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  • C12N2750/10034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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  • C12N2750/10061—Methods of inactivation or attenuation
  • C12N2750/10062—Methods of inactivation or attenuation by genetic engineering

Definitions

• the present invention provides the formation and characterization of the neutralizing conformational epitope of the CAV protein VPl, which is required for eliciting a protective immune response in vaccinated animals.
• Various vaccine vectors against CAV infections are disclosed. All these vaccines are less pathogenic than CAV. All these vaccine vector are shown to produce the required neutralizing conformational epitope of CAV.
• One vector provides a subunit vaccine, based on a recombinant baculovirus expressing both VPl and VP2 in the same cell.
• a second type of vaccine is a live virus vector, comprising a Marek's disease vector stably harboring the coding sequences for VPl and VP2.
• the third CAV vaccine comprises genetically attenuated CAV strains with a reduced cytopathogenic effect.
• Chicken anemia virus is a small virus of a unique type with a particle diameter of 23 to 25 nm and a genome consisting of a circular single-stranded (minus-strand) DNA (Gelder blom et al., 1989, Noteborn et al., 1991 and Todd et al., 1990). This DNA multiplies in infected cells via a circular double-stranded replicative intermediate, which was recently cloned and fully sequenced. The CAV genome is 2319 nucleotides long (Noteborn and De Boer, 1990). DNA analysis of CAV strains isolated in different continents revealed only minor differences among the various isolates (Meehan et al.
• CAV has been placed into the novel virus family of Circoviridae.
• CAV is not related to the other members of this virus family consisting of animal single-stranded circular-DNA viruses, such as porcine circovirus and psittacine beak-and-feather-disease virus (Noteborn and Koch, 1995) .
• the CAV genome contains one evident promoter/enhancer region (Noteborn et al.
• the major transcript from the CAV genome is an unspliced polycistronic mRNA of about 2100 nucleotides encoding three proteins of 51.6 kDa (VPl), 24.0 kDa (VP2) and 13.3 kDa (VP3 or apoptin (Noteborn and Koch, 1995, Noteborn et al., 1992 and Phenix et al., 1994). All three predicted CAV proteins are synthesi zed in CAV-infected cells (Noteborn and Koch, 1995).
• CAV causes clinical and subclinical disease in chickens, and is recognized as an important avian pathogen worldwide (Mcllroy et al. , 1992 and McNulty et al . , 1991).
• Day-old chicks are especially susceptible to CAV infections.
• anorexia and anemia are observed from 10 days after inoculation with CAV.
• After infection mortality may increase to a maximum of 50%.
• the resistance also increases (Jeurissen et al. , 1992).
• the hematocrit values of chicks that had been infected with CAV at an age of 1-3 days are decreased.
• CAV infections of 1-21 days old chicks result in a depletion of in particular the thymus cortex.
• CAV infection in older chickens can be determined by the occurence of seroconversion.
• the depletion of the cortical thymocytes is considered to cause immunodeficiency resulting in enhanced concurrent infections and to vaccination failures
• CAV-neutralizing antibodies were detected in yolk of eggs produced by hens that had been inoculated with lysates of cells that had been co-infected with CAV-recombinant baculovirus and produced all three CAV proteins, or mainly VPl and VP2. Specific clinical signs did not develop in CAV-challenged progeny that hatched from these eggs (Koch et al. , 1995) .
• the recombinant CAV proteins synthesized by means of the baculovirus expression system can be used as a subunit vaccine.
• the recombinant CAV proteins VPl and VP2 have been proven to protect chicks by maternal immunity (Koch et al, 1995). Since the baculovirus vector is an. insect-specific virus, known to be non-pathogenic for vertebrates, it can be cultured and supplied to chicken without undue risks (Vlak and Keus, 1990) . In general, live-virus vaccines induce a better immune response and are less expensive than sub-unit vaccines.
