EP1483287A1 - Nouvelle variante du virus de la bursite infectieuse - Google Patents

Nouvelle variante du virus de la bursite infectieuse

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Publication number
EP1483287A1
EP1483287A1 EP03743327A EP03743327A EP1483287A1 EP 1483287 A1 EP1483287 A1 EP 1483287A1 EP 03743327 A EP03743327 A EP 03743327A EP 03743327 A EP03743327 A EP 03743327A EP 1483287 A1 EP1483287 A1 EP 1483287A1
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Prior art keywords
ibdv
variant
vaccine
virus
chickens
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Adriaan Antonius Wilhelmus Maria Van Loon
Paul Guntram
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Intervet International BV
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Akzo Nobel NV
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5252—Virus inactivated (killed)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
    • C12N2720/00011—Details
    • C12N2720/10011—Birnaviridae
    • C12N2720/10021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
    • C12N2720/00011—Details
    • C12N2720/10011—Birnaviridae
    • C12N2720/10022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
    • C12N2720/00011—Details
    • C12N2720/10011—Birnaviridae
    • C12N2720/10034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention is concerned with a variant infectious bursal disease virus (IBDV) and a vaccine comprising such a variant IBDV.
  • IBDV infectious bursal disease virus
  • IBDV Infectious bursal disease virus
  • Birnaviridae family Viruses in this family have a very similar genomic organisation and a similar replication cycle. The genomes of these viruses- consist of 2 segments (A and B) of double-stranded (ds) RNA.
  • segment A encodes a polyprotein, which is cleaved by autoproteolysis to form mature viral proteins VP2, VP3 and VP4.
  • VP2 and VP3 are the major structural proteins of the virion.
  • VP2 is the major host-protective immunogen of birnaviruses, and contains the immunogenic regions responsible for the induction of neutralising antibodies.
  • IBDV For IBDV, two serotypes exist, serotype 1 and 2. The two serotypes can be differentiated by virus neutralisation (VN) tests. Serotype 1 viruses have been shown to be pathogenic to chickens, while serotype 2 IBDV only causes sub-acute disease in turkeys.
  • IBD Infectious Bursal disease
  • Gumboro disease is an acute, highly-contagious viral infection in chickens that has lymphoid tissue as its primary target with a selective tropism for cells of the bursa of Fabricius. The morbidity rate in susceptible flocks is high, with rapid weight loss and moderate to high mortality rates.
  • Chicks that recover from the disease may have immune deficiencies because of the destruction of the bursa of Fabricius, which is essential to the defence mechanism of the chicken.
  • the IBD-virus causes severe immunosuppression in chickens younger than 3 weeks of age and induces bursal lesions in chicks up to 3 months old.
  • the disease could be prevented by inducing high levels of antibodies in breeder flocks by the application of an inactivated vaccine, to chickens that had been primed with attenuated live IBDV vaccine. This has kept economic losses caused by IBD to a minimum. Maternal antibodies in chickens derived from vaccinated breeders prevent early infection with IBDV and diminish problems associated with immunosuppression. In addition, attenuated live vaccines have also been used successfully in commercial chicken flocks after maternal antibodies had declined.
  • IBD viruses Historically, IBD viruses consisted of only one type that is known as "classic" IBD virus. However, in the mid-1980s acute disease in flocks vaccinated with vaccines based on classic IBDV was observed, in particular in the US. It was found that this disease was caused by IBD viruses that had a different immunogenic make-up. These new viruses probably emerged as a result of an antigenic shift. The emergence of these so-called "variant" IBDV strains required the design of new IBD vaccination programmes, because the classic IBDV vaccine strains 03 01905
  • serotype 1 IBDVs identified in the past were the Delaware-E, GLS, RS/593 and DS326 variants.
  • a panel of virus neutralising monoclonal antibodies (Moab) is presently used in the art in an antigen-capture enzyme immuno assay (AC-ELISA) to identify the various IBDV types.
