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Journal of Virology, October 2002, p. 10346-10355, Vol. 76, No. 20
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.20.10346-10355.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Exchange of the C-Terminal Part of VP3 from Very Virulent Infectious Bursal Disease Virus Results in an Attenuated Virus with a Unique Antigenic Structure

Hein J. Boot,* A. Agnes H. M. ter Huurne, Arjan J. W. Hoekman, Jan M. Pol, Arno L. J. Gielkens, and Ben P. H. Peeters

Institute for Animal Science and Health, Lelystad, The Netherlands

Received 8 April 2002/ Accepted 8 July 2002


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ABSTRACT
 
Infectious bursal disease virus (IBDV) is the major viral pathogen in the poultry industry. Live attenuated serotype 1 vaccine strains are commonly used to protect susceptible chickens during their first 6 weeks of life. Wild-type serotype 1 IBDV strains are highly pathogenic only in chickens, whereas serotype 2 strains are apathogenic in chickens and other birds. Here we describe the replacement of the genomic double-stranded RNA (dsRNA) encoding the N- or C-terminal part of VP3 of serotype 1 very virulent IBDV (vvIBDV) (isolate D6948) with the corresponding part of serotype 2 (isolate TY89) genomic dsRNA. The modified virus containing the C-terminal part of serotype 2 VP3 significantly reduced the virulence in specific-pathogen-free chickens, without affecting the distinct bursa tropism of serotype 1 IBDV strains. Furthermore, by using serotype-specific antibodies we were able to distinguish bursas infected with wild-type vvIBDV from bursas infected with the modified vvIBDV. We are currently evaluating the potential of this recombinant strain as an attenuated live vaccine that induces a unique serological response (i.e., an IBDV marker vaccine).


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INTRODUCTION
 
The relatively unknown infectious bursal disease virus (IBDV) is currently responsible for a huge economic impact on the worldwide poultry industry (33). IBDV, an avibirnavirus, is the causative agent of a highly contagious disease among chickens known as Gumboro disease (13). The genome of IBDV consists of two segments of double-stranded RNA (dsRNA) (16). The largest dsRNA segment (the A segment, 3,260 bp) contains two partly overlapping open reading frames (ORFs). The first, smaller ORF encodes the nonstructural viral protein 5 (VP5) (145 to 149 amino acids, 17 kDa). The second ORF encodes a polyprotein (1.012 amino acids, 110 kDa) that is autocatalytically cleaved to yield the viral proteins pVP2 (also known as VPX) (48 kDa), VP4 (29 kDa), and VP3 (33 kDa). During in vivo virus maturation, pVP2 is processed into VP2 (41 to 38 kDa), probably resulting from site-specific cleavage of pVP2 by a host cell-encoded protease (23). The smaller B segment (2,827 bp) contains one large ORF, encoding VP1 (877 to 881 amino acids, 91 kDa). VP1 is the RNA-dependent RNA polymerase and is present both in its free form and covalently linked to the 5' ends of the genomic RNA segments (viral protein genome linked [VPg]) (14). In vivo expression of the polyprotein (VP2-VP4-VP3) results in the formation of virus-like particles, consisting of VP2 and VP3, which have the same dimension as mature virions (60 nm). This indicates that neither VP1 nor viral dsRNA is essential for the formation of the viral capsid (18).

Two different serotypes of IBDV (serotypes 1 and 2) have been described (27). The pathogenic wild-type serotype 1 IBDV isolates specifically infect developing B-lymphoid cells in the bursa of Fabricius. Serotype 1 isolates are subdivided into classical, antigenic variant, and very virulent isolates. Antigenic variant IBDV isolates appeared to have single amino acid changes in a specific region of the VP2 protein (the hypervariable region) that lead to a partial change in antigenicity (31). Very virulent IBDV (vvIBDV) isolates, which were first isolated in Europe, have the same antigenic structure as classical strains but have an increased virulence (12). Amino acid differences between viral proteins of vvIBDV and classical IBDV isolates are scattered throughout all viral proteins, although most of them are found in the hypervariable region of VP2 (10). Specific mutations in VP2 result both in a change of cell tropism (28) and in attenuation (36). Although VP2 is a key factor for virulence, we have recently shown that it is not the sole determinant for the very virulent phenotype (4).

Unlike serotype 1 isolates, wild-type serotype 2 isolates do not have a specific B-lymphoid cell tropism. Serotype 2 isolates are able to replicate naturally in different tissues of birds and can even be propagated on cell lines. Serotype 2 isolates, usually recovered from turkeys, are apathogenic in turkeys (21) and in chickens (20). Conformation-dependent, virus-neutralizing epitopes of both serotype 1 and 2 isolates are present in the capsid protein VP2. The other abundant viral capsid protein (VP3) does not contain virus-neutralizing epitopes, although a rapid immune response to linear VP3 epitopes is found after both vaccination and infection (17). Both group- and serotype-specific epitopes have been described for VP3 (25, 29, 34).

