Fig. ). This illustrates the importance of the four hydrophilic loops, propagated 780 times by the T=13 icosahedral symmetry of the virion, with the likely ability to bind neutralizing antibodies.
The identification of several residues in loops PBC and PHI associated with antigenic variability prompted us to analyze how single and multiple substitutions in these loops can modulate antibody binding. This study was focused on the amino acids determining the binding of MAbs 57, R63, 67, B69, and 10. It should be noted that all these epitopes were present only during experiments using immunofluorescence (Fig. ) and immunoprecipitation (data not shown). The lack of reactivity in Western blot experiments (data not shown) suggests that they are confirmation dependent. Structural models of selected VP2 forms (D78, D78-PS, Mut3) with different MAb binding patterns are shown in Fig. .
FIG. 4. Structural modeling of amino acid changes influencing the binding of MAbs 57 and 67. Exposed loops from domain P are highlighted with the same color scheme as that for Fig. on a cartoon representation of the VP2 trimer. (A) Side chains (more ...) Single and multiple amino acid substitutions in both loops PBC and PHI of VP2 control reactivity to MAbs.
Substitutions at positions 222, 318, 321, and 323, either singly or in combination, were engineered on the D78 segment A backbone to identify residues associated with the different antigenic patterns (Table ). Substitution of aa 330 was also included due to the presence of serine at this position in all IBDV strains compared except for strain D78. Immunoreactivity was analyzed by indirect immunofluorescence after cotransfection of a pD78A derivative with pP2B cRNA.
(i) MAb 67 epitope.
The reactivity of the MAb 67 epitope involves specific amino acids in the two loops PBC and PHI. The presence of both a serine/threonine at position 222 (PBC loop) and an alanine at position 321 (PHI loop) is necessary for the recognition of this epitope, showing that this epitope is defined by amino acids that are 100 residues apart on the VP2 primary sequence. Substitutions at positions 318, 323, and 330 had no effects on the binding of MAb 67.
The critical role of positions 222 and 321 for recognition by MAb 67 is illustrated with D78-PS in Fig. . Although these residues are distant in the VP2 primary sequence, they are located in loops PBC and PHI, respectively, which are in close proximity at the tip of VP2 spike. The amino acid at position 222 is exposed to a solvent and could therefore interact directly with MAb 67. As a consequence, serine or threonine at position 222 is likely to be part of the epitope. On the other hand, alanine 321 is unlikely to be a critical part of the epitope, and loss of recognition by MAb 67 with a glutamate at position 321 probably results from the masking of surrounding residues of loop PHI as observed by computer modeling (data not shown).
(ii) MAb 57 epitope.
Recognition of the MAb 57 epitope involves the PHI loop and a particular residue in position 330 but not the PBC loop. A glutamic acid at position 321 located in the PHI loop is a requisite for the binding of MAb 57 to this epitope (Table ), as shown for Mut3 in Fig. . An aspartic acid at position 323, instead of a glutamic acid, also contributes to the binding of MAb 57. With one notable exception (pMut11 and one of its derivatives), a glutamic acid at position 323 correlated with binding of MAb 57. For pMut11, asparagine at position 318, instead of glycine or glutamic acid at position 321, appears to compensate for the lack of aspartic acid at position 323 (see the difference in reactivity between pMut7 and pMut11 [Table ]). In addition, serine 330 is generally not tolerated, with some notable exceptions: pMut6-R330S was recognized by MAb 57. Thus, the reactivity of the MAb 57 epitope appears to depend on at least one critical residue, glutamic acid 321, and on two more-dispensable residues, aspartic acid 323 and arginine 330.
While E321 is exposed and could constitute part of the MAb 57 epitope, residues 323 and 330 seem to have an indirect role in altering the backbone of VP2. Modeling analysis suggests that a D323E mutation could break a stabilizing salt bridge with lysine 316 within the PHI loop (data not shown). Further experiments need to be performed to confirm this hypothesis. Residue 330 is located at the interface between subunits in the trimeric spike, away from the main antigenic site formed by loops PBC and PHI. Arginine 330 appears to stabilize a bulge by interaction with glutamates 212 and 213 of the PB β-strand (data not shown). Thus, it is possible that the R330S mutation leads to subtle alterations in the jelly roll, ultimately leading to a different conformation of loop PBC that is incompatible with the binding of MAb 57.
Finally, these data provide a rationale for understanding why IBDV variants recognized by MAb 67 are not recognized by MAb 57, and vice versa. This restriction is due to a single substitution at position 321 at the tip of the VP2 spike: a glutamic acid at this position allows MAb 57 binding, while an alanine allows MAb 67 binding.
