Cs.N. Drén1, A. Kant2, D.J. Van Roozelaar2,
L. Hartog2, M.H.M. Noteborn3 & G. Koch2
1 Veterinary Medical Research Institute, Hungarian Academy of Sciences,
Budapest, P.O. Box 18., Hungary
2 DLO-Institute of Animal Science and Health, P.O. Box 365, 8200
AJ Lelystad, Netherlands
3 Laboratory for Molecular Carcinogenesis, Leiden University, Leiden,
The Netherlands
The pathogenesis of chicken infectious anaemia virus (CAV) infection was studied in 6-week-old and one-day-old SPF chickens inoculated with graded doses of Cux-1 strain (106-102 TCID50/chicken). Viraemia, viral shedding, development of virus neutralising (VN) antibodies and CAV distribution in the thymus were studied by virus isolation, PCR, immunohistochemistry (IP) and in situ hybridization respectively, until 28 days after infection (PID). In 6-week-old chickens infected with high dose of CAV (106 TCID50), viraemia and VN antibodies could be detected at 4 PID and onward without virus shedding or contact transmission to sentinel birds. Viral shedding and contact transmission was demonstrated in one-day-old infected chickens. In samples from chickens infected with 104-105 TCID50, VN antibodies could be detected at 4 PID, low level viraemia at 14 PID with (104 Group) or without (105 Group) transient viral shedding. Serum samples from chickens infected with lower doses of CAV (102-103 TCID50) were negative at 4 PID, developed VN antibodies by 14 PID, varying degree of viraemia with (102 Group) or without (103 Group) virus shedding. In 6-week-old chickens few cells of the thymus cortex were positive with VP3-specific mAb, whereas all thymus follicles of 1-day-old inoculated group were positive. Cells with positive in situ hybridization signal were fewer and scattered throughout of the thymus tissue as compared to IP positive cells. These data suggest that the immune response induced by CAV in 6-week-old chickens curtails viral replication and prevents viral shedding in a dose dependent mode.
B.M. Adair
Veterinary Sciences Division, Stormont , Belfast BT4 3SD, Northern Ireland
Studies on the effects of CAV on immune function, combined with investigations of the cells targeted by the virus following infection of 1-day-old and 21-day-old chickens, give a good indication of how the immunopathogenesis of the infection proceeds. These studies have shown that the virus appears to target erythroid and lymphoid progenitor cells in the bone marrow and thymus respectively. Destruction of erythroid progenitors in bone marrow results in severe anemia, and depletion of granulocytes and thrombocytes.
The T lymphocyte progenitor cells in the thymus are particularly susceptible to CAV infection and appear to be a major target for the virus. B cells and their precursors are not susceptible to infection and no substantial depletion of B cell numbers comparable to the dramatic depletion in numbers of T cells is observed following infection. Double labelling studies confirmed that precursor T cells were selectively infected and there was evidence for selective infection of CD8+ cells. Functional studies demonstrated adverse effects on lymphocyte and macrophage functions, including depression of cytokine production, all of which suggested that CAV infection was followed by a profound immunosuppression. The effects of the virus on immune function, contribute to increased susceptibility to, and enhancement of the pathogenicity of secondary infectious agents, and sub-optimal antibody responses, which have been reported as features of CAV-associated disease in the field.
A.A.H.M. ter Huurne, G.L. Kok, A. Kant , G. Koch , & J.M.A.
Pol.
DLO-Institute for Animal Science and Health, P.O. Box 65, NL-8200 AB Lelystad,
The Netherlands
Infectious bursal disease is caused by an avibirnavirus, infectious bursal disease virus (IBDV). The virus is extremely lymphocidal with an affinity for actively dividing B lymphocytes resulting in atrophy of the bursa of Fabricius. Three-weeks old SPF White Leghorn chickens were inoculated intraocularly and intranasally with 103 ELD50 of a very virulent IBDV isolate (D6948). We studied the early pathogenesis of this very virulent isolate by using immunohistochemistry and a Mab directed against VP2. The bursa, spleen, thymus, cecal tonsils and liver were collected from 2 h p.i. onwards.
Antigen-positive cells could be detected in the bursa from 12 h p.i., whereas the cecal tonsils became positive from18 h p.i, the spleen and liver from 24 h p.i., while the thymus was still negative at this latter time point. B-cells in the medulla of only a few bursal follicles were the first cells that became positive. After massive replication and cell lysis had occurred in these few bursal follicles, viral antigen was detected in the interfollicular connective tissue and follicles mainly directly adjacent to the heavily affected follicles and to a lesser extent other follicles. Up to 25% of the follicles was positive at 24 h p.i., up to 90% at 36 h p.i, whereas at 48 h p.i viral antigen was detected in necrotic cell masses in all follicles. Viral antigens were detected in B-cells expressing Bu-1 and IgM, in macrophages and in reticular cells but not in T-cells. The early spread of this highly virulent IBDV isolate in the bursa and from the bursa is under investigation.
1Oláh, I., 1Magyar, A., 1Nagy,
N., 2Nagy,E. & 2Horváth, E.
