Veterinary Microbiology, 33 ( 1992 ) 175-183 Elsevier Science Publishers B.V., Amsterdam
175
Infectious bursal disease of poultry: antigenic structure of the virus and control H. Miiller ~, D. Schnitzler ~, F. Bernstein a, H. Becht a, D. Cornelissen b and D.H. Liittickenb alnstitut J~r Virologie, Justus-Liebig- Universitiit Giessen, Frankfurter Strasse 107, D-6300 Giessen, Germany blntervet International B. V., Boxmeer, Netherlands (Accepted 26 June 1992 )
ABSTRACT M~iller, H., Schnitzler, D., Bernstein, F., Becht, H., Cornelissen, D. and Liitticken, D.H., 1992. Infectious bursal disease of poultry: antigenic structure of the virus and control. Vet. Microbiol., 33:175183. The present knowledge of genome organisation, structural basis of pathogenicity and antigenicily of infectious bursal disease virus (IBDV) are briefly reviewed. The current situation of IBDV infection in various countries is slated and recommendations for improved vaccination schemes are given.
INTRODUCTION
Infectious bursal disease (IBD) is a highly contagious disease of young chickens (Gumboro disease). The aetiological agent, infectious bursal disease virus (IBDV), has a selective tropism for cells of the bursa of Fabricius (BF). Economic losses in poultry industries can result from high mortality rates due to an acute course of the disease, or from consequences of a B-celldependent immunodeficiency. Despite vaccination programmes used on a large scale, IBD outbreaks are reported from all over the world. Antigenic variants and very virulent IBDV strains have been held responsible for acute disease in vaccinated broiler flocks or progeny from vaccinated parents. VIRUS STRUCTURE AND GENOME ORGANISATION
IBDV is a member of the family Birnaviridae. The virion is a non-enveloped, single-shelled particle with a diameter of about 60 nm. The icosahedral Corresponding author." Dr. Hermann Miiller, Institut ftir Virologie, Justus-Liebig-Universitat Giessen, Frankfurter Strasse 107, D-6300 Giessen, Germany.
0378-1135/92/$05.00 © 1992 Elsevier Science Publishers B.V. All rights reserved.
176
H.MOLLERETAL.
capsid is composed of the structural polypeptides VP1 (Mr 90 kilodalton, kDa), VP2 (Mr 40 kDa), VP3 (Mr 32 kDa), and VP4 (Mr 28 kDa); it includes the viral genome consisting of two segments of double-stranded (ds) RNA. In the case of the virulent IBDV strain Cu-1 (Nick et al., 1976) the size of the smaller segment B has been determined at 2796 bp (Bernstein et al., unpublished). This encodes VP1 (Azad et al., 1985; Miiller, unpublished) in a single open reading frame (ORF). VPI is firmly linked to the ends of the two genome segments which are circularized (Miiller and Nitschke, 1987); this complex is responsible for replicase and transcriptase activities associated with IBDV (Spies et al., 1986). Furthermore, guanylyltransferase and methyltransferase activities have been shown to be associated with VPI (Spies and Mtiller, 1990). In accordance, consensus sequences with RNA-dependent RNA polymerases and GTP-binding proteins can be delineated from the nucleotide sequence of segment B. Thus, VPI represents a "multifunctional" polypeptide. The larger segment A of IBDV strain Cu-1 has been determined at 3214 bp. This segment contains two major ORFs (Spies et al., 1989). A smaller ORF (nucleotides 62 to 497) codes for 145 amino acids among which are 7 cysteine molecules, but only an amino-terminal methionine; a corresponding protein with anapprox. Mr of about 16.5 kDa has not been specified (U. Spies, personal communication ). The large ORF (nucleotides 96 to 3132 ) codes for a single large polypeptide composed of 1012 amino acids, on which the capsid proteins are arranged in the order VP2, VP4 and VP3 (Hudson et al., 1986; Spies et al., 1989). This "polyprotein" is very rapidly cleaved co- or posttranslationally; a second proteolytic modification has been observed late in virus maturation, when VP2 is processed further to the final structural polypeptide (MiJller and Becht, 1982 ). ANTIGENICSTRUCTURE Besides pathogenic IBDV strains which replicate preferentially in lymphoid cells of the BF at its maximal stage of development, a second serotype has been isolated from turkeys (McFerran et al., 1980; Jackwood et al., 1982). This seroytpe II does not have a selective tropism for the bursa and is not pathogenic for chickens. The two serotypes can be distinguished by virus neutralization (VN) test. Neutralizing antibodies are induced by a conformation-dependent antigenic domain, located on VP2 (Azad et al., 1987; Becht et al, 1988 ). It is composed of at least three independent epitopes which have recently been defined by the selection of escape mutants (t3ppling et al., 1991a). Two independent non-overlapping epitopes were demonstrated on VP3 by non-neutralizing monoclonal antibodies (mAbs); both serotypes have
ANTIGENIC STRUCTURE AND CONTROL OF IBDV
177