A common way of producing IBDV vaccines is adaptation of wild-type virus by propagation in chicken embryos or in cell culture with primary chicken embryo cells or cell lines of either avian or mammalian origin. Adapted wild-type IBDV always has reduced virulence (attenuation) (35, 37). Differentiation between live IBDV vaccines and field IBDV isolates recovered during field outbreaks is difficult and laborious, as the genetic structure of the IBDV isolate in question has to be determined. This is either done by sequence analysis (38) or restriction fragment length polymorphism (22) of the cDNA. In our search for an live IBDV vaccine that can easily be distinguished from IBDV field isolates, we recently replaced the complete VP3 protein or the N- or C-terminal part of the VP3 protein of an attenuated IBDV isolate (CEF94) with the corresponding part of a serotype 2 isolate (TY89). Analysis of these rescued serotype 1 IBDV recombinants showed that this chimeric strain has a slightly reduced release of progeny virus when grown on QM5 cells, while the final titer was similar to that of unchanged CEF94 (6). The yield of the mosaic virus in which either the complete VP3 or the N-terminal part of VP3 was replaced was reduced (final titers were about 10-fold lower) compared to that of unmodified CEF94, indicating a more severe effect on replication due to the exchange of the N-terminal part of VP3.

In this paper we describe the replacement of the genomic RNA encoding the C-terminal part of VP3 of vvIBDV (isolate D6948) with the corresponding part of serotype 2 (isolate TY89) genomic RNA. Compared to unmodified vvIBDV, this modified vvIBDV strain significantly reduces the mortality and morbidity rates of specific-pathogen-free (SPF) chickens. Furthermore, by using serotype-specific antibodies we were able to distinguish between bursas infected with wild-type vvIBDV and bursas infected with the modified vvIBDV.


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MATERIALS AND METHODS
 
Viruses, cells, and antibodies. The classical IBDV isolate CEF94 is a derivative of PV1 that is able to replicate in non-B-lymphoid cells (5). The D6948 strain is a very virulent field isolate (Poultry Health Service, Doorn, The Netherlands; 1989) that grows only in primary B-lymphoid cells (4). IBDV strain TY89 is the prototype serotype II IBDV strain (27) and is able to grow naturally on avian-derived fibroblast cell lines such as QM5. Recombinant fowlpox virus containing the T7 polymerase gene (FP-T7) (9) was generously provided by M. Skinner (Compton Laboratory, Berks, United Kingdom). QM5 cells (1) were maintained by using QT35 medium (Gibco/BRL) supplemented with 5% fetal calf serum and 2% antibiotic solution (4) (QM5+ medium). Primary bursa cells were isolated from 14-day-old SPF chicken embryos and were maintained in Eagle's modified minimal essential medium supplemented with 15% fetal calf serum, 0.125% lactoalbumin hydrolysate, and 1,000 U of penicillin and 1 mg of streptomycin per ml. The VP3-specific monoclonal antibodies (MAb) B10A (25), IVS1 (25), VII S2 (25), and I/G4 S1 (29) were kindly given to us by H. Müller, University of Leipzig (Leipzig, Germany), while MAb 9.7 was prepared in our laboratory by using purified CEF94 as an antigen (6).

Construction of chimeric VP3 cDNAs. The construction of hybrid CEF94 and TY89 plasmids has been described before (6). To construct plasmid pHB60-s2VP3N, we generated five different PCR fragments, using Pwo polymerase in 20 reaction cycles. PCR-1N was generated by using pHB-60 as the template and oligonucleotides AC3 (GGTAGCCACA TGTGACAG) and HY3MR (CCAGTCCCGC GGATTGTGAGG) at 56°C, yielding a 1,614-bp fragment (nucleotides [nt] 731 to 2346). PCR-2N was generated by using pHB36-s2VPN as the template and oligonucleotides HY3P (AACGTTTTCC TCACAATCCG CGGGACTGGG) and M13F-17 (GTAAAACGAC GGCCAGT) at 56°C, yielding a 1,251-bp fragment (nt 2318 to 3566). PCR-3N was generated by using gel-purified PCR-1N and PCR-2N fragments as the template and oligonucleotides AC4 (ACCCAGCCAA TCACATCC) and AGTM (GAGACTCCCA GGTACCTCAC TC) at 54°C, yielding a 2,151-bp fragment (nt 1057 to 3208), which was subsequently digested with ScaI (nt 2799), yielding PCR-3sN. PCR-4N was generated by using pHB-60 as the template and oligonucleotides AC9 (CTCAAAGAAG ATGGAGACC) and M13F-24 (CGCCAGGGTT TTCCCAGTCA CGAC) at 54°C, yielding an 869-bp fragment (nt 2727 to 3593). PCR-5 was generated by using gel-purified PCR-3sN and PCR-4 fragments as the template and oligonucleotides AC5 (AAGGCCTTCA TGGAGGTGGC CG) and M13F-17 (GTAAAACGAC GGCCAGT) at 54°C, yielding a 2,143-bp fragment (nt 1423 to 3566), which was subsequently digested with XhoI and XbaI, yielding a 1,889-bp PCR-5xxN fragment (nt 1606 to 3495). The PCR-5xxN fragment was subsequently used to replace the corresponding part of pHB-60 by using a rapid DNA ligation kit and transformation of Escherichia coli cells. The DNA sequence of the selected plasmid clone of pHB60-s2VP3N was determined (nt 1600 to 3325). It appeared to have the intended mosaic D6948-TY89 cDNA sequence, as shown in Fig. 1.



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  		FIG. 1. Schematic representation of the plasmids containing the full-length (hybrid) A-segment cDNA sequences. Although the T7 promoter sequence, the hepatitis delta virus ribozyme (HDVR or HDR), and the T7 terminator are indicated only for plasmids pHB-36W and pHB-60, they are present in each plasmid. Open boxes, CEF94 cDNA; shaded boxes, D6948 cDNA; dashed boxes, TY89 cDNA. UTR, untranslated region.

