HK1095161B - Porcine circoviruses, nucleic acids, polypeptides and vaccines - Google Patents

Description

The present invention relates to novel porcine circovirus (PCV) strains responsible for post-weaning multisystemic wasting syndrome (PMWS), reagents and methods for their detection, vaccination methods and vaccines, and methods for the production of such reagents and vaccines.

PCV was originally detected as a non-cytopathogenic contaminant in PK/15 pig kidney cell lines. This virus was originally thought to be a Circoviridae with Chicken Anemia Virus (CAV) and PBFDV (Pscittacin Beak and Feather Disease Virus). These are small (15-24 nm) unenveloped viruses with the common feature of containing a genome in the form of a single circular DNA strand of 1.76 to 2.31 kb. This genome was originally thought to be coded for a polypeptide of about 30 kDa (Todd et al., 1991, Virol 117: 129-135). However, work has shown a more complex transcription (Meehan et al., 781-227: 22), and no other known sequence of three types of viruses has been reported.

PCV from PK/15 cells is considered non-pathogenic. The sequence is known from B. M. Meehan et al., J Gen Virol 1997 (78) 221-227. It is only very recently that authors have thought that PCV strains could be pathogenic and associated with PMWS syndrome (Gupi P. S. Nayar et al., Can Vet J, vol. 38, 1997: 385-387 and Clark E. G., Proc Am Assoc Prac Swine 1997: 499-501).

PMWS as detected in Canada, the United States and France is clinically characterised by progressive weight loss and manifestations such as tachypnea, shortness of breath and jaundice. Pathologically, it is characterised by lymphocytic or granulomatous infiltrations, lymphadenopathies and, more rarely, by lymphocytic or granulomatous hepatitis and nephritis (Clark E. G., Proc. Am. Assoc. Swine. 1997: 499-601; La Semaine Vétérinaire no. 26, supplement to La Semaine Vétérinaire 1996 (834) La Semaine Vétérinaire 1997 (857): 54 Prac G. S. Nayar et al., 1997: 385-38; J. Can., vol. 3887; J. Can. 1997: 385-37).

The applicant was successful in isolating five new PCV strains from pulmonary or ganglion samples from holdings in Canada, the United States (California) and France (UK), hereinafter referred to as circoviruses according to the invention. These viruses were detected in lesions in pigs with PMWS but not in healthy pigs.

The applicant also sequenced the genomes of four of these strains, namely the strains from Canada and the United States and two French strains. The strains have a very strong homology at the nucleotide level with each other exceeding 96%, and much less with the PK/15 strain, about 76%. The new strains can therefore be considered as representative of a new type of porcine circovirus, here referred to as type II, type I being represented by PK/15.

The present invention is defined by the claims that the porcine circovirus group II, as defined above, isolated or in purified form is described.

Any porcine circovirus which can be isolated from a physiological sample or tissue sample, including lesions, from a sick pig with PMWS syndrome, including by the method described in the examples, in particular type II circovirus, shall also be described.

The present invention describes in particular purified preparations of five strains, which were filed with the European Collection of Cell Cultures, Centre for Applied Microbiology & Research, Porton Down, Salisbury, Wiltshire SP4 OJG, United Kingdom on Thursday 2 October 1997: The Commission has not received any information from the Member States on the application of the provisions of the Directive. and, on Friday 16 January 1998: access number V98 011608 (hereinafter referred to as Imp 1011-48285)

The invention is intended to consider porcine circoviruses isolated from a sick pig and/or circoviruses having a significant serological affinity with the invention strains and/or circoviruses having cross-hybridation with the invention strains under strict conditions such that there is no hybridation with PCV PK/15 strain.

Viral strains isolated from a physiological sample or tissue sample, including a lesion, from a pig with PMWS syndrome can be advantageously propagated on cell lines such as pig kidney cell lines, in particular PK/15 cells free from contamination (in particular for PCV, as well as for pestiviruses, porcine adenoviruses and porcine parvoviruses) for multiplication or specifically for the production of antigen, whole (e.g. viruses) and/or subunits (e.g. polypeptides).

Remarkably and unexpectedly, these isolates have been shown to be highly productive in PK/15 cell culture, which has undeniable advantages for virus or antigen production, especially for inactivated vaccine production.

Circovirus preparations isolated from cell transitions, including cell lines, e.g. PK cells, cultured in vitro by infection with at least one of the circoviruses described herein or with any porcine circovirus that may be isolated from a physiological sample or tissue sample, including lesions, from a pig with PMWS syndrome, including surgical swabs or culture extracts, where appropriate purified by standard techniques, and in general any antigenic preparation obtained from in vitro cultures, are also described.

Immunogenic active substances and vaccines containing at least one antigen as defined above are also described.

These may be immunogenic active substances based on attenuated whole live viruses, or vaccines prepared with these active substances, with attenuation carried out by the usual methods, e.g. cell-passage, preferably pig cell-passage, including lineages, such as PK/15 cells (e.g. 50 to 150, especially 100 passages). These vaccines generally include a veterinary acceptable vehicle or diluent, possibly a veterinary acceptable adjuvant and possibly a freeze-drying stabilizer.

These antigenic preparations and vaccines will preferably include 103 to 106TCID50.

They may also be immunogenic active substances or vaccines based on the invention's circovirus antigen in the inactivated state. Vaccines may also include a veterinary acceptable vehicle or diluent, with a veterinary acceptable adjuvant if necessary.