• CAV or other recombinant-virus vectors such as avian herpes viruses (Nakamura et al. , 1992, Morgan et al., 1993) or fowlpox virus (Nazerian et al. , 1992, Boyle and Heine, 1993).
• avian herpes viruses Nema et al. , 1992, Morgan et al., 1993
• fowlpox virus Newcastle et al. , 1992, Boyle and Heine, 1993.
• These recombinant viruses should express the CAV protein VPl and VP2, in addition to their own proteins, and may be applicable as vaccine for both layer breeders and broiler breeders to protect their offspring against infectious anaemia.
• such vectors may be used to protect maternally immune broilers against subclinical disease.
• the present invention relates to production and analyses of the neutralizing conformational epitope structure of chicken anemia virus (CAV) .
• CAV chicken anemia virus
• neutralizing monoclonal antibodies directed against CAV is disclosed. It is shown that these neutralizing antibodies are directed against a conformational epitope of CAV protein
• VP2 For the formation of the neutralizing conformational epitope of VPl, the synthesis of VP2 within the same cell is required. Recombinant baculovirus expressing only VPl in insect cells only does not react with neutralizing antibodies directed against CAV, but these neutralizing antibodies will react when VPl and VP2 are synthesized in one cell. In purified CAV capsids, however, only VPl is present. This invention discloses that during the synthesis of VPl, and most likely during its complex formation resulting in the CAV capsids, VP2 binds temporarily to VPl. Denaturation of the CAV capsids results in the destruction of the neutralizing epitope, indicating that the neutralizing epitope is an conformational one.
• the invention relates to vaccines and compositions for preventing or treating virus infections in poultry, in particular infections with CAV.
• the invention relates to vaccines that are less pathogenic than the CAV itself but are still capable of producing the neutralizing conformational epitope. Vaccination of chickens with these type of vaccines will lead to the generation of neutralizing antibodies and thus protect the animals and their progeny against CAV infections.
• the invention relates to a baculovirus vector which contains separately on its genome the coding sequences for VPl or VP2. This recombinant baculovirus is able to synthesize VPl and VP2 in the same cell, resulting in the formation of the neutralizing conformational epitope of CAV.
• the invention also relates to the construction of a Marek's " disease virus (MDV) vectors containing CAV sequences encoding VPl, VP2 proteins.
• MDV disease virus
• the invention describes the formation of various attenuated CAV strains, which reveals a reduced cytopathogenic effect in chicken T cells in comparison to a a wild-type-derived CAV strain.
• the attenuated CAV strains were made by introducing point mutations in a cloned CAV DNA genome, in particular a sequence within the promoter/enhancer region has been mutated.
• the attenuated CAV mutant strains are able to produce the neutralizing conformational epitope.
• Processes for the prophylaxis or control of CAV infections, in particular in chickens, and processes for the preparation of recombinant parts of CAV comprising sequences, and processes for the preparation of vaccine are also subjects of the invention.
• reiterating the invention provides a neutralizing antibody or an antibody fragment or derivative thereof reacting with a conformational epitope of viral protein 1 (VPl) of chicken anaemia virus (CAV) recognized by a monoclonal antibody designated as 132-1, 132-2 or 132-3, as produced by the hybridoma's deposited under no. ' s xxxxxx, yyyyyy, zzzzzz at the Insitut Pasteur, Paris, France.
• a workable experiment for determining whether an antibody is an antibody according to the invention is to see if it cross-reacts (or competes) with an antibody from the deposited hybridomas.
• the invention also provides a conformational neutralizing epitope of viral protein 1 of chicken anaemia virus recognized by an antibody as disclosed above.
• the epitope may be part of a larger molecule, it may for instance be bound to a carrier or the epitope may be repeated (at intervals) in a polypeptide chain.
• the epitope is part of a larger part of VPl, because such a molecule will have the right conformation more easily.
