Institute of Molecular Biology, Friedrich-Loeffler-Institutes, Federal Research Centre for Virus Diseases of Animals, D-17498 Insel Riems, Germany1
Intervet International BV, NL-5830AA Boxmeer, The Netherlands2
Author for correspondence: Egbert Mundt. Fax +49 38351 7151. e-mail Egbert.Mundt{at}rie.bfav.de
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Abstract |
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Introduction |
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Serotype I strains are pathogenic, since they cause lesions by lymphocytic depletion in the bursa of Fabricius (BF) of susceptible chickens. Serotype II strains lack the ability to induce lesions in the BF and are therefore not pathogenic for susceptible chickens (Ismail et al., 1988 ). The reasons for these different biological properties are unknown. Nieper & Müller (1996)
showed binding of serotype I as well as serotype II virus particles to lymphoid cells such as bursal, thymic and spleen cells. This indicates that restriction of IBDV to lymphoid B cells might not be determined by the presence of specific receptors, but might be due to alternative regulatory elements, possibly involved in regulation of replication. The 5'-noncoding regions (NCRs) of segment A of strains of the two serotypes differ in 13 of 96 nucleotides and the 3'-NCRs differ in four of 93 nucleotides (Mundt & Müller, 1995
). The differences in the 5'-NCRs led to a different calculated RNA secondary structure (Mundt & Müller, 1995
), which might represent such a regulatory element involved in the regulation of replication. In members of the Picornaviridae, changes in the 5'-NCR can influence attenuation (Kawamura et al., 1989
) and can also enhance cap-independent translation in vivo (Martinez-Salas et al., 1993
). An influenza A virus with a chimeric neuraminidase gene containing the 5'- and 3'-NCRs of influenza B virus showed markedly reduced mortality in mice (Muster et al., 1991
). The influence of the different NCRs of segment A of IBDV was investigated by the generation of chimeric viruses containing exchanged NCRs of segment A by using the reverse genetics system for IBDV (Mundt & Vakharia, 1996
). Virus that was recovered was subsequently characterized in vitro and in vivo.
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Methods |
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Construction of IBDV chimeras of segment A of serotypes I and II.
For construction of a chimeric segment A, a full-length clone of segment A of strain D78 (pAD78/EK; Mundt et al., 1997 ) was used. Fragments containing the 5'- and 3'-NCRs of segment A of serotype II strain 23/82 were generated by PCR with plasmid pAD78/EK and two primer pairs (Table 1
). For the 5'-NCR of segment A, an oligonucleotide (5'-STIIA; Table 1
) specifying the complete NCR of serotype II strain 23/82 up to the start codon of VP5 (Mundt & Müller, 1995
) and a second oligonucleotide (A15; Table 1
) localized at the 5'-part of the polyprotein gene were used for PCR amplification. The resulting PCR fragment was cloned blunt-ended and sequenced. A plasmid containing an appropriate insert (p5'-STII-D78A) was used for a second PCR to place the T7 RNA polymerase promoter immediately upstream of the viral sequence. To this end, an oligonucleotide containing the promoter sequence and parts of the 5'-NCR of segment A (FKA5'; Table 1
) was combined with oligonucleotide A15 for PCR amplification. The PCR fragment was cloned and sequenced and an appropriate plasmid (pT75'-STII-D78A) was selected. A unique restriction endonuclease cleavage site (RsrII) present in segment A and a unique EcoRI cleavage site in the oligonucleotide FKA5' as well as in pAD78/EK were used for insertion of the appropriate part of pT75'-STII-D78A into pAD78/EK, resulting in plasmid pFL5'-D78A. The 3'-NCR of segment A of serotype II strain 23/82 was generated by PCR with an oligonucleotide containing the serotype II 3'-NCR (STIIA-3'; Table 1
) and a second oligonucleotide (A40; Table 1
) localized at the 3'-part of the polyprotein gene. After cloning and sequencing of the PCR fragment (pD78A-STII-3'), an appropriate insert was ligated into BglII/BsrGI-cleaved pAD78/EK (pFLD78A-3'). An appropriate fragment of pD78A-STII-3' was cloned into pFL5'-D78A to generate a plasmid containing the 5'- and 3'-NCRs of serotype II strain 23/82 and the coding region of serotype I strain D78 (pFL5'-D78A-3'). The plasmids are depicted in Fig. 1
.
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Propagation of chimeric virus.