Circoviruses, with any fractions that may be present, are inactivated by techniques known to the professional. Inactivation will preferably be carried out by chemical means, e.g. by exposure of the antigen to a chemical agent such as formaldehyde (formol), paraformaldehyde, β-propiolactone or ethylene imine or its derivatives. The preferred method of inactivation here will be exposure to a chemical agent and in particular to ethylene imine or ß-propiolactone.

Preferably, disclosed inactivated vaccines will be adjuvanted, preferably in the form of emulsions, e.g. water-in-oil or oil-in-water, according to techniques well known to the professional.

Examples of adjuvants that may be used include alumina hydroxide, saponins (e. g. Quillaja saponin or Quil A; see Vaccine Design, The Subunit and Adjuvant Approach, 1995, edited by Michael F. Powel and Mark J. Newman, Plennum Press, New-York and London, p. 210), Avridine® (Vaccine Design p. 148), DDA (Dimethyldioctadecylammonium bromide, Vaccine Design p. 167), lephosphazene (Vaccine Design p. 204), or also depending on the water-based emulsions of mineral oil, squid emulsion (e. g. SPT, squid emulsion in Vaccine Design p. 147 or P. 107A. 2007-59), or a combination of the preferred combination of Vaccine Design M-A, or Vaccine Design P. 1049 or M-A, as well as a combination of water-based emulsions of M-bol-O-E (e. g.

These vaccines will preferably include 106 to 108 TCID50.

The adjuvants of the live vaccine may be selected from those given for the inactivated vaccine. Emulsions are preferred. In addition to those indicated for the inactivated vaccine, those described in WO-A-9416681 may be added.

As freeze-drying stabilizers, examples include SPGA (Bovarnik et al., J. Bacteriology 59, 509, 950), carbohydrates such as sorbitol, mannitol, starch, sucrose, dextran or glucose, proteins such as albumin or casein, derivatives of these compounds, or buffers such as buffers of alkaline metal phosphates.

The applicant also obtained the genome of four of the isolates, identified as SEQ ID NO: 1 to 4 and possibly 6.

A DNA fragment containing all or part of one of these sequences is also described. Equivalent sequences, i.e. sequences that do not change the functionality or strain specificity of the described sequence or the polypeptides encoded by this sequence, are automatically overlaid. Sequences that differ by code degeneration are of course included.

Equivalent sequences are also covered in that they are capable of hybridizing with the above sequence under high stress conditions and/or have a strong homology with the strains of the invention and belong to the group defined above.

These sequences and fragments are used for the in vitro or in vivo expression of polypeptides using appropriate vectors.

In particular, open reading frames, forming DNA fragments according to the invention, which can be used for this purpose, have been identified in the genome sequence of circoviruses type II. Any polypeptide containing at least one such open reading frame (corresponding amino acid sequence) is disclosed. The invention concerns a protein formed mainly by COL4, COL7, COL10 or COL13.

The coding sequence (s) or their fragments are incorporated into the genome of the baculovirus (e.g. Autographa californica Nuclear Polyhedrosis Virus AcNPV) and the baculovirus is then propagated to insect cells, e.g. Spodoptera frugiperda Sf9 (ATCC CRL 1711 repository). The subunits can also be produced in eukaryotic cells such as yeast (e.g. Saccharomyces cerevisiae) or mammalian cells (e.g. CHOH, BK).

The invention also concerns polypeptides which will be produced in vitro by these means of expression and then possibly purified by conventional techniques. It also concerns subunit vaccines containing at least one polypeptide as obtained or fragment in a veterinary acceptable vehicle or diluent and possibly a veterinary acceptable adjuvant.

In vivo expression for the production of recombinant live vaccines, the coding sequence (s) or their fragments are inserted into an appropriate expression vector under conditions which allow expression of the polypeptide (s). Live viruses, preferably capable of multiplying in pigs, which are not pathogenic to pigs (naturally non-pathogenic or rendered as such), may be used as suitable vectors, according to techniques well known to the profession. In particular, pig herpesviruses such as Aujeszky's disease virus, swine avianovirus, smallpox viruses, including vaccine virus, smallpox, canarypox, swine pox. DNA can also be used as plastic vectors (WOWO-A-9092, WOWO-A-9093, WOWO-A9602, WOWO-A9792, WOWO-A9602, WOWO-A9602, WOWO-A9602, WOWO-A9602, WOWO-A9602, WOWO-A9602, WOWO-A9602, WOWO-A9602, WOWO-A9603, WOWO-A9602, WOWO-A9602, WOWO-A93, WO-A9602, WO-A93, WO-A93, WO-A93, WO-A93, WO-A93, WO-A93, WO-A93, WO-A93, WO-A93, WO-A93, WO-A93, WO-A93, WO-A93, WO-A94, WO-A94, WO-A95, WO-A95, WO-A95, WO-A95, WO-A95, WO-A95, WO-A95, WO-A95, WO-A95, WO-A95, WO-A95, WO-A95, WO-A95, WO-A95, WO-A95, WO-A95, WO-A95, WO-A95, WO-A93, WO-A93, WO-A93, WO-A93, WO-A93, WO-A93, WO-A93, WO-A93, WO-A93, WO-A93, WO-A93, WO-A93, WO

The invention therefore also covers vectors and recombinant or plasmidic live vaccines (polynucleotide or DNA vaccines) thus produced, vaccines also including a veterinary acceptable vehicle or diluent.

Vaccines according to the invention (subunits, recombinant organisms, plasmidics) may include one or more active ingredients (antigens) of one or more (2 or 3) circoviruses according to the invention.

