VP5 and the N terminus of VP2 are not responsible for the different pathotype of serotype I and II infectious bursal disease virus

Anja Schröder1, Adriaan A. W. M. van Loon2, Danny Goovaerts2, Jens Peter Teifke3 and Egbert Mundt1

Institute of Molecular Biology1 and Infectiology3, Friedrich-Loeffler-Institutes, Federal Research Centre for Virus Diseases of Animals, D-17498 Insel Riems, Germany
Intervet International BV, NL-5830 AA Boxmeer, The Netherlands2

Author for correspondence: Egbert Mundt. Fax +49 38351 7151. e-mail Egbert.Mundt{at}rie.bfav.de


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
Two serotypes have been identified in infectious bursal disease virus (IBDV), a member of the family Birnaviridae. A reverse genetics system was used for generation of chimeras in genome segment A of the two serotypes, in which the complete viral VP5 gene and 3' noncoding region (NCR), or parts thereof, were exchanged. The engineered viruses were characterized in vitro and in vivo in comparison to serotype I and II IBDV. Our results show that IBDV chimeras exhibit a different phenotype in cell culture compared to the wild-type viruses. In in vitro-cultivated bursal-derived cells, chimeric viruses infected B lymphocytes, as does serotype I IBDV. Surprisingly, serotype II virus was also able to infect in vitro-cultivated bursal cells, but these were neither B lymphocytes nor macrophages. After infection of susceptible chickens all chimeras replicated in the bursa of Fabricius (BF), and three chimeric viruses caused mild depletion of bursal cells. In contrast, after infection of chickens with a chimeric IBDV containing exchanged VP5 as well as 3'-NCR, no depletion was detectable. The serotype II strain did not replicate in the BF nor did it cause depletion of bursal cells. Thus, the origin of VP5 does not explain the different pathotype of IBDV serotype I and II.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
Infectious bursal disease of chickens was first described in chickens by Cosgrove (1962) . The causative agent of this highly contagious immunosuppressive disease is infectious bursal disease virus (IBDV). Serotype I IBDV are pathogenic for chickens, but individual strains differ markedly in their virulence. A second serotype (serotype II), isolated from fowl, turkeys and ducks (McFerran et al., 1980 ), is apathogenic for chickens. The two serotypes can be differentiated by cross-neutralization assay (McFerran et al., 1980 ) and ELISA using monoclonal antibodies (Öppling et al., 1991 )

IBDV belongs to the genus Avibirnavirus of the family Birnaviridae (Murphy et al., 1995 ). The genome consists of two segments, A and B, of double-stranded RNA, which are enclosed within a nonenveloped icosahedral capsid approximately 60 nm in diameter. Mundt & Müller (1995) determined the terminal sequences of three serotype I and one serotype II strains of IBDV and completed the sequence of four strains. The larger segment A encodes a polyprotein of approximately 110 kDa (Hudson et al., 1986 ). The polyprotein is autoproteolytically processed by the cis-acting viral protease VP4 into the viral proteins (VP) VP2, VP3 and VP4 as shown recently (Birghan et al., 2000 ). A second open reading frame (ORF) preceding and partially overlapping the polyprotein gene (Spies et al., 1989 ; Bayliss et al., 1990 ) encodes VP5 (Mundt et al., 1995 ), a protein which is not essential for virus replication in vitro (Mundt et al., 1997 ) or in vivo (Yao et al., 1998 ). Segment B encodes the 97 kDa VP1, which represents the putative viral RNA-dependent RNA polymerase (Spies et al., 1987 ).

Serotype I strains are pathogenic since by lymphocytic depletion they cause lesions in the bursa of Fabricius (BF) of susceptible chicken strains. No lesions in the BF were detected after infection of susceptible chickens with serotype II strains, which are therefore considered nonpathogenic (Ismail et al., 1988 ). The reasons for these different biological properties are unknown. Exchange of the noncoding region (NCR) of segment A of serotype II strains with that of serotype I strains did not alter the pathotype of chimeric viruses (Schröder et al., 2000 ). Therefore, the NCR did not cause the different phenotype as hypothesized by Mundt & Müller (1995) . Yao et al. (1998) showed that inactivation of VP5 expression by a pathogenic serotype I strain resulted in complete attenuation of the virus. Although the exact function of VP5 is still unknown, it might influence the different pathotype of both serotypes (Yao et al., 1998 ).