  • AC-ELISA antigen-capture enzyme immuno assay
  • Table 1 The reactivity pattern of these Moabs with the existing IBDV strains is summarised in Table 1 below.
  • the molecular basis of the antigenic variation between IBDV strains has been elucidated by Vakharia et al. (Virus Research 31, 265-273, 1994).
  • the region in the VP2 protein that displays most of the amino acid differences between IBDV strains and the putative amino acids involved in the formation of virus neutralising epitopes were identified.
  • the different variant IBDV strains as determined by the Moab panel pattern VN Moabs R63 and B69 neutralize classic IBDV strains to high titres and Moab B69 specifically binds to classic strains.
  • Moab BK9 uniquely binds to Delaware variant-E strains.
  • a positive reaction by Moab 57 can be used to separate the GLS- and DS326 strains from classic- and Delaware variant strains.
  • These and other Moabs are generally used in the field to distinguish between IBDV (variant) strains by determining the reaction pattern of the panel of available Moabs.
  • hybridomas secreting the Moabs are also available from the ATCC (Rockville, USA) under the following accession numbers: R63 (HB-9490), 8 (HB-10174), B29 (HB-9746), BK-9 (HB-10157), 67 (HB-11122), 57 (HB-10156), B69 (HB-9437) and 179 (HB- 10158).
  • Variant IBDV strains and hybridomas are also available from the Collection Nationale de Cultures de Microorganismes of the Institute Pasteur, Paris, France under the following accession no.'s: DS 326 (1-910), GLS (I-792 and I-793) and Moab 10 (1-2812).
  • a new variant IBDV has been identified that was isolated from an outbreak of the disease that was not fully prevented by the administration of a classic vaccine.
  • the new variant IBDV displaying an altered reaction pattern with the existing VN
  • Moabs disclosed for the first time herein, provides a basis for the preparation of new vaccines and diagnostic methods to prevent and monitor the disease caused by this new IBDV variant.
  • the present invention provides a variant infectious bursal disease virus
  • IBDV immunoglobulin deficiency virus
  • IBDV characterised in that the virus binds with monoclonal antibody 10 and 67, secreted by hybridoma cell lines 1-2812 and HB-11122, deposited at the Collection Nationale de Cultures de Microorganismes of the Institute Pasteur, Paris, France and the ATCC, Rockville, USA, respectively.
  • IBDV variant subtypes are well defined in the art, e.g. by means of their reaction pattern with monoclonal antibodies.
  • the present variant IBDV lacks the 57 and BK9 epitopes that are characteristic for GLS- and variant-E strains, respectively.
  • an IBDV according to the invention is a virus that comprises both the 10 and 67 epitopes. Moab 10 usually reacts with classical- and GLS-Iike strains, whereas Moab 67 only reacts with variant-E like strains.
  • the reaction of a Moab with the variant IBDV is determined by means of an AC-ELISA that is commonly used in the art for this purpose, such as described by Snyder et al. (1992, supra) or US patent no. 5,518,724.
  • the AC-ELISA as described by van der Marel et al. (Dtsc . Tierartzl. Wschr. 97, 81-83, 1990) is used.
  • This test basically comprises three steps. In a first step the antigenic mass of the IBDV antigen under investigation is examined by a Moab-ELISA using a Moab that reacts with IBDV, e.g. Moab B29.
  • bursal homogenates of chickens infected with the IBDV are tested in an ELISA for their reactivity with a (relevant) panel of Moabs under standardized conditions.
  • absorption ratios between the antigenic mass-Moab and the other Moabs are calculated.
  • the present invention provides a variant IBDV as defined above that comprises codons for amino acids Ser (position 222), His (position 249), Ala (position 256) and Ser (position 278) in the VP2 coding region of segment A of the IBDV genome.
  • the numbers indicating the amino acid- and nucleotide positions herein are based on the complete sequence of segment A of the IBDV genome as described by Mundt and M ⁇ ller (J. Gen. Virol. 77, 437-442, 1995; NCBI accession no. X84034).
  • the variant IBDV according to the invention comprises a VP2 coding region that encodes a VP2 protein having an amino acid sequence of the variable region variable region (aa 212-332) as shown in SEQ ID no. 1, more in particular, a VP2 coding region that encodes a VP2 protein having an amino acid sequence as shown in SEQ ID no. 1.
