Tissue culture infectivity of different strains of infectious bursal disease virus is determined by distinct amino acids in VP2

Egbert Mundt1

Institute of Molecular Biology, Friedrich-Loeffler-Institutes, Federal Research Centre for Virus Diseases of Animals, D-17498 Insel Riems, Germany1

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 types of strains of serotype I of infectious bursal disease virus (IBDV) have been described, on the basis of their ability (IBDV-TC) or inability (IBDV-BU) to infect chicken embryonic cells in culture. However, both types infect B lymphocytes in the bursa of Fabricius of young chickens. To determine the molecular basis for tissue culture infectivity, virus recombinants with chimeric segments A were constructed from IBDV-TC and IBDV-BU by reverse genetics. The region responsible for the different phenotypes was located in VP2. Site-directed mutagenesis identified single amino acids that are responsible for the restriction in infectivity. However, the appropriate amino acid exchanges are strain-specific.


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

IBDV belongs to the genus Avibirnavirus of the family Birnaviridae (Dobos et al., 1995 ). The genome consists of two segments, A and B, of double-stranded RNA, which are localized within a single-shelled icosahedral capsid of 60 nm diameter. Recently, the complete genomic sequences of both segments of three serotype I strains and one serotype II strain of IBDV were determined (Mundt & Müller, 1995 ). The larger segment, A, encodes a polyprotein of approximately 110 kDa that is autoproteolytically cleaved (Hudson et al., 1986 ) to form the virus proteins VP2, VP3 and VP4. A second open reading frame (ORF), preceding and partially overlapping the polyprotein gene, has been identified (Bayliss et al., 1990 ; Spies et al., 1989 ). A protein encoded by this ORF, designated VP5, has been detected in IBDV-infected chicken embryo cells (CEC) as well as in bursal cells of IBDV-infected chickens (Mundt et al., 1995 ). Comparison of segment A sequences of different serotype I strains showed an overall high homology. However, a variable region is localized in VP2 (Bayliss et al., 1990 ). Genome segment B encodes a 97 kDa protein, designated VP1, that represents the putative viral RNA-dependent RNA polymerase (Spies et al., 1987 ).

Serotype I field strains can cause serious problems in the poultry industry. Several serotype I strains have been adapted to tissue culture, Cu-1 (Nick et al., 1976 ), PGB98 (Baxendale, 1976 ), P2 (Schobries et al., 1977 ), OKYMT, TSKMT (Yamaguchi et al., 1996 ), STC and IN (Hassan et al., 1996 ). After adaptation, these strains showed reduced in vivo pathogenicity (Cursiefen et al., 1979 ; Lange et al., 1987 ; Yamaguchi et al., 1996 ; Hassan et al., 1996 ). Usually, pathogenic field strains (bursa derived) are not easily adapted to cell culture, a process which requires extensive passaging either in cell culture (Hassan et al., 1996 ) or in the chorioallantoic membrane (CAM) as well as in the yolk sac of embryonated eggs (Yamaguchi et al., 1996 ). Several field isolates failed to become adapted to cell culture (McFerran et al., 1980 ).

The aim of this study was to determine specific structural requirements for the ability to infect cultured cells. Conversion of IBDV strains unable to grow in cultured cells into cell culture-infectious viruses by the use of the established reverse genetics system of IBDV (Mundt & Vakharia, 1996 ) is described. Furthermore, the application of the reverse genetics system to generation of IBDV segment A chimeras and their functional analysis is described.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Virus and cells.
The serotype I strain D78 (Intervet, Boxmeer, Netherlands) was propagated in CEC. The serotype I strain GLS, obtained from the bursa of Fabricius of infected chickens, (GLS-BU) and the tissue culture-adapted form (GLS-TC) were a generous gift from Dr A. van Loon (Intervet). CEC derived from embryonated specific pathogen-free (SPF) eggs (VALO, Lohmann, Cuxhaven, Germany) were grown in Dulbecco's minimal essential medium (DMEM) supplemented with 10% foetal calf serum (FCS) and were used for transfection, propagation of recovered virus, passaging of transfection supernatants and immunofluorescence assays (IFA). Transfection experiments and IFA were performed by using quail muscle cells (QM-7; ATCC no. CRL 1962) grown in medium 199 supplemented with 10% FCS.