For each of the types of vaccines described above, vaccination against porcine circovirus is described as being also associated with vaccination against other porcine pathogens, in particular those that may be associated with PMWS syndrome. The vaccines disclosed, including inactivated ones, may therefore include another valence corresponding to another porcine pathogen. These other porcine pathogens may include PRRS (Pig Reproductive and Respiratory Syndrome) (professional may refer to WO-A-93/07898, WO-A-94/18311, FR-A-2 709 966; C. Chyreare et al., Proceedings of the 15th IPVS Congress, Birmingham, 5 July 1998, pp. 139-95; for porcine pollen, including cholera and/or mycoplasma; and for porcine pollen, may refer to EPV, including cholera and/or mycoplasma; and for other relevant porcine diseases, see EPV, EPV, P. 585, pp. 47-57, and EPV, P. 587, and other relevant reports on the European Commission's influenza and influenza.

A method for inducing an immune response in pigs to the described circoviruses is also disclosed, in particular to develop an effective vaccination method in pigs.

This method involves the administration of one or more vaccines to pigs, and it is also possible to combine several types of vaccines in one vaccination protocol.

This method is not only used in adult pigs, but also in young pigs and pregnant piglets, and the vaccination of the latter gives passive immunity to the newborn piglets (mother's antibodies).

It also describes the possibility of diagnosing the presence of the circoviruses of the invention in pigs.

Knowledge of the sequences of the different circoviruses allows the definition of common sequences that allow the production of reagents capable of recognising all known porcine circoviruses.

The professional may also select sequence fragments from regions with little or no homology to the corresponding PK/15 circovirus sequence to enable a specific diagnosis.

The sequence alignments allow the craftsman to choose a reagent according to his wishes.

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A second reagent consists of polypeptides coded by these sequences from the virus or expressed by means of a vector (see above), or chemically synthesized by conventional peptide synthesis techniques.

A third and fourth reagents consist of polyclonal and monoclonal antibodies respectively which may be produced by the usual techniques from the virus, polypeptides or fragments, extracted or coded by DNA sequences.

These second, third and fourth reagents may be used in a diagnostic method in which the presence of a specific antigen of a circovirus of the invention is sought in a sample of physiological fluid (blood, plasma, serum, etc.) or tissue (ganglia, liver, lungs, kidneys, etc.) from a pig to be tested, by seeking to detect either the antigen itself or antibodies directed against this antigen.

Antigen and antibody can be used in all known laboratory diagnostic techniques.

However, it is preferable to use them in techniques which can be used directly in the field by the veterinarian, the breeder or the owner of the animal.

The diagnostic techniques which will be used in preference are Western Blot, immunofluorescence, ELISA and immunochromatography.

In the case of the implementation of immunochromatographic methods, the expert may refer in particular to Robert F. Zurk et al., Clin. Chem. 31/7, 1144-1150 (1985) and to the patents or patent applications WO-A-88/08 534, WO-A-91/12528, EP-A-291 176, EP-A-299 428, EP-A-291 194, EP-A-284 232, US-A-5 120 643, US-A-5 030 558, US-A-5 266 497, US-A-4 740 468, US-A-5 266 497, US-A-4 855 240, US-A-5 451 504, US-A-5 141 850, US-A-5 232 835 and US-A-5 238 652.

Thus, it is preferable to detect specific antibodies in the sample by indirect testing, by competition or by displacement. To do this, the antigen itself is used as a diagnostic reagent, or a fragment of this antigen, preserving the recognition of antibodies.

The antigen itself can also be detected in the sample by means of an antibody specifically marked for that antigen.

Antigen-specific antibodies, particularly those for use in competition or travel or for the detection of the antigen itself, are monoclonal and polyclonal antibodies specific to the antigen, fragments of these antibodies, preferably Fab or F (((ab) '2 fragments.

The production of antibodies, whether polyclonal or monoclonal, specific to the antigen of the invention is also described, and these antibodies may then be used, inter alia, as diagnostic reagents for the detection of the antigen in a sample of physiological fluid or tissue, or even for the detection of antibodies present in such a sample or sample.

Antibodies may be prepared by the usual techniques, such as Antibodies, A Laboratory Manual, 1988, Cold Spring Harbor Laboratory, USA or J.W. Goding, Monoclonal Antibodies: Principles and Pratice, Academic Press Inc., the contents of which are incorporated herein by reference.

In particular, as is well known, it will be possible to fuse splenic cells from mice immune to the antigen or at least one of its fragments with appropriate myeloma cells.

A preparation, preferably pure or partially purified, or even raw, of antigen-specific monoclonal or polyclonal antibodies, including antibodies from mice or rabbits, is also described.

It is also possible to determine epitopes of interest, particularly on the basis of the DNA sequences described here, whether they are epitopes of vaccine interest or epitopes of diagnostic interest. From the DNA sequence of the circovirus genome according to the invention, the professional is able to determine epitopes using known methods e.g. appropriate computer program or PEPSCAN. Epitopes are immune-dominant regions of proteins and are therefore regions exposed to the surface of proteins. They can therefore be recognized by antibodies and thus be particularly used in the field of diagnosis either for the preparation of antibodies for diagnostic purposes or for the production of corresponding peptides that are reactive to titers.

At a minimum, an epitope is a peptide with 8 to 9 amino acids, and a minimum of 13 to 25 amino acids is usually preferred.

The professional is therefore able, using one or more of these techniques as well as other available techniques, to find epitopes for the implementation of peptides or antibodies for diagnostic purposes.