  • a most preferred virus according to the invention is IBDV isolate GB02, a sample of which is deposited at the Institute Pasteur, Paris, France under accession no. 1-2811.
  • Isolate GB02 PP represents plaque purified GB02 IBDV and is deposited at the Institute Pasteur,
  • a variant IBDV according to the invention can be obtained from the Depository Institute or can be isolated from the field and identified by the above-mentioned Moabs as described in Example 1.
  • the present invention provides an isolated polynucleotide comprising a nucleic acid sequence encoding a VP2 protein of a variant IBDV according to the present invention as defined above.
  • the polynucleotide comprises a coding sequence of the VP2 variable region having an amino acid sequence as shown in SEQ ID no. 1.
  • the polynucleotide encodes the VP2 protein having an amino acid sequence as shown in SEQ ID no. 1.
  • the polynucleotide according to the invention may also comprise the coding sequences for the other structural proteins encoded by segment A of the IBDV genome, i.e. VP3 and VP4.
  • cDNA clones containing the entire coding region of the segment A of the new variant IBDV can be prepared according to standard cloning procedures described in the prior art for this purpose (Vakharia et al., Avian
  • the present invention also provides an isolated polypeptide comprising the amino acid sequence of the VP2 protein of a variant IBDV according to the present invention, preferably as shown in SEQ ID no. 1.
  • This polypeptide can be obtained by recombinant DNA expression using the polynucleotide defined above in a standard expression system.
  • the VP2 protein of the new variant IBDV can be used in the form of a subunit protein or in the form of a virus-like- particle (VLP) when co-expressed with a VP3 and VP4 protein, preferably by a recombinant baculovirus, for the purpose of preparing an IBDV vaccine.
  • VLP virus-like- particle
  • a polynucleotide according to the invention may be cloned into an appropriate expression system, such as a bacterial expression system (e.g. Ecoli DH5 ⁇ ), a viral expression system (e.g. Baculovirus), a yeast system (e.g. S. Cerevisiae, Pichia) or eukaryotic cells (e.g. Cos, BHK, MDCK, MDBK, HeLa, PK15 cells).
  • an appropriate expression system such as a bacterial expression system (e.g. Ecoli DH5 ⁇ ), a viral expression system (e.g. Baculovirus), a yeast system (e.g. S. Cerevisiae, Pichia) or eukaryotic cells (e.g. Cos, BHK, MDCK, MDBK, HeLa, PK15 cells).
  • bacterial expression system e.g. Ecoli DH5 ⁇
  • a viral expression system e.g. Baculovirus
  • yeast system e.g. S
  • the present invention therefore relates to a recombinant vector comprising a polynucleotide according to the invention.
  • Suitable vectors are for example cosmids, bacterial or yeast plasmids, wide host range plasmids and vectors derived from combinations of plasmid and phage or virus DNA.
  • suitable cloning vectors are plasmid vectors such as pBR322, the various pUC, pEMBL and Bluescript plasmids.
  • a recombinant vector according to the present invention may further comprise an expression control sequence operably linked to the nucleic acid sequence coding for the protein.
  • the variant IBDV according to the invention displays an immunogenic make-up that is not observed before.
  • the new variant IBDV may form the basis of a new type of IBDV vaccine that can effectively protect poultry against disease conditions resulting from the infection by the new variant IBDV. Therefore, another aspect of this invention is a vaccine-for use in the protection of poultry against disease caused by IBDV infection, characterised in that the vaccine comprises a variant IBDV as defined above, together with a pharmaceutical acceptable carrier or diluent.
  • the variant IBDV can be incorporated into the vaccine as live attenuated or inactivated virus.
  • a vaccine according to the invention can be prepared by conventional methods such as for example commonly used for the commercially available live- and inactivated IBDV vaccines. Briefly, a susceptible substrate is inoculated with a variant IBDV according to the invention and propagated until the virus replicated to a desired infectious titre after which IBDV containing material is harvested, optionally inactivated, and mixed with a pharmaceutical acceptable carrier or diluent.