1Department of Human Morphology and Developmental Biology, Semmelweis
University, Medical School, Hungary
2State Control Institute for Veterinary Biologicals, Drugs and Foods,
Budapest, Hungary
The cortico-medullary (CM) border of the bursal follicle has a peculiar histological structure. The CM epithelial cells are separated by basement membrane from the cortex and form arcades towards the medulla. These arcades seem to be filled with mainly medium-sized lymphocytes which proliferate and continously enter the medulla through the openings of the arcades. We followed the changes of cellular composition and structure of the CM arcades during infectious bursal disease virus (IBDV) infection.
CVL 52/70 IBD virus strain was introduced to four week old SPF chickens of SPAFAS strain by eyedrops. Short and long term effects of IBDV infection were monitored by immunohistochemical and histological studies. The histological changes in the arcades after IBDV infection are comparable with those of emetin, an antiparasitic drug and macromolecular synthesis inhibitor that drastically reduces the number of the cells in the bursa by apoptosis and induces histiocytosis-like monocyte-macrophage accumulation. By day two postincetcion the vimentin intermediate filament positive bursal secretory dendritic cells (BSDC) migrate out of the medulla and appeare in the cortex, surface epithelium and even in the lumen of the bursa. The BSDCs which migrate out of the medulla lose their surface IgG. The follicular loss of BSDCs is followed by histological changes in the CM arcades. The number of medium-sized lymphocytes rapidly decreases in the arcades and histiocyte-like cells appeare everywhere in the bursa. Similar to emetin treatment, bursal follicle regeneration occured, if medium-sized lymphocytes remained in the arcades and vice versa if the arcades are completely emptied no regeneration has been observed. We believe that the CM border is fundamental compartment for the development of the clinical symptomes of IBDV infection.
1Nagy, N., 1Magyar, A., 2Horváth,
E., 2Kovács, A. & 1Oláh, I. I
1Department of Human Morphology and Developmental Biology, Semmelweis
University, Medical School, Hungary
2State Control Institute for Veterinary Biologicals, Drugs and Foods,
Budapest, Hungary
We have suggested that the condition of cortico-medullary (CM) border is crucial for the bursal regeneration after acute involution caused by either chemical agents or infectious bursal disease virus (IBDV). The epithelial cells of the CM border enclose medium-sized lymphocytes which are depleted during acut bursal involution resulting in disappearance of the follicles. During the cell depletion of the CM border remarkable number of monocyte-macrophage-like cells of unknown origin emerge in the bursal follicle and connective tissue of the folds. The purpose of this study to throw some light on the origin of these "macrophages" after IBDV infection.
At hatching the SPF chickens of SPAFAS strain were separated into two groups (20 birds/group). In group one chickens were infected with CVL/70 IBD virus strain by eyedrops and two days later 15 birds were surgically bursectomized. In group two the chickens were surgically bursectomized at hatch and two days later infected with IBDV. No definitive clinical symptomes appeared in any groups. Both groups were sacrified 7 days after infection, namely at 7 and 9 days of age. Tissue samples from the infected birds (bursa, spleen and thymus) were taken out for histological and immunohistochemical studies.
In group one large number of phagocytic cells loaded with phagosomes appeared in the bursas two days postinfection. At day seven few vacuolated cells occured in the red pulp of the spleen but not in the thymus. The bursal folds and splenic red pulp of infected but not bursectomized chickens contained remarkable number of vacuolated phagocytic cells. 7 days postinfection the bursal folds did not show follicular arrangement. Our monoclonal antibody (1E5) which recognizes basement membrane of the CM border in normal follicle, indicates the deterioration of epithelio-mesenchymal border and possibly there some alterations also in the extracellular matrix. In group two (bursectomized first and infected at day two) no vacuolated phagocytic cell emerged in the spleen and thymus, at all. This observation suggests that the histiophagocytic cells after IBDV infection is coming from the bursa and they could be "virus modified or transformed" bursal secretory dendritic cells.
H.J. Boot, A.A.H.M. ter Huurne, B.P.H. Peeters & A.L.J. Gielkens
Institute for Animal Science and Health (ID-Lelystad), Department of Avian Virology,
PO. Box 65, NL-8200 AB Lelystad, The Netherlands
To study the mechanism of replication of IBDV, and to determine factors on the IBDV RNA which are involved in viral replication, we used cloned full length cDNA to generate infectious IBDV. We evaluated the effect of the length of the 3’-terminus of the A-segment plus strand of IBDV. No difference in virus yield was found between cDNA containing 3 to 6 adjacent cytosine residues at the 3’-terminus, although we found that wild-type virus predominantly contained 4 cytosines. If the 3’-untranslated terminus was shorter than 3 cytosines the ability to generate infectious IBDV from cDNA was reduced, but IBDV could be recovered in all cases examined. The sequence of the 5’-terminus of the non-coding strand of the A-segment from several rescued viruses was determined and appeared to be the same as we found for wild-type IBDV.
K. Zierenberg; H. Nieper and H. Müller
Institute of Virology, Faculty of Veterinary Medicine, University of Leipzig,
An den Tierkliniken 29, D-04103 Leipzig, Germany; Tel.: +49/341/9738200, Fax.:
+49/341/9738219, e-mail: virology@vetmed.uni-leipzig.de
IBDV infection of chicken results i) in the destruction of the bursa of Fabricius (BF) which is accompanied with high morbidity and mortality ("Gumboro Disease“), ii) in a subclinical or chronical course associated with a B-cell-dependent immunsuppression. Infections result in considerable economic losses in poultry industry.