A diagnostic kit containing this antigen and/or specific polyclonal or monoclonal antibodies to this antigen is also described, in particular diagnostic kits corresponding to the diagnostic techniques described above.

Non-limiting examples of implementation are now described, taken with reference to the drawing in which: Figure 1: DNA sequence from the genome of the Imp 1011-48121 strainFigure 2: DNA sequence from the Imp 1011-48285 strainFigure 3: DNA sequence from the Imp 999 strainFigure 4: DNA sequence from the Imp 1010 strainFigure 5: alignment of the 4 sequences from Figures 1 to 4 with the PCV PK/15 strainFigure 6: DNA sequence from the Imp 999 strain as defined in the first filing in France on 3 October 1997Figure 7 : Sequence alignments from Figure 6 to PK/15

List of sequences of the SEQ ID

SEQ ID NO: 1 DNA sequence from the genome of the Imp 1011-48121SEQ ID NO: 2 DNA sequences from the Imp 1011-48285SEQ ID NO: 3 DNA sequences from the Imp 999SEQ ID NO: 4 DNA sequences from the Imp 1010SEQ ID NO: 5 DNA sequences from the PK/15SEQ ID NO: 6 DNA sequences from the Imp 999 strain genome as defined in the first filing in France of 3 October 1997.

Examples Example 1: culture and isolation of porcine circovirus strains:

Tissue samples were collected in France, Canada and the USA from piglets' lungs and lymph nodes, which showed clinical signs typical of post-weaning generalized wasting syndrome. To facilitate virus isolation, tissue samples were frozen at -70°C immediately after autopsy.

For viral isolation, suspensions containing approximately 15% of tissue sample were prepared in a minimum medium containing earl salts (EMEM, BioWhittaker UK Ltd., Wokingham, UK), penicillin (100 Ul/ml) and streptomycin (100 μg/ml) ((MEM-SA medium), by grinding the tissue with sterile sand using a sterile mortar and pestle. This ground preparation was then taken back into MEM-SA and then centrifuged at 3000 g for 30 minutes at + 4°C to harvest the supernatant.

Prior to seeding the cell cultures, a volume of 100 μl of chloroform was added to 2 ml of each surfactant and continuously mixed for 10 minutes at room temperature. This mixture was then transferred to a microcentrifuge tube, centrifuged at 3000 g for 10 minutes, and then the surfactant was harvested. This surfactant was then used as an inoculum for viral isolation experiments.

All viral isolation studies were performed on PK/15 cell cultures known to be free from porcine circovirus (PCV), pestiviruses, porcine adenoviruses and porcine parvovirus (Allan G. et al. Pathogenesis of porcine circovirus experimental infections of colostrum-deprived piglets and examination of pig-foetal material, Vet. Microbiol, 1995. 44. 49-64).

The isolation of porcine circoviruses was carried out using the following technique:

PK/15 monolayers were separated by trypsination (with a trypsin-versene mixture) from the confluent cultures and taken up in MEM-SA medium containing 15% of uncontaminated pestivirus (= MEM-G medium) calf foetal serum at a final concentration of approximately 400,000 cells per ml. Aliquot fractions of 10 ml of this cell suspension were then mixed with aliquot fractions of 2 ml of the inoculums described above, and the final mixtures were aliquoted in 6 ml volumes in two 25 cm Falcon vials. These cultures were then incubated at +37 °C for 18 hours in an atmosphere containing 10% CO2.

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Example 2: Preparation of cell culture samples for the detection of porcine circoviruses by immunofluorescence or in situ hybridisation.

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Example 3: Techniques for detecting PCV sequences by in situ hybridisation

In situ hybridisation was carried out on tissues taken from sick pigs and fixed with formaldehyde and also on inoculated cell culture preparations for viral isolation (see example 2) and fixed on glass lamellae.

Complete genomic probes corresponding to porcine circovirus PK/15 (PCV) and chicken anemia virus (CAV) were used. The plasmid pPCV1, containing the replicative form of the PCV genome cloned as a single 1.7 kilopari base (kpb) insert (Meehan B. et al. Sequence of porcine circovirus DNA: affinities with plant circoviruses. J. Gen. Virol. 1997. 78. 221-227) was used as a source of specific PCV DNA. An analogue plasmid, pCAA1, containing the replicative form 2,3b of the CAV negative avian circovirus was used as a control. The glycogen stocks of these two plasmids were used for the production of plasmids and for the purification of Jambro alkali (J.S.Err1:Expecting ',' delimiter: line 1 column 513 (char 512)

The digoxygenine-labelled probes were recovered under a volume of 50-100 pcs sterile water before use for in situ hybridisation.

Formaldehyde-bound tissue samples from sick pigs, including paraffin, and formaldehyde-bound cultures of infected cells were prepared for PCV nucleic acid detection using the following technique:

5 pm-thick sections were cut from the blocks of tissue included in the paraffin, deparaffined, and then rehydrated in successive solutions of decreasing alcohol concentration. The tissue sections and cell cultures fixed to formaldehyde were incubated for 15 minutes and 5 minutes at +37°C respectively in a 0.5% proteinase K solution in Tris-HCl buffer 0.05M, EDTA 5 mM (pH 7.6). The blades were then placed in a 1% glycine solution in autoclaved distilled water for 30 seconds, washed twice with a PBS buffer 0.01 M (pH 7.2), and then sterilized for 5 minutes. Finally, they were rescued and placed in contact with the probes.