Serotype I as well as serotype II virus particles bound to different lymphoid cells of chickens (Nieper & Müller, 1996 ), which indicates that restriction of IBDV to B cells might not be due to presence or absence of specific cellular receptors, but might be due to factors involved in regulation of replication.

The influence of VP5 on virulence was investigated by using a reverse genetics system (Mundt & Vakharia, 1996 ) to generate chimeric viruses containing either partly or completely exchanged VP5 genes. Recovered virus was subsequently characterized in vitro and in vivo.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Virus and cells.
The serotype II strain 23/82 (a generous gift from H. Becht, University of Giessen, Germany) and the chimeric IBDV/EK (Schröder et al., 2000 ) were propagated in Vero cells [Collection of Cell Lines in Veterinary Medicine (CCLV), Insel Riems, Germany, RIE 136]. Chicken embryo culture cells (CEC) derived from embryonated SPF eggs (VALO, Lohmann, Cuxhaven, Germany) grown in Dulbecco’s Minimal Essential Medium (DMEM) supplemented with 10% foetal calf serum (FCS) were used for establishment of growth kinetics, passaging of transfection supernatants and immunofluorescence assays. Transfection experiments were performed on quail muscle cells (QM-7, CCLV, RIE 466) grown in medium 199 supplemented with 10% FCS. Buffalo green monkey cells (BGM, CCLV, RIE 136) grown in DMEM supplemented with 10% FCS were used for plaque assays. Recovered viruses were propagated in Vero cells grown in DMEM supplemented with 10% FCS.

UV inactivation of the virus was performed by irradiation (30 W, 254 nm, 1 h) of 2 ml of virus suspension placed in one well of a six-well tissue culture dish. The infectivity of the inactivated virus was tested by plaque assay on BGM cells.

{blacksquare} Construction of chimeric IBDV.
For construction of chimeric A segments a plasmid (pD78A) containing the full-length cDNA sequence of segment A of strain D78 was used. For cloning the VP5 gene of serotype II strain 23/82 virus was propagated in CEC and purified by ultracentrifugation as described (Müller et al., 1986). After proteinase K (0·5 mg/ml)–SDS (0·5%) digestion genomic viral RNA was purified (Mundt & Müller, 1995 ), reverse transcribed into cDNA, and amplified by PCR following standard procedures using oligonucleotides FKA5' and FKA1R (Table 1). The amplification product was cloned blunt-ended, and plasmids containing appropriate PCR fragments (pA23part) were sequenced. After sequence analysis pA23part was cleaved with EcoRI/RsrII and EcoRI/NdeI. After electroelution the EcoRI–RsrII fragment was ligated into appropriately digested pD78A to obtain p5'-R-D78A, containing the 5'-end of the overlapping ORF encoding the N-terminal part of VP5 as well as VP2 of serotype II strain 23/82 (Fig. 1). Exchange of the complete ORF encoding VP5 and, due to the gene overlap, a part of the ORF of VP2, was achieved by EcoRI/NdeI cleavage of pA23part and ligation into appropriately cleaved pD78A to obtain p5'-N-D78A. Exchange of the 3'-NCR was performed as described previously (Schröder et al., 2000 ) to obtain p5'-R-D78A-3' and p5'-N-D78A-3', respectively. Maps of the plasmids are depicted in Fig. 2.


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Table 1. Oligonucleotides used for amplification of serotype II strain 23/82 sequences

 


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Fig. 1. Alignment of partial nucleotide and amino acid sequences of segment A of serotype I strain D78 and serotype II strain 23/82. (A) Alignment of the 5'-NCR. Dashes indicate residues identical to the sequence of strain D78. The start codon of VP5 and the polyprotein are underlined. (B) Alignment of the amino acid sequence of VP5. The restriction enzyme cleavage site where exchange of sequences of segment A of serotype I strain D78 and serotype II strain 23/82 occurred within the nucleotide sequence of VP5 is highlighted by an arrow. (C) Alignment of the amino acid sequence of the exchanged part of the polyprotein. The restriction enzyme cleavage site where exchange of sequences of segment A of serotype I strain D78 and serotype II strain 23/82 occurred and the location of the VP5 stop codon within the nucleotide sequence of the polyprotein are marked by an arrow. Numbering of nucleotides and amino acids is according to the published sequence of strain P2 (Mundt & Müller, 1995 ).

