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
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Abstract |
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Introduction |
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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.
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Methods |
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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.
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 EcoRIRsrII 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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Propagation of the chimeric viruses in Vero cells was as described by Schröder et al. (2000) . Virus stocks were stored at -70 °C.
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.
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 RTPCR 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).
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 haematoxylineosin (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 Mayers haematoxylin the sections were mounted in Eukitt (Hecht). As negative control a polyclonal rabbit anti-bovine papillomavirus serum (Dako) was used.
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 75100% of follicular lymphocytes.
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 acetonemethanol (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 antibodycell 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.
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Results |
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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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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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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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Discussion |
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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 RNAprotein 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.
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Acknowledgments |
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References |
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Received 26 July 2000;
accepted 6 October 2000.
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