• the conformational epitope will only be present on VPl if it is produced in one cell, preferably from one vector together with VP2. Therefore the.
• invention provides a method for producing a viral protein 1 comprising a conformational epitope as disclosed above, comprising the expression of said viral protein 1 in one cell together with viral protein 2 of said CAV whereby the genetic information encoding VPl and the genetic information encoding VP2 are separately present on one recombinant vector.
• the invention also includes vectors for use in a method just described comprising as two separate coding sequences the genetic information encoding VPl and the genetic information encoding VP2.
• a vector is based on Marek' s disease virus vector so that a vaccine can be produced giving protection against two pathogens.
• a very safe and efficient expression system for the viral proteins according to the invention are vectors which are based on a baculo virus vector. As explained before Baculovirus is generally regarded as safe for use in vertebrates since it cannot infect them.
• the invention also provides another way of arriving at vaccines or vaccine components which do have the very important neutralizing comformational epitope, but have reduced pathogenicity.
• a viral protein 1 comprising a neutralizing conformational epitope according to the invention, comprising expressing at least a functional part of VPl and VP2 from genes encoding them, at least one of which genes is under control of a regulatory sequence derived from the CAV sequence upstream of the transcription intitiation site which regulatory sequence is modified to reduce its efficiency.
• a regulatory sequence derived from the CAV sequence upstream of the transcription intitiation site which regulatory sequence is modified to reduce its efficiency.
• virus particles are also part of the invention.
• the modification is preferably in the promoter/enhancer region and most preferably the modification is in the 12 base pair insert in the promoter enhancer region.
• Recombinant virus particles obtainable by any method according to the invention are also part of the invention, as are nucleic acids for use in any method according to the invention, for instance vectors comprising a gene encoding at least a functional part of VPl and a gene encoding a functional part of VP2, at least one of the genes being inder control of a regulatory sequence which is modified to reduce its efficiency.
• the antibodies and epitopes according to the invention are also useful in a diagnostic test kit for detecting or determining the presence of CAV or antibodies to CAV in a sample
• vaccines for the treatment or prophylaxis of CAV associated disease comprising an antibody or an epitope according to the invention together with a suitable adjuvans and/or a suitable vehicle for administration are also provided herewith as are vaccines for the treatment or prophylaxis of CAV associated disease comprising recombinant virus particles according to the invention together with a suitable adjuvans and/or a suitable vehicle for administration.
• mice were injected with purified CAV particles.
• the supernatant of a 1-liter culture of CAV-infected MDCC-MSBl cells was fourty times concentrated by means of a MILLITAN 300-kDa filter (Millipore, USA) .
• the supernatant was dialyzed against lOmM Tris(ph 8.7)-lmM EDTA (TE) buffer.
• TE lOmM Tris(ph 8.7)-lmM EDTA
• SDS sodium dodecyl sulphate
• the CAV capsids were pelleted on a 30% sucrose cushion.
• the pellet containing the CAV capsids was resuspended in 1 ml TE buffer. Mice were twice injected with 100 ⁇ ul CAV-capsid suspension.
• the supernatants of the candidate monoclonal antibodies were diluted 1:2 and then a two-fold dilution series was made.
• the diluted supernatants were incubated for 1 hour with 104-105 TCID50 CAV-Cux-1 (Von Bulow et al., 1983, Von Biilow, 1985).
• TCID50 CAV-Cux-1 Von Bulow et al., 1983, Von Biilow, 1985.
• Approximately one hundred thousand cells of the T cell line MDCC-MSBl transformed by Marek's disease virus were infected with this mixture of diluted supernatant and virus (Yuasa, 1983, Yuasa et al. 1983) .
• Microtiter wells (Greiner, FRG) were coated with the CAV-specific neutralizing monoclonal antibody 132.1, which was 1:10,000 diluted in 50 mM sodiumbicarbonate pH 9.6. Wash the wells three times with tapped water containing 0.05% Tween 80. Saturate the wells with 100 ⁇ ul phosphate-buffered saline containing 4% horse serum, 51 gram/liter NaCl, and 0.05% Tween 80 (saturation buffer) for 30 minutes at 37 * C. Wash the wells three times with tap water containing 0.05% Tween 80.