For the construction of pHB60-s2VP3C3, we first transferred a 1,735-bp fragment of pHB36-s2VP3C into pHB-60 by using the unique SacII (nt 1670) and XbaI (nt 3495) sites, yielding pHB60-s2VP3C1. The 3' untranslated region of CEF94 was subsequently replaced by the corresponding cDNA sequence of D6948. Fragment PCR-3UTR (385 bp, nt 3184 to 3566) was generated by using primers AGTP (CTTGAGTGAG GTACCTGGGAG) and M13F-17 (GTAAAACGAC GGCCAGT), pHB-60 as the template, Pwo polymerase, and 20 reaction cycles at a hybridization temperature of 52°C. This PCR-3UTR fragment was purified and digested with KpnI and XbaI. The resulting fragment (315 bp, nt 3198 to 3519) was gel purified and used to replace the corresponding part of pHB60-s2VP3C1 (digested with KpnI and XbaI) by means of rapid ligation and transformation of E. coli cells, yielding pHB60-s2VP3C2. Next, fragment PCR-3N (1187 bp, nt 1423 to 2607) was generated by using oligonucleotides AC5 (AAGGCCTTCA TGGAGGTGGC CG) and vvVP3CM (GAGAAAATTT CGCATCCGATG), pHB-60 as the template, Pwo polymerase, and 20 reaction cycles at a hybridization temperature of 54°C. This PCR-3N fragment was purified (High Pure PCR purification; Boehringer Mannheim) and subsequently digested with SacII (nt 1760) and ApoI (nt 2576). The resulting 816-bp fragment was used to replace the corresponding part of pHB60-s2VP3C2 (digested with ApoI [nt 2576] [partially] and SacII [nt 1760]), by means of rapid ligation and transformation of E. coli cells, yielding pHB60-s2VP3C3. The DNA sequence of the selected plasmid clone of pHB60-s2VP3C3 was determined (nt 1600 to 3275), and it appeared to have the intended mosaic D6948-TY89 cDNA sequence, as shown in Fig. 1.

Transfection of QM5 cells. QM5 cells were grown to 80% confluency in a 35-mm-diameter culture dish and infected with FP-T7 (multiplicity of infection of 3). After 1 h, the cells were washed twice with 3 ml of QT-35 medium and covered with 3 ml of Optimem 1 (Gibco/BRL). Meanwhile, DNA (1.0 µg) was mixed with 12.5 µl of Lipofectamine (Gibco/BRL) in 250 µl of Optimem 1 and kept at room temperature for at least 30 min. Next, 2 ml of fresh Optimem 1 was added to the QM5 cells, followed by the DNA-Lipofectamine mixture. Transfection was performed overnight (18 h) in an incubator at 37°C (5.0% CO2). The transfected monolayer was rinsed once with phosphate-buffered saline (PBS), fresh QM5+ medium (2.5 ml) was added, and the plates were further incubated at the 37°C (5.0% CO2). After 24 h of incubation, the plates were freeze-thawed once and the supernatant was filtered through a 200-nm-pore-size filter (Acrodisc; Gelman Sciences) to remove fowlpox virus and cellular debris. If not used immediately for further analysis, the cleared supernatant was stored at -70°C.

Western blotting. Monolayers of QM5 cells transfected with different full-length A-segment plasmids (Table 1) were collected by scraping the surface of the dish in the presence of 0.5 ml of PBS. Cells were collected by centrifugation (5 min, 1,500 x g) and resuspended in 0.2 ml of PBS supplemented with 20 mM EDTA and 0.5 mg of leupeptin (Sigma) per ml. Cells lysates were prepared by three cycles of freeze-thawing and centrifugation for 1 min at 13,000 x g. The supernatant (whole-cell lysate) was transferred to a clean tube and stored at -20°C. Samples of these lysates were separated in a sodium dodecyl sulfate (SDS)-12% polyacrylamide gel and blotted onto nitrocellulose (Protean BA85; Schleicher and Schuell). Detection of VP3 on the membrane was done by the ECL detection method (Amersham) with VP3-specific MAb and peroxidase-conjugated rabbit anti-mouse serum (Dako) as the secondary antibody.


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  		TABLE 1. Description of the viruses used

Detection of infectious rIBDV. To detect the presence of recombinant IBDV (rIBDV) after cotransfection of (modified) A and B segments, we inoculated a 96-well tissue culture plate containing a near-confluent monolayer of QM5 cells with 10-fold dilutions of the cleared transfection supernatant. VP3 expression in infected cells was visualized, after 48 h of incubation, in an immunoperoxidase monolayer assay (IPMA) with MAb 9.7, a group-specific VP3 antibody (5). Rescued IBDV (e.g., rD6948) with the inability to replicate on QM5 monolayers was transferred to a monolayer of primary bursa cells grown in vitro for 24 h in a 35-mm-diameter tissue culture dish in a CO2 incubator (5%) at 39°C. IBDV-specific proteins in infected B-lymphoid cells were detected by using the IPMA described above.

Virulence of the chimeric rIBDV strains. The virulence of the rIBDV strains was evaluated in SPF layer-type chickens. Rescued IBDV was first propagated on embryonated eggs by inoculating supernatant from a transfection experiment into 11-day-old embryonated eggs via the chorioallantoic membrane route. After five days of incubation, the embryos (dead or alive) were recovered, homogenized in a Sorval Omni-mixer (three times for 10 s each at maximum speed), clarified by centrifugation (6,000 x g, 10 min), and subsequently stored in aliquots at -70°C. The virus titers (50% embryo lethal dose [ELD50]) in these samples were determined with 11-day-old embryonated eggs.

