Intervet International BV, PO Box 31, 5830 AA Boxmeer, The Netherlands1
Institute of Molecular Biology, Friedrich-Loeffler-Institutes, Federal Research Center for Virus Diseases of Animals, D-17498 Insel Riems, Germany2
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 (Leong et al., 2000 ). The genome consists of two segments, designated A and B, of double-stranded RNA, which are localized within a single-shelled icosahedral capsid of 60 nm diameter. The larger segment, A, encodes a polyprotein of approximately 110 kDa, which is proteolytically cleaved in cis by the viral protease VP4 (Birghan et al., 2000
) to form the viral proteins (VP) VP2, VP3 and VP4. A second open reading frame encodes VP5 (Mundt et al., 1995
). Genome segment B encodes a 98 kDa protein, designated as VP1, which represents the putative viral RNA-dependent RNA polymerase (Spies et al., 1987
).
Usually, pathogenic bursal-derived field strains are not easily adapted to cell culture, a process which requires extensive passaging either in cell culture (Hassan et al., 1996 ) or on the chorioallantoic membrane (CAM) as well as in the yolk sac of embryonated eggs (Yamaguchi et al., 1996a
). Several field isolates failed to become adapted to cell culture (McFerran et al., 1980
). Recently, Lim et al. (1999)
and Mundt (1999)
showed the adaptation of serotype I strains to tissue culture by the reverse genetics approach. Lim et al. (1999)
showed for the very virulent (vv) IBDV strain HK46 that aa 279 and 284, located in the variable region of VP2 (Bayliss et al., 1990
), are responsible for infection of tissue culture. Mundt (1999)
showed for variant strains that aa 253 and 284, or aa 284 alone, can be responsible for infection of tissue culture, depending on the strain used. Serotype I strains adapted to tissue culture by serial passaging showed reduced in vivo virulence in infected chicken (Cursiefen et al., 1979
; Lange et al., 1987
; Yamaguchi et al., 1996a
; Hassan et al., 1996
). The reason for this altered virulence in chicken is unknown.
We report here the adaptation to tissue culture of vvIBDV strain UK661 by site-directed exchanging of certain amino acids using a reverse genetics system (Mundt & Vakharia, 1996 ), verifying the amino acids responsible for tissue culture adaptation of IBDV as described recently (Mundt, 1999
). The influence of the adaptation to tissue culture on virulence was investigated by animal experiments.
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Methods |
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Construction of full-length cDNA clones.
IBDV strain UK661 was propagated in five SPF chickens. Three days after infection the bursae Fabricii (BF) were removed, homogenized and virus was purified as described (Müller et al., 1986 ). Pelleted virus particles were incubated overnight with proteinase K (0·5 mg/ml) and SDS (0·5%) at 37 °C. The genomic viral dsRNA was extracted with phenolchloroform, recovered by ethanol precipitation and used for amplification of viral cDNA. The extreme terminal sequences of both segments were determined by the 5'-RACE method using oligonucleotides as described earlier (Mundt & Müller, 1995
). Based on these results and on the data published earlier for the UK strain 661 (Brown & Skinner, 1996
) oligonucleotides were designed to construct full-length cDNA clones of both segments. Following reverse transcription the full-length genome consisting of segments A and B was amplified in three pieces using Deep Vent polymerase (New England Biolabs; Fig. 1
). For amplification of the 5' fragment (A1), the middle fragment (A2) and the 3' fragment (A3) of segment A primer pairs 661AF1/661AR1, 661AF2/661AR2 and 661AF3/661AR3 were used. Amplified fragments were cloned into SmaI-cleaved vector pUC18 (Pharmacia) to obtain appropriate plasmids (pA1, pA2, pA3). After sequencing, a unique restriction enzyme cleavage site (NaeI) located in the overlapping region of A1 and A2 and the BsrGI cleavage site created by the oligonucleotides were used to obtain p661A12. pA3 was cleaved with SacII/BsrGI to excise the 3' fragment and ligated into the appropriately cleaved p661A12 to obtain the full-length cDNA clone of segment A of strain UK661 (p661A) under control of the T7 promotor. For construction of a full-length clone of segment B, primer pairs 661BF1/661BR1, 661BF2/661BR2 and 661BF3/661BR3 were used for amplification of three fragments of segment B (B1, B2, B3) encompassing the full-length cDNA sequence. These three fragments were cloned blunt-ended to obtain pB1 (5' fragment), pB2 (middle fragment) and pB3 (3' fragment), respectively. After sequencing, a unique LspI cleavage site located in the overlapping region of the middle- and 3' fragment was used together with the EcoRI cleavage site located in the plasmid, respectively, to ligate the middle fragment (B2) into EcoRI/LspI-cleaved pB3 to obtain plasmid p661B23. pB1 was cleaved with DraIII/EcoRI, the fragment was excised, eluted and ligated into EcoRI/DraIII-cleaved p661B23 to obtain a full-length segment B under control of the T7 promotor (p661B). Oligonucleotides used are listed in Table 1
, and the construction of p661A and p661B is diagrammatically represented in Fig. 1
.