• the recombinant baculovirus AcRP23-lacZ (Bishop, 1992) was obtained from Dr. R. Possee, NERC Institute of Virology, Oxford, England, and the genomic DNA was purified as described by Summers and Smith (1987).
• Spodoptera frugiperda (Sf9) cell were obtained from the American Tissue Culture Collection (no. CRL 1711) .
• Baculovirus stocks were grown in confluent monolayers and suspension cultures in TC-100 medium (GIBCO/BRL) containing 10% fetal calf serum, as described by Summers and Smith (1987) .
• DNA transformations were carried out in the E.coli strain HB101. All plasmid were multiplied in large culture under agitation, purified on CsCl gradients, and then by filtration over Sephacryl-S500 columns or by filtrations on QIAGEN-chromatography columns.
• Recombinant transfer vector pAcVPl/VP2 DNA was transfected with linearized recombinant baculovirus AcRP23-lacZ DNA, in Sf9 cells. After homologous recombinantion baculoviruses were obtained, which had incorporated in the polyhedrin unit instead of the lacZ the two CAV proteins VPl and VP2 under regulation of the promoter of the plO or polyhedrin gene, respectively.
• the recombinant CAV viruses were characterized for the absence of beta-galactosidase activity in plaques of baculovirus-infected insect cells. Further the integration of CAV DNA sequences in the baculovirus genome was determined by means of a CAV-specific DNA probe in a hybridization experiment.
• I ⁇ rnunop ecipitfltiQn assay Two days after infection, the cells were incubated with Promix label (ICN, USA) and four hours later, the cells were lysed in ElA buffer (50 mM Tris (pH7.5), 0.1% Triton-X-100, 250 mM NaCl, 50 mM NaF, and 5 mM EDTA) and incubated with monoclonal antibody 111.1 directed against VP2 for 2 hours at 4'C, washed with ElA buffer and separated on a PAA-SDS gel.
• ElA buffer 50 mM Tris (pH7.5), 0.1% Triton-X-100, 250 mM NaCl, 50 mM NaF, and 5 mM EDTA
• a CAV-specific serum neutralization test showed that three of these monoclonal antibodies obtained, had a neutralizing activity against CAV. These three CAV-specific neutralizing monoclonal antibodies were called 132.1; 132.2 and 132.3. (deposited under no's xxxx,yyyy, zzzz at the Institut Pasteur, France.
• Electron-microscopic analysis was carried out with purified CAV particles incubated with neutralising antibodies against CAV (132.1) or with monoclonal antibodies 111.1 (against VP2) or 111.3 (against VP3).
• Todd et al. (1990) have reported that purified CAV capsids contain onlya 50-kDa protein, which is most likely VPl.
• the various monoclonal antibodies were detected by immunogold labeling. Only the neutralizing monoclonal antibodies 132.1 were found to bind to CAV particles. Binding of the monoclonal antibody 132.1 to a CAV particle resulted in its lysis.
• CAV capsids which were lysed due to incubation with the neutralising monoclonal antibody 132.1, showed no binding with monoclonal antibo dies directed against VP2 or VP3.
• These results reveal the mechanism by which the neutralizing monoclonal antibodies act: They cause the lysis of the virus capsids, by doing so causing non-infectious particles.
• these data show that purified CAV particles contain (almost) only VPl.
• Neutralizing monoclonal antibodies are directed a ⁇ ainst an conformational epitope on VPl.