 


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Fig. 2. Construction of chimeric cDNA clones of segment A of IBDV. A map of the genomic organization of IBDV segment A is shown at the top of the figure. Sequences of segment A of strain D78 are depicted by an open box and sequences of serotype II strain 23/82 are marked by shaded boxes. Restriction enzymes cleavage sites used for cloning are indicated. Full-length cDNAs were constructed under control of the T7 RNA polymerase promotor. Numbering of nucleotides is according to the published sequence of strain P2 (Mundt & Müller, 1995 ).

 
For in vitro transcription plasmids p5'-R-D78A, p5'-R-D78A-3', p5'-N-D78A, p5'-N-D78A-3' (Fig. 2) and pBP2 (Mundt & Vakharia, 1996 ) were linearized by cleavage with BsrGI and PstI. Further treatment of linearized DNA, and transcription and transfection of RNA into QM-7 cells were carried out as described by Mundt (1999) . Two days after transfection cells were freeze–thawed, centrifuged at 700 g to remove cellular debris, and the resulting supernatants were further clarified by filtration through 0·45 µm filters and stored at -70 °C. Virus progeny recovered from QM-7 cells transfected with RNA of p5'-R-D78A/pBP2 (5'-R-IBDV), p5'-R-D78A-3'/pBP2 (5'-R-IBDV-3'), p5'-N-D78A/pBP2 (5'-N-IBDV) or p5'-N-D78A-3'/pBP2 (5'N-IBDV-3') were passaged once on Vero cells and the presence of IBDV antigen was detected by immunofluorescence assay using rabbit anti-IBDV serum (Mundt et al., 1995 ).

Propagation of the chimeric viruses in Vero cells was as described by Schröder et al. (2000) . Virus stocks were stored at -70 °C.

{blacksquare} Characterization of chimeric viruses in cell culture.
Growth kinetics in cell culture were established for investigation of the replication properties of chimeric viruses and the serotype II strain 23/82. Confluent secondary CEC grown in a 24-well tissue culture dish were infected with strain 23/82, IBDV/EK, 5'-R-IBDV, 5'-R-IBDV-3', 5'-N-IBDV or 5'-N-IBDV-3' at an m.o.i. of 1. Following incubation at room temperature for 1 h inoculum was removed, cells were rinsed twice with PBS, and overlaid with 1 ml DMEM. Immediately thereafter the supernatant from one well was removed and stored at -70 °C [0 h post-infection (p.i.)]. Remaining wells were further incubated; 8, 12, 24, 36 and 48 h p.i. supernatants were removed and stored at -70 °C. Supernatants were then centrifuged and titrated on BGM cells.

{blacksquare} Characterization of chimeric viruses in chicken.
In the first experiment, 92 2-week-old specific pathogen free (SPF) chickens (Intervet, Boxmeer, The Netherlands) were randomly divided into six groups. Each group was maintained in negative pressure filtered air isolators. Chickens were infected via eye drop with 104·7 TCID50 of IBDV/EK, 5'-R-IBDV-3', 5'-R-IBDV-3', 5'-N-IBDV or 5'-N-IBDV-3'. Uninoculated hatchmates were used as controls. At 3, 7 and 13 days p. i. five chickens from each group were bled, euthanized and the BF of each chicken was removed. Based on the results of the first animal experiment, in a second experiment chimeric IBDV 5'-N-IBDV and 5'-N-IBDV-3' were compared with the serotype II strain 23/82. To this end 100 2-week-old SPF chickens (Intervet) were randomly divided into four groups. After infection via eye drop with 105·3 TCID50 per animal five chickens from each group were bled and euthanized at 3, 7, 13, 17 and 24 days p.i., and the BF was removed. The BF obtained from both experiments were divided into two parts. One part was used for virus reisolation, and the second part was fixed in 10% neutral-buffered formalin for histology.

To confirm the identity of the chimeric virus RNA from the reisolated virus was amplified by RT–PCR using oligonucleotides as shown in Table 1. Cloned PCR fragments were sequenced and the sequences were analysed using the Wisconsin package, Version 8 (Genetics Computer Group, Madison, WI, USA).