  • Every substrate which is able to support the replication of IBDVs can be used to prepare a vaccine according to the present invention, including primary (avian) cell cultures, such as chicken embryo fibroblast cells (CEF) or chicken embryo liver cells (CEL), mammalian cell lines such as the VERO cell line or the BGM-70 cell line, or avian cell lines such as QT-35, QM-7 or LMH.
  • primary (avian) cell cultures such as chicken embryo fibroblast cells (CEF) or chicken embryo liver cells (CEL)
  • mammalian cell lines such as the VERO cell line or the BGM-70 cell line
  • avian cell lines such as QT-35, QM-7 or LMH.
  • the variant IBDV can also be propagated in embryonated chicken eggs. Attenuation of the variant IBDV can be obtained by standard serial passaging of the virus in cell cultures, for example in the primary cell cultures or established cell lines mentioned above (Bayyari et al., Avian Diseases 40, 516-532, 1996; Tsai et al., Avian diseases 36, 415-422, 1992).
  • the variant IBDV can be propagated in vivo in infected chickens followed by the isolation of the bursa of Fabricius from these infected animals, mixing it with diluent and homogenizing the mixture. IBDV propagated in this way commonly forms the basis of an inactivated vaccine.
  • the vaccine according to the invention containing the live virus can be prepared and marketed in the form of a suspension or in a lyophilised form and additionally contains a pharmaceutically acceptable carrier or diluent customary used for such compositions. Carriers include stabilisers, preservatives and buffers.
  • Suitable stabilisers are, for example SPGA, carbohydrates (such as sorbitol, mannitol, starch, sucrose, dextran, glutamate or glucose), proteins (such as dried " milk serum, albumin or casein) or degradation products thereof.
  • Suitable buffers are for example alkali metal phosphates.
  • Suitable preservatives are thimerosal, merthiolate and gentamicin.
  • Diluents include water, aqueous buffer (such as buffered saline), alcohols and polyols (such as glycerol).
  • the live vaccines according to the invention may contain an adjuvant.
  • suitable compounds and compositions with adjuvant activity are the same as mentioned below.
  • the vaccine is preferably administered by an inexpensive mass application route commonly used for IBDV vaccination.
  • IBDV vaccination this route includes drinking water, spray and aerosol vaccination.
  • the present invention provides a vaccine comprising the variant IBDV in an inactivated (killed) form.
  • An advantage of an inactivated IBDV vaccine is the high levels of protective antibodies of long duration that can be obtained.
  • the aim of inactivation of the viruses harvested after the propagation step is to eliminate reproduction of the viruses. In general, this can be achieved by chemical or physical means well known in the art.
  • a vaccine containing the inactivated variant IBDV can, for example, comprise one or more of the above-mentioned pharmaceutically acceptable carriers or diluents suited for this purpose.
  • an inactivated vaccine according to the invention comprises one or more compounds with adjuvant activity.
  • Suitable compounds or compositions for this purpose include aluminium hydroxide, -phosphate or -oxide, oil-in-water or. water-in-oil emulsion based on, for example a mineral oil, such as Bayol F® or Marcol 52® or a vegetable oil such as vitamin E acetate, and saponins.
  • the vaccine according to the invention comprises an effective dosage of the variant IBDV as the active component, i.e. an amount of immunising IBDV material that will induce immunity in the vaccinated birds against challenge by a virulent virus.
  • Immunity is defined herein as the induction of- a significant higher level of protection in a population of birds after vaccination compared to an unvaccinated group.
  • the live vaccine according to the invention can be administered in a dose of
  • R 1 ⁇ Inactivated vaccines may contain the antigenic equivalent of 10 -10 TCID50 per animal.
  • Inactivated vaccines are usually administered parenterally, e.g. intramuscularly or subcutaneously.
  • the IBDV vaccine according to the present invention may be used effectively in chickens, also other poultry such as turkeys, guinea fowl and partridges may be successfully vaccinated with the vaccine.
  • Chickens include broilers, pullets, reproduction stock and laying stock.