Each fabric/probe preparation was coated with a clean, degreased lamella, then placed in an oven at +90°C for 10 minutes, then exposed to an ice pack for 1 minute, and finally incubated for 18 hours at +37°C. The preparations were then briefly immersed in a sodium citrate (SSC) 2X (pH 7.0) salt buffer to remove the protective lamellae, then washed twice for 5 minutes in SSC 2X buffer and finally washed twice for 5 minutes in PBS buffer.

After these washes, the preparations were immersed in a solution of 0.1 M maleic acid, NaCl 0.15 M (pH 7.5) (maleic buffer) for 10 minutes and then incubated in a 1% solution of blocking reagent (Cat # 1096176, Boehringer Mannheim UK, Lewis, East Sussex, UK) in maleic buffer for 20 minutes at + 37 °C.

The preparations were then incubated with 1/260 anti-digoxygen monoclonal antibody (Boehringer Mannheim) solution, diluted in a blocking buffer for 1 hour at + 37 °C, washed in PBS and finally incubated with a biotinyled anti-mouse immunoglobulin antibody for 30 minutes at + 37 °C. The preparations were washed in PBS and the endogenous peroxidase activity was blocked by treatment with a 0.5% hydrogen peroxide solution in PBS for 20 minutes at room temperature. The preparations were re-tested in PBS and treated with a 3-amino-9-diethylcarbazole (AEC) substrate (Cambridge Bioscience, UK) prepared ex-temperature.

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Example 4: Immunofluorescence technique for the detection of PCV

Initial screening of all acetone-bound cell culture preparations was performed by an indirect immunofluorescence (IFI) technique using a 1/100 dilution of a pool of adult pig serum. This pool of serums includes serums from 26 adult sows from Northern Ireland and is known to contain antibodies against a wide variety of pig viruses, including PCV: porcine parvovirus, porcine adenovirus, and PRRS virus. The IFI technique was performed by contacting the serum (dilute in PBS) with the cell cultures for one hour at + 37°C, followed by two washes in PBS ultraviolet. The coloured cells then with a 1/80 dilution of PBS antigen are stained with PBS antigen in conjunction with a PBS immunoglobulin in a microscopic swab and then stained in PBS glucose swab during the observation.

Example 5: Results of in situ hybridisation on diseased pig tissues

In situ hybridization using a PCV genomic probe, performed on tissues taken from French, Canadian and California piglets with formaldehyde-bound generalized decay lesions, revealed the presence of PCV nucleic acids associated with lesions in several of the lesions studied. No signals were observed when the PCV genomic probe was used on tissues taken from unsick pigs or when the CAV probe was used on tissues from sick pigs. The presence of PCV nucleic acid was identified in the cytoplasm and nucleus of many mononuclear cells infiltrating the lesions in California piglets. The presence of PCV nucleic acid was also found in small cells, lymphocytes, bronchioles, endothelial cells and bronchioles, and in the epithelial cells of the California piglets.

In sick French pigs, PCV nucleic acid was detected in the cytoplasm of many follicular lymphocytes and in intrasynuclear mononuclear cells of lymph nodes. PCV nucleic acid was also detected in occasional syncytias. Based on these detection results, samples of California pig lungs, French pig mesenteric lymph nodes, and Canadian pig organs were selected for the purpose of isolating the new porcine circovirus strains.

Example 6: Results of cell culture of new porcine circovirus strains and immunofluorescence detection

No cytopathic effect (ECP) was observed in inoculated cell cultures with samples taken from French piglets (strain Imp.1008), California piglets (strain Imp.999) and Canadian piglets (strain Imp.1010) showing clinical signs of generalized wasting syndrome. However, immuno-marking of preparations from inoculated cell cultures, after attachment to acetone and with a pool of porcine polyclonal serums, revealed nuclear fluorescence in many cells in cultures inoculated from California piglets' lungs (strain Imp.999), from French piglets' lymph nodes (strain Imp.1008), and from porcine canine organs (strain Imp.109).

Example 7: Extraction of genomic DNA from porcine circoviruses

Replicative forms of the new porcine circovirus (PCV) strains were prepared from cultures of infected PK/15 cells (see example 1) (10 75 cm2 Falcons) harvested after 72-76 hours of incubation and treated with glucosamine, as described for cloning the replicative form of the CAV (Todd. D. et al. Dot blot hybridization assay for chicken anemia agent using a cloned DNA probe. J. Clin. Microbiol. 1991. DNA 29 933-939).

Example 8: Map of restriction of the replicative form of the genome of porcine circovirus strain Imp.999.

The DNA (1-5 μg) extracted by Hirt's technique was treated by S1 nuclease (Amersham) as recommended by the supplier, then this DNA was digested by different restriction enzymes (Boehringer Mannheim, Lewis, East Sussex, UK) and the products of digestion were separated by electrophoresis on 1.5% agarose gel in the presence of ethidium bromide as described by Todd et al. (Purification and biochemical characterization of chicken anemia agent. J. Gen. Virol. 1990. 71. 819-823). - The DNA extracted from cultures of the strain Ecocov.999 has a single site, 2 sites and therefore no site Pstl. This restriction profile is different from the restriction profile presented by the PCRI PKV and Pstl. Sequence of Pstl. B. and B. Sequence of Pstl. B. and Pstl. 78. 221-227.