{blacksquare} Analysis of sequences of segment A of IBDV.
To determine the amino acids that are responsible for the ability of IBDV to infect CEC, sequences of strains that are able to infect CEC were compared with sequences of strains that are unable to do so. Amino acid sequences (aa 251–360), comprising the variable regions of sequences of VP2 from tissue culture-infecting strains published by Yamaguchi et al. (1996) (strains J1, accession no. D16677; K, D16678; OKYMT, D83985; and TKSMT, D84071), Bayliss et al. (1990) (strains PGB98, D00868; and Cu-1, D00867) and Mundt & Müller (1995) (strain P2, X84034) and from strain D78 (V. N. Vakharia, personal communication), were compared to obtain a consensus sequence. Sequences of bursa-derived IBDV strains have been published by Vakharia et al. (1992) (strain E/Del, D10065), Bayliss et al. (1990) (52/70, D00869), Thiry et al. (1992) (Edgar, A33255), Kibenge et al. (1990) (STC, D00499), Yamaguchi et al. (1996) (OKYM, D49706; and TKSM, D84072), Brown et al. (1994) (DV86, Z25482; and UK661, X92760), Hudson et al. (1986) (002-73, X03993), Vakharia et al. (1994) (GLS, M97346), Yamaguchi et al. (1997) (LukertBP, D16679), Lana et al. (1992) (variant A strain, M64285), Pitcovski et al. (1998) (KS, L42284) and Heine et al. (1991) (variant E strain, D10065). Unpublished sequences from strains U-26 (AF091099), Miss (AF091098) and 3212 (AF091097) (H. S. Sellers, P. N. Villegas, D. J. Jackwood & B. S. Seal, unpublished results) were also included in the comparison. In this manuscript, a consensus sequence was deduced and compared with the consensus sequence of tissue culture-infecting strains. Sequences were analysed with the GCG software package, version 8 (Genetics Computer Group, Wisconsin, USA).

{blacksquare} Construction of a full-length cDNA clone of segment B of strain D78.
For cloning of the full-length cDNA of segment B of serotype I strain D78, virus was propagated in CEC and purified by ultracentrifugation as described previously (Müller et al., 1986 ). Genomic viral RNA of strain D78 was purified (Mundt & Müller, 1995 ), reverse-transcribed into cDNA and amplified by PCR by standard procedures, using oligonucleotides described previously (Mundt & Vakharia, 1996 ). The amplification product was cloned blunt-ended and plasmids containing appropriate PCR fragments were sequenced. The cloning procedure to obtain a plasmid containing the full-length cDNA of segment B (pD78B) under control of the T7 RNA polymerase promoter corresponded to the procedure described by Mundt & Vakharia (1996) for segment B of strain P2.

{blacksquare} Construction of chimeric IBDV plasmids.
A prerequisite for the following site-directed mutagenesis was the modification of the plasmid pUC18. To this end, pUC18 was cleaved with NdeI and BamHI, electroeluted, blunt-ended by Klenow polymerase and religated to obtain pUC18/{Delta}NB. Plasmid pAD78/EK (Mundt et al., 1997 ) was cleaved with EcoRI and KpnI to obtain the full-length sequence of segment A of serotype I strain D78, including the T7 RNA polymerase promoter site. This fragment was ligated into EcoRI/KpnI-cleaved pUC18/{Delta}NB to obtain pD78A. Plasmid pD78A was used as the backbone for the following cloning and site-directed mutagenesis procedures.