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Characterization of virus mutants in chicken.
In an animal experiment, 123 1-day-old SPF White Leghorn chickens were allocated into three groups of 35 chickens and one group of 18 chickens. All chickens were housed in negative pressure isolators. Food and water were available ad libitum. At 14 days of age 35 chickens were each infected by eye drop (0·1 ml) with 102·0 EID50 of the wild-type IBDV strain UK661 (bursa material) and 35 chickens with UK661rev (bursa material), respectively. The third group of 35 chickens were each infected by the eye-drop method with 104·4 TCID50 of the tissue culture-adapted IBDV strain UK661-QH-AT. Eighteen animals of the fourth group were not infected and served as challenge controls. Three, 4, 7, 10 and 14 days post-infection (p.i.) and 3 and 10 days post-challenge, 510 chickens per group were euthanized with CO2 and necropsied. Chickens which showed severe clinical signs (3, 4, 7 and 10 day after infection) were killed for ethical reasons. At 2 weeks p.i., remaining chickens were challenged via the eye-drop route with virulent IBDV strain Faragher 52/70 (102·0 CID50 per chicken). From all killed chickens BF were isolated and split into two parts. One part was used for amplification of viral RNA and for the determination of the presence of IBD viral antigen by means of an ELISA. The second part was fixed in 10% neutral-buffered formalin for histology. Furthermore, the serological response to IBDV infection was assayed by the virus-neutralizing (VN) test (Schröder et al., 2000 ), 14 days post-exposure. During the course of the experiment animals were observed daily for clinical signs and mortality.
To confirm the identity of virus and to investigate whether changes in the amino acid sequence occurred during passaging viral RNA of IBDV obtained from BF of chicken before challenge was amplified by RTPCR using oligonucleotides (A44, UNI1R; Table 1). Cloned PCR fragments were sequenced and obtained sequences were analysed using the Wisconsin package, version 8 (Genetics Computer Group, Madison, WI, USA).
Histopathology.
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. The severity of bursal follicular necrosis was recorded using the bursa lesion score (BLS) as described earlier (Schröder et al., 2000 ).
Detection of viral antigen in BF.
Bursae were isolated and homogenized with 1 ml glass pearls (diameter 2 mm) and 1 ml PBS (pH 7·2±0·2) in a Retsch MM2 homogenizer (Retsch) for 20 min at maximum speed. The presence of virus in the bursal homogenate was determined with a sandwich ELISA which incorporated mAb-8. This mAb recognizes all strains of IBDV serotype I and has previously been described by Snyder et al. (1992) . The ELISA was based on that described by van Loon et al. (1994)
. In brief, 125 µl of mAb-8 ascitic fluid was mixed with 625 µl distilled water and cooled to 0 °C. Then 31 µl 0·2 M H2SO4 was carefully added. After 1 h incubation at 0 °C, 250 ml coating buffer (7·5 mM NaH2PO4, 34 mM Na2HPO4, 145 mM NaCl, 0·01% NaN3; pH 7·2±0·2) was added. Polystyrene microtitre plates (96-well, tissue culture grade; Greiner) were filled with 100 µl per well of pre-treated mAb-8. The plates were incubated for 12 h at 37 °C and overnight at 4 °C. Next, the plates were washed twice with washing buffer (500 mM NaCl, 0·15% Tween 20 in PBS; pH 7·2±0·2), 100 µl per well of the bursa homogenate were added and incubation was continued at 37 °C for 1·5 h. After another washing cycle, the wells of the plate were filled with an anti-IBDV polyclonal rabbit serum (1:2000 diluted) and the plates were further incubated at 37 °C. After 1·5 h the plates were washed three times and incubated with goat anti-rabbit IgG conjugated to horseradish peroxidase (Nordic; diluted 1:15000) for 30 min at 37 °C. After a final wash, substrate (0·014 mg/ml urea peroxide; Organon Teknika) in the presence of 0·11 mg/ml 3,3',5,5'-tetramethylbenzidine (Fluka Chemie) was added. After 510 min of incubation, the substrate reaction was stopped by adding 2 M H2SO4. Absorption was measured at 450 nm in an ELISA reader (Titretek, multiscan plus MK II, ICN).