In experiment 1, groups of chickens (21 days old, housed separately in isolators) were administered, intranasally and intraocularly, 1,000 ELD50 of (r)IBDV in PBS or only PBS (negative control group). The animals were monitored for clinical signs daily, and dead chicks were removed and necropsied. In experiment 1 most birds were bled (5 ml) and euthanatized for necropsy at 9 days postinfection (p.i.), while some birds were left until day 15 p.i. to be bled and euthanatized (see Table 3). In experiment 2, all birds were bled and euthanatized for necropsy at day 13 p.i. Bursa and body weights were determined for all chicks euthanatized.


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  		TABLE 3. Experimental data from the chicken infection experiments

Some samples from the bursa of Fabricius taken at necropsy were fixed in 10% neutral buffered formalin for histopathology (experiments 1 and 2), and some samples were snap frozen in liquid nitrogen and preserved at -70°C for immunohistochemical examination (only experiment 1). Initial analyses of the bursa/body weight ratios showed that standard deviations and means within groups were approximately linearly related. Therefore, group effects were introduced on the logarithmic scale and variances were assumed to be proportional to the squares of the means. Effects were estimated by maximum-quasilikelihood estimation. The dispersion variable (proportionality constant in the variance function) was estimated from Pearson's chi-square statistics (26). Pairwise comparisons were based on approximate normality of estimated group effects on the log scale. All calculations were performed with Genstat 5 (Genstat 5 release 3 reference manual, 1993; Nag Ltd., Oxford, United Kingdom), employing facilities for generalized linear models (gamma distribution and logarithmic link function).

Virus neutralization assay. Serum samples from chickens in both animal experiments were used to determine the amount of IBDV-neutralizing antibodies. Diluted sera were incubated with 30 to 300 50% tissue culture infective doses of IBDV strain CEF94 at 38°C with 5% CO2 for 1 h. After incubation, the mixture was transferred to monolayers of QM5 cells in flat-bottom 96-well plates (Greiner, Frickenhausen, Germany) and incubated for 24 h at 38°C with 5% CO2. Thereafter the monolayers were washed with PBS, dried overnight, and fixed in 4% paraformaldehyde for 10 min at 4°C. Infective centers were detected in an IPMA using VP3-specific antibodies (see above).

Histopathology and immunohistochemistry. Formalin-fixed bursa samples were dehydrated, embedded in paraffin wax, sectioned, and stained with hematoxylin-eosin. The histopathologic bursal lesion score (HBLS) was determined by microscopic analysis of the bursa. The HBLS was determined on a scale of 1 to 5 as described by Bayyari et al. (2): 1, normal bursa; 2, scattered or partial follicle damage; 3, 50% or less follicle damage; 4, 50 to 75% follicle damage; and 5, 75 to 100% follicle damage. Frozen bursa samples were sectioned for immunohistology on a cryostat at 8-µm thickness and taken up on glass slides (Superfrost). The sections were fixed with acetone for 10 min, air dried, and stored at -20°C until used. Immunoperoxidase staining was performed as described by Pol et al. (30). Briefly, endogenous peroxidase in sections was eliminated by 0.01% H2O2. Nonspecific reactions were blocked with 0.2% bovine serum albumin. MAb 9.7, I/G4SI, IVSI, or VIIS2 was used as the primary antibody, a 1:100 dilution of rabbit anti-mouse antibody conjugated to peroxidase (Dako) was used as the secondary antibody, and diaminobenzidine was used as the substrate.

Genetic stability of the chimeric IBDV strain mDT-VP3C. To determine the genetic stability of the mDT-VP3C strain, we inoculated 10 chickens (7 days old) with 1,000 ELD50 each. At 3 days p.i. the bursas were recovered and pooled. After addition of 3 volumes of tryptose phosphate broth (3%), the mixture was homogenized in a Sorval Omni-mixer (3 times for 10 s each at maximum speed) and subsequently clarified by centrifugation (6,000 x g, 10 min.) and stored in aliquots at -70°C. The IBDV titer (ELD50) of the clarified bursa suspension was determined on embryonated SPF eggs. This inoculation-extraction procedure was repeated four times, each time using the recovered virus to start the next round of infection (serial passages). After the first, third, and fifth round of infection, viral dsRNA was extracted from the bursa homogenate by using a QIAamp DNA minikit (Qiagen). Amplification of the 3' part of the A segment was achieved by performing a reverse transcription step with primer ANC1 as described before (7). The reverse-transcribed cDNA was used as the template in a PCR with primers AGTM (GAGACTCCCA GGTACCTCACTC) and AC9 (CTCAAAGAAG ATGGAGACC). The PCR was performed with 10 pmol of each primer, 2.5 mM MgCl2, 1x Taq buffer (Perkin-Elmer), 50 µM each deoxynucleoside triphosphate, and 3 U of Taq polymerase (Perkin-Elmer) in a total volume of 50 µl. The amplification was performed by running 30 cycles through temperature levels of 92°C (30 s), 58°C (30 s), and 72°C (50 s). The resulting PCR fragment of 484 bp was agarose gel purified (High Pure purification kit; Boehringer Mannheim) and used for direct sequence analysis in a cycle sequencing reaction (BigDye terminator kit; PE Applied Biosystems) and an ABI310 apparatus (PE Applied Biosystems).