one of these epitopes in common, whereas the second epitope is distinct for serotype I and serotype II (Oppling et al., 1991b). PATHOGENICITYOF IBDV Pathogenicity of IBDV, which in the case of the chicken strains (serotype I) means replication in lymphoid cells of the BF, is not governed by one of the two viral genome segments. Reassortant IBDV strains which possessed segment A from the pathogenic serotype I-strain Cu-l and segment B from the non-pathogenic serotype II-strain 23/82 were no longer lethal and produced only slight clinical manifestations; lesions in the BF were comparable to those caused by some vaccine strains (MiJller, 1987). This indicates that both segments contribute to an optimal replication in the BF, and it might be speculated that some of the segment A gene products are operative as "transactivators" during viral replication, either alone or in concert with cell-type specific proteins. A comparative analysis of segment A nucleotide sequences of several serotype I-strains indicated that five amino acid changes in VP2 differentiated between the virulent strain Cu-l and the apathogenic vaccine strain PBG 98 (Bayliss et al., 1990). To identify the exact sequences involved in pathogenicity, the nucleotide sequences of the two genome segments of a highly attenuated stable Cu-l variant strain Cu-lM were determined (unpublished results). In contrast to "wild-type" virus, this strain forms only small plaques in tissue culture (Cursiefen et al., 1979a; Lange et al., 1987) and has been in use as a live-vaccine for many years (Cursiefen et al., 1979b). A schematic comparison of this attenuated strain with those of tissue culture-adapted Cu1 (Spies et al., 1990; Bayliss et al., 1990) is shown in Fig. 1. In segment A, two amino acid changes are located in a "variable" region (Bayliss et al., 1990 ) in VP2; two point mutations were determined in the 5'- noncoding region. In segment B, there are three amino acid changes within the coding region for VP 1; they are located upstream of the consensus sequences known for RNAdependent RNA polymerases and GTP-binding proteins. The significance of these changes for attenuation remains to be determined. It has to be taken into account, that in both strains the complete 5'- and 3'ends of the two genome segments have not yet been determined. These noncoding regions contain inverted terminal repeats and palindromic sequences and thus form extensive secondary structures (unpublished results). Structures like these may function in the regulation ofgenome replication and transcription; they play an important role in assortment and packaging of the viral genome during maturation, and they may determine cell type-specificity. It has been observed that the attenuated strain Cu-1M replicates much more slowly than Cu-l "wild type" and that virus remains predominantly cell-associated (Cursiefen et al., 1979a); in addition, Cu-1M has a strong tendency
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to forming "incomplete" virus particles (Miiller et al., 1987). These observations might support the speculations on the significance of the non-coding regions mentioned above. STRUCTURAL BASIS OF ANTIGENIC VARIABILITY
A comparison of the sequences of segment A of four serotype I-strains identified a variable region in VP2 (Bayliss et al., 1990). This region, encoded by nucleotide sequences located within AccI and SpeI restriction sites, is flanked by hydrophilic amino acids and has been suspected to form the conforma-
ANTIGENIC STRUCTURE AND CONTROL OF IBDV
179
tional epitope for neutralizing antibodies. Indeed, it has been shown to represent a binding-site for neutralizing mAbs (Azad et al., 1987, 1988; Schnitzler, unpublished). To get more insight into the significance of nucleotide changes in this region in variant strains obtained under defined experimental conditions, the nucleotide sequences of Cu-1 escape mutants characterized previously (0ppiing et al., 1991a) were analyzed. Since these mutants replicate in the presence of a neutralizing mAb (mAb-resistant variants) the genetic region coding for this particular epitope must have individual nucleotide exchanges. All of the five escape mutants under investigation have different amino acid exchanges which are located within the hydrophilic regions of the AccISpeI-fragment (Table 1 ). These results substantiate the hypothesis that this region codes for the conformational epitope. As mentioned before, an individual escape mutant resists neutralization by the corresponding mAb. It has to be taken into account, however, that all of these escape mutants are still very efficiently neutralized by chicken convalescent sera or rabbit polyclonal antibodies prepared with purified virus particles (0ppling et al., 199 la). This may indicate frequent minor antigenic variations during virus replication, and it can be supposed that mutations like these are readily controlled by use of the current vaccine strains. This contrasts with the situation in variant strains isolated recently in the field (Rosenberger and Cloud, 1985; Snyder et al., 1988). One of these strains has been analyzed (Heine et al., 1991 ), and two amino acid changes in either one of the two hydrophilic regions have TABLE 1
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H. MI~LLER ET AL.