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Results |
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Adaptation of very virulent UK661 to tissue culture
To adapt UK661rev to tissue culture plasmids resulting from site-directed mutagenesis experiments (p661AQ253H, p661AD279N, p661AA284T, p661AQ253H-A284T, p661AD279N-A284T) and p661B were used. Transcribed cRNA of the different A segments was cotransfected with cRNA of segment B into QM-7 cells. 48 h after transfection cells were freezethawed and resulting supernatants were passaged onto primary CEC. 24 h and 48 h p.i. CEC were processed for immunofluorescence. Infectious virus was generated after transfection of p661AA284T/p661B (UK661-AT), p661AQ253H-A284T/p661B (UK661-QH-AT) and p661AD279N-A284T/p661B (UK661-DN-AT). Virus was not recovered after transfection experiments using cRNA of p661AQ253H and p661AD279N, respectively, in combination with cRNA of p661B.
The obtained titres after rescue and passaging of the virus showed striking differences. Supernatants were passaged twice on CEC to test the efficiency of virus replication. UK661-AT and UK661-DN-AT resulted in titres of 104 TCID50/ml. In contrast, after an additional two passages in CEC UK661-QH-AT contained 105·25 TCID50/ml.
Exchange of aa 253 and 284 attenuated vvIBDV in chicken
To investigate the properties of the generated IBDV in chickens animal experiments were performed. All animals infected with UK661 or UK661rev showed severe clinical signs of IBD before challenge. All chickens which had been examined at days 3, 4, 7 and 10 p.i. died or were euthanized for ethical reasons after the occurrence of severe clinical signs following infection with UK661 and UK661rev. The mortality rates were 80% (28/35) for UK661 and 49% (17/35) for UK661rev. In contrast, none of the animals infected with UK661-QH-AT died or showed clinical signs of IBD.
Bursae of chickens infected with the different IBDV strains showed depletion of bursal cells in bursal follicles but with remarkable differences (Table 3). UK661 and UK66rev induced severe lesions (complete lymphocytic depletion, BLS of 5), only 3 days after infection. The lesions induced by these strains were very persistent. 14 days after infection total lymphocyte depletion (BLS of 5) was still observed in the BF. In contrast, 3 to 14 days after infection with UK661-QH-AT only mild to moderate lesions (BLS between 1 and 2) were induced in the BF of infected chickens.
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Sequence analysis of the RTPCR products based on the virus reisolated from the chickens confirmed the identity of the IBDV used. Furthermore, no amino acid substitutions in comparison to the sequence of the used plasmids (p661A, p661AQ253H-A284T) were found within the region flanked by oligonucleotides used for RTPCR, proving the genetic stability of the virus during chicken passage.
Detection of viral antigen in the BF
To determine the presence of viral antigen on different days after infection and challenge infection the mAb-8 ELISA was used (Table 4). In animals infected with UK661 or UK661rev viral antigen was detected from 3 to 10 days after infection. Single chickens infected with UK661-QH-AT contained viral antigen in the BF between 7 and 14 days after infection. Three days after challenge infection no virus was detected when the animals had been infected with UK661, 661rev or UK661-QH-AT, indicating that 100% protection was achieved. In the non-infected control group 3 days after challenge infection viral antigen was detected in all BF investigated.