Example 9 : Cloning of the genome of porcine circovirus strain Imp.999

The restriction fragment of approximately 1.8 kpb generated by digestion of the double stranded replicative form of the PCV Imp.999 strain with the EcoRI restriction enzyme was isolated after electrophoresis on 1.5% agarose gel (see example 3) using a commercial Qiagen (QIAEXII Gel Extraction Kit, Cat # 20021, QIAGEN Ltd., Crawley, West Sussex, UK). This EcoRI-EcoRI restriction fragment was then ligated to the pGEM-7 vector (Promega, Medical Supply Company, Dublin, Ireland), previously digested by the same restriction enzymes and phosphorylated, following standard cloning techniques (Sambrook J. et al. Molecular cloning: A Laboratory Manual).The resulting plasmids were transformed into a host strain of Escherichia coli JM109 (Stratagene, La Jolla, USA) using standard techniques. The EcoRI-EcoRI restriction fragment of the PCV Imp.999 strain was also cloned at the pBlueScript SK+ vector EcoRI site (Stratagene Inc. La Jolla, USA). From the clones obtained for each host strain, at least 2 clones containing the expected size fragments were selected. The resulting clones were then cultured and the plasmids containing the complete Imp strain genome were cultured.999 were purified in small volume (2 ml) or large volume (250 ml) using standard plasmid preparation and purification techniques.

The following is a list of the most commonly used methods for the determination of the DNA sequence of the PCV strain Imp.999.

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The complete sequence (EcoRI-EcoRI fragment) originally obtained from the pGEM-7/8 clone (SEQ ID NO: 6) is shown in Figure 6.

The sequencing was then optimized and the SEQ ID NO: 3 (Figure 3) gives the total sequence of this strain, which was started arbitrarily at the beginning of the EcoRI site, i.e. G as the first nucleotide.

A similar procedure was followed for the sequence of the other three isolates according to the invention (see SEQ ID NO: 1, 2 and 4 and Figures 1, 2 and 4).

The genome size of these four strains is: - What?

Imp 1011-48121	1767 nucléotides
Imp 1011-48286	1767 nucléotides
Imp 999	1768 nucléotides
Imp 1010	1768 nucléotides

Example 11 - Sequence analysis of the PCV Imp.999 strain

When the sequence generated from Imp.999 strain was used for a homology search with sequences in the GenBank database, the only significant homology detected was a homology of approximately 76% (at the nucleic acid level) with the PK/15 strain sequence (access numbers Y09921 and U49186) (see Figure N°5).

At the amino acid level, the search for homology of the 6-phase sequence translation with the data banks (BLAST X algorithm on the NCBI server) revealed a 94% homology with the open reading frame corresponding to the theoretical replicase of the BBTV virus similar to plant circoviruses (identification number GenBank 1841515) encoded by the GenBank sequence U49186.

No other sequence in the databases shows significant homology with the sequence generated from PCV Imp.999.

Analysis of sequences obtained from Imp.999 strain cultured from lesions taken from California piglets with clinical signs of generalized decay syndrome clearly shows that this virus isolate is a new strain of porcine circovirus.

Example 12 - Comparative analysis of sequences

The nucleotide sequences of the four new PCV strains were aligned with the sequence of the PK/15 PCV strain (Figure 5). A homology matrix taking into account the four new and the previous PK/15 strain was obtained. - What? 1 Imp 1011-48121 is the 2 Imp 1011-48285 and Imp 3: 999 4: Imp 1010 5: PK/15 - What?

1	1,0000	0,9977	0,9615	0,9621	0,7600
2		1,0000	0,9621	0,9632	0,7594
3			1,0000	0,9949	0,7560
4				1,0000	0,7566
5					1,0000

The homology between the two French strains Imp 1011-48121 and Imp 1011-48285 is more than 99%.

The homology between the two North American strains Imp 999 and Imp 1010 is also over 99%.

The homology of all these strains with PK/15 falls to a value of between 75 and 76%.

It follows that the strains of the invention are representative of a new type of porcine circovirus, distinct from the type represented by the PK/15 strain. This new type, isolated from pigs with PMWS syndrome, is referred to as porcine circovirus type II, with PK/15 representing type I. The strains of this type show remarkable homogeneity in nucleotide sequence, even when isolated from very distant geographical regions.

Example 13: Analysis of the proteins encoded by the genome of the new PCV strains.

The nucleotide sequence of the Imp. 1010 isolate was considered representative of other circovirus strains associated with generalized decay syndrome. This sequence was further analysed using the BLASTX algorithm (Altschul et al. J. Mol. Biol. 1990. 215. 403-410) and a combination of programs from the MacVector 6.0 software package (Oxford Molecular Group, Oxford OX4 4GA, UK). 13 frames or reading vertices (or COLs) of more than 20 amino acids in size were detected on this sequence (circular COLs). These 13 COLs are as follows: - What?

Nom	Début	Fin	Brin	Taille du COL (nucléotides (nt))	Taille protéine (acides aminés (aa))
COL1	103	210	sens	108 nt	35 aa
COL2	1180	1317	sens	138 nt	45 aa
COL3	1363	1524	sens	162 nt	53 aa
COL4	398	1342	sens	945 nt	314 aa
COL5	900	1079	sens	180 nt	59 aa
COL6	1254	1334	sens	81 nt	26 aa
COL7	1018	704	antisens	315 nt	104 aa
COL8	439	311	antisens	129 nt	42 aa
COL9	190	101	antisens	90 nt	29 aa
COL10	912	733	antisens	180 nt	59 aa
COL11	645	565	antisens	81 nt	26 aa
COL12	1100	1035	antisens	66 nt	21 aa
COL13	314	1381	antisens	702 nt	213 aa

The beginning and end positions of each LOC refer to the sequence shown in Figure 4 (SEQ ID No 4) of the genome of strain 1010. The boundaries of LOCs 1 to 13 are identical for strain 999. - What?