Virus progeny recovered from QM-7 cells transfected with pAD78/EK/pBP2 (IBDV/EK), pFLD78A-3'/pBP2 (IBDV-3'), pFL5'-D78A/pBP2 (5'-IBDV) or pFL5'-D78A-3'/pBP2 (5'-IBDV-3') were passaged once on Vero cells and the presence of IBDV antigen was detected by immunofluorescence assay (IFA) by using rabbit anti-IBDV serum (Mundt et al., 1995 ). For propagation of the chimeric viruses, transfection supernatants were passaged once on Vero cells until a CPE was visible. Vero cells were frozen and thawed and scraped off the flask and the supernatants obtained after low-speed centrifugation were titrated on BGM cells as described previously (Hassan et al., 1996
). Virus stocks were stored at -70 °C.
Characterization of chimeric viruses in cell culture.
To assay the replication of chimeric viruses, growth kinetics were established. Confluent secondary CEC grown in a 24 well tissue-culture dish were infected with IBDV/EK, IBDV-3', 5'-IBDV or 5'-IBDV-3' at an m.o.i. of 1. After incubation at room temperature for 1 h, the inoculum was removed and the cells were rinsed twice with PBS and overlaid with 1 ml DMEM. The supernatant was removed immediately from one well and stored at -70 °C [0 h post-infection (p.i.)]. The remaining wells were incubated and supernatants were removed at 8, 12, 24, 36 and 48 h p.i. and stored at -70 °C. Supernatants were centrifuged and titrated on BGM cells.
Characterization of chimeric viruses in chickens.
Seventy-five 2-week-old SPF chickens (Intervet) were divided randomly into five groups. Each group was maintained in negative-pressure, filtered-air isolators. Chickens were infected via eye drop with 104·7 TCID50 IBDV/EK, IBDV-3', 5'-IBDV or 5'-IBDV-3'. Non-inoculated hatchmates were used as controls. At 3, 7 and 13 days p.i., five chickens from each group were bled and euthanized. The BF of each chicken was removed and subdivided into two parts. One part was fixed in 10% neutral-buffered formalin. The second part of the bursa was weighed, homogenized and tested for the presence of virus in the BF. To this end, QM-7 cells were infected with the supernatant obtained and assayed for the presence of IBDV antigen 24 h p.i. by using indirect IFA (Mundt et al., 1995 ). Supernatants that tested positive were titrated on BGM cells. Titres were calculated per gram bursal tissue.
Histopathology of bursae.
Formalin-fixed samples of BF were embedded in paraffin (Paraplast), sectioned and stained with haematoxylin and eosin (H&E). Microscopic bursal lesion score (BLS) was determined by histopathological analysis of the bursa. BLS was evaluated on a scale of 0 to 5 as follows: 0, no abnormalities; 1, 120%; 2, 2140%; 3, 4160%; 4, 6180%; and 5, 81100% lymphocyte depletion.
Analysis of chicken serum.
Chicken sera were analysed by Western blot. For Western blot, virus particles of IBDV serotype I strain P2 (Schobries et al., 1977 ) were used that had been purified by ultracentrifugation as described previously (Müller et al., 1986
). Two µl purified IBDV particles was separated by SDSPAGE and blotted onto nitrocellulose membrane (Schleicher & Schüll). Groupwise-pooled chicken sera were used as the first antibody in different dilutions. After incubation with the second antibody (alkaline phosphatase-conjugated anti-species serum, Sigma), the reaction was visualized by using BCIP/NBT (Roth).
To test the ability of the chimeric viruses to induce neutralizing antibodies, a virus-neutralizing (VN) test was performed. The VN test was based on the tests described by Skeeles et al. (1979) and Lütticken & Cornelissen (1981)
with modifications. Briefly, serum was serially diluted twofold in a constant amount (750 TCID50/100 µl) of virus (strain D78, Intervet). To this end, 100 µl of the diluted virus solution, containing 750 TCID50/100 µl, was placed in each well of a microtitre plate with the exception of the first well in each row, which was left empty. Next, 100 µl serum was added to the empty first well of each row. One hundred µl of a virus suspension containing 1500 TCID50/100 µl was then added to the serum-containing wells in the first well of each row. After mixing the virus and serum, resulting in a virus concentration of 750 TCID50/100 µl in the first well of each row, serial dilutions were made by transferring 100 µl per well to the next well of each row. After incubation for approximately 1·5 h, 100 µl per well of a CEC suspension (0·51·0x106 cells/ml) was added and the microtitre plates were covered and incubated at 37 °C for 57 days. After incubation, wells were scored for the presence of a CPE. The end-point of the VN test for a serum sample was determined to be the reciprocal of the highest dilution, expressed in log2, in which there was no visible CPE. Positive and negative controls, as well as internal serum standards with different concentrations of antibodies against IBDV, were run at all times.