Whereas all pathogenic strains are members of the serotype I (SI), serotype II (SII) strains are considered apathogenic, because they do not cause lesions of the BF. A study was undertaken in order to investigate the importance of segments A and B for pathogenicity. Using a reverse genetics system the recombinant viruses of the parental strains Cu-1 (SIr) and 23/82 (SIIr) as well as the reassortant viruses R1 (segment A strain Cu-1 / segment B strain 23/82) and R2 (segment A strain 23/82 / segment B strain Cu-1) were constructed.
Full length clones of segments A and B of the pathogenic strain Cu-1 and the apathogenic strain 23/82 were constructed. These clones were in vitro transcribed and the RNA transfected in chicken embryo cells (CEC). Resulting infectious viruses were characterized by immunofluorescence and restriction enzyme analyses. The viruses were passaged three times in CEC and the infectivity was determined by plaque assays. Variants in plaque size and infectivity were obtained. The plaque sizes of SII, R1 and SIr were identical to each other, and smaller than those of SI, but larger than the plaque sizes of SIIr and R2. Compared with SI and SII the infectivity of SIr and SIIr was decreased. Similar amounts of infectious virus were obtained from R1 and SIr as well as R2 and SIIr. One-step-growth curves were established for the recombinant/reassortant viruses.
D. Hot1 and M. A. Skinner
Institute for Animal Health, Compton, Newbury, Berks., RG20 7NN, U. K.
1Current address: Microbiologie des écosystčmes, Laboratoire de virologie,
Institut Pasteur de Lille, Rue du professeur Calmette, 59000 Lille, France.
The VP1 protein of the birnaviruses is the putative RNA-dependent RNA-polymerase (RdRp) of the virus and is, therefore, likely to interact with the viral genomic RNA. The interaction of VP1 with the viral genome has, however, never been investigated. The capacity of infectious bursal disease virus (IBDV) RdRp to bind to single-stranded (ss) RNA was studied in vitro. A poly-histidine tagged VP1 was expressed using a newly constructed recombinant fowlpox virus (FWPV) vector, pFTM2. The recombinant protein was purified to homogeneity using a nickel charged column and the binding to viral ssRNA was studied using filter-binding assays. It was found that the binding is co-operative with respect to the polymerase concentration. The obtained saturation-binding curve indicates an apparent dissociation constant (Kd) around 15 nM and a Hill coefficient of 1.76. The co-operative nature of the binding was supported by the observation of VP1-VP1 complexes in SDS-PAGE under non-reducing conditions and using gel filtration. A region of VP1 sequence (734-761) was predicted to exhibit a coiled-coil structure, which could account for the VP1-VP1 complex formation. This demonstration of the RNA binding activity of VP1, together with the recent demonstration of its interaction with VP3 indicates that, in addition to its RdRp activity, VP1 may play a significant role in encapsidation of the viral RNA.
A. Jungmann, H. Nieper and H. Müller
Institute of Virology, Faculty of Veterinary Medicine, University of Leizig,
An den Tierklinikein 29, D-04103 Leipzig, Germany;
Tel.: +49/341/9738200, Fax- +49/341/9738219; e-mail: virology@vetmed.uni-leipzig.de
Chicken embryo cells (CEC) were infected with serotype I strain Cu-1 IBDV and investigated for the presence of viral antigens and the induction of apoptosis. Cellular DNA was isolated and the characteristic DNA fragmentation was first observed at 12 hours post infection. The number of apoptotic cells in infected CEC increased from 9% at 8 hours to 78% at 48 hours after infection as monitored by flow cytometry.
With a double labelling method apoptotic cells were detected around those expressing vital antigens, whereas cells positive for antigens seemed to be protected from apoptosis. In later stages of the infection also double labelled cells were observed. The same results were obtained by investigation of the bursa of Fabricius (BF) of infected chickens. Furthermore we showed that viral replication is a prerequisite for the induction of apoptosis, since viral particles inactivated by incubation with VP2-specific neutralizing mAb or treatment with UV-light did not induce apoposis.
These results suggest the production of a soluble factor in the productively infected cells triggering apoptosis in surrounding cells. However, such a substance alone seems not to be sufficient to induce apoptosis, since treatment of CEC with supernatant of infected cells incubated with neutralizing antibodies did not induce apoprtotic cell dean
The higher number of apoptotic cells compared to the productively infected cells have to be considered as a reason for the strong depletion of the cells in the BF, playing an important role in the pathogenesis of the disease. In further studies viral and cellular components involved in the apoptotic pathway will be investigated.
H.R. Juul-Madsen1, O.L. Nielsen2, T.K.