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RESULTS
 
Construction and analysis of chimeric A-segment plasmids. To assess the viability, virulence, and antigenic properties of IBDVs containing a chimeric A-segment dsRNA, we made several full-length A-segment cDNA plasmids encoding a hybrid polyprotein (Fig. 1 and Table 1). Apart from A-segment plasmids based upon CEF94 cDNA and encoding a VP3 that is (partially) derived from the TY89 serotype 2 isolate, we also constructed three chimeric plasmids that were based on the A-segment plasmid of D6948, a very virulent serotype 1 IBDV isolate. Two of these plasmids (pHB60-s2VP3C1 and -VP3C3) encode a polyprotein in which the C-terminal part of VP3 is derived from serotype 2, while the other (pHB60-s2VP3N) encodes a polyprotein in which the N-terminal part of VP3 originates from serotype 2 IBDV. T7 RNA polymerase-driven transcription of these plasmids results in positive-stranded A-segment IBDV RNA which mimics the viral form of positive-stranded RNA exactly, except for the absence of the viral protein genome-linked molecule (VP1), which is most likely present at the 5' end of the viral mRNA (15).

The plasmids were used to transfect cells of a quail fibroblast cell line (QM5) that were infected with a recombinant fowlpox virus expressing T7 RNA polymerase prior to transfection. No replication of the resulting A-segment positive-stranded RNA can occur in transfected cells because of the absence of RNA-dependent RNA polymerase (VP1), which is encoded by the B segment. After transfection, the cells were washed and total cell extracts were assessed for the presence of (chimeric) VP3 by Western blot analysis (Fig. 2). Four different MAb known to react with VP3 were used, and their reactivities are summarized in Table 2. MAb B10A, an IBDV group-specific MAb, reacted with VP3 originating from all of the plasmids used. The serotype 1-specific MAb IVS1 reacted only with VP3 that possessed the C-terminal part of serotype 1. The serotype 1-specific MAb I/G4S1 reacted only with the C-terminal part of VP3 of the cell culture-adapted classical serotype 1 strain (CEF94) and not with the corresponding part of the very virulent serotype 1 strain (D6948). The recognition pattern of the serotype 2-specific MAb VIIS2 was the opposite of that of MAb IVS1; it recognized only VP3 with the serotype 2 C-terminal part. A small but distinct difference in migration of chimeric VP3 due to the presence of the N-terminal part of serotype 2 VP3 was observed in the Western blot analysis. We always find this difference in migration behavior of VP3 of the different serotypes in SDS-polyacrylamide gel electrophoresis analysis (6), despite the predicted molecular weights of VP3 of serotypes 1 and 2 being the same. Most likely this difference in migration behavior is due to a difference in the overall structure of the N-terminal part of VP3.



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  		FIG. 2. Western blot analysis of protein samples isolated 24 h after transfection from FP-T7-infected QM-5 cells transfected with pHB-36W (lane 1), pHB36-s2VP3 (lane 2), pHB36-s2VP3N (lane 3), pHB36-s2VP3C (lane 4), pHB-60 (lane 5), pHB60-s2VP3N (lane 6), pHB60-s2VP3C1 (lane 7), or pHB60-s2VP3C3 (lane 8). Four SDS-polyacrylamide gels containing equal amounts of cell extract were blotted onto nitrocellulose, and VP3 was detected by using the four indicated MAb. Sizes markers (Rainbow marker; Amersham) are indicated in kilodaltons on the left.


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  		TABLE 2. Reactivities of the MAb with different virusesa

Amino acid sequence comparison. Based on the Western blot analysis, we concluded that the VP3 epitopes that are recognized by all four of the MAb used are most probably linear. Furthermore, it is clear that the serotype-specific epitopes of MAb IVS1, I/G4S1, and VIIS2 are present in the C-terminal part (amino acids 850 to 1012) of VP3. An alignment of the deduced amino acid sequences of the VP3 part of the polyprotein revealed differences between the two serotype 1 isolates CEF94 and D6948 (i.e., at amino acids 981 and 1005) (Fig. 3). The fact that MAb I/G4S1 recognized only CEF94 and not D6948 is probably because of one of these amino acid differences. Furthermore, MAb I/G4S1 was also unable to interact with the serotype 2-derived VP3, which has the same amino acid mutation as D6948 at position 981 (L981P) (Fig. 3) but lacks the mutation at position 1005. These data strongly suggest that the epitope for I/G4S1 is located around position 981. The exact positions of the epitopes for IVS1 and VIS2 in the C-terminal part of VP3 are presently unknown.



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  		FIG. 3. Comparison of the amino acid sequences of the C-terminal parts of the polyproteins of the classical, cell culture-adapted serotype 1 strain CEF94 (GenBank accession no. AF194428), the very virulent serotype 1 strain D6948 (AF240686), and the wild-type serotype 2 strain TY89 (AF312793). The amino acid sequence of the CEF94 polyprotein is presented, while only those amino acids that differ from the CEF94 sequence are given for D6948 and TY89. The position of the ScaI restriction site (transition between the N- and C-terminal parts of the VP3) in the corresponding cDNA (see Fig. 1) is indicated.