  • the age of the animals receiving a live or inactivated vaccine according to the invention is the same as that of the animals receiving the conventional live- or inactivated IBDV vaccines.
  • broilers free of maternally derived antibodies-MDA
  • broilers with high levels of MDA are preferably vaccinated at 2-3 weeks of age.
  • Laying stock or reproduction stock with low levels of MDA may be vaccinated at 1-10 days of age followed by booster vaccinations with inactivated vaccine on 6-12 and 16-20 weeks of age.
  • the invention also includes combination vaccines comprising, in addition to the variant IBDV described above, one or more vaccine components of other pathogens infectious to poultry.
  • the combination vaccine additionally comprises one or more vaccine strains of Mareks Disease virus (MDV), infectious bronchitis virus (IBV), Newcastle disease virus (NDV), egg drop syndrome (EDS) virus, turkey rhinotracheitis virus (TRTV) or reovirus.
  • MDV Mareks Disease virus
  • IBV infectious bronchitis virus
  • NDV Newcastle disease virus
  • EDS egg drop syndrome
  • TRTV turkey rhinotracheitis virus
  • the combination vaccine additionally comprises an IBDV vaccine strain of different immunogenic make-up, such as a classic-, variant E or GLS vaccine strain.
  • the new virus was isolated from bursas of diseased animals according to the following procedure:
  • a chicken flock vaccinated with classic vaccine showed wet litter, general immunosuppression, low weight gain but no high mortality (1-2%).
  • Chickens from this flock were offered for post-mortem examination. Some muscle bleedings were seen and some small bursae. Bursae were isolated, PBS and antibiotics added. Next, the bursae were homogenized with glass pearls and sterile PBS. The homogenates were tested using the Moab panel test. IBDV material that showed a deviating panel pattern was passaged further in animals. 0.1 ml of this homogenate was applied via the in eye-drop route to 14 days old SPF white leghorns. Three-4 days after inoculation the presence of IBDV in the bursa of Fabricius was investigated using the Moab panel test.
  • CEF Primary chicken embryo fibroblasts
  • IBDV GB02 after 3 plaque purification rounds on CEF was cultured for 2 passages on VERO cells. After the second incubation on VERO cells for 10 days at 37°C the infected cells suspension was filtered through a sterile cheese cloth.
  • Antigen-capture enzyme immuno assay AC-ELISA
  • IBDV was characterised by means of an ELISA using different monoclonal antibodies according to van der Marel et al. (Dtsch. Tierartzl. Wschr. 97, 81-83, 1990): 1. Moab B29 was coated on 96 well microtiterplates. After coating, the plates were washed and the wells of the plates were filled with two- or threefold dilution series of bursal homogenate samples and a standard antigen of known antigenic mass. After incubation and washing of the plates, the plates were incubated with a hyperimmune rabbit anti-lBDV serum.
  • the plates were washed again and incubated with conjugate (goat-anti-rabbit immunoglobulin coupled to horse-radish-peroxidase). Finally, the plates were incubated with substrate solution. The enzymatic reaction was stopped with 4N H2SO4. The absorption in the wells was read at 450nm with an ELISA reader. The antigenic mass content of the bursal samples relative to the standard antigen was calculated by regression analysis. The antigenic content of the bursal samples is expressed as B29 EU/ml. 2. The subsequent procedure was similar to the one described for the B29 ELISA with the exception of: 1. The plates were coated with different Moabs, 2. The bursa homogenate samples were diluted to a concentration of 5000 B29 EU/ml. Then a single dilution was dispensed in the wells coated with the different Moabs. The subsequent steps were the same as for the B29 ELISA. 3. The absorption ratios were calculated according to the following formula
  • Obtained bursae of chicken were homogenized.
  • the homogenate was centrifuged at 13000 x g for 5 min to eliminate cellular debris.
  • One volume of chloroform were added to the obtained supernatant and vortexed. After centrifugation at 13000 x g for 5min to the resulting supernatant proteinase K and sodium dodesylsulphate was added to a final concentration of 1mg/ml and 0.5%, respectively, and incubated at 56°C for 1 hour.