been identified. This particular amino acid constellation may represent the prototype of a rare major antigenic variation which could lead to an antigenic shift which would explain the fact that such variant strains circumvent control by vaccination. RELEVANCEOF ANTIGENICVARIABILITYFOR VACCINATION As mentioned above, escape mutants prepared in the laboratory by cultivating IBDV under the pressure of a neutralizing mAb are completely neutralized by polyclonal serotype I antisera. Very virulent strains of IBDV have been isolated recently on the Delmarva peninsula (U.S.A.); they were defined as variants by the fact that they had been isolated from vaccinated flocks and by a specific reaction pattern with mAbs (Rosenberger and Cloud, 1985; Snyder et al., 1988). Incorporation of variants into vaccines has been discussed although strains displaying the reaction pattern of these isolates have not yet been demonstrated in Europe (Van der Marel et al., 1990; Oppling et al., 1991a). Protection against IBD in the field has to be monitored by serological methods or by challenge infections of field birds moved to isolation facilities. If the progeny of vaccinated breeders is to be tested, infection history of the flock is often unknown, a fact which complicates interpretation of infection studies. We have tried to mimic field situations in a series of infection and vaccination experiments with SPF breeder birds held in isolation. Progeny of the infected or vaccinated breeders was subjected to a challenge infection with virulent IBDV isolates. Protection induced by "natural" infection with the three prototype viruses D78 (classic), Variant E (Delaware) and GLS-5 was compared with protection induced by vaccination with monovalent (Variant E ) or trivalent (D78 + Variant E + GLS-5 ) inactivateded vaccines. Results of two studies are presented. In both experiments protection by passively transferred maternal antibodies has been measured. Breeders were SPF white leghorn type birds held in isolation units with filtered air. Eggs were collected and hatched in isolators for challenge of the chickens at the age of l and 3 weeks. All challenge viruses were administered as bursa homogenates at a dose of about 50 TCIDso per bird by eyedrop. Protection was measured by virus detection in the bursa (histology and ELISA ), or by bursa body weight ratio determination. Only protection data based on histology are shown. In Experiment 1 three groups of SPF breeders were vaccinated twice with live IBDV strains representing the classic strain D78, the Delaware variant strain Variant E, and the GLS type variant strain GLS-5, at 17 and 22 weeks of age. Six weeks after the second infection, eggs were hatched and the progeny subjected to challenge infections at 7 and 21 days of age. As shown in Fig. 2, infection of SPF breeders with three variants of serotype I IBDV induced
181
ANTIGENIC STRUCTURE AND CONTROL OF IBDV
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Fig. 2. Protection of progeny of birds vaccinated with one of 3 live strains of IBDV (D78, GLS5 variant strain, or Delaware strain variant E) when challenged with one of 3 challenge strains.
different degrees of cross protection. Only birds infected with Variant E virus transferred complete protection against homologous and heterologous challenge for a short period of seven days. Surprisingly, infection with GLS virus induced only low levels of protection, even in the homologous system.This has been confirmed by VN tests of the sera (data not shown). In Experiment 2, two groups of SPF breeders were vaccinated once with an inactivated monovalent vaccine containing Variant E or a trivalent vaccine containing D78+Variant E+GLS-5. All viruses were propagated in tissue culture, inactivated with formalin and calibrated by an antigen mass ELISA based on mAbs (Van der Marel et al., 1990). Six months after vaccination eggs were collected and the progeny subjected to a challenge infection at 14 and 21 days. The results of this experiment (Fig. 3 ) confirm the field experience that vaccination of breeders with an inactivated vaccine will produce a better protection for the progeny than "natural" infection of the breeders as simulated in Experiment 1. It could also be confirmed that the cross-protection induced by Variant E is more effective against standard virus than against the GLS subtype which supports the assumption that GLS-5 represents a distinct variant virus. To induce complete protection all three antigens have to be present in the inactivated vaccine. In areas where the GLS subtype prevails, GLS antigen has to be incorporated in the vaccine, in areas where only
182
n. MOLLER ET AL.
Protection of progeny after Challenge % 100 90 80 70 60 50 40 30 20 10 0
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standard virus a n d / o r the Variant E (Delaware) occur, an inactivated vaccine based on Variant E or on Variant E + s t a n d a r d virus should be used for breeder vaccination.