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Discussion |
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The adaptation to tissue culture of strain UK661 showed that the alteration of aa 284 (Thr to Ala) was necessary and sufficient for adaptation to tissue culture. These results are in agreement with data of Mundt (1999) , who showed that the exchange of either two amino acids (Q253H, A284T) of the variant E strain E/Del, or of one amino acid (A284T) of the GLS strain was sufficient to adapt those strains to tissue culture. In contrast, Lim et al. (1999)
claimed that the exchange of two amino acids (D279N, A284T) of the very virulent strain HK46 was necessary for adaptation to tissue culture. However, single alterations of aa 284 and 279 were not tested. Based on the sequence data encoding VP2 of the HK46 strain, which differed from UK661 by only one amino acid (HK46-T485A-UK661) it is likely that the amino acid exchange A284T could also be sufficient for adaptation of strain HK46 to tissue culture. For vvIBDV Yamaguchi et al. (1996b
) suggested that the exchange of aa 279 and 284 is important for infection in tissue culture. Taken together, it is likely that aa 284 plays a central role in infection of tissue culture. However, dependent on the surrounding amino acids other amino acids are also important for the ability of the virus to productively infect tissue culture. A single exchange (A284T) and the double exchange (D279N, A284T) resulted in UK661 derivatives (UK661-AT, UK661-DN-AT) which grew only to low titres in cell culture in comparison to the UK661-QH-AT. Why the additional exchange of aa 253 (Q to H) enhanced the amount of obtained virus after passaging is not clear. It is possible that this exchange gave the virus an advantage in virus replication by an unknown mechanism. These data are in contrast to the data of Lim et al. (1999)
where the tissue culture-adapted HK46 strain containing two amino acid exchanges (D297N, A284T) grew to very high titres.
The failure of rescue of UK661 able to infect tissue culture and the successful rescue of infectious virus in chickens using the same tissue culture supernatant confirmed previous experiences of difficulties to adapt virulent IBDV strains to tissue culture (Yamaguchi et al., 1996a ) and stressed the importance of single amino acids for adaptation to tissue culture as described recently (Lim et al., 1999
; Mundt, 1999
). This was a prerequisite for the determination of the influence of tissue culture adaptation on the virulence of the virus. Since the mutants UK661-AT and UK661-DN-AT replicated only to low titres in tissue culture, UK661-QH-AT was used for animal experiments. The examination of the BF of chicken infected with UK661-QH-AT clearly showed an attenuated phenotype. In addition, the tissue culture-adapted UK661-QH-AT induced 100% protection against a classical virulent strain. Whether this virus protects against the vvIBDV requires further investigation. Since all known IBDV strains adapted to tissue culture by classical methods contained an Asn and Thr at position 253 and 284 (Mundt, 1999
), respectively, the experiments presented here reflect the natural situation. It was suggested earlier that aa 279 and 284 were important for virulence in chicken (Yamaguchi et al., 1996b
). However, this has not been proven since other exchanges of amino acids were also found, e.g. in VP3 and VP4. As described, we show that VP2 was responsible for the attenuated phenotype of the tissue culture-adapted IBDV strain UK661. UK661-QH-AT showed a comparable phenotype to other vvIBDV strains adapted to tissue culture by classical methods (Yamaguchi et al., 1996b
). Thus, the attenuated phenotype of the tissue culture-adapted UK661-OH-AT was the result of the exchange of only two amino acids in VP2. One question remains: what is the reason for the attenuated phenotype in chicken? Both UK661rev and UK661-QH-AT are able to infect and subsequently destroy B-lymphocytes in the BF, resulting in a loss of the follicle architecture. Thus, the use of a different receptor is unlikely. It is possible that the entry of the tissue culture-adapted IBDV is delayed. However, it is also possible that UK661rev replicates more efficiently in the BF, resulting in a higher degree of follicle destruction. Infection of the B-lymphocyte with UK661rev results in a more extensive negative influence on the cell life-cycle than infection with UK661-QH-AT. If this is the case VP2 has to interfere with cellular processes. Further investigations are under way to address these questions.