(iii) MAb R63 epitope.
When an alanine is present at position 321, there is always reactivity with MAb R63, showing that loop PHI controls the binding of this MAb. However, alanine at position 321 is not an absolute requirement for reactivity. Depending on adequate substitution at some other positions, even the presence of alanine 321 is dispensable (see data for pMut10-R330S and pMut11-R330S constructs in Table ). Thus, the reactivity of MAb R63 appears to be less restricted than that of MAb 67, which requires an alanine at position 321 and a serine or threonine at position 222, as described above.
(iv) MAb 10 epitope.
Two residues appear to be critical for the presence of the MAb 10 epitope: glycine at position 318 and aspartic acid at position 323. The reactivity of MAb 10 was not influenced by any of the substitutions carried out at other positions (positions 222, 321, and 330).
(v) MAb B69 epitope.
None of the substitutions carried out on the pD78A backbone at positions 222, 318, 321, 323, and 330 altered the reactivity of the MAb B69 epitope, suggesting that neither the PBC nor the PHI loop is involved. To determine if a third loop (PDE loop [Fig. ]) present at the top of the VP2 projection could contribute to this epitope, a series of additional single or multiple substitutions were engineered in the pD78A backbone at positions 249, 254, and 270. None of the single (Q249K, G254S, or T270A) or combined (Q249K G254S, Q249K T270A, or Q249K G254S T270A) substitutions were able to alter the epitope defined by MAb B69, indicating that this third loop probably is not associated with the presence of this epitope (data not shown).
(vi) The unique antigenic pattern of IBDV-Belg.
The results obtained also suggested that the reactivity of IBDV-Belg with MAb 67 was caused by a single substitution at position 222 (P222S). To provide additional evidence that the serine in position 222 is critical for MAb 67 binding, this position was mutated in the IBDV-Belg VP2 backbone. Thus, a plasmid (pD78A-Belg) coding for segment A and containing the complete VP2 gene segment of the Belgian isolate mutated at position 222 (S222P) was constructed. While VP2 encoded by pD78A-Belg was reactive with MAb 67, the corresponding S222P mutant was not (Table ).
Neutralization assay of IBDV mutants using a polyclonal serum and MAbs
Analysis of the immunoreactivity of the corresponding recombinant viruses by a neutralization assay.
Ten different cDNAs encoding VP2 mutants with unique antigenic and/or sequence patterns were chosen to produce mutants by reverse genetics and to confirm by a neutralization assay the importance of the PBC and PHI loops in VP2 antigenicity. Virus mutants were produced as described in Materials and Methods, and their ability to be neutralized by each MAb was measured (Table ). In most cases, when a virus mutant was recognized by a MAb in the immunofluorescence assay, it was efficiently neutralized by the same MAb. One exception was observed with Mut11, which was recognized in the immunofluorescence assay by MAb 57 but was not neutralized. The fact that Mut10, which differs from Mut11 by a single substitution at position 323, was neutralized by MAb 57 suggests that the aspartic acid at position 323 contributes to the reactivity of this epitope. Confirmation of the relative importance of position 222 in MAb 67 binding was provided by this neutralization assay using D78-Belg and D78-BelgS222P.
Analysis of growth in cell culture.
To test whether the amino acid substitutions influenced the replication of mutated viruses in cell culture, several IBDV mutants generated by reverse genetics were analyzed in comparison to the recombinant D78 virus (Fig. ). Mutation of aa 222 from proline to serine did not influence replication in cell culture. In contrast, all amino acid changes in the PHI loop influenced the replication of the corresponding mutants. These mutants grew to lower titers at all time points investigated, indicating that the three residues of the PHI loop in positions 318, 321, and 323 are important parameters for replication in cell culture.
FIG. 5. Replication kinetics of recombinant IBDV in cell culture. Embryonic chicken cells were infected with IBDV D78, PS-D78, Mut1, PS-Mut1, Mut2, PS-Mut2, Mut10, or Mut11 as described in Materials and Methods. Supernatants removed at the indicated times (hours) (more ...)
To exclude the possibility that the virus used for the growth curve and the neutralization assays exhibited revertant mutations, RT-PCRs using the IBDVFP1-IBDVRP3 primer pair (available from author upon request) and Deep Vent polymerase (New England Biolabs) were carried out on the propagated viruses. Sequence analysis of cloned PCR products revealed either no mutations or only silent mutations.