Hansen1, P. Sorensen1, P.H. Jřrgensen3 and
T.S. Dalgaard1
1Danish Institute of Agricultural Sciences, P.O. Box 50, DK-8830
Tjele, Denmark
2Royal Veterinary and Agricultural University, DK-1870 Frederiksberg,
Denmark
3Danish Veterinary Laboratory, DK-8200 Aarhus, Denmark
The influence of B haplotypes on the IBDV vaccination response in chickens was investigated in three different chicken lines containing four different B haplotypes. Two B haplotypes were present in each of the lines. The B19 haplotype is present in all three lines, whereas BW1 is present exclusively in line 1, B131 is present exclusively in line 131, and finally B21 is present exclusively in line 21. The haplotypes B19 and B21 are standard international haplotypes isolated from White Leghorn, whereas the B131 haplotype originates from a commercial line of meat type White Cornish. The BW1 haplotype originates from the Red Jungle Fowl, Gallus Gallus. The chickens were vaccinated with live attenuated vaccine at the age of 3 weeks followed by an infection with IBDV at 6 weeks of age.
In this study, we found a notable B haplotype effect on the specific antibody response to IBDV. This result was further supported by the bursa of Fabricius recovery from the disease measured by histological scorings of the bursa of Fabricius. Furthermore, there was a remarkable line effect on other immunological parameters in the line containing leftover Red Jungle Fowl genes.
C.A. Butter and T.F. Davison
Institute for Animal Health, Compton, Newbury, Berkshire, RG20 7NN, U.K.
The recombinant vaccine fpIBD1 contains the entire VP2 coding region of IBDV, expressed as a b-galactosidase fusion protein, in the FP9 fowlpox virus vector. Previous work in this laboratory has identified differences between inbred lines of White Leghorns in their response to this vaccine. Line 15 birds (B15 haplotype) are not protected by the vaccine, and we have hypothesized that this may be due to the lack of peptides of the VP2 molecule that can be bound by the MHC Class I molecule expressed by this line. In contrast, line C.12 birds expressing the B12 MHC haplotype are protected at high efficiency. Computer models suggest that the B12 haplotype can bind up to nine peptides from VP2.
Line 6 birds (B2 haplotype) are protected by fpIBD1, but at low challenge doses the fowlpox vector alone affords the same protection. We have suggested that a non-specific cell-mediated response might explain this finding, and that protection in this line might be MHC class I independent.
Two backcrosses were produced:
1. line 15 (line 15 x C.12) The population was therefore 75% line 15 background, and approximately 50% MHC B 15,15 and 50% MHC B12,15.
2. line 15 (line 15 x line 6), giving 75% line 15 background and approximately 50% B15,15 and 50% B2,15.
All animals, together with pure line controls were then vaccinated at one and three weeks of age with fpIBD1 and challenged with 101.3 EID50 of a virulent strain of IBDV (52/70). Pathology was assessed at three days post-infection by bursal score.
The results are highly suggestive of protection dependent on the MHC region in C.12 birds, the difference between the B 15,15 and B12,15 components of the 15(15 x C.12) backcross being highly significant. In contrast, the B15,15 and B2,15 components of the 15 (15 x 6) backcross showed no significant differences, suggesting an MHC independent method of protection.
Y.Q. Wang, M. Korkeamäki and O. Vainio
Department of Medical Microbiology, Turku University, Kiinamyllynkatu 13, FIN-20520
Turku, Finland
T cells are crucially important for both the humoral and cell-mediated responses of adaptive immunity. The conventional method for the quantification of cytotoxic T cell activity is based on chromium-release from loaded target cells. There has been two major problems with the chromium-release assay in chicken: inefficient labelling of the target cells and a high spontaneous release. Additionally, chromium-release assay is a radioactive method with potential health and environmental hazards.
The cytotoxicity assay based on flow cytometric analysis uses fluorescent labels to stain live target cells and propidium iodide (PI) nuclear dye intercalation into killed target cell DNA to detect the dead cells. The damaged pre-labelled target cells appear double stained as their membranes become permeable to the nuclear stain during incubation. The percentage of cytotoxicity at various effector : target cell ratios is discerned using flow cytometric analysis of PI-positive cells after 50 min of incubation.
Our studies exploit as a model system H.B15 White Leghorn chickens immunized with fixed reticuloendotheliosis virus (REV) transformed MHC-compatible cells. 30x106 cells were given i.v. 4 times at 5d interval. Peripheral blood lymphocytes (PBL) and spleen cells (SPL) from the immunized chickens were used as the effector and REV-cells as the target cells.
The assay showed a 16.4±2.1% specific killing against REV-transformed cells three days after the third and a 31.3±2.9% killing three days after the fourth immunization by using the PBL as effector cells. The specific killing was as high as 44.5±1.1% when SPL were used as the effectors one week after the fourth immunization.
Multicolor flow cytometric assay also enables phenotyping of the effector cells. In this way, we have shown that CD8-positive cells are the cytotoxic cells in this system. More detailed phenotyping is in progress.
The method developed in this study will be useful for the assessment of cytotoxicity in chicken induced by viruses or parasites.
Y.Q. Wang1, J.L. Martínez-Torrecuadrada2,
I. Casal2, J.F. Rodriguez3 and O. Vainio1
1Department of Medical Microbiology, Turku University, Kiinamyllynkatu
13,FIN-20520 Turku, Finland.
2INGENASA. Hnos. García Noblejas 41. 28037 Madrid. Spain.