REFERENCES Azad, A.A., Barrett, S.A. and Fahey, K.J., 1985. The characterization and molecular cloning of the double stranded RNA genome of an Australian strain of infectious bursal disease virus. Virology, 143: 35-44. Azad, A.A., Jagadish, M.N., Brown, M. and Hudson, P., 1987. Deletion mapping and expression in Escherichia coil of the large segment ofa birnavirus. Virology, 161:145-152. Azad, A.A., Jagadish, M.N. and Fahey, K.J., 1988. IBDV VP2 epitope recognised by virus neutralising and protective monoclonal antibodies. International Patent Application, PCT/ AU88/00206. Bayliss, C.D., Spies, U., Shaw, K., Peters, R.W., Papageorgiou, A., Miiller, H. and Boursnell, M.E.G., 1990. A comparison of the sequences of segment A of four infectious bursal disease virus strains and identification of a variable region in VP2. J. Gen. Virol., 71, 1303-1312. Becht, H., Miiller, H. and Miiller, H.K., 1988. Comparative studies on structural and antigenic properties of two serotypes of infectious bursal disease virus. J. Gen. Virol., 69, 631-640. Cursiefen, D., K~iufer, I., and Becht, H., 1979a. Loss of virulence in a small plaque mutant of the infectious bursal disease virus. Arch. Virol., 59: 39-46.
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Cursiefen, D., Vielitz, E., Landgraf, H. and Becht, H., 1979b. Evaluation of a vaccine against infectious bursal disease in field trials. Avian Pathol., 8:341-351. Heine, H.-G., Haritou, M., Failla, P., Fahey, K. and Azad, A.A., 1991. Sequence analysis and expression of the host-protective immunogen VP2 of a variant strain of infectious bursal disease virus which can circumvent vaccination with standard type I strains. J. Gen. Virol., 72: 1835-1843. Hudson, P.J., McKern, N.M., Power, B.E. and Azad, A.A., 1986. Genomie structure of the large RNA segment of infectious bursal disease virus. Nucleic Acids Res., 14: 5001-5012. Jackwood, D.F., Saif, Y.M. and Hughes, J.H., 1982. Characteristics and serological studies of two serotypes of infectious bursal disease virus in turkeys. Avian Dis., 26: 871-882. Lange, H., Miiller, H., K~ufer, I. and Becht, H., 1987. Pathogenic and structural properties of wild type infectious bursal disease virus (IBDV) and virus grown in vitro. Arch. Virol., 92: 187-196. McFerran, J.B., McNulty, M.S., Killop, E.R., Connor, T., McCracken, R.M., Collins, P.S. and Allan, G.M., 1980. Isolation and serological studies with infectious bursal disease virus from fowl, turkeys and ducks: Demonstration of a second serotype. Avian Pathol., 8: 395-404. Miiller, H.K., 1987. Strukturelle und pathogene Eigenschaften der beiden Serotypen sowie einer Reassortanten des Virus der infektiiSsen Bursitis (IBDV). Doctoral Thesis, Justus-LiebigUniversit~it Giessen. Miiller, H. and Becht, H., 1982. Biosynthesis of virus-specific proteins in cells infected with infectious bursal disease virus and their significance as structural elements for infectious virus and incomplete virus particles. J. Virol., 44: 384-392. MUller, H. and Nitsehke, R., 1987. The two segments of the infectious bursal disease virus genome are circularized by a 90,000-Da protein. Virology, 159:174-177. Nick, H., Cursiefen, D. and Becht, H., 1976. Structural and growth characteristics of infectious bursal disease virus. J. Virol., 18: 227-234. Oppling, V., Miiller, H. and Becht, H., 1991a. Heterogeneity of the antigenic site responsible for the induction of neutralizing antibodies in infectious bursal disease virus. Arch. Virol., 119:211-223. Oppling, V., Mtiller, H. and Becht, H., 1991b. The structural polypeptide VP3 of infectious bursal disease virus carries group- and serotype-specific epitopes. J. Gen. Virol., 72: 22752278. Rosenberger, J.K. and Cloud, S., 1985. Isolation and characterization of variant infectious bursal disease viruses. Abstr. 123rd Am. Vet. Med. Assoc. Meet., p. 357. Snyder, D.B., Lana, D.P., Savage, A.P., Yancey, F.S., Mengel, S.A. and Marquardt, W.W., 1988. Differentiation of infectious bursal disease viruses directly from infected tissues with neutralizing monoclonal antibodies: evidence of a major antigenic shift in recent field isolates. Avian Dis., 32: 535-539. Spies, U. and Miiller, H., 1990. Demonstration of enzyme activities required for cap structure formation in infectious bursal disease virus, a member of the birnavirus group. J. Gen. Virol., 71: 977-981. Spies, U., Miiller, H. and Becht, H., 1987. Properties of RNA polymerase activity associated with infectious bursal disease virus and characterization of its reaction products. Virus Res., 8: 127-140. Spies, U., Miiller, H. and Becht, H., 1990. Nucleotide sequence of infectious bursal disease virus genome segment A delineates two major open reading frames. Nucleic Acids Res., 17: 7982. Van der Marei, P., Snyder, D. and Liitticken D., 1990. Antigenic characterization of IBDV field isolates by their reactivity with a panel of monoclonal antibodies. Deutsche Tier~irz. Wochensch., 97:81-83.