{blacksquare} Histopathology and Immunohistochemistry.
Tissue samples of BF were fixed immediately after necropsy in 10% neutral-buffered formalin for 24 h and paraffin-embedded. Serial sections (4 µm) were mounted on organosilane-coated slides, dewaxed and stained with haematoxylin–eosin (H&E) or used for immunohistochemistry (IHC) or in situ hybridization (ISH). The severity of bursal follicular necrosis was scored using the Bursa-Lesion-Scale (BLS) as described (Schröder et al., 2000 ). IHC was performed according to a standardized protocol as described previously (Teifke et al., 1998 ). In brief, for detection of IBDV-antigen tissue sections were incubated overnight with a polyclonal rabbit anti-IBDV serum (Mundt et al., 1995 ), diluted 1:1000 in PBS. The sections were incubated consecutively with a biotinylated anti-rabbit IgG antibody and avidin-biotinylated horseradish peroxidase complex (Vector Laboratories). Staining was done with diaminobenzidine tetrahydrochloride (DAB). After counterstaining with Mayer’s haematoxylin the sections were mounted in Eukitt (Hecht). As negative control a polyclonal rabbit anti-bovine papillomavirus serum (Dako) was used.

{blacksquare} In situ hybridization.
Preparation of a VP-4 gene-specific digoxigenin-labelled probe, ISH and detection reaction were done as described recently (Nieper et al., 1999 ). As control a probe specific for chelonid herpesvirus DNA was used (Teifke et al., 2000 ). Intensity of hybridization signals in 100 bursal follicles was scored on a scale of - to +++ as follows: -, no hybridization signal in the bursal follicle; +, hybridization signals in <25% of follicular lymphocytes; ++, hybridization signals in <75% of follicular lymphocytes; +++, hybridization signals in 75–100% of follicular lymphocytes.

{blacksquare} Infection of bursal cells in vitro.
Three- to six-week-old SPF chickens (VALO) were bled, euthanized, and the BF were removed. Single bursal cells were obtained by stirring for 15 min at room temperature after the bursal tissue was minced with a sterile scalpel in 40 ml Hahn medium (Hirai & Calnek, 1979 ): 8 ml of the cell suspension was layered onto 4 ml Ficoll-paque, and centrifuged (625 g, 4 °C, 30 min). Bursal cells were collected from the interface, washed twice, and resuspended in Hahn medium; 3 ml of the single cell suspension was pipetted into a six-well tissue culture plate, infected at an m.o.i. of 1, incubated for 7 h at 37 °C, and fixed with acetone–methanol (1:1) in a 1·5 ml Eppendorf tube. After a wash in PBS, cells were incubated with either a mixture of rabbit anti-IBDV serum/AV20 (monoclonal anti-B cell antibody; a generous gift from F. Davison, Compton, UK) or rabbit anti-IBDV serum/CVI-68.1 (monoclonal anti-mononuclear phagocytes antibody, ID-IDLO, Lelystad, The Netherlands). The rabbit anti-IBDV serum was described by Mundt et al. (1995) , the AV20 monoclonal antibody by Rothwell et al. (1996) and CVI-68.1 by Jeurissen et al. (1988) . The antibody–cell mixture was incubated for 30 min, washed twice in PBS and incubated with a mixture of DTAF-conjugated goat anti-rabbit IgG and Cy3-conjugated goat anti-mouse IgG (Dianova). After 30 min cells were washed twice, resuspended in PBS, and pipetted onto a poly-L-lysine-treated coverslip. After 5 min to allow cells to sediment onto the surface of the coverslip the supernatant was carefully decanted and the coverslip was dried. The cells were examined by confocal laser scan microscopy using a Zeiss LFM510.


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
Recovery of chimeric IBDV
For transfection experiments full-length cDNA clones of chimeric segments A (p5'-R-D78A, p5'-R-D78A-3', p5'-N-D78A or p5'-N-D78A-3') were transcribed into plus-strand cRNA and cotransfected with segment B (pBP2) full-length plus-strand cRNA into QM-7 cells. The generation of infectious chimeric IBDV was verified by infection of secondary CEC with transfection supernatant. At 24 h p.i. acetone-fixed cells were incubated with rabbit anti-IBDV serum and fluorescein-conjugated secondary antibody. IBDV antigen was detected in the cytoplasm of cells infected with supernatants of all cRNA-transfected cells. Mock-infected cells showed no specific fluorescence (data not shown). Resulting virus progeny (5'-R-IBDV, 5'-R-IBDV-3', 5'-N-IBDV and 5'-N-IBDV-3') were passaged once on Vero cells and titrated. Titres were 3·6x106 p.f.u./ml for 5'-R-IBDV, 2·3x106 p.f.u./ml for 5'-R-IBDV-3', 1·5x106 p.f.u./ml for 5'-N-IBDV and 1·3x106 p.f.u./ml for 5'-N-IBDV-3'. In parallel serotype II strain 23/82 was propagated once on Vero cells; the titre was 6·5x107 p.f.u./ml.