3Centro Nacinal de Biotecnología (CSIC), Universidad Autóma de Madrid,
28049 Madrid, Spain.
T cell proliferation assay for specific-IBDV proteins may help define the involvement and relative importance of different IBDV proteins in the protective response against the virus. For this purpose we immunized H.B15 (B-haplotype B15) chickens with 25 µg of a particular soluble recombinant IBDV protein i.m. twice at 14 days interval. Lymphocytes from peripheral blood were prepared by a low speed centrifugation method.
100 ml of PBL suspension (7x105 cells) and 100 ml antigen dilution per well were added in triplicate into a 96 flat-bottom microtiter plate. Cells were cultured for 5 days at 39°C in 5% CO2–air humidified atmosphere and the uptake for the last 18 hours before harvesting was measured.
The results showed that VP1F1 (149 amino acids long recombinant protein from the N-terminus of VP1) has weaker T cell proliferation response than that of other two IBDV encoded proteins. Only a small enhancement (stimulation index 4.65) was found when the in vivo VP1F1 primed T cells were stimulated with 5 µg/ml of the same antigen in vitro. In contrast, VPX induced strong a T cell proliferation response; two weeks after a single dose of in vivo immunization with 25 µg of VPX, the stimulation index was 22.0 in response to 0.1 µg/ml of VPX in vitro. VP3 also induces a vigorous T cell proliferation response but obviously much more antigen was needed in vitro. 5 µg/ml of VP3 induced the same proliferation response in vitro as 0.1 µg/ml of VPX. Our data also show that multiple immunizations increase the in vitro proliferation responses.
C. Birghan1 , E. Mundt1 , A. E. Gorbalenya2
1Institut for Molecular Biology, Friedrich-Loeffler-Institutes, Federal
Research Centre for Viral Diseases of Animals, D-17498 Insel Riems.
2Biomedical Supercomputer Center, Frederick, MD 21702-1201, USA
The genome of infectious bursal disease virus (IBDV) consists of two segments of double-stranded RNA. Segment A encodes a polyprotein (PP) which is proteolytically processed into the viral Proteins VP2, VP4 and VP3. A second open reading frame (ORF) precedes and partially overlaps the polyprotein gene. Segment B encodes the putative RNA-dependent RNA polymerase (VP1). The mechanism of the PP processing is not clear and the proteolytic activity seemed to be associated with VP4.
The processing of the PP was studied by using bioinformatics, biochemical, and molecular genetic tools. Bioinformatical analysis of the birnaviral VP4-sequences lead to the identification of a domain distantly related to prokaryotic proteases. In this domain two amino acids (aa) - serine and lysine - have the potential to play a role in peptide bond cleavage. These potential catalytic residues were studied in a cell-free transcription/translation system by using mutants of a full-length cDNA-construct of segment A of strain P2. Replacement of these two aa by site-directed mutagenesis resulted in a restriction of the PP processing. Uncleaved PP expressed from mutated plasmids was used as substrate in a trans-cleavage-assay. Using this assay the aa responsible for the proteolytic activity of VP4 were characterized in analogy to other proteolytic enzymes with a serine-lysine-dyade, e. g. E. coli leader peptidase, UmuD, LexA.
N. Lejal, S. Petit, B. Da Costa, J.-C. Huet1 and B.
Delmas
Unité de Virologie et Immunologie moléculaires, 1Unité de Biochimie
et Structure des Protéines, Institut National de la Recherche Agronomique, Domaine
de Vilvert, F-78350 Jouy-en-Josas, France
The polyproteins of infectious bursal disease virus (IBDV) and infectious pancreatic necrosis virus (IPNV), two members of the Birnaviridae, are processed by the viral protease VP4 to generate three polypeptides: VPX (also named pVP2), VP3 and VP4. First, we have undertaken a mutational analysis to identify active-site residues of the IPNV VP4 domain. In all, 42 single-amino-acid substitutions were created in a cloned IPNV A segment, and the effect of each mutation on the extent of self-cleavage of the polyprotein precursor at the pVP2-VP4 and VP4-VP3 junctions was assayed in vitro. Twelve mutations almost completely or completely inhibited protease activity, fifteen decreased cleavage, mainly at the VP4-VP3 junction, and others yielded wild-type level of activity. Results allowed the characterization of the IPNV VP4 catalytic site. Next, self-cleavage sites were determined at the pVP2-VP4 and the VP4-VP3 junctions by N-terminal sequencing of different forms of 5’-truncated polyproteins expressed in Escherichia coli. Cleavage sites were subsequently probed by mutagenesis. A set of additional candidate cleavage sites in the C-terminal domain of pVP2 was identified by sequence homologies and analyzed by mutagenesis. Finally, taking advantage of the identification of critical residues associated to the catalytic and cleavage sites on IPNV polyprotein, the replacement of the homologuous residues on the IBDV polyprotein was carried out on a cloned IBDV A segment. The replacement of the residues of the predicted catalytic site confirmed their indispensability. The substrate cleavage sites of IBDV VP4 were determined by N-terminal sequencing of VP3 and VP4 proteins and were subsequently probed by mutagenesis. Additional candidate cleavage sites were also identified and analyzed by mutagenesis in the C-terminal domain of VPX. Collectively, our data characterize the birnavirus VP4 proteases as members of a novel group of eucaryotic protease that shares properties with some bacterial peptidases in terms of catalytic and substrate cleavage sites.