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Acknowledgments |
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References |
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Birghan, C., Mundt, E. & Gorbalenya, A. E. (2000). A non-canonical Lon proteinase deficient of the ATPase domain employs the SerLys catalytic dyad to impose broad control over the life cycle of a double-stranded RNA virus. EMBO Journal 19, 114-123.
Boot, H. J., ter Huurne, A. A., Peeters, B. P. & Gielkens, A. L. (1999). Efficient rescue of infectious bursal disease virus from cloned cDNA: evidence for involvement of the 3'-terminal sequence in genome replication. Virology 265, 330-341.[Medline]
Boot, H. J., ter Huurne, A. A., Hoekman, A. J. W., Peeters, B. P. & Gielkens, A. L. (2000). Rescue of very virulent and mosaic infectious bursal disease virus from cloned cDNA: VP2 is not the sole determinant of the very virulent phenotype. Journal of Virology 74, 6701-6711.
Box, P. (1989). High maternal antibodies help chicks beat virulent strains. World Poultry 53, 17-19.
Brown, M. D. & Skinner, M. A. (1996). Coding sequences of both genome segments of a European very virulent infectious bursal disease virus. Virus Research 40, 1-15.[Medline]
Bygrave, A. C. & Faragher, J. T. (1970). Mortality associated with Gumboro disease. Veterinary Record 86, 758-759.
Chettle, N., Stuart, J. C. & Wyeth, P. J. (1989). Outbreak of virulent infectious bursal disease in East Anglia. Veterinary Record 125, 271-272.[Medline]
Cosgrove, A. S. (1962). An apparently new disease of chickens avian nephrosis. Avian Diseases 6, 385-389.
Cursiefen, D., Käufer, I. & Becht, H. (1979). Loss of virulence in a small plaque mutant of the infectious bursal disease virus. Archives of Virology 59, 39-46.[Medline]
Di Fabio, J., Rossini, L. I., Eterradossi, N., Toquin, M. D. & Gardin, Y. (1999). European-like pathogenic infectious bursal disease viruses in Brazil. Veterinary Record 145, 203-204.
Eterradossi, N., Picault, J. P., Drouin, P., Guittet, M., LHospitalier, R. & Bennejean, G. (1992). Pathogenicity and preliminary antigenic characterization of six infectious bursal disease virus strains isolated in France from acute outbreaks. Journal of Veterinary Medicine B 39, 683-691.[Medline]
Eterradossi, N., Arnauld, C., Tekaia, F., Toquin, D., Le Coq, H., Rivallan, G., Guittet, M., Domenech, J., van den Berg, T. P. & Skinner, M. A. (1999). Antigenic and genetic relationship between European very virulent infectious bursal disease viruses and an early West African isolate. Avian Pathology 28, 36-46.
Hassan, M. K., Nielsen, C. K., Ward, L. A., Jackwood, D. J. & Saif, Y. M. (1996). Antigenicity, pathogenicity, and immunogenicity of small and large plaque infectious bursal disease virus clones. Avian Diseases 40, 832-836.[Medline]
Kunkel, T. A., Roberts, J. D. & Zakour, R. A. (1987). Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods in Enzymology 154, 367-382.[Medline]
Lange, H., Müller, H., Käufer, I. & Becht, H. (1987). Pathogenic and structural properties of wild type infectious bursal disease virus (IBDV) and virus grown in vitro. Archives of Virology 92, 187-196.[Medline]
Leong, J. C., Brown, D., Dobos, P., Kibenge, F. S. B., Ludert, J. E., Müller, H., Mundt, E. & Nicholson, B. (2000). Family Birnaviridae. In Virus Taxonomy. Seventh Report of the International Committee on the Taxonomy of Viruses , pp. 481-490. Edited by M. H. V. van Regenmortel, C. M. Fauquet, D. H. L. Bishop, E. B. Carstens, M. K. Estes, S. M. Lemon, J. Maniloff, M. A. Mayo, D. J. McGeoch, C. R. Pringle & R. B. Wickner. San Diego:Academic Press.