Replication of chimeric IBDV in CEC
To characterize replication of the different chimeric IBDV in more detail growth kinetics were analysed (Fig. 3). Supernatants of confluent secondary CEC infected with strain 23/82, IBDV/EK, 5'-R-IBDV, 5'-R-IBDV-3', 5'-N-IBDV or 5'-N-IBDV-3' were removed at 0, 8, 12, 24, 36 and 48 h p.i. and titrated on BGM cells. The titres determined showed no significant differences between serotype I IBDV/EK and serotype II 23/82. However, all chimeric IBDV containing a part or the complete VP5 of the serotype II strain 23/82 replicated less well than IBDV/EK and 23/82 during the observed time period. No significant titre differences between the chimeric IBDV were observed for any of the assayed growth kinetics.



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Fig. 3. Replication kinetics of chimeric IBDV. CEC were infected with 23/82, IBDV/EK, 5'-R-IBDV, 5'-R-IBDV-3', 5'-N-IBDV or 5'-N-IBDV-3', as described in Methods. Supernatants removed at the indicated times p.i. were used for determination of the virus titre (p.f.u./ml). Values are plotted exponentially on the vertical axis. Average titres and standard deviation (error bars) from three independent experiments are indicated.

 
Replication of chimeric IBDV in bursal cells
To analyse whether the exchange of VP5 or part of it influenced the ability of IBDV to replicate in B lymphocytes, bursal cells were infected and investigated by indirect immunofluorescence using double labelling. All experiments were performed in parallel. After infection of bursal cells with serotype I IBDV/EK (Fig. 4 A) or 5'-N-IBDV-3' (Fig. 4 C) viral antigen was detected in the cytoplasm of cells, which was also labelled by the AV20 antibody. However, after infection with IBDV/EK (Fig. 4B) or 5'-N-IBDV-3' (not shown) no IBDV antigen was detected in cells labelled with antibody CVI-68.1. 5'-R-IBDV, 5'-R-IBDV-3' and 5'-N-IBDV showed the same pattern as IBDV/EK (data not shown). In contrast, after infection of bursal cells with the serotype II strain 23/82 IBDV antigen was only visible in cells which were neither labelled by AV20 (Fig. 4 D) nor by CVI-68.1 (Fig. 4 E). To confirm that the detected IBDV antigen was indeed the result of virus replication, UV-inactivated virus was used for infection of bursal cells. Here, no IBDV antigen was detectable in the cytoplasm (data not shown). The noninfectivity of the UV-inactivated virus used was confirmed by plaque assay.



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Fig. 4. Infection of bursal-derived cells with IBDV. Cells were infected with serotype I IBDV/EK (A, B), 5'-N-IBDV-3' (C) or serotype II strain 23/82 (D, E). Cells were fixed cells and incubated with monoclonal antibody AV20 (A, C, D) or CVI-68.1 (B, E). In all samples IBDV antigen was detected by using rabbit anti-IBDV serum. The reactivity of the monoclonal antibodies was detected with goat anti-mouse Cy3-conjugated antibodies and the rabbit antibodies were detected with goat anti-rabbit DTAF-conjugated antibodies.

 
Replication of chimeric IBDV in chickens
Two animal experiments were performed to test the properties of chimeric IBDV in chickens. During both experiments neither clinical signs nor macroscopic abnormalities of the BF on necropsy were observed. In the first experiment chimeric IBDV (5'-R-IBDV, 5'-R-IBDV-3', 5'-N-IBDV and 5'-N-IBDV-3') were compared to the serotype I virus IBDV/EK as described by Schröder et al. (2000) . Bursal sections were stained with H&E and analysed to determine a bursal lesion score (BLS). Histopathological lesions in the cloacal bursa were consistent with earlier findings (Schröder et al., 2000 ). Degeneration and necrosis of lymphocytes starting in the medulla of bursal follicles and followed by replacement with heterophils, pyknotic cellular debris and proliferating reticuloendothelial cells were observed. At 3 days p.i. bursae of chicken infected with IBDV/EK showed acute inflammation with interfollicular oedema mixed with numerous heterophils and phagocytic cells in single follicles resulting in a BLS of 1. Single bursae obtained from animals infected with 5'-R-IBDV, 5'-R-IBDV-3' or 5'-N-IBDV showed different extents of follicular necrosis on day 7 p.i. (BLS of 1). At day 13 p.i. increasing numbers of macrophages and the beginning of fibroplasia were observed, leading to a predominance of interfollicular connective tissue, typical of the chronic stage of disease (IBDV/EK, 5'-R-IBDV, 5'-R-IBDV-3' and 5'-N-IBDV). Interestingly, in bursae of animals infected with 5'-N-IBDV-3' lesions were not detectable. They were also absent in the negative controls. Virus was reisolated on days 3 and 7 p.i. from nearly all BF of chickens infected with the chimeric virus. No virus was isolated from day 13 p.i. onwards from all BF of chicken infected with the chimeric virus or the uninfected controls.