For substitution of IBDV-specific sequences, a plasmid containing the complete coding region of the USA variant E strain E/Del was used (p10-22, a generous gift from Dr A. van Loon, Intervet). p10-22 was cleaved with restriction enzymes NdeI and SalI at nucleotides 647 and 1725, respectively (numbering follows the full-length sequence of strain P2, NCBI accession no. X84034), to obtain a 1078 bp fragment encompassing coding sequences of the variable region of VP2 and partial sequences of VP4 of strain E/Del. After ligation into NdeI/SalI-cleaved pD78A, a chimeric full-length plasmid pD78A-E/Del, containing sequences of segment A of strain D78 as well as E/Del, was established. Plasmids pD78A and pD78A-E/Del were used for site-directed mutagenesis and are depicted in Fig. 1.



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Fig. 1. 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. Coding sequences of segment A of strain D78 are depicted by an open box. E/Del sequences are marked by shaded boxes and sequences of strain GLS are shown as a black box. Restriction enzymes and their cleavage sites used for cloning are indicated. Full-length cDNAs were constructed under the control of the T7 RNA polymerase promoter. Locations of mutated amino acids are indicated and named by the single letter code. Numbering of nucleotides and amino acids are according to the published sequence of strain P2 (Mundt & Müller, 1995 ).

 
A further pair of plasmids was constructed, containing the variable regions of GLS-BU and GLS-TC. For cloning of the variable region, GLS-TC was propagated in CEC and purified by ultracentrifugation as described previously (Müller et al., 1986 ). A bursal homogenate of GLS-BU was purified by low-speed centrifugation and the supernatant was further processed. After 0·5 mg/ml proteinase K/0·5% SDS digestion, viral RNA was purified (Mundt & Müller, 1995 ), reverse-transcribed into cDNA and amplified by PCR by standard procedures, using oligonucleotides A14 and A44 (Table 1). The amplification product was cloned blunt-ended and plasmids containing appropriate PCR fragments (pGLS-TC, pGLS-BU) were sequenced. For construction of chimeric segments A, the full-length clone pD78A-E/Del was used. pGLS-TC and pGLS-BU were digested with SacI and SpeI, respectively. Electroeluted fragments were subsequently ligated into SacI/SpeI-digested pD78A-E/Del to obtain pD78A-E/Del-GLS-TC and pD78A-E/Del-GLS-BU, respectively. Maps of both plasmids are depicted in Fig. 1.


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Table 1. Oligonucleotides used for the construction of full-length cDNA clones of IBDV segment A containing amino acid substitutions

 
{blacksquare} Site-directed mutagenesis.
Site-directed mutagenesis was performed by PCR. Oligonucleotides contained restriction enzyme cleavage sites and mutations leading to amino acid exchanges (Table 1). For the establishment of full-length clones of segment A of plasmid pD78A containing mutated codons, an NdeI–HindIII fragment of pD78A was subcloned into NdeI/HindIII-cleaved pUC19 (pUC19/{Delta}NH) to obtain single restriction enzyme sites for the cloning procedures (pUC19/{Delta}NH-D78A, Fig. 1). Plasmids based on pUC19/{Delta}NH-D78A, containing the mutated codons for residues 253 (H to Q), 284 (T to A), 330 (R to S) and all three amino acids, were cleaved with NdeI and SacII, appropriate fragments were electroeluted and ligated into pD78. Mutations in pD78-E/Del were generated by following standard procedures. Here, substitutions of residues 253 (Q to H), 284 (A to T) and 330 (S to R) were performed in all seven combinations (Table 2). After PCR, amplification fragments were cloned blunt-ended and sequenced. Fragments containing the mutated codons were ligated into plasmids using restriction enzyme cleavage sites as described in Fig. 1. Oligonucleotides, plasmids used for PCR and the resulting plasmids are summarized in Table 2. Sequences of the exchanged parts of final plasmids were analysed with the GCG software package, version 8.


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Table 2. Summary of oligonucleotides and plasmids used for site-directed mutagenesis

 
{blacksquare} In vitro translation of segments A.
To analyse processing of viral proteins of the mutated segments A, in vitro transcription/translation was performed by using the TNT-coupled reticulocyte lysate system (Promega) in accordance with the manufacturer's instructions. Plasmid DNA for the in vitro transcription was obtained by using a plasmid miniprep kit (Quantum Prep, Bio-Rad).