Cliff J. Luke1 and Michael A. Skinner
>Institute for Animal Health, Compton, Newbury, Berks., RG20 7NN, U. K.
1Current address: Joint Program in Neonatology, Children's Hospital,
300 Longwood Ave, Enders 970, Boston, MA, 02115, USA
Infectious bursal disease virus (IBDV) is a member of the Birnavirus family and has 2 segments of dsRNA for its genome. The larger of these 2 segments, segment A, encodes a polyprotein that is co-translationally cleaved to form 3 products, VPX, VP4 and VP3. The VPX protein is then further cleaved to form a mature VP2 protein. VP2 and VP3 make up the main constituents of the virion.
It has been shown that VP4 was responsible, at least for the initial cleavage events of the polyprotein processing. Initial database searches showed that VP4 had no significant homology to any known proteases, however, more in depth analysis revealed a weak relationship between VP4 and the Lon protease from E. coli. This homology existed over the active site serine of the yes"> protease, however, the Lon protease is much larger than that of VP4 and may be structurally very different. In order to address which part of VP4 and which residues were important for the processing, we employed 2 expression systems; yes"> the in vitro coupled transcription/translation system from the T3 promoter and the E. coli expression system from the lac promoter. Using these 2 systems we analysed the affect of large deletions and specific mutagenised residues within VP4 that were conserved within the Birnavirus family. In total we created 4 deletion mutants, 2 frameshift mutations and 30 site directed mutants and analysed the yes"> ability of each VP4 to cleave the polyprotein.
Of the mutations made, 11 affected the processing of the polyprotein significantly. The most dramatic of these mutations was the serine at position 652 in the polyprotein. Mutation S652A almost abolished the polyprotein processing, whereas S652C and S652T had a less dramatic consequence. This is similar to other serine proteases where substituted cysteines or threonines can act as nucleophiles giving partial proteolytic activity. Two other residues (H546 & D589) previously postulated to form, with S652, a catalytic triad proved not to be crucial to protease activity.
Particularly noteworthy was the significant inhibition caused by the mutation of K692. This residue, completely conserved in the birnaviruses, aligns with a residue identified as part of a catalytic serine-lysine dyad in the E. coli leader peptidase & LexA. The spacing between S652 and K692 is compatible with that in the prokaryotic proteases. Inhibition was also observed on mutagenesis of H697, which falls in the same motif, conserved between the prokaryotic proteases and the birnaviruseses, as K692.
In conclusion, VP4 appears to form a novel class of viral serine protease, resembling bacterial leader proteases with a ser-lys catalytic dyad rather than the more common proteases with his-asp-ser catalytic triads.
D. Todd1, A.N.J. Scott2, B.M. Meehan2,
J.L. Creelan1, T.J. Connor1 and M.S. McNulty1
1 Department of Agriculture for Northern Ireland, Veterinary Sciences
Division, Stormont, Belfast BT4 3SD, Northern Ireland
2 Department of Veterinary Science, the Queen’s University of Belfast,
Veterinary Sciences Division, Stormont, Belfast BT4 3SD, Northern Ireland
Chicken anaemia virus (CAV) is a non-enveloped, icosahedral virus, which, on the basis of its small, circular ssDNA genome (2.3kb), has been classified within the virus family, Circoviridae. yes"> The genome organisation and gene expression strategy of CAV differ from all known animal viruses and these differences will probably lead to its eventual taxonomic separation from other circoviruses. The ability to grow CAV in MDCC-MSB1 cells and the cloning of the circular, ds replicative form (RF) DNA, which can be isolated from infected cells, have provided the basis for elucidating CAV’s molecular biology. yes"> Thus, only 1 of the strands comprising CAV RF is transcribed to produce a single polycistronic mRNA (~2.1kb) that contains 3 partially-overlapping ORFs encoding proteins of 52kDa, 26kDa and 13kDa, all of which are expressed in infected cells. The non-transcribed region is rich in transcription-factor binding motifs and exhibits promoter/enhancer activity. An understanding of how the CAV genome functions coupled with the ability to produce infectious RF clones have provided a means by which those sequence changes that attenuate CAV can be identified. In our laboratory, the molecular basis of the attenuations exhibited by cloned isolates, selected from virus pools, that have received multiple cell culture passages, are being investigated using a chimeric virus approach. Elsewhere, CAV RF sequences with known genome function are being altered by genetic manipulation in an attempt to produce attenuated isolates by rational design.
K. Wark and M. A. Skinner
Institute for Animal Health, Compton, Newbury, Berks., RG20 7NN, U. K.
The infectious bursal disease virus (IBDV) genome yes"> consists of two segments of double stranded RNA which encode five proteins. Segment B (2.8kb) encodes yes"> a 98kDa protein, VP1 which is the RNA dependant RNA polymerase (RdRp). The larger segment A (3.2kb) contains a large open reading frame (ORF) encoding a 109 kDa polyprotein (pp) which is autoproteolytically yes"> cleaved, yielding VPX, VP4 and VP3. VPX undergoes further modifications to produce VP2, the mature form present in the capsid. An additional ORF overlapping that of segment A codes for VP5 (17kDa), which is non essential for replication.