Rescue of infectious mIBDV. The full-length hybrid A-segment plasmids were transfected into FP-T7-infected QM5 cells in combination with a full-length B-segment plasmid (cotransfection). The presence of infectious mosaic IBDV (mIBDV) 24 h after transfection was shown by using either fresh QM5 cells (for rCEF94 and derivatives thereof) or primary bursa cells (for rD6948 and derivatives thereof). We were unable to detect infectious virus when plasmid pHB60-s2VP3N was transfected in combination with plasmid pHB-55 containing the full-length B segment of D6948. All rescued mIBDV and parental wild-type strains were amplified on embryonated SPF eggs, harvested from the infected embryos, and stored at -70°C.

Infection of SPF chickens with m IBDV. In an earlier study we showed that mCEF94-s2VP3C had a slightly reduced release of progeny virus when grown on QM5 (6). We assessed the virulence of this strain and the mIBDV strains rescued in this study by inoculating (intranasally and intraocularly) groups of SPF chickens with 1,000 ELD50 of wild-type or mIBDV strains. Several virulence parameters during the living phase and postmortem were determined (Table 3). In the first experiment (experiment 1) we observed mortality during the first 5 days after inoculation only in those groups inoculated with unmodified vvIBDV (D6948 or rD6849). No mortality occurred in this experiment with the vvIBDV derivative mDCT-VP3C. This was in contrast to the second experiment (experiment 2), where one chicken (out of 15) died from an infection with mDCT-VP3C. No mortality was induced in experiment 2 by mDT-VP3C, whereas we observed a high mortality rate for both the wild-type and rescued D6948 (53 and 23%, respectively). The bursa/body weight ratio of each surviving chicken was determined after euthanization. This ratio is a good indication of the damage to the bursa caused by an IBDV infection. Cell culture-adapted IBDV strains (e.g., CEF94), which do not specifically infect B-lymphoid cells in the bursa of Fabricius, induced only a minor reduction of this ratio, while non-cell culture-adapted IBDV strains (e.g., D6948), which specifically infect the B-lymphoid cells of the bursa of Fabricius, induced a large reduction of the bursa/body weight ratio (Table 3). The introduction of the C-terminal part of the serotype 2 VP3 did not alter the ratio dramatically in experiment 1, consistent with the fact that bursa tropism correlates with the origin of VP2 (4). In experiment 2, the bursa/body weight ratio with mDCT-VP3C differed significantly from that with the wild type or rescued D6948. Whether this difference is specific to this combination of D6948, CEF94, and TY89 cDNA sequences or is due to the intrinsic variation in animal experiments (see also Discussion) is unclear.

Histopathology of infected bursas and presence of viral antigen. Sections of the bursa of Fabricius of IBDV-infected chickens (experiment 2) were examined after euthanization to determine the severity of bursa damage. Bursa damage was classified as described previously (2), where an HBLS of 1 indicates a normal bursa and an HBLS of 5 indicates 75 to 100% follicle damage (see Material and Methods). All viruses containing VP2 of CEF94 induced only minor bursa damage (<2.0) (Table 3), while all viruses containing VP2 of D6948 induced severe bursa damage (4.7 to 5.0) (Table 3). Based upon this HBLS, it would seem that not much difference in bursa damage is present after infection with mDCT-VP3C, mDT-VP3C or (r)D6948. However, careful examination revealed some clear differences between the damage induced in the bursa follicles by the different D6948-based viruses (Fig. 4). Both D6948 and rD6948 completely destroyed the follicular structure in the bursa and induced cystic formation, while the follicular structure of the bursas of mDT-VP3C-infected chickens was still present. Although some bursa follicles of mDT-VP3C-infected chickens have the same appearance as those found in bursas of mock-infected chickens, the diameter of these follicles is smaller than that of the wild-type follicles. The differences between bursas infected with D6948 and mDT-VP3C are also clear from the detection of viral antigen (Table 4). No viral antigen was detected in an VP2-based enzyme-linked immunosorbent assay (ELISA) at 9 days p.i. of mDT-VP3C, while a large amount of viral VP2 was present in bursas of chickens infected with D6948 (Table 4). There was also a clear difference in the presence of IBDV infectious particles (about a 100-fold reduction of the ELD50) between chickens infected with mDT-VP3C (titer of 2.6) and with D6948 (titer of 6.0) (Table 4).



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  		FIG. 4. Representative hematoxylin-eosin-stained sections of the bursa of Fabricius recovered at day 13 p.i. of SPF chickens either mock infected or infected with wild-type vvIBDV D6948 or mDT-VP3C (experiment 2). The HBLS (2) was determined for at least eight birds infected with the same virus (see Table 3).


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  		TABLE 4. Presence of virus in the bursas of SPF chickens at 9 days p.i. (experiment 1)

Immunohistochemistry of infected bursas. Sections of the bursa of Fabricius of IBDV-infected chickens (experiment 1) were examined for the presence of viral antigen after infection. MAb, either group specific (MAb 9.7) or serotype specific (MAb IVS1, I/G4S1, or VIIS2), were used to detect the distribution of viral antigen throughout the infected bursa and to assess the usefulness of the C-terminal part of the serotype 2 VP3 as an immunological marker. VP3 was detected in all three examined bursas of chickens infected with rD6948 or mDCT-VP3C when group-specific MAb 9.7 was used. As expected, we did not detect any viral antigen when MAb IVS1 was used after infection with mDCT-VP3C and when MAb VIIS2 was used with sections of rD6948-infected bursas (Fig. 5). Consistent with the results of the Western blot analysis, we found no reaction of MAb I/G4 with sections of bursas infected with either rD6948 or mDCT-VP3C.



