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
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Abstract |
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Introduction |
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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.
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Methods |
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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 251360), 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).
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.
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/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/
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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Products of the TNT reaction were radioimmunoprecipitated with rabbit anti-IBDV antiserum and separated by SDSPAGE under standard conditions (Sambrook et al., 1989 ).
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 phenolchloroform 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 freezethawed 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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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,.
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.
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Results |
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RTPCR 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 SDSPAGE 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 15) and pD78A-E/Del and derivatives (lanes 613). 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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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.
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Discussion |
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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.
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Acknowledgments |
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References |
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Received 22 March 1999;
accepted 10 May 1999.