From in vitro experiments in which segment A was expressed and from analysis of a series of deletion mutants in E coli, VP4 was identified as the viral protease responsible for polyprotein processing. Relationships between VPX-related proteins yes"> have been confirmed by pulse chase experiments, yes"> tryptic peptide maps and indirectly yes"> by reactivity yes"> with VP2-specific monoclonal antibodies. However the precursor-product relationship between the polyprotein and its three major cleavage products has not been directly demonstrated in a pulse chase in IBDV.
Here we use rabbit polyclonal serum together with monoclonals against VP2 and VP3 to characterise the relationship between the polyprotein and its putative cleavage products. Surprisingly, we found not only VPX but also VP3 present after only three minutes of labelling. Even more surprisingly, after a 20 second pulse, only yes"> VP3 was detected. VPX was not detected until 100 second later in the chase.
These results will be considered alongside the various possible translation strategies for IBDV and will be related to the viral replication cycle and morphogenetic pathway.
J.I. Casal1, J.L. Martínez-Torrecuadrada1,
J.R. Castón2, J.L. Carrascosa2 and J.F. Rodríguez2
1 INGENASA. Hnos. García Noblejas 41. 28037 Madrid. Spain.
2Centro Nacional de Biotecnología. Universidad Autónoma de Madrid.
Cantoblanco. 28049 Madrid. Spain.
Infectious bursal disease virus (IBDV) is a highly contagious birnavirus, which causes a major disease in the poultry industry. The large RNA segment A encodes a precursor polyprotein in the order 5´-VPX-VP4-VP3-3´. The precursor polyprotein is processed through different proteolytic steps to yield mature virion capsid proteins VP2 and VP3, and the viral protease VP4. VP2 and VP3 are the major structural components of the IBDV virion. VP4 has been defined as the virus-coded protease responsible for the processing of the precursor polyprotein. However, the precise events implicated in this proteolytic process remain unknown.
INGENASA is currently working on the development of diagnostic kits for IBDV based on the use of recombinant IBDV virus-like particles (VLPs) expressed in insect cells using the baculovirus system. To learn more about the processing of the polyprotein and factors affecting the correct assembly of the viral capsid in vitro, different constructions of the polyprotein and IBDV structural proteins were expressed in insect cells using the baculovirus system. The starting virus was the Soroa strain, isolated in Cuba. Expression of the complete polyprotein lead to the formation of proteins VPX, VP2, VP3 and VP4. Purification of these preparations in sucrose gradients allowed the isolation of viral capsids and different types of particles, which contained variable amounts of the viral structural proteins. In general, only low amounts of VLPs were really made in insect cells. This low amount correlated quite well with minor amounts of VP2. Therefore, the processing of VPX to VP2 seems to be necessary for a proper formation of VLPs. This was clearly confirmed by the EM analysis.
Single expression of VP3 resulted in a massive expression of the protein. Subsequently, we carried out individual expression of VP2 and VPX. The sequences were selected from previous publications or unpublished data. Viral proteins were expressed at high level, purified and tested for diagnostic purposes. However, in no case IBDV VLPs were recovered from these new constructions. Initial results indicate that VPX and VP3 could be suitable candidates for diagnostics, but not VP2, probably due to an incorrect folding.
E. Lombardo1, I. Espinosa2, A. Maraver1,
A. Fernández-Arias2 and J. F. Rodríguez1.
1Departamento de Biología Molecular y Celular, Centro Nacional de
Biotecnología (CSIC), Madrid, Spain.
2Departamento de Biología Molecular, Centro de Investigación en Sanidad
Animal, La Habana, Cuba.
VP5 is a 17 kDa non-structural polypeptide encoded by the ORF1 of IBDV. It has been recently shown that although it is not essential for virus replication, VP5 plays a key role for virus dissemination (Mundt et al, 1997, J. Virol. 71, 5647). We have characterised the expression of VP5 in IBDV-infected CEF, BSC-1 cells infected with a VP5-vaccinia virus (VV) recombinant, and COS cells transfected with a VP5-expression plasmid. In these three cell systems, VP5 was shown to be associated to the plasma membrane. Expression of VP5 in both BSC-1 and COS cells triggered cell death, a response that appeared to be directly related to the disruption of the plasma membrane. The results obtained strongly suggest that VP5 might act during the last stages of the replication cycle "permeabilising" the plasma membrane and thus facilitating the release of the virus progeny.
Poster session
B. Lambrecht1, M. Gonze1, R Schuurmans1,
J. Lowenthal2, G. Meulemans1 & T.P. van den Berg1
1Section of Avian Virology, Veterinary and Agrochemical Research
Centre, Brussels, Belgium
2Australian Animal Health Laboratory, CSIRO Animal Health, Geelong,
Victoria, Australia
Interferon-gamma (IFN-g), a cytokine produced by stimulated T lymphocytes and Natural Killer cells, acts principally as an immuno-modulatory factor. This cytokine plays a pivotal role in determining the extent and type of immune response generated following vaccination or infection. While IFN-g preferentially induces a TH1 (or cell-mediated) type of immune response, it also enhances the humoral response, activates the macrophages and possesses an anti-viral activity.