Products of the TNT reaction were radioimmunoprecipitated with rabbit anti-IBDV antiserum and separated by SDS–PAGE under standard conditions (Sambrook et al., 1989 ).

{blacksquare} Virus recovery from cRNA in tissue culture.
For in vitro transcription of RNA, plasmids containing segment A (Table 3) and pD78B were linearized by cleavage with either BsrGI or PstI. Further treatment of linearized DNA and transcription were carried out as described by Mundt & Vakharia (1996) with two exceptions: (i) the transcription mixtures were not purified by phenol–chloroform extraction and (ii) QM-7 cells and CEC were used for transfection experiments as described previously (Mundt & Vakharia, 1996 ). Two days after transfection, cells were freeze–thawed and centrifuged at 700 g to eliminate cellular debris and the resulting supernatants were clarified further by filtration through 0·45 µm filters and stored at -70 °C. For immunofluorescence studies, cells were grown on sterile coverslips.


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Table 3. Summary of transfection experiments using intergeneric full-length cDNA clones of IBDV segment A and segment B of strain D78

 
{blacksquare} Virus recovery from cRNA after transfection of CAM.
For recovery of viruses that did not infect QM-7 cells or CEC, an alternative method was applied. Transcription mixtures were transfected onto the CAM of 11-day-old embryonated SPF eggs. Six days after transfection, CAM were harvested and homogenized. Supernatants were clarified by low-speed centrifugation and used for Western blot, ELISA and passaging onto CEC.

{blacksquare} Detection of IBDV antigen.
IBDV antigen was detected by indirect IFA and Western blot with rabbit anti-IBDV antiserum (Mundt et al., 1995 ). Supernatants of transfected QM-7 cells, CEC and CAM were passaged onto CEC. For IFA, QM-7 cells as well as CEC grown on coverslips were incubated with supernatants resulting from the passaging experiments. After 16 h, cells were acetone-fixed and processed for IFA. For examination of IBDV replication after transfection, QM-7 cells and CEC grown on coverslips were incubated for 24 and 48 h, acetone-fixed and processed for IFA.

To detect IBDV antigen in transfected CAM, clarified supernatants of homogenized CAM were analysed by Western blot with a rabbit anti-IBDV antiserum. An ELISA (van Loon et al., 1994 ) was performed to confirm the Western blot results,.

{blacksquare} Analysis of tissue culture-infecting virus.
IBDV able to infect tissue cultures was passaged onto CEC and purified by ultracentrifugation and the resulting pellets were digested with proteinase K/SDS as described above. After precipitation, genomic RNA was reverse-transcribed into cDNA and amplified by PCR under standard conditions with oligonucleotides A14 and A44 (Table 1). PCR fragments were cloned blunt-ended and three clones resulting from each virus were sequenced. Sequences were analysed using the GCG software package, version 8.


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
Transfection experiments with chimeric cRNA
For transfection experiments, a full-length cDNA clone of segment A of strain D78 (pD78A) and the chimeric segment A pD78A-E/Del were transcribed into synthetic cRNA and cotransfected with segment B (pD78B) full-length cRNA into QM-7 cells, as well as into CEC in parallel. Two days after transfection, cells were freeze–thawed and the resulting supernatants were passaged twice on CEC. CEC were incubated for up to 5 days after infection in each passage. After freeze–thawing, transfection and passage, supernatants were tested for IBDV antigen by IFA with CEC and QM-7 cells. Transfection experiments were repeated three times. Virus was generated after transfection of cRNA from plasmid pD78B in combination with pD78A, leading to strain D78r (Fig. 2B). In contrast, after transfection experiments with cRNA from pD78A-E/Del and pD78B, no tissue culture-infecting virus could be isolated (Fig. 2 D). To analyse whether transfection was followed by replication, transfection was carried out using cells growing on coverslips. In both cases ,virus antigen was detected by IFA 24 h after transfection (Fig. 2 A, C). Thus, virus replication ensued in both cases, but it was only possible to generate tissue culture-infecting IBDV in the case of D78r.