COL3	1432-1539, sens, 108 nt, 36aa
COL13	314-1377, antisens, 705 nt, 234 aa.

Of these 13 LCOs, 4 show significant homology with analogous LCOs located on the genome of cloned PCV PK-15 virus. Each of the open reading frames present on the genome of all circovirus isolates associated with GFS was analysed. These 4 LCOs are: - What?

Nom	Début	Fin	Bdn	Taille du COL (nt)	Taille protéine (acides aminés)	Masse moléculaire
COL4	398	1342	sens	945 nt	314 aa	37,7 kDa
COL7	1018	704	antisens	315 nt	104 aa	11,8 kDa
COL10	912	733	antisens	180 nt	59 aa	6,5 kDa
COL13	314	1381	antisens	702 nt	233 aa	27,8 kDa

The start and end positions of each LCO refer to the sequence shown in Figure 4 (SEQ ID No 4).

Comparison of the genomic organization of PCV Imp. 1010 and PCV PK-15 isolates has led to the identification of 4 conserved LCOs in the genome of both viruses. - What?

COL4/COL1	86 %
COL13/CO12	66,4 %
COL7/COL3	61,5 % (au niveau du recouvrement (104 aa)
COL10/COL4	83 % (au niveau du recouvrement (59 aa)

The highest sequence identity was observed between COL4 Imp. 1010 and COL1 PK-15 (86% homology), which was expected since this protein is likely involved in viral DNA replication and is essential for viral replication (Meehan et al. J. Gen. Virol. 1997. 78. 221-227; Mankertz et al. J. Gen. Virol. 1998. 79. 381-384).

Sequence identity between COL13 Imp. 1010 and COL2 PK-15 is less strong (66.4% homology), but each of these two COLs does have a highly conserved N-terminal baseline region, which is identical to the N-terminal region of the major structural protein of avian circovirus CAV (Meehan et al. Arch. Virol. 1992. 124.301-319). Larger differences are observed between COL7 Imp. 1010 and COL3 PK-15 and between COL10 Imp. 1010 and COL4 PK-15. In each case, there is a high deletion of the C-terminal region of COL7 and COL10 homology of the isolate 1010 when compared to the PKL3 and PKL4 regions of PCLV-15.

It appears that the genomic organization of the porcine circovirus is quite complex due to the extreme compactness of its genome. The major structural protein is likely to be the result of splicing between multiple reading frames on the same porcine circovirus genome strand. Therefore, it can be considered that any open reading frame (COL1 to COL13) as described in the table above may represent all or part of an antigenic protein encoded by the porcine circovirus type II and is therefore potentially a pre-protein for specific diagnosis and/or vaccination. The invention therefore concerns a protein comprising at least one of these COLs. Preferably, the invention concerns a protein formed primarily by COL7, COL10, COL13 or COL4.

Example 14: Infectious character of PCV genome cloned from new strains.

The pGEM-7/8 plasmid containing the complete genome (replicative form) of the Imp.999 isolate was transfected into PK/15 cells using the technique described by Meehan B. et al. (Characterization of viral DNAs from cells infected with chicken anemia agent sequence analysis of the cloned replicative form and transfection capabilities of cloned genome fragments. Arch. Virol. 1992. 124. 301-319). Immunofluorescence analysis (see example 4) performed on the first pass after transfection on undiagnosed PK/15 cells showed that the pGEM7/8 clone plasmid was capable of inducing the production of infectious PCV viruses. The availability of a clone containing an infectious PCV gene material on the PCV genome allows any useful virus to be produced (or used for the production of viruses or viruses) or to be manipulated in the production of viruses or viruses (such as vaccines, vaccines, or antibiotics).

Example 15: Production of PCV antigens by in vitro culture

The culture of uncontaminated PK/15 cells and viral multiplication are carried out in the same manner as in example 1.Infected cells are harvested after trypsination after 4 days of incubation at 37 °C and numbered.The next step is inoculated with 400 000 infected cells per ml.

Example 16: Inactivation of viral antigens

At the end of viral culture, the infected cells are harvested and lysed by ultrasound (Branson Sonifier) or using a rotor-stator-type colloidal mill (UltraTurrax, IKA). The suspension is then centrifuged at 3700 g for 30 minutes. The viral suspension is inactivated by 0.1% ethylene imine for 18 hours at + 37°C or by 0.5% beta-propiolactone for 24 hours at + 28°C. If the titer of the virus before inactivation is insufficient, the viral suspension is concentrated by ultrafiltration using a membrane with a cutting threshold of 300 kTM (Dalipore PK300). The inactivated viral suspension is kept at + 5°C.

Example 17: Preparation of the vaccine as a mineral oil emulsion.

The vaccine is prepared according to the following formula: Suspension of inactivated porcine circovirus: 250 mlMontanide® ISA 70 (SEPPIC): 750 ml The suspension is administered as a single dose.

The aqueous phase and the oily phase are sterilised separately by filtration.

One dose of vaccine contains approximately 107.5 DICT50 The volume of one dose of vaccine is 0.5 ml for intradermal administration and 2 ml for intramuscular administration.

Example 18: Preparation of the vaccine as an emulsion based on a metabolizable oil.