Lim, B. L., Cao, Y., Yu, T. & Mo, C.-W. (1999). Adaption of very virulent infectious bursal disease virus to chicken embryonic fibroblasts by site-directed mutagenesis of residues 279 and 284 of viral coat protein VP2. Journal of Virology 73, 2854-2862.
McFerran, J. B., McNulty, M. S., Killop, E. R., Connor, T. J., McCracken, R. M., Collins, P. S. & Allan, G. M. (1980). Isolation and serological studies with infectious bursal disease virus from fowl, turkeys and ducks: demonstration of a second serotype. Avian Pathology 9, 395-404.
Müller, H., Lange, H. & Becht, H. (1986). Formation, characterization and interfering capacity of a small plaque mutant and of incomplete virus particles of infectious bursal disease virus. Virus Research 4, 297-309.[Medline]
Mundt, E. (1999). Tissue culture infectivity of different strains of infectious bursal disease virus is determined by distinct amino acids in VP2. Journal of General Virology 80, 2067-2076.
Mundt, E. & Müller, H. (1995). Complete nucleotide sequences of 5'- and 3'-noncoding regions of both genome segments of different strains of infectious bursal disease virus. Virology 209, 209-218.
Mundt, E. & Vakharia, V. N. (1996). Synthetic transcripts of double-stranded birnavirus genome are infectious. Proceedings of the National Academic of Sciences, USA 93, 11131-11136.
Mundt, E., Beyer, J. & Müller, H. (1995). Identification of a novel viral protein in infectious bursal disease virus-infected cells. Journal of General Virology 76, 437-443.[Abstract]
Nakamura, T., Otaki, Y. & Nunoya, T. (1992). Immunsuppressive effect of a highly virulent infectious bursal disease virus isolated in Japan. Avian Diseases 36, 891-896.[Medline]
Öppling, V., Müller, H. & Becht, H. (1991). The structural polypeptide VP3 of infectious bursal disease virus carries group- and serotype-specific epitopes. Journal of General Virology 72, 2275-2278.[Abstract]
Schröder, A., van Loon, A. A. W. M., Goovaerts, D. & Mundt, E. (2000). Chimeras in noncoding regions between serotypes I and II of segment A of infectious bursal disease virus are viable and show pathogenic phenotype in chickens. Journal of General Virology 81, 533-540.
Snyder, D. B., Vakharia, V. N. & Savage, P. K. (1992). Naturally occurring-neutralizing monoclonal antibody escape variants define the epidemiology of infectious bursal disease virus in the United States. Archives of Virology 127, 89-101.[Medline]
Spies, U., Müller, H. & Becht, H. (1987). Properties of RNA polymerase activity associated with infectious bursal disease virus and characterization of its reaction products. Virus Research 8, 127-140.[Medline]
Spies, U., Müller, H. & Becht, H. (1989). Nucleotide sequence of infectious bursal disease virus segment A delineates two major open reading frames. Nucleic Acids Research 17, 7982.[Medline]
van den Berg, T. P., Gonze, M. & Meulemans, G. (1991). Acute infectious bursal disease in poultry: isolation and characterization of a highly virulent strain. Avian Pathology 20, 133-143.
van Loon, A. A. W. M., Lütticken, D. & Snyder, D. B. (1994). Rapid quantification of infectious bursal disease (IBD) challenge, field or vaccine virus strains. Proceedings of the International Symposium on Infectious Bursal Disease and Chicken Infectious Anaemia, pp. 179187. World Veterinary Poultry Association and Institut für Geflügelkrankheiten, Giessen, Germany, 2124 June 1994.
Yamaguchi, T., Kondo, T., Inoshima, Y., Ogawa, M., Miyoshi, M., Yanai, T., Masegi, T., Fukushi, H. & Hirai, K. (1996a). In vitro attenuation of highly virulent infectious bursal disease virus: some characteristics of attenuated strains. Avian Diseases 40, 501-509.[Medline]
Yamaguchi, T., Ogawa, M., Inoshima, Y., Miyoshi, M., Fukushi, H. & Hirai, K. (1996b). Identification of sequence changes responsible for the attenuation of highly virulent infectious bursal disease virus. Virology 223, 219-223.[Medline]
Received 27 June 2001;
accepted 23 August 2001.