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  		FIG. 5. Viral antigen detection in bursas recovered 9 days after infection of SPF chickens with either rD6948 or mDCT-VP3C (experiment 1). MAb for VP3 known to react group specifically (MAb 9.7) or serotype specifically (MAb IVSI, I/G4SI, and VIIS2) were used as primary antibodies on acetone-fixed cryostat slices.

Determination of the nucleotide sequence of mDT-VP3C after infection. To determine the genetic stability of mDT-VP3C after infection of SPF chickens, we inoculated chickens with this recombinant virus five times sequentially. Viral dsRNA was isolated at 3 days p.i. from the bursas of the infected chickens after the first, third, and fifth sequential passages. Following reverse transcription-PCR amplification, we directly sequenced the region of the A segment of mDT-VP3C corresponding to the complete genomic dsRNA region originating from the serotype 2 strain TY89. Sequence alignment of the mDT-VP3C-produced reverse transcription-PCR fragments with the original cDNA plasmid pHB60-s2VP3C3 revealed that no nucleotide mutations were present in the genome of mDT-VP3C after the sequential passages.


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DISCUSSION
 
Outbreaks of vvIBDV pose a continuing threat to the poultry industry worldwide. Live vaccines currently in use to prevent vvIBDV outbreaks are based on adapted field isolates of vvIBDV, the molecular basis of which is unknown. Moreover, the residual virulence of these so-called hot vaccine strains is still considerably high, hampering official registration and leading to uncontrolled usage of wild-type vvIBDV strains as hot vaccines during an outbreak. Our search for a new live vvIBDV vaccine with limited residual virulence that would also serve as an immunological marker for differentiation between the vaccine and field strains culminated in our obtaining the first engineered recombinant vvIBDV strain to possess both properties. The rescued mosaic vvIBDV described in this paper encodes a VP3 that is based partly upon the serotype 1 genome and partly upon the serotype 2 genome. Introduction of the VP3 sequence of serotype 2 results in attenuation, as is clear from a large reduction or even absence of mortality in highly susceptible SPF chickens as well as a reduction in bursa damage. Furthermore, by using specific MAb in an immunohistochemical analysis, we have proved that we can distinguish between bursas infected with wild-type (r)IBDV and bursas infected with the engineered mIBDV.

Amino acid sequence comparison of IBDV serotype 1 and 2 strains reveals that the hypervariable region of VP2 (amino acids 224 to 314; identity of less than 70%) is the most diverse. This hypervariable region is involved both in receptor recognition and in binding of neutralizing antibodies. Sequence identity in the other parts of the polyprotein (i.e., the remaining parts of VP2, VP4, and VP3) is above 90%. The identity between VP3 of serotype 1 and 2 is 96%, and four mutations are clustered in the first 33 amino acids of VP3 (Fig. 3). We failed to rescue mosaic vvIBDV containing these four mutations and one additional mutation located more downstream (mDT-VP3N [Table 1]). This failure might have been due to the inviability of the engineered virus or to the inefficient rescue of severely crippled mosaic vvIBDV.

Although we believe that our cDNA transfection method is more efficient than the mRNA transfection method (3), we also tried to rescue virus from pHB60-s2VP3N after cotransfection of capped mRNA. However, several independent cotransfection experiments with mRNAs derived from pHB60-s2VP3N and pHB-55 did not result in infectious mDT-VP3N (data not shown). The fact that SDS-polyacrylamide gel electrophoresis migration of chimeric VP3 proteins alters when the serotype 2 N-terminal part is present (Fig. 2) indicates that the overall properties of this hybrid VP3 are different from those of wild-type serotype 1 VP3. Furthermore, we discovered that both the rescue efficiency and final titers of the corresponding cell culture-adapted variant of this virus (mCEF94-s2VP3N) were considerably less than those of the wild-type virus (6).

The rescued IBDV strains were analyzed in two different animal experiments with essentially the same experimental setup. Postmortem analysis of the bursa of Fabricius of infected chickens indicated persistent bursa tropism of mosaic vvIBDV viruses, as the bursa/body weight ratio was considerably less than that for the noninfected chickens or (m)CEF94-infected chickens. Despite the bursa tropism, no mortality was induced by infection with mDCT-VP3C in experiment 1. However, in experiment 2 one chick died, indicating some residual pathogenicity. The other mosaic vvIBDV tested in experiment 2 (mDT-VP3C) did not induce mortality. This result was unexpected, as mDT-VP3C resembles wild-type D6948 more than mDCT-VP3C does (Fig. 1 and Table 1). We believe that the mDCT-VP3C-induced mortality in experiment 2 and not in experiment 1 is because of the variation that is intrinsic in animal trials. Variation was also observed between the two different animal trials with wild-type and rescued D6948 virus infections. The virus preparations used to inoculate the SPF chickens in animal experiments 1 and 2 had exactly the same history (only the storage time at -70°C was different) and the same formulation (diluted in PBS). Despite this, we found considerable variation in mortality during the first days after infection (30 and 53% for D6948 and 50 and 23% for rD6948 in experiments 1 and 2, respectively). This variation made it difficult for us to use differences in mortality rates in SPF chickens as a parameter for the exact classification of the virulence of (modified) vvIBDV strains.