• CTB complex-trapping-blocking
• Serum from CAV-infected chickens contains antibodies which will block all epitopes on the CAV " capsids substituteor recombinant VP1/VP2. This means that the CAV capsid or recombinant VP1/VP2 will not bind to the coated monoclonal antibody 132.1. Negative serum, howe ver, will allow binding of CAV capsids or recombinant VP1/VP2 to the coated 132.1. A signal smaller than 0.5 of the signal detected with a negative control serum will be examined as positive.
• the detection level of our CTB-ELISA are titers of 24 to 25 a determined in a serum neutralization test, which is very sensitive. More than 400 sera were analyzed. Comparison to the serum neutralization test revealed that 96.5% of the positive sera within the serum neutrali zation test were positive within the CTB-ELISA, and 98.3% of the negative sera within the serum neutralization test were negative within the CTB-ELISA.
• the coding sequences for the CAV proteins VPl and VP2 were cloned into the baculovirus transfer vector pAcUW51 (cat. no: 21205P) , which was commercially obtained from PharMingen, San Diego, USA.
• This vector is shown in Figure 3 and contains the polyhe drin flanking region, within their midst the baculovirus polyhdrin promoter and the plO promo ter and for both transcription units, the required 3 ' -non-coding transcrioptional sequences including the polyadenylation signals.
• the transfer vector contains prokaryotic sequences for multiplication in bacteria.
• the plasmid pET-16b-VP2 (Noteborn et al.
• This CAV DNA fragment contains the coding region for VP2 flanked by 484 bp 3 ' -non-coding CAV DNA sequences. 106 bp downstream of the start codon for VP2 the start codon for VP3 is found in another reading frame.
• the plasmid pET-16b-VP2 was treated with the restriction enzymes Ndel and Nhel, and the sticky ends were filled by means of Klenow polymerase. A 0.8 kb CAV DNA fragment was isolated.
• the plasmid pAcUW51 was linearized with BamHI, the sticky ends filled by means of Klenow polymerase and finally treated with alkaline phosphatase (CIP) .
• CIP alkaline phosphatase
• the 0.8 kB CAV DNA fragment was ligated at the linearized pAcUW51 DNA.
• the orientation of VP2 in pAcUW51 was determined by restriction enzyme analysis . This construct was called pUW-VP2.
• the plasmid pET-16b-VPl (Noteborn et al., data not published) contains CAV DNA sequences of positions 853 to 2319.
• the CAV DNA insertion contains the complete coding region for the protein VPl flanked by 117 bp 3 ' -non-coding CAV DNA sequences.
• the plasmid pET-16b-VPl was treated with the restriction enzymes Ndel and EcoRI, and the sticky ends were filled by means of Klenow polymerase. A 1.45 kb CAV DNA fragment was isolated.
• the plasmid pUW-VP2 was linearized by EcoRI, the sticky ends filled by means of Klenow polymerase and finally treated by CIP.
• the 1.45 kb CAV DNA fragment was ligated at the linearized pUW-VP2.
• the orientation of VPl opposite of the plO promoter unit was determined by restriction-enzyme analysis, and the final construct pAcVPl/VP2 is shown in Figure 3.
• the open reading frames encoding VPl and VP2 were separately cloned into a single baculovirus transfervector, viz pAcUW51.
• the CAV sequences encoding VPl are under the regulation of the baculovirus plO promoter and VP2 under the regulation of the baculovirus poyhedrin promoter.
• Transfection of Sf9 insect cells occurred with 'naked' baculovirus DNA and transfervector DNA.
• homologous recombinantion baculovirues were obtained which had incorporated the VP1/VP2 expression unit, integrated in the polyhedrin region of the recombinant baculovirus, instead of the lacZ unit.
• the recombinant baculoviruses were characterized for the absence of beta-galactosidase activity and integration of CAV DNA sequences.
• CAV-specific products could be detected as radioactively labeled protein band or Coomassie-brilliant blue stained protein band. The latter result indicates that both products are produced in relatively high levels in recombi nant-VPl/VP2-baculovirus-infected insect cells. Sf9 cells infected with a recombinant-lacZ baculovirus did not contain these CAV-specific proteins.