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Fig. 2. Immunofluorescence with rabbit anti-IBDV antiserum of QM-7 cells 24 h after transfection (A, C) and after passaging of the supernatants of transfected cells (B, D). Cells were transfected with in vitro-transcribed cRNA of either pD78A (A) or pD78A-E/Del (C) together with cRNA of pD78B. Transfection supernatants of pD78A (B) and pD78A-E/Del (D) were passaged once in CEC and tested for IBDV antigen. Magnification, x300.

 
Comparison of viral sequences
As a result of this observation, and assuming that VP2 plays an important role during virus entry, amino acid sequences comprising the variable region of VP2 of different IBDV were compared. Sequences were divided into two groups before alignment on the basis of their origin. One group contained sequences of tissue culture-adapted (TC) strains, whereas the second group consisted of sequences of IBDV not able to infect tissue culture (bursa-derived strains, BU). After alignment of amino acid sequences (aa 241–360) of TC and BU groups, the consensus sequences of both groups were compared (Fig. 3A). Differences in amino acids were found in the following positions (the residue found in TC strains is given first): 241 (V->I), 253 (H->Q), 270 (T->A), 279 (N->D), 284 (T->A) and 330 (R->S). Within the TC group, residues V241 and H253 were conserved in 6 of 8 and 7 of 8 strains, respectively, whereas N279 and T284 were present in all strains. Amino acids T270 (5 of 8) and R330 (4 of 8) showed the lowest degree of conservation within the consensus sequence of the TC group. Residues Q253 (16 of 17), A284 (14 of 17) and S330 (17 of 17) were the positions most conserved in the BU group. Residues I241 (9 of 17), A270 (12 of 17) and D279 (10 of 17) showed a degree of identity lower than 73%. The amino acid sequence of the tissue culture-adapted strain D78 was 100% identical to the TC consensus sequence. Comparison of the sequence of the E/Del (BU) strain with those of the TC group sequence revealed five amino acid exchanges (TC sequence given first), at positions 249 (Q->K), 254 (G->S), 286 (T->I), 318 (G->D) and 323 (D->E), in addition to the amino acid substitutions between the TC and BU group. In contrast, amino acids 241 and 279 were not different. Taken together, residues 253 and 284 were highly conserved in both groups, whereas S330 was the only amino acid fully conserved within the BU group.



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Fig. 3. Comparison of deduced amino acid sequences of the variable region of VP2 of IBDV. (A) The consensus sequence of tissue culture-adapted (TC) strains is compared with the consensus sequence of bursa-derived strains (BU) and the sequences of strains D78 and E/Del. (B) Sequences of the bursa-derived strain GLS (GLS-BU) and the tissue culture-adapted variant GLS-TC are aligned. Differences are indicated. Identical amino acids are marked by dashes. Numbering of amino acids is according to the published sequence of strain P2 (Mundt & Müller, 1995 ).