  • Nucleic acids were obtained after extraction of the digested proteins using one volume phenol/chloroform. After one additional phenol extraction using one volume chloroform nucleic acids were precipitated by adding of 1/10 volume of 3M sodium acid (pH5.2) and 2.5 volume absolute ethanol.
  • RNA was reverse transcribed into cDNA, and amplified by polymerase chain reaction (PCR) following standard procedures using the methods as described by Mundt and Vakharia (PNAS 93, 11131-11136, 1996).
  • Appropriate primer pairs (BelgFP/BelgRP nucleotides 608-629 and 1201-1222) were added to dissolved nucleic acids.
  • the primer-nucleic acid mixture was added to the reaction mixture [reaction buffer (Invitrogen), DTT (Invitrogen), dNTP mixture (Promega), Superscript II (Invitrogen)], The amplification of the appropriate cDNA fragments were performed using the primer pair BelgFP/BelgRP resulting in the RT-PCR fragment BelgApart.
  • the PCR was performend by using Deep Vent polymerase (New England Biolabs) and 2 ⁇ l of the RT-reaction. Amplification product was cloned blunt ended and plasmids containing appropriate PCR fragments were sequenced.
  • the cloning procedure to obtain a plasmid containing the segment A under control of the T7- RNA polymerase promotor corresponded to the procedure described by Mundt and Vakharia (1996, supra).
  • plasmids resulting from cloning of each PCR fragment were analysed by sequencing of the DNA in both directions. Cloned PCR fragments were sequenced and obtained sequences were analysed using the Wisconsin package, Version 8 ( Genetics Computer Group, Madison, Wis., USA). The amino nucleotide sequence as well as amino acid sequence alignments were performed using ClustalW (http://www.ebi.ac.uk clustalw). Virus neutralisation (VN) assay
  • VN virus neutralisation
  • Sera were examined for the presence/absence of IBDV antibodies in a virus neutralisation test according to the procedure outlined below.
  • Table 2B Panel pattern of different IBDVs with different Moabs.
  • epitopes that specifically react with the various Moabs are located on the VP2 protein, in particular, in the so-called variable regions that generally spans amino acids 212 to 332 (see Vakharia et al., 1994, supra).
  • the compared region showed a variety of amino acid exchanges to all sequences used for the sequence comparison. This is further evidence that the newly isolated virus represents a new type of IBDV not described so far. The highest number of exchanges was detected in the VP2 variable region (aa 212-332). The comparison of the percentage homology of the nucleotide- and amino acid sequences (92.3% to 95.9%) showed that the sequences of the new virus were as much different to the subtypes of IBDV as the subtypes were different to each other.
  • the mean IBDV antibody titres determined in the sera collected 4 weeks post infection are outlined in the table below:
  • IBDV variant vaccine Variant IBDV (isolate GB02) was produced on VERO cells and subsequently inactivated with formaldehyde.
  • the inactivated IBDV GB02 antigen was emulsified in a W/O emulsion so that each dose contained 10 EU (R63 ELISA based) of IBDV GB02.
  • Chickens were vaccinated each with 0.5 ml of the IBDV GB02 vaccine via intramuscular route in the leg muscle.
  • Classical IBDV (strain D78) was produced on VERO cells and subsequently inactivated with formaldehyde.
  • the inactivated IBDV D78 antigen was emulsified in a W/O emulsion so that each dose contained 10 EU (R63 ELISA based) of IBDV D78.
  • Chickens were vaccinated each with 0.5 ml of the IBDV D78 vaccine via intramuscular route in the leg muscle.
  • IBDV GB02 was plaque, purified on CEF and subsequently passaged in SPF chickens. Chickens were challenged each with a calculated infectivity titre of 10 5'1 TCID 50 of GB02 challenge virus via the ocular route.
  • Sera were examined for the presence/absence of IBDV antibodies in a virus neutralisation test according to the procedure outlined below.
  • Results obtained with the detection of challenge virus in the bursae removed at 4 days post challenge are outlined in the table below.
  • the results obtained in this experiment indicate that the inactivated IBDV GB02 vaccine provides chickens with a solid protection against infection with the IBDV GB02 field isolate.
  • the results indicate that a vaccine based on a classical IBDV does not provide chickens with a solid protection against infection with the new variant IBDV.