• VP2 is a non-structural protein that at some stage of infection is required for virus assembly and/or the correct conformation of VPl, which result(s) in the formation of the neutralizing epitope(s).
• VP2 acts as a scaffold protein that is necessary during the assembly of the virion but absent in the final product (Leibowitz and Horwitz, 1975) .
• scaffold proteins are the IVa2 and 39kDa proteins of adenovirus (D'Halluin et al. , 1978; Persson et al., 1979). These proteins act as scaffolds for the formation of the so-called light capsid, but are removed in the next step.
• VP2 might function in a similar way during the formation of CAV virions. However, at this stage, we cannot entirely exclude that (very) small amounts of VP2 that remained undetected in electroblots of purified CAV preparations (Todd et al.
• VPl and VP2 interact temporarily with each other.
• the neutralizing epitope of VPl is only formed when VP2 is present, and therefore is a conformationally distinct epitope. This implies that VPl and VP2 associate with each other during a short time period.
• Sf9 insect cells were infected with recombinant baculoviruses, which synthesized VPl, VP2, or VPl plus VP2.
• the recombinant CAV products which will be used for vaccination of laying-hens, can be synthesized by means of the baculovirus system.
• the CAV proteins can also be. synthesized by means of other expression systems, such as yeast cells, via (retro)- viral infection or gene amplification (CHO-dhfr system) in mammalian cell systems.
• the expression of fragments of VPl in combination with VP2 or VP2 and VP3 may be sufficient for the induction of a protective immune response.
• VPl, VP2 or VP3, separately, but then within the context of a living virus vector, is also suitable for the induction of a protective immune response against CAV infections.
• the expression of fragments of one of the abo ve-mentioned CAV proteins by living virus vectors may be sufficient for the induction of a protective immune response.
• VP3 causes apoptosis in, i.e. chicken mononuclear cells (PCT/NL94/00168; Noteborn et al. , 1994a) favors to construct living virus vectors not expres sing VP3.
• the replication of i.e. Marek_s disease virus might be negatively influenced by VP3-induced apoptosis.
• recombinant-VPl/VP2 MDV Construction of recombinant-VPl/VP2 MDV.
• recombinant transfer vector pMD-US10-SV-VPl/VP2 DNA was transfected with MDV (Rispens isolate) DNA, in CEF cells.
• MDV Rospens isolate
• recombinant MDVs were obtained, which had incor porated the CAV coding sequences in the the US10 region (Nakamura et al. , 1992).
• the recombinant-VPl/VP2 MDVs were characterized for the presence of the expression of VP2.
• in-parallel plaque purification and immuno-peroxidase analyis using a monoclonal antibody directed against VP2 several 100%-purified recombinant-VPl/VP2 MDV independent strains were obtained.
• the monoclonal antibody 132.1 is directed against a neutralizing epitope of VPl.
• the CAV-EcoRI clone is used for the production of viable infectious CAV particles.
• the complete CAV insert the 2,319-bp EcoRI fragment has to be isolated from the bacterial sequences and recircularized by DNA polymerase treatment.
• MDCC-MSBl cells are transfected with the recircularized EcoRI fragments and after several days wild-type CAV will be produced.
• wild-type CAV in principle one can produce mutant CAV and if the mutant is less pathogenic, attenuated CAV.
• the complete strategy for the production of attenuated CAV is shown in Figure 5.
• CAV genomes containin ⁇ mutated promo er/enh ncer regions
• a remarkable feature of the CAV promoter/enhancer region is a sequence of 4 or 5 near-perfect 21-bp repeats, interrupted by an insert of 12-bp (Noteborn et al. , 1991). This region is involved in the regulation of the CAV transcription (Noteborn et al., 1994b).