 
Transfection experiments with mutated cRNA
On the basis of the results of the sequence comparison, several different mutated full-length cDNA clones were established by site-directed mutagenesis. Mutated plasmids of pD78A-E/Del were generated containing substitutions at amino acid positions 253, 284 and 330 in all possible combinations (Table 3). Transfection experiments and passaging were performed three times in parallel on CEC and QM-7 cells. The supernatants obtained were analysed for infectivity by IFA. Infectious virus could not be isolated after transfection of cRNA of plasmids pD78A-E/Del, pD78A-E/Del-QH, pD78A-E/Del-AT, pD78A-E/Del-SR, pD78A-E/Del-AT-SR and pD78A-E/Del-QH-SR in combination with cRNA of pD78B into QM-7 cells or CEC. Transfection of cRNA obtained from pD78A-E/Del-QH-AT or pD78A-E/Del-QH-AT-SR led to generation of infectious virus (D78A-E/Del-QH-AT and D78A-E/Del-QH-AT-SR). Specificity was confirmed by IFA on CEC as well as QM-7 cells (data not shown). This indicated that VP2 of IBDV plays a critical role in tissue culture infection. Amino acid substitutions Q253->H and A284->T were necessary and sufficient to generate IBDV infectious for the tissue cultures used. To confirm these results, a second set of plasmids was constructed by using pD78A for site-directed mutagenesis to obtain plasmids containing substitutions of either a single residue (pD78A-HQ, pD78A-TA, pD78A-RS) or of all three amino acids (pD78A-HQ-TA-RS). These four plasmids were used for transfection experiments in combination with pD78B as described above. Infectious IBDV could be generated after transfection of cRNA from pD78A-RS (D78-RS) as detected by IFA. Again, residue 330 did not have any influence on the ability of the virus generated to infect tissue culture, but single substitutions at positions 253 (H to Q) or 284 (T to A) were sufficient to prevent generation of tissue culture-infecting IBDV. All constructs were tested by IFA for replication after transfection. IBDV antigen could be detected 24 and 48 h after transfection, showing typical large, intensely stained aggregates within the cytoplasm (data not shown).

RT–PCR was performed to examine whether changes in the mutated part of the viral genome occurred during replication of D78r, D78-RS, D78A-E/Del-QH-AT or D78A-E/Del-QH-AT-SR. Sequence analysis of the cloned PCR fragments revealed no differences from the sequences of the plasmids used for cRNA synthesis (data not shown).

In vitro transcription and translation of cDNA constructs of segment A
To exclude the possibility that polyprotein processing had been altered by cloning manipulation or amino acid substitution, each full-length clone of segment A was transcribed and translated in vitro. Products were immunoprecipitated with rabbit anti-IBDV antiserum and analysed after SDS–PAGE and fluorography (Fig. 4). As expected, the viral polyprotein was processed into VP2, VP3 and VP4. The apparent molecular masses of VP3 (32 kDa) and VP4 (28 kDa) showed no differences between the two sets of full-length clones, pD78A and derivatives (lanes 1–5) and pD78A-E/Del and derivatives (lanes 6–13). The apparent molecular mass of VP2 differed between the two sets of full-length clones as well as within the sets. For example, exchange of R330 in VP2 encoded by pD78A (lane 1) to serine in pD78A-RS (lane 4) seemingly led to an increase of the apparent molecular mass of approximately 2 kDa.



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Fig. 4. Radioimmunoprecipitation of in vitro-translated products of different segments A of IBDV. Plasmids used are: pD78A (lane 1), pD78A-HQ (2), pD78A-TA (3), pD78A-RS (4), pD78A-HQ-TA-RS (5), pD78A-E/Del (6), pD78A-E/Del-QH (7), pD78A-E/Del-AT (8), pD78A-E/Del-SR (9), pD78A-E/Del-QH-AT (10), pD78A-E/Del-AT-SR (11), pD78A-E/Del-QH-SR (12) and pD78A-E/Del-QH-AT-SR (13). Translated proteins were precipitated by using rabbit anti-IBDV serum. The locations of viral proteins and molecular mass markers are indicated.

 
Transfection of chimeric cRNA containing strain GLS sequences
To confirm the results of the transfection experiments with chimeric as well as mutated plasmids, we took advantage of a naturally occurring pair of IBDV strains. The variable regions of VP2 of the bursa-derived strain GLS (GLS-BU) and the tissue culture-adapted variant GLS-TC were amplified, cloned and analysed. Comparison of the amino acid sequences of the two strains obtained from pGLS-BU and pGLS-TC revealed one amino acid exchange, at position 284, from A in GLS-BU to T in GLS-TC (Fig. 3 B). Residues 253 (Q) and 330 (S) were identical to those of the BU group, as described above. To analyse whether the exchange at position 284 (A->T) was sufficient for the generation of tissue culture-infectious virus, two plasmids (pD78A-E/Del-GLS-TC and pD78A-E/Del-GLS-BU) were constructed containing the variable regions of VP2 from the two variants. cRNA from pD78A-E/Del-GLS-TC and pD78A-E/Del-GLS-BU was transfected with cRNA of pD78B into QM-7 cells and CEC. After passaging of the supernatants in tissue culture, infectious virus could be detected by IFA and CPE occurred after transfection of cRNA of pD78A-E/Del-GLS-TC. In several attempts, transfection of cRNA from pD78A-E/Del-GLS-BU failed to produce tissue culture-infectious IBDV. In vitro transcription/translation of both plasmids showed complete processing of the polyproteins (data not shown). Viral antigen was detected by IFA after transfection of cRNA of both plasmids together with cRNA from pD78B. Thus, both chimeras proved to be replication-competent (data not shown). In summary, a single amino acid exchange at position 284 of VP2 of strain GLS was sufficient to generate cell culture-infectious chimeric IBDV (Table 3).