To verify the results of the first experiment and to compare the phenotype of 5'-N-IBDV-3' with the serotype II strain 23/82 a second animal experiment was performed. The period of observation was extended for better assessment of the properties of the inoculated virus. Histological examination of the BF confirmed the result from the first animal experiment. At no times p.i. were histological abnormalities like inflammation or depletion of bursal cells detectable in BF of chickens infected with 5'-N-IBDV-3' or strain 23/82. Only BF of chickens infected with 5'-N-IBDV showed a BLS of 1 at days 7 and 13 p.i., but appeared normal at 17 and 24 days p.i. Reisolation of virus was successful from chickens infected with 5'-N-IBDV (3 and 7 days p.i.) and 5'-N-IBDV-3' (7 days p.i.). No virus was isolated from 13 days p.i onwards nor from all BF of chicken infected with serotype II strain 23/82 or the uninfected controls. Uninoculated control chickens were examined in parallel. Neither clinical signs nor gross or histological lesions were observed. Data summarizing the animal experiments are shown in Table 2.


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Table 2. Summary of results of animal experiments for testing different chimeric IBDV

 
The analysis of the sequences resulting from the amplified RT–PCR product based on the virus reisolated from the chickens confirmed the identity of the chimeric IBDV used.

In situ hybridization and immunohistochemistry
To determine whether there was any correlation between histopathological findings, IBDV antigen demonstration and viral nucleic acid detection serial sections of single bursae of selected chickens obtained from the first animal experiment were analysed. Strong hybridization signals specific for IBDV-RNA were confined to the cytoplasm of bursal follicular cells (Fig. 5, 1A). In general, intensity and location of hybridization signals correlated well with the staining intensity and distribution of IBDV-antigen in the different groups (Table 3; Fig. 5, 1A and 1B). However, by using methods for the detection of viral RNA or antigen, the number of positive follicles detected was greater than the number of necrotic follicles detected in bursae of chickens infected with IBDV/EK, 5'-R-IBDV, 5'-R-IBDV-3' or 5'-N-IBDV (data not shown). However, infectious virus was reisolated from bursae of chickens (Table 3) which showed neither hybridization signals (Fig. 5, 2A) nor viral antigen (Fig. 5, 2B).



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Fig. 5. Formalin-fixed paraffin-embedded sections of BF from chickens infected with 5'-R-IBDV-3' (1) or 5'-N-IBDV-3' (2), and killed 3 days p.i. Serial sections were examined by ISH (A) and IHC using polyclonal rabbit anti-IBDV serum (B), respectively. Bars represent 50 µm.

 

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Table 3. Comparison of different methods for virus detection

 

   Discussion
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Abstract
Introduction
Methods
Results
Discussion
References
 
The genus Avibirnavirus of the family Birnaviridae contains two serotypes (serotype I and II) which are distinguishable by conventional serology or monoclonal antibodies. The two serotypes show a striking difference, in that serotype I strains are pathogenic and serotype II strains are apathogenic in chickens. The molecular basis for this difference in phenotype is unknown. IBDV particles of both serotypes bind to lymphoid cells (Nieper & Müller, 1996 ), which indicates that the restriction of IBDV to lymphoid B cells might not be determined by the presence of a specific receptor, but cis-acting elements might play a role in restriction of virus replication in B-lymphocytes. Experiments described recently showed that the different NCRs of segment A of both serotypes did not influence pathogenicity in chickens (Schröder et al., 2000 ). By using the reverse genetics system for IBDV (Mundt & Vakharia, 1996 ) different chimeric IBDV were produced in order to investigate a possible influence of VP5 of serotype I and II on virus replication in vitro and in vivo.