Analysis of NCRs of segment A of virus reisolated after passage in chickens.
For investigation of the NCRs of segment A after passage in chickens, virus-containing supernatants of CEC showing CPE during passage of the bursa homogenate were analysed. For this purpose, groupwise-pooled aliquots of bursa homogenates were used for infection of CEC. After complete CPE had developed, cells were frozen and thawed and virus was purified by ultracentrifugation as described previously (Müller et al., 1986 ). Genomic viral RNA was purified (Mundt & Müller, 1995
) and the sequences of the complete NCRs of segments A of the four chimeric viruses were determined by two methods, 5'-RACE using oligo(dGTP) tailing for the 5'-NCR and poly(A) tailing followed by RTPCR for the 3'-NCR (Mundt & Müller, 1995
), using oligonucleotides shown in Table 1
. The resulting PCR fragments were cloned blunt-ended and sequenced. Sequences were analysed by using the Wisconsin package, version 8 (Genetics Computer Group, Madison, WI, USA).
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Results |
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Replication of chimeric IBDV in CEC
In order to compare replication of the different chimeric IBDV in more detail, growth kinetics were analysed (Fig. 2). Supernatants of confluent secondary CEC infected with IBDV/EK, IBDV-3', 5'-IBDV or 5'-IBDV-3' were removed at 0, 8, 12, 24, 36 and 48 h p.i. and titrated on BGM cells. Titres produced by IBDV containing the 5'-NCR of serotype I (IBDV/EK, IBDV-3') were about 0·5 log10 higher than those of the IBDV containing the 5'-NCR of serotype II (5'-IBDV, 5'-IBDV-3') at 12 h p.i. From 12 h p.i. onwards, no significant differences could be observed between the four chimeric IBDV analysed.
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Analysis of NCRs
In order to confirm the identity of the chimeric virus in tissue culture supernatants, reisolated viruses were analysed by RTPCR. Sequence analysis of the amplified fragments revealed that clones specific for both ends differed in size. Therefore, sequencing of cDNA clones was performed until at least four clones with the longest nucleotide sequence had been analysed. Sequence analysis showed that nearly all IBDV sequences were 100% identical to the parental plasmids (Fig. 4). No nucleotide substitutions was detected in the 5'-NCRs of IBDV/EK or IBDV-3'. Single clones contained single nucleotide substitutions, e.g. in the 3'-NCR of IBDV-3', one of 10 clones showed a C3226A substitution. One mutation detected in the in the 3'-NCRs of IBDV/EK and 5'-IBDV (C3227T) represented a mutation from C (serotype I strain D78) to T (serotype II strain 23/82). However, no substitution was a revertant mutation to the serotype I sequence. Since plus-sense RNAs transcribed from plasmids containing segment A have four additional nucleotides at the 3' end after linearization with BsrGI, it was interesting to analyse whether the vector-derived nucleotides were retained. Sequence data for the 3' ends of all four genomic segments A showed that the vector-derived nucleotides were not part of the viral genomic RNA.
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Discussion |
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The determination of the 5'- and 3'-NCR sequences of the four genomic segments A showed that the substitutions detected were point mutations present in the minority of the plasmids analysed. Single mutations in the NCRs are expected for RNA viruses. No mutations from serotype II-specific to serotype I-specific nucleotides were detected. One mutation detected in the 3'-NCR (C3227T) of IBDV/EK and 5'-IBDV was present in the sequence of the plasmids analysed. But this mutation was also present in the minority of the plasmids analysed and therefore the importance of this mutation for the virus replication is not clear. The 3' end of segment A was trimmed exactly before being packaged into the virion, as described recently for infectious pancreatic necrosis virus (Yao & Vakharia, 1998 ).
In summary, exchange of the NCRs of segment A of IBDV has no significant influence on virus replication in cell culture. The restriction of replication of IBDV in lymphoid cells of BF is not determined by the NCRs of segment A. The binding of the virus to cells and the ability to replicate within these cells seem to be two different processes. By using other engineered chimeric viruses, it is hoped that the viral genomic element responsible for the different pathotypes of the two serotypes in the natural host will be identified.
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Acknowledgments |
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References |
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Received 12 July 1999;
accepted 16 September 1999.