Recovery of chimeric virus from CAM
CAM of 11-day-old embryonated eggs were used for transfection of in vitro-transcribed cRNAs. Supernatants harvested from the homogenized CAM were analysed by Western blot and ELISA. Antigen could be detected 6 days after transfection of CAM with pD78A, pD78A-HQ, pD78A-TA, pD78A-RS, pD78A-HQ-TA-RS, pD78A-E/Del, pD78A-E/Del-QH, pD78A-E/Del-AT or pD78A-E/Del-SR in conjunction with pD78B. Because the concentration of IBDV antigen was low, the results of Western blot experimentswere confirmed by ELISA. No IBDV-specific antigen could be detected by both Western blot and ELISA after transfection of CAM with pD78A-E/Del-QH-AT, pD78A-E/Del-QH-SR, pD78A-E/Del-AT-SR or pD78A-E/Del-QH-AT-SR in conjunction with pD78B. Furthermore, supernatants of transfected CAM were passaged onto CEC. Testing of CEC supernatants by IFA resulted in detection of IBDV antigen for D78r, D78-RS, D78-E/Del-QH-AT and D78-E/Del-QH-AT-SR. Data from the CAM experiments are summarized in Table 3.


   Discussion
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Methods
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Discussion
References
 
Two types of serotype I strains have been described for IBDV. Strains of the first type are able to infect and replicate in B lymphocytes located in the bursa of Fabricius (bursa-derived) but not in CEC. In contrast, serotype I strains of the second type infect and replicate in both B lymphocytes and CEC (adapted strains). In this report, the structural requirements for either phenotype were identified at the molecular level. The viral protein responsible for the difference in tissue tropism was determined after exchange of parts of VP2 and VP4 between bursa-derived and adapted strains that led to amino acid exchanges in VP2. Furthermore, mutational analysis identified specific residues in VP2 that were responsible for the alteration of tropism from bursa-derived to tissue culture-adapted strains. Two amino acid exchanges, (BU) Q253->H (TC) and (BU) A284->T (TC), in VP2 of the bursa-derived E/Del strain were necessary for the ability to infect CEC as well as QM-7 cells. This was confirmed by the results of single amino acid exchanges in the tissue culture-adapted strain D78, where substitutions at positions 253 (H to Q) or 284 (T to A) resulted in IBDV that was not able to infect tissue culture cells. In contrast, only one amino acid exchange, (BU) A284->T (TC), was sufficient for the alteration of the tissue tropism of GLS-BU to GLS-TC. Interestingly, residue 253 was conserved as glutamine in both GLS strains, as in most of the bursa-derived IBDV strains. In the case of the GLS strain, the exchange of residue 284 (A to T) during natural adaptation was sufficient to obtain tissue culture infectivity. Comparison of the amino acid sequence of strain GLS-BU with the sequence of strain D78 revealed eight amino acid exchanges (K249->Q, Q253->H, S254->G, S269->T, A270->T, A284->T, E321->A and S330->R). Why a single amino acid exchange in the GLS strains was sufficient to generate infectious GLS-TC is not clear. However, each amino acid exchange may lead to alterations within the structure of VP2 that together lead to acquisition of the property of tissue culture infectivity.