• For the production of attenuated CAV we have introduced mutational changes in the 12-bp insert/direct-repeat region of the CAV-EcoRI clone (Noteborn et al. , 1991).
• the mutated promoter/enhancer region of the wild-type (wt) and the various CAV mutants are shown in Fig. 6.
• the Nae-mutant contains no
• All other mutants contain 4 original direct repeats instead of 5, as is the case for the CAV-EcoRI clone.
• the various CAV mutants called '6b'p, '12bp', '24bp' contain changed 12-bp inserts.
• the CAV mutants * 6bp' , '12bp' , '18bp', '24bp' and 'wt in Nae' contain all additional sequences flanking the 12-bp insert/direct-repeat region.
• the linker 5' -GCCCATGT-3 ' and at the 3 '-site the linker 5 ' -GATCCGCC-3 ' has been introduced.
• VP3 To determine whether the mutated CAV genomes could produce live CAV particles, firstly the synthesis of VP3 was examined. At several timepoints after transfection, a part of the various transfected MDCC-MSBl-cell cultures were aceton-fixed and analysed by indirect immunofluorescence using a monoclonal antibody directed against VP3. In parallel, 1 ml of each culture was added to 9 ml of fresh RPMI-medium, which was supplemented with 10% fetal bovine serum. Approximately 15% and 90% of the cultured MDCC-MSBl cells, transfected with 'wt' recircularized CAV genome DNA, contained VP3 after 6 days and 11 days (the cells were one time passaged after transfection), respectively.
• the CAV mutant genome 'Nae' lacking the complete 12-bp insert/direct-repeat region, was shown not to produce infectious virus particles.
• a similar percentage is obtained with expression plasmid pRSV-VP3 (Noteborn et al . , 1994b) encoding VP3 only, and which are known not to replicate in eukaryotic cells. After 3 weeks and several passages, no VP3 was present anymore in the Nae-DNA transfected MDCC-MSBl cell cultures.
• CAV mutants have a reduced cytopat oqenic effect.
• the transfected cells were analysed whether they had become apoptotic.
• CAV is known to induce apoptosis, 2-3 days after infection.
• the cells were stained with propidium iodide, which stains 'normal' DNA strongly, but apoptotic DNA weakly and/or irregularly.
• PMC-MSBl cells transfected with 'wt' DNA were shown to be apoptotic, whereas the majority of the cells, transfected with the various mutated CAV-DNA genome were not.
• the capacity for inducing apoptosis was shown to be mostly reduced for the CAV mutants ' 6bp' and '18bp' .
• Attenuated CAV synthesizes the CAV-specific neutralizing epitope-
• Figure 1 shows the pepscan analysis of the neutralizing monoclonal antibodies of type 132.1 with peptides (12-mers) derived from VPl.
• Figure 2 shows the pepscan analysis of the neutralizing monoclonal antibodies of type 132.1 with peptides (12-mers) derived from VP2.
• Figure 3 shows the diagrammatic representation of the recombinant transfer pUW-VPl/VP2.
• Figure 4 shows the diagrammatic representation of the recombinant transfer vector pMD-US10-SV-VPl/VP2..
• Figure 5 shows the strategy for the production of attenuated CAV-mutants, based on the plasmid pCAV/EcoRl containing 2319 bp of wild-type CAV.
• Figure 6 shows the schematic structure of the CAV promoter/enhancer region of the various mutated and wild-type CAV strains .
• Figure 7 shows the VP3-expression rates obtained for the various mutant-CAVs and wild-type CAV after tranfection of mutant or wild-type circularized DNA.
• the VP3-expression rate are given as percentage of VP3-positive MDCC-MSBl cells.
• FIG. 8 gives the sequence of CAV.
• CAV chicken anemia virus
• ORF-3 gene and the development of an ELIA for the detection of serum antibody to CAV.
• Anaemia-dermatitis of broilers field observations on its occurrence, transmission and prevention. Avian Pathology 17, 113-120.

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