The ratio between the weight of the bursa of Fabricius and the weight of the body (bursa/body weight ratio) is an objective and commonly used way of measuring IBDV pathogenicity. In our two animal experiments, the bursa/body weight ratio gives a clear-cut difference between viruses that possess B-lymphoid cell tropism (i.e., D6948-derived viruses [bursa/body weight ratio, <3.0]) and those that do not (i.e., CEF94-derived viruses [bursa/body weight ratio, >3.0]). Variation between the two animal experiments was found in the case of mDCT-VP3C (the bursa/body weight ratios were 1.9 [standard deviation, 0.6] and 2.7 [standard deviation, 0.7]) in experiments 1 and 2, respectively). The slightly different experimental setups in experiment 1 (the bursa/body weight ratio was determined at 9 days p.i.) and experiment 2 (the bursa/body weight ratio was determined at 13 days p.i.) and the repopulation of the partly damaged bursa by new B-lymphoid cells might be the reason for the different bursa/body weight ratios. Although repopulation is an important criterion after clearance of the IBDV infection, 14 days p.i. would be very early for a considerable repopulation to occur.

Determination of the histological damage of the bursa of Fabricius after infection is another way of measuring the virulence of IBDV strains. Using the classification developed previously (2), we found no difference between the severe damage induced by infection with vvIBDV (D6948 and rD6948) and the modified derivatives (mDCT-VP3C and mDT-VP3C). The method used to measure the level of damage in IBDV-infected bursas is not standardized. The overall damage is influenced by the reduction in percentage of B-lymphoid cells per follicle, the number of completely depopulated follicles, and the amount of necrosis and cystic formation. Furthermore, the lack of repopulation of the depleted follicles with new B-lymphoid cells seems to be the most important factor accounting for the observed subclinical immunosuppression associated with an IBDV infection. Thus, despite the equal HBLS values for wild-type IBDV and mIBDV, we found a clear difference between the induced bursa damage after infection with mIBDV and infection with unmodified vvIBDV (Fig. 4). Whether there is a difference in subclinical immunosuppression between the wild-type vvIBDV and the mIBDV strains will need to be evaluated in additional animal experiments, for instance, by challenge of IBDV-vaccinated SPF chickens with Newcastle disease virus and measurement of the neutralizing antibody response.

The present lack of objective virulence parameters for IBDV makes it difficult to quantify residual virulence of modified vvIBDV strains. An objective and quantitative classification of the virulence of recombinant IBDV strains will become increasingly more important as more genetically altered vvIBDV strains become available.

Despite the fact that the wild-type vvIBDV strain D6948 and the mosaic vvIBDV strain mDT-VP3C differ by only a mere six amino acids, mDT-VP3C was found to be clearly less virulent. VP3 has interactions at least with itself (trimer formation) and with two other viral proteins (i.e., VP2, and VP1). The direct interaction between VP3 and VP1 (and VPg) has been shown indirectly by analysis in the yeast two-hybrid system (32) and directly by specific immunoprecipitations (24, 32). The interaction of the trimeric VP3 with the trimeric VP2 at the interface of the outer capsid layer (VP2) and inner capsid layer (VP3) has been shown by electron cryomicroscopy (8, 11). Mutations in the C-terminal part of VP3 might thus influence one or multiple protein-protein interactions.

VP3 is also postulated to interact with the viral genome. The basic amino acids in the C-terminal part of VP3 are the prime candidates for this interaction (8, 19). Recently, we showed that VP3 is indeed able to interact with the viral dsRNA (32a). The fact that the capsid protein VP3 interacts with two essential components of the genome replication machinery (i.e., the dsRNA and the RNA-dependent RNA polymerase [VP1]) suggests that VP3 has a multifunctional role in replication of the dsRNA. The three-dimensional structure of VP3 of birnaviruses is not yet known, making it difficult to allocate the different homotypic or heterotypic protein- and dsRNA-interacting domains to specific positions on VP3 and to predict the effect of amino acid mutations in the C-terminal part on VP3 on these interactions.

Despite replication of mDT-VP3C being somewhat impaired in comparison to that of wild-type virus, no major selection pressure on the introduced part of the serotype 2 genome of the chimeric mDT-VP3C3 seemed to be present. Sequential passage of this virus (five times) did not lead to any back mutation of the introduced nucleotide changes.

Immunohistological examination shows that specific MAb can be used to differentiate between bursas infected with wild-type vvIBDV and bursas infected with our engineered mIBDV and vice versa. A proven approach to distinguish between wild-type-(vv)IBDV-infected chickens and vaccinated chickens is to make use of a difference in the antibody response after vaccination and infection. We are currently developing an indirect competition ELISA to evaluate whether the different antigenic structure of mDT-VP3C indeed induces an antibody response that can be differentiated from that of wild-type-(vv)IBDV-infected chickens.


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ACKNOWLEDGMENTS
 
We thank Stephanie Vastenhouw and Jos Dekker for assistance with plasmid construction and Arie Kant and Ralph Kok for assistance with the animal experiments and postmortem analysis.


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FOOTNOTES
 
* Corresponding author. Mailing address: ID-Lelystad, P. O. Box 65, NL-8200 AB Lelystad, The Netherlands. Phone: 31 320 238 695. Fax: 31 320 238 668. E-mail: H.J.Boot{at}id.wag-ur.nl. Back


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Journal of Virology, October 2002, p. 10346-10355, Vol. 76, No. 20
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.20.10346-10355.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.




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