The vaccine is prepared according to the following formula: Suspension of inactivated porcine circovirus: 200 mlDehymuls HRE 7 (Henkel) is 60 mlRadia 7204 (Oleofina) is 740 ml

The aqueous phase and the oily phase are sterilized separately by filtration.

One dose of vaccine contains approximately 107.5 DICT50 The volume of one dose of vaccine is 2 ml for intramuscular administration.

Example 19: Results of indirect immunofluorescence against US and French PCV strains and contaminant PK/15 with hyperimmune serum (PCV-T), a panel of F99 monoclonal antibodies prepared from PK/15 and a hyperimmune serum prepared from the Canadian strain (PCV-C)

VIRUS
	PK/15	USA	France
PCV-T antiserum	≥ 6 400	200	800
PCV-C antiserum	200	≥ 6,400	≥ 6,400
F99 1H4	≥ 10 000	< 100	100
F99 4B10	≥ 10000	< 100	< 100
F99 2B7	≥ 10 000	100	< 100
F99 2E12	≥ 10 000	< 100	< 100
F99 1C9	≥ 10 000	< 100	100
F99 2E1	≥ 10 000	< 100	< 100
F99 1H4	≥ 10 000	100	< 100

* inverse de la dernière dilution du sérum ou de l'anticorps monoclonal qui donne une réaction positive en immunofluorescence indirecte.

Claims (20)

1. Expression vector comprising a nucleotidic sequence of a type II porcine circovirus (PCV II) responsible for post-weaning multisystemic wasting syndrome (PMWS), wherein said vector said nucleotidic sequence comprises an open reading frame (ORF) of the type II porcine circovirus (PCV II) said open reading frame being selected from the group consisting of:

   - the open reading frame 4 (ORF 4) comprised of nucleotides 398 to 1342 of the sense strand of the sequence entered under the reference SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:6,

   - the open reading frame 13 (ORF 13) comprised of nucleotides 314 to 1377 of the antisense strand of the sequence entered under the reference SEQ ID NO: 1 or SEQ ID NO:2 or of nucleotides 314 to 1381 of the antisense strand of the sequence entered under the reference SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:6,

   - the open reading frame 7 (ORF 7) comprised of nucleotides 1018 to 704 of the antisense strand of the sequence entered under the reference SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:6, and

   - the open reading frame 10 (ORF 10) comprised of nucleotides 912 to 733 of the antisense strand of the sequence entered under the reference SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:6,

   said expression vector expressing in vitro or in vivo said open reading frame of the type II porcine circovirus.
2. Expression vector according to claim 1, wherein said vector the nucleotidic sequence comprises the open reading frame 4 (ORF 4) or the open reading frame 13 (ORF 13).
3. Expression vector according to claim 1 or claim 2, wherein the vector is a baculovirus or a plasmid.
4. Expression vector according to claim 1 or claim 2, wherein the vector is suitable for expressing in vivo.
5. Expression vector according to claim 4, wherein the vector is a live virus or a plasmid.
6. Expression vector according to claim 5, wherein the vector is selected from among porcine herpes viruses, porcine adenovirus and poxviruses.
7. Expression vector according to claim 6, wherein the vector is selected from among Aujeszky's disease virus, vaccinia virus, avipox virus and swinepox virus.
8. Expression vector according to claim 7, wherein the vector is the canarypox virus.
9. Expression vector according to any of claims 4 to 8, wherein the expression vector is used to induce an immune response in pigs against a type II porcine circovirus (PCV II).
10. A cell expressing in vitro a polypeptide of a type II porcine circovirus (PCV II), the cell comprising an expression vector according to any of claims 1 to 3.
11. Cell according to claim 10, wherein the cell is an E. coli cell or a eukaryotic cell.
12. Cell according to claim 11, wherein the eukaryotic cell is a yeast.
13. Cell according to claim 11, wherein the eukaryotic cell is a mammalian cell.
14. Cell according to claim 11, wherein the eukaryotic cell is an insect cell.
15. Polypeptide encoded by a DNA fragment comprising an open reading frame (ORF) of a type II porcine circovirus (PCV II), the open reading frame being selected from the group consisting of:

    - the open reading frame 4 (ORF 4) comprised of nucleotides 398 to 1342 of the sense strand of the sequence entered under the reference SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:6,

    - the open reading frame 13 (ORF 13) comprised of nucleotides 314 to 1377 of the antisense strand of the sequence entered under the reference SEQ ID NO:1 or SEQ ID NO:2 or of nucleotides 314 to 1381 of the antisense strand of the sequence entered under the reference SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:6,

    - the open reading frame 7 (ORF 7) comprised of nucleotides 1018 to 704 of the antisense strand of the sequence entered under the reference SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:6, and

    - the open reading frame 10 (ORF 10) comprised of nucleotides 912 to 733 of the antisense strand of the sequence entered under the reference SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:6.
16. Polypeptide according to claim 15, wherein the open reading frame of PCV II is the open reading frame 4 (ORF 4) or the open reading frame 13 (ORF 13).
17. Polypeptide according to claim 15 or claim 16, wherein the polypeptide is used to induce an immune response in pigs against a type II porcine circovirus (PCV II).
18. Subunit vaccine comprising at least one polypeptide according to claim 15 or claim 16, in veterinarily acceptable vehicle or diluent.
19. Subunit vaccine according to claim 18 further comprising a veterinarily acceptable adjuvant.
20. Vaccine comprising at least one expression vector according to any of claims 4 to 8 in a veterinarily acceptable vehicle or diluent.