Monoclonal antibodies (mAbs) specific for chicken IFN-g (ChIFN-g were generated by gene gun immunization and were utilized to develop a mAb-based capture ELISA specific for ChIFN-g. In addition to being more rapid and easier to perform than classical cell-mediated immunity tests, this ELISA has excellent sensitivity and improved specificity. The use of a specific rabbit polyclonal serum as revealing antibody could further increase the sensitivity of the detection down to 0.5 ng/ml of ChIFN-g. Furthermore, we demonstrated that this ELISA detects only biologically active forms of ChIFN-g, thereby allowing a direct quantification of functional cytokine.
This ELISA opens a broad range of applications in the field of IBDV:
1) First sets of experiments could demonstrate that the sensibility of our capture ELISA test is adequate to measure the in vitro release of ChIFN-g by T cells in response to specific recall antigen or mitogen activation.
2) Although the immunosuppression caused by IBDV is principally directed towards B lymphocytes, an effect on cell-mediated immunity (CMI) has also been demonstrated. This can be demonstrated in vitro by using proliferation tests or by measuring cytokine (ChIFN-g) release after mitogen activation of T cells using the same ELISA.
3) Furthermore, cytokines might play an important role in the pathogenesis of IBD. Particularly, the acute phase of infection is correlated with increased levels of macrophage-derived pro-mediators. This macrophage activation could be mediated by increasing levels of ChIFN-g during infection and the ELISA test could therefore be used to measure the level of circulating ChIFN-g in the serum of chicken. We have produced and characterized recombinant ChIFN-g the baculovirus and prokaryotic (E. coli) expression systems and their activity was compared to that of naturally ChIFN-g produced by mitogen-activated splenic cells. The half life of ChIFN-g recombinants in the chicken serum after different injections (i.v, i.m) has also been investigated.
J. Mast, B.M. Goddeeris
Laboratory of Physiology and Immunology of Domestic Animals, Faculty of Agricultural
and Applied Biological Sciences, KULeuven, Kardinaal Mercierlaan 92, B-3001
Heverlee, Belgium
By immunohistochemical staining, it was demonstrated that the structural organisation of chicken spleen is not completely mature before the end of the first week of life. Although, subpopulations of T-cells, B-cells, macrophages and ellipsoid-associated reticular cells (EARC) could be demonstrated early in the development of chicken spleen, the typical structures of the spleen, such as the peri-arteriolar lymphoid sheath (PALS) and the ellipsoids with their surrounding ring of macrophages, were only formed around embryonic day (ED) 20. These structures and especially the B-cell compartment, i.e. the peri-ellipsoid lymphoid sheath (PELS) gradually matured during the first week post–hatch. This implies, assuming a strong relationship between structural organisation and function, that the immune function of the late embryonic and neonatal spleen may not entirely be developed.
In a series of experiments, we analysed the ontogeny of the humoral response against thymus-dependent antigens, such as bovine serum albumin (BSA) and thyroglobulin (THY). Broilers were immunised in ovo (ED16 and ED18) and post-hatch (at 1, 7, 12 and 14 days of age) and subsequent antigen-specific total immunoglobulin (Ig) and IgM and IgG responses were examined. The in ovo immunisations had no effect on the relative growth rate of the immunised chickens, while hatchability was only slightly reduced.
In all experiments, the primary Ig-responses of chicks immunised in ovo or in the first week of life were low and did not show the characteristic kinetic pattern observed in chicks immunised later (7 to 14 days). When antigen-specific Ig-responses were followed up to one month of age, peak IgG responses were always observed in the fourth week of live regardless whether the chickens were immunised at 1, 4, 7 or 14 days of age, and this with BSA as well as with THY. This suggests that the Ig-responses of chickens immunised at early age were delayed until the immune system had matured enough to respond. Moreover, the IgG response of chicks immunised at one day of age tended to be lower and was not increased by a booster immunisation on 29 days of age in contrast to the response of chicks immunised at 7 and 14 days of age. This would rather suggest tolerance induction.
In conclusion, our results indicate that vaccination at one day of age does not effectively activate the B-cell response resulting in antibody production and support the idea that the immune function of the late embryonic and neonatal chickens is not entirely developed due to the incomplete structural organisation of their secondary immune organs.
T. Farkas1, A. Tanaka2, K. Kai2
and M. Kanoe2
1Veterinary Medical Research Institute of the Hungarian Academy of
Sciences, H-1581 Budapest, P.O. Box 18., Hungary,
2Department of Veterinary Microbiology, Faculty of Agriculture, Yamaguchi
University, Yamaguchi-city 753, Japan
The complete genomic sequence of chicken anaemia virus (CAV) TK-5803 strain was determined. Compositions of sequence data showed 37 nucleotide differences between TK-5803 and Cux-1, 38 nucleotide differences between TK-5803 and 26P4, and 48 nucleotide differences between TK-5803 and 82-2. There were 65 nucleotide differences in the largest open reading frame (ORF3) between TK-5803 and one Australian isolate. Base changes introduced amino acid changes at 6 position in the C-terminal half of the VP3, at 4 positions in the C-terminal quarter of VP2 and at 17 positions in VP1 among strains. These indicate that the N-terminal half of VP3 and the N-terminal three quarter of VP2 are well conserved, and might sustain essential function of these proteins. The amino acid changes in VP1, which thought to be the capsid protein may influence the antigenic character of different VP1s.