Analysis of replication in tissue culture revealed significant differences between the chimeric viruses and serotype I as well as serotype II IBDV. Chimeric viruses replicated with a delay and to lower titres compared to wild-type IBDV. This was surprising since after exchange of the NCRs only no significant differences were observed in vitro (Schröder et al., 2000 ). The difference in phenotype in cell culture seems to be caused by the exchange of the N-terminal parts of VP5 (four amino acid substitutions) and VP2 (two amino acid substitutions), as shown in Fig. 1, since the phenotype did not change after exchange of the 5'-NCR only (Schröder et al., 2000 ). Nucleotide substitutions located between the start codon of VP5 and the start codon of the polyprotein could also result in different phenotypes in cell culture (see Fig. 1). However, an altered phenotype in cell culture (Fig. 3) of the chimeric viruses did not result in the loss of pathogenicity in chicken, since chimeric viruses were able to replicate in the BF as shown by virus reisolation. In contrast, no infectious serotype II virus could be detected in the BF of chickens after infection via the eye drop route at any time-point investigated (Table 2). Our findings seem to support earlier findings on the characterization of serotype II strains where no bursal lesions were detected (Jackwood et al., 1982 ; Ismail et al., 1988 ). However, these researchers did not attempt virus reisolation. The discrepancy between both experiments concerning virus isolation rate from chicken infected with 5'-N-IBDV-3' was possibly caused by animal variation since the flock was not inbred.

Three chimeric IBDV (5'-R-IBDV; 5'-R-IBDV-3' and 5'-N-IBDV) caused bursal lesions whereas 5'-N-IBDV-3' did not. Here, the additional exchange of the 3'-NCR seems to have an effect on pathogenicity. Therefore, the replication rate in cell culture does not faithfully reflect the pathogenicity of IBDV. This effect is dependent on the complete VP5 since bursal lesions were observed after infection with 5'-R-IBDV-3'. A possible RNA–protein interaction between the serotype II VP5 and 3'-NCR may play a role in bursal cells. However, VP5 of serotype II can functionally replace VP5 of serotype I and alone is not responsible for the different pathotypes of IBDV.

To determine whether the chimeric IBDV are able to infect B lymphocytes a double labelling technique was established. It was clearly shown that all mutants and, as expected, serotype I IBDV/EK infected B lymphocytes. This result supported the finding that infection of B lymphocytes is not restricted by VP5 of serotype II. Serotype II strain 23/82 was also able to infect bursal-derived cells, but these were neither mononuclear phagocytes nor B lymphocytes. This was surprising since in animal experiments no serotype II virus was reisolated from the infected animal. The identity of these cells remains unknown and requires further investigation.

Comparison of histological examination, ISH and IHC with virus reisolation results (see Table 3) demonstrated that virus reisolation from the bursal homogenates was more sensitive that the other methods for detection of viral antigen (IHC) or viral RNA (ISH). Results of the ISH correlated with the detection of antigen (IHC). This finding contrasts with Lui et al. (2000) who reported that ISH was superior to IHC. But ISH and IHC were clearly better than detection of bursal lesions by histological examination. Thus, detection of bursal lesions was the least sensitive method for assessing replication of IBDV in bursal tissue. Furthermore, the results support the finding that serotype II strain 23/82 is not able to infect bursal tissue after eye drop infection, which mimics the natural route of infection.

In summary, neither the NCRs (Schröder et al., 2000 ) nor VP5 nor the N terminus of VP2 up to nucleotide 647 are responsible for the different pathotype of IBDV serotypes I and II. Now, the most probable candidate for causing this different phenotype is VP2, which contains epitopes for neutralization of the virus in cell culture as well as the functional domains responsible for infection of cultured cells (Mundt, 1999 ). Appropriate experiments for the generation of IBDV chimeric in VP2 between serotype I and II are under way. Nieper & Müller (1996) showed that IBDV of both serotypes bound to bursal as well as cultivated cells. In the light of the data presented here, this binding does not seem to be sufficient for productive infection.


   Acknowledgments
 
We thank Thomas Mettenleiter for helpful discussions and critical reading of the manuscript and Dietlind Kretzschmar for excellent technical assistance. This study was supported by DFG grant MU 1244/1-2 and Intervet International, Boxmeer, The Netherlands.


   References
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Abstract
Introduction
Methods
Results
Discussion
References
 
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Received 26 July 2000; accepted 6 October 2000.



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