After comparison of amino acid sequences of two tissue culture-adapted IBDV strains with sequences of their parental strains, Yamaguchi et al. (1996) suggested that residues N279 and T284 are important for the ability to infect CEC as well as for virulence. Comparison of the amino acid sequences of strains D78, E/Del, GLS-BU and GLS-TC with the sequences reported by Yamaguchi et al. (1996) revealed that the amino acid of the proposed tissue-culture type (glutamine) was located at position 279 in each case. Whether residue 279 indeed plays an important role in infection of CEC remains unclear. N279 is conserved in nearly all tissue culture-adapted strains, with the exception of the vaccine strain Bursine 2, which has aspartate at position 279 (Eterradossi et al., 1998 ). Here, the amino acid sequence (H253 and T284) was of the tissue culture type. This contrasts with the proposal of Yamaguchi et al. (1996 ). Based on the data obtained in this study, residue 284 appears to play a critical role in the infectivity of IBDV in tissue culture, since all tissue culture-adapted strains analysed have threonine at this position. Obviously, residue S330 does not play any role in tissue culture infection. The sequence motif SWSAS330GS has been identified as being conserved in segment A of virulent strains (Heine et al., 1991 ; Lin et al., 1993 ; Vakharia et al., 1994 ), as well as in very virulent strains in Europe and Japan (Lin et al., 1993 ; Brown et al., 1994 ). These strains are not able to infect tissue culture. In summary, an amino acid exchange at position 330 is not necessary for CEC infection but may play a role in the virulence of IBDV in chickens. On the basis of these data, it can be suggested that the ability to infect CEC is determined by the variable region of VP2. It seems that infection of CEC is not associated with conserved amino acid alterations, since the amino acids responsible are specific for the strain used. Therefore, any prediction of effects caused by a particular amino acid alteration is impossible without detailed knowledge of the three-dimensional structure of VP2 and identification of the domains involved in tissue culture tropism. Data regarding the three-dimensional structure of VP2 are not available. Furthermore, the use of theoretical models based on the amino acid sequence is not possible, since the carboxy terminal amino acid of VP2 is unknown. However, the amino acids responsible for infection of CEC can be determined by amino acid substitution, accomplished by using the IBDV reverse genetics system.

During the early part of this study, it was not possible to generate a chimeric virus based on the exchanged part of the bursa-derived E/Del strain. IBDV antigen could be detected by using IFA after transfection of viral cRNA, proving that transcription and translation occurred. To overcome this problem, it was necessary to develop a system that allowed generation of progeny of bursa-derived IBDV in the laboratory. The successful transfection of CAM of embryonated eggs provided a new way of generating infectious IBDV, even from isolates that were not able to infect tissue culture. IBDV able to infect tissue culture was not detected by Western blot and ELISA, due to the low virus yield, although the virus was present, as shown by CEC passage. In contrast, in only two cases of IBDV that was unable to infect tissue culture could antigen not be detected by both ELISA and Western blot (D78A-E/Del-AT-SR, D78A-E/Del-QH-SR). These two may not be viable or the amount of antigen may have been too low. However, both are able to produce virus after transfection in tissue culture, as detected by IFA, which indicates that the system for generation of IBDV in CAM needs to be optimized. Problems associated with this system included reproducibility and the difficulty of detection of virus by Western blot due to low yields. Here, the use of a highly sensitive ELISA system (van Loon et al., 1994 ) was helpful. With all these constraints, it was important to show that viable chimeric as well as mutated IBDV could be generated.

The results obtained show new methods for the characterization of structural elements of IBDV that are responsible for replication and infectivity. The generation of chimeric IBDV between TC and BU strains makes `tailor-made' vaccines a possibility for the future.


   Acknowledgments
 
I thank Thomas Mettenleiter for helpful discussions and critical reading of the manuscript, Dietlind Kretzschmar for excellent technical assistance and A. van Loon for performing the ELISA. This study was supported in part by DFG grant MU 1244/1-1.


   References
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
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Received 22 March 1999; accepted 10 May 1999.