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Abstract

L'invention concerne une variante du virus de la bursite infectieuse qui se distingue des (sous)types du virus de la bursite infectieuse, tels que le virus de la bursite infectieuse classique, GLS et sa variante E.
EP03743327A 2002-03-01 2003-02-25 Nouvelle variante du virus de la bursite infectieuse Withdrawn EP1483287A1 (fr)

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EP03743327A EP1483287A1 (fr) 2002-03-01 2003-02-25 Nouvelle variante du virus de la bursite infectieuse

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EP02075881 2002-03-01
EP02075881 2002-03-01
EP02079827 2002-11-21
EP02079827 2002-11-21
EP03743327A EP1483287A1 (fr) 2002-03-01 2003-02-25 Nouvelle variante du virus de la bursite infectieuse
PCT/EP2003/001905 WO2003074552A1 (fr) 2002-03-01 2003-02-25 Nouvelle variante du virus de la bursite infectieuse

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WO2005049794A2 (fr) * 2003-11-13 2005-06-02 University Of Georgia Research Foundation, Inc. Procedes de caracterisation du virus de la bursite infectieuse
US7338661B2 (en) * 2004-03-12 2008-03-04 Wyeth Infectious bursal disease virus antigenic isolates and vaccines
ES2310062B1 (es) 2005-07-15 2009-11-13 Bionostra, S.L. Particulas pseudovirales vacias quimericas derivadas del virus causante de la enfermedad de la bursitis infecciosa (ibdv), procedimiento de obtencion y aplicaciones.
CN103232974B (zh) * 2013-03-18 2014-09-03 江苏省农业科学院 分泌高中和活性传染性法氏囊病毒单克隆抗体的杂交瘤细胞
CN109320594B (zh) * 2018-11-13 2021-09-28 四川大学 一种禽传染性支气管炎和新城疫的病毒样颗粒、制备方法及应用
CN112501129B (zh) * 2020-12-14 2022-12-02 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） 抗ibdv vp2蛋白的单克隆抗体组合及其在鉴别检测ibdv中的用途
CN112500458B (zh) * 2020-12-15 2022-12-16 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） 鸡传染性法氏囊病病毒新型变异株亚单位疫苗、其制备方法及应用

Citations (1)

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WO1995026196A1 (fr) * 1994-03-29 1995-10-05 The University Of Maryland College Park CLONES D'ADNc CHIMERES DU VIRUS DE LA BURSITE INFECTIEUSE, PRODUITS D'EXPRESSION ET VACCINS A BASE DESDITS CLONES

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EP1170302B1 (fr) * 2000-07-07 2006-06-21 Intervet International BV Vaccin a base des mutants du virus de la bursite infectieuse

Patent Citations (1)

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WO1995026196A1 (fr) * 1994-03-29 1995-10-05 The University Of Maryland College Park CLONES D'ADNc CHIMERES DU VIRUS DE LA BURSITE INFECTIEUSE, PRODUITS D'EXPRESSION ET VACCINS A BASE DESDITS CLONES

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Title
BERG T P ET AL: "Acute infectious bursal disease in poultry: Immunological and molecular basis of antigenicity of a highly virulent strain.", AVIAN PATHOLOGY : JOURNAL OF THE W.V.P.A DEC 1996 LNKD- PUBMED:18645896, vol. 25, no. 4, December 1996 (1996-12-01), pages 751 - 768, ISSN: 0307-9457 *
DARAL J. JACKWOOD: "Molecular identification of infectious bursal disease virus strains", VINELAND UPDATE, vol. 62, 1 November 1998 (1998-11-01), XP055020609 *
DATABASE UNIPROT [online] 1 March 2001 (2001-03-01), "SubName: Full=Viral protein 2 variable region; Flags: Fragment;", retrieved from EBI accession no. UNIPROT:Q9EM22 Database accession no. Q9EM22 *
DATABASE UNIPROT [online] 1 November 1996 (1996-11-01), "SubName: Full=VP2 capsid protein; Flags: Fragment;", retrieved from EBI accession no. UNIPROT:Q82633 Database accession no. Q82633 *
DATABASE UNIPROT [online] 1 October 2002 (2002-10-01), "SubName: Full=Viral protein 2; Flags: Fragment;", retrieved from EBI accession no. UNIPROT:Q8JYA4 Database accession no. Q8JYA4 *
JACKWOOD D J ET AL: "AMINO ACID COMPARISON OF INFECTIOUS BURSAL DISEASE VIRUSES PLACED IN THE SAME OR DIFFERENT MOLECULAR GROUPS BY RT/PCR-RFLP", AVIAN DISEASES, AMERICAN ASSOCIATION OF AVIAN PATHOLOGISTS, KENNET SQ., PA, US, vol. 45, no. 2, 1 April 2001 (2001-04-01), pages 330 - 339, XP008054150, ISSN: 0005-2086 *
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SHAHID AMIN ET AL: "Comparative sensitivity of different tests in the diagnosis of infectious bursal disease in broilers", INTERNATIONAL JOURNAL OF AGRICULTURE & BIOLOGY, vol. 1, no. 2, 1 January 1999 (1999-01-01), pages 48 - 50, XP055020603 *

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PL209994B1 (pl) 2011-11-30
AU2003227026A1 (en) 2003-09-16
WO2003074552A1 (fr) 2003-09-12
PL372453A1 